The invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway. The invention additionally provides a method for producing 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid. The method can include culturing a 6-aminocaproic acid, caprolactam or hexametheylenediamine producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid.
1. Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from 2-oxoheptane-1,7-dioate (2-OHD), using at least one biocatalyst, wherein the method further comprises converting adipate semialdehyde to the 6-aminocaproic acid or converting the 2-OHD to 2-aminoheptane-1,7-dioate (2-AHD), using at least one biocatalyst, wherein the biocatalyst comprises an enzyme capable of catalysing a transamination and/or a reductive amination or a decarboxylation.
2. Method according to
3. Method according to
α-ketoglutarate aminotransferases, β-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipate aminotransferases (EC 2.6.1.39), 5-aminovalerate aminotransferases (EC 2.6.1.48), 2-aminohexanoate aminotransferases (EC 2.6.1.67), lysine:pyruvate 6-aminotransferases (EC 2.6.1.71), and lysine-6-dehydrogenases (EC 1.4.1.18).
4. Method according to
5. Method according to
(i) an enzyme from Vibrio fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus subtilis, or Pseudomonas aeruginosa that catalyses the conversion of adipate semialdehyde to 6-aminocaproic acid;
(ii) an enzyme from Vibrio fluvialis, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus subtilis, Rhodobaeter sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas aeruginosa, or Rhodopseudomonas palustris that catalyses the conversion of 2-OHD to 2-AHD; or
(iii) a gene product of gabT from Escherichia coli, puuE from Escherichia coli, abat from Mus musculus, gabT from Pseudomonas fluorescens, or abat from Sus scrofa.
6. Method according to
7. Method according to
8. Method according to
9. Method according to
10. Method according to
(i) an enzyme from Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, Lactococcus lactis or Mycobacterium tuberculosis that catalyses the conversion of 2-OHD to adipate semialdehyde or 2-AHD to 6-aminocaproic acid; or
(ii) a gene product of pdc from Zymomonas mobilis, pdc l from Saccharomyces cerevisiae, pdc from Acetobacter pasteurians, pdc l from Kluyveromyces lactis, mdIC from Pseudomonas putida, mdIC from Pseudomonas aeruginosa, dpgB from Pseudomonas stutzeri, ilvB-1 from Pseudomonas fluorescens, kgd from Mycobacterium tuberculosis, kgd from Bradyrhizobium japonicum, kgd from Mesorhizobium loti, kdcA from Lactococcus lactis, BCKDHB from Homo sapiens, BCKDHA from Homo sapiens, BCKDHB from Bos taurus, BCKDHA from Bos taurus, panD from Escherichia coli K12, panD from Corynebacterium glutamicum or panD from Mycobacterium tuberculosis.
11. Method according to
12. Method according to
13. Method according to
14. Method for preparing caprolactam, comprising cyclising the 6-aminocaproic acid prepared by a method according to
15. Method according to
16. Method according to
17. Method according to
18. Method according to
19. Method according to
20. Method according to
21. Method according to
22. Method according to
23. Method according to
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This application is a continuation of U.S. application Ser. No. 12/776,365, filed May 7, 2010, which claims the benefit of priority of U.S. Provisional Application Ser. No. 61/176,196, filed May 7, 2009, U.S. Provisional Application Ser. No. 61/219,365, filed Jun. 22, 2009, U.S. Provisional Application Ser. No. 61/244,844, filed Sep. 22, 2009, U.S. Provisional Application Ser. No. 61/246,973, filed Sep. 29, 2009, and U.S. Provisional Application Ser. No. 61/247,533, filed Sep. 30, 2009, each of which the entire contents are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 11, 2012, is named Sequence_Listing.txt and is 35,150 bytes in size.
The present invention relates generally to biosynthetic processes, and more specifically to organisms having adipate, hexamethylenediamine, 6-aminocaproic acid and caprolactam biosynthetic capability.
Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. It can be used is to produce nylon 6,6, a linear polyamide made by condensing adipic acid with hexamethylenediamine. This is employed for manufacturing different kinds of fibers. Other uses of adipic acid include its use in plasticizers, unsaturated polyesters, and polyester polyols. Additional uses include for production of polyurethane, lubricant components, and as a food ingredient as a flavorant and gelling aid.
Historically, adipic acid was prepared from various fats using oxidation. Some current processes for adipic acid synthesis rely on the oxidation of KA oil, a mixture of cyclohexanone, the ketone or K component, and cyclohexanol, the alcohol or A component, or of pure cyclohexanol using an excess of strong nitric acid. There are several variations of this theme which differ in the routes for production of KA or cyclohexanol. For example, phenol is an alternative raw material in KA oil production, and the process for the synthesis of adipic acid from phenol has been described. The other versions of this process tend to use oxidizing agents other than nitric acid, such as hydrogen peroxide, air or oxygen.
In addition to hexamethylenediamine (HMDA) being used in the production of nylon-6,6 as described above, it is also utilized to make hexamethylene diisocyanate, a monomer feedstock used in the production of polyurethane. The diamine also serves as a cross-linking agent in epoxy resins. HMDA is presently produced by the hydrogenation of adiponitrile.
Caprolactam is an organic compound which is a lactam of 6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocaproic acid). It can alternatively be considered cyclic amide of caproic acid. One use of caprolactam is as a monomer in the production of nylon-6. Caprolactam can be synthesized from cyclohexanone via an oximation process using hydroxylammonium sulfate followed by catalytic rearrangement using the Beckmann rearrangement process step.
Methods for effectively producing commercial quantities of compounds such as hexamethylenediamine, 6-aminocaproic acid, levulinic acid and carpolactamare described herein and include related advantages.
The invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam or hexametheylenediamine pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway. The invention additionally provides a method for producing 6-aminocaproic acid, caprolactam or hexametheylenediamine. The method can include culturing a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid.
The present invention is directed to the design and production of cells and organisms having biosynthetic production capabilities for 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The results described herein indicate that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in Escherichia coli and other cells or organisms. Biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis, including under conditions approaching theoretical maximum growth.
As disclosed herein, a number of metabolic pathways for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid are described. Two routes, the reverse adipate degradation pathway and the 3-oxoadipate pathway, were found to be beneficial with respect to: (i) the adipate yields (92% molar yield on glucose), (ii) the lack of oxygen requirement for adipate synthesis, (iii) the associated energetics, and (iv) the theoretical capability to produce adipate as the sole fermentation product. Metabolic pathways for adipate production that pass through a-ketoadipate or lysine are also described but are lower yielding and require aeration for maximum production. A pathway for producing either or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is also disclosed herein.
As disclosed herein, a number of exemplary pathways for biosynthesis of adipate are described. One exemplary pathway involves adipate synthesis via a route that relies on the reversibility of adipate degradation as described in organisms such as P. chrysogenum (see Examples I and II). A second exemplary pathway entails the formation of 3-oxoadipate followed by its reduction, dehydration and again reduction to form adipate (see Examples III and IV). The adipate yield using either of these two pathways is 0.92 moles per mole glucose consumed. The uptake of oxygen is not required for attaining these theoretical maximum yields, and the energetics under anaerobic conditions are favorable for growth and product secretion. A method for producing adipate from glucose-derived cis,cis-muconic acid was described previously (Frost et al., U.S. Pat. No. 5,487,987, issued Jan. 30, 1996)(see Example V). Advantages of the embodiments disclosed herein over this previously described method are discussed. Metabolic pathways for adipate production that pass through α-ketoadipate (Example VI) or lysine (Example VII) precursors are lower yielding and require aeration for maximum production. A pathway for producing either or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is described (see Example VIII and IX). Additional pathways for producing adipate are described in Examples X and XI. Pathways for producing any one, two, three or all four of 6-aminocaproate, caprolactam, hexamethylenediamine and levulinic acid from succinyl-CoA and acetyl-CoA are described in Examples XII, XXVIII. Several pathways for the production of 6-aminocaproate from succinic semialdehyde and pyruvate are described in Example XIX. Several pathways for the production of hexamethylenediamine from 6-aminocaproate are described in Examples XX and XXVII. A pathway for producing either or both 6-aminocaproate and hexamethylenediamine from glutamate is described in Examples XXIV and XXV. Several pathways for the production of hexamethylenediamine from glutaryl-CoA and at least one pathway for production of 6-aminocaproate from glutaryl-CoA are described in Examples XXIV and XXV. A pathway for producing 6-aminocaproate from homolysine is described in Example XXV. Pathways for producing hexamethylenediamine from 2-amino-7-oxosubarate are described in Example XXIV. Several pathways for producing 6-aminocaproate are described in Example XXV. Exemplary genes and enzymes required for constructing microbes with these capabilities are described as well as methods for cloning and transformation, monitoring product formation, and using the engineered microorganisms for production.
As disclosed herein, six different pathways for adipic acid synthesis using glucose/sucrose as a carbon substrate are described. For all maximum yield calculations, the missing reactions in a given pathway were added to the E. coli stoichiometric network in SimPheny that is similar to the one described previously (Reed et al., Genome Biol. 4:R54 (2003)). Adipate is a charged molecule under physiological conditions and was assumed to require energy in the form of a proton-based symport system to be secreted out of the network. Such a transport system is thermodynamically feasible if the fermentations are carried out at neutral or near-neutral pH. Low pH adipic acid formation would require an ATP-dependant export mechanism, for example, the ABC system as opposed to proton symport. The reactions in the pathways and methods of implementation of these pathways are described in Examples I-XI.
As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes within a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway.
A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.
As used herein, the term “isolated” when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.
As used herein, the terms “microbial,” “microbial organism” or “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
As used herein, the term “CoA” or “coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
As used herein, “adipate,” having the chemical formula —OOC—(CH2)4-COO— (see
As used herein, “6-aminocaproate,” having the chemical formula —OOC—(CH2)5-NH2 (see
As used herein, “caprolactam” (IUPAC name azepan-2-one) is a lactam of 6-aminohexanoic acid (see
As used herein, “hexamethylenediamine,” also referred to as 1,6-diaminohexane or 1,6-hexanediamine, has the chemical formula H2N(CH2)6NH2 (see
As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
As used herein, the term “osmoprotectant” when used in reference to a culture or growth condition is intended to mean a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, for example, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine.
As used herein, the term “growth-coupled” when used in reference to the production of a biochemical is intended to mean that the biosynthesis of the referenced biochemical is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.
As used herein, “metabolic modification” is intended to refer to a biochemical reaction that is altered from its naturally occurring state. Metabolic modifications can include, for example, elimination of a biochemical reaction activity by functional disruptions of one or more genes encoding an enzyme participating in the reaction. Sets of exemplary metabolic modifications are described herein (see Example XXX).
As used herein, the term “gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention.
“Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.
The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.
Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5′-3′ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.
In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. In gene disruption strategies, evolutionally related genes can also be disrupted or deleted in a host microbial organism, paralogs or orthologs, to reduce or eliminate activities to ensure that any functional redundancy in enzymatic activities targeted for disruption do not short circuit the designed metabolic modifications.
Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.
Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
Disclosed herein are non-naturally occurring microbial organisms capable of producing adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. For example, an adipate pathway can be a reverse adipate degradation pathway (see Examples I and II). For example, a non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. In addition, an adipate pathway can be through a 3-oxoadipate pathway (see Examples III and IV). A non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.
Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase and transaminase (see Examples VIII and IX). Alternatively, 6-aminocaproate dehydrogenase can be used to convert adipate semialdehyde to form 6-aminocaproate (see
As disclosed herein, a 6-aminocaproic acid or caprolactam producing microbial organism can produce 6-aminocaproic acid and/or caprolactam from an adipyl-CoA precursor (see
In addition, as shown in
An additional exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see
Yet another exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see
As disclosed herein, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; steps A/B/C/D/K/L/M of
In another embodiment, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase (see Examples XII and XV; steps K/L of
In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps A/B/C/D of
In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a 2-oxohept-4-ene-1,7-dioate (OHED) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, an adipate semialdehyde oxidoreductase (aminating), a 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate (6-OHE) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase, a 2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), a 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a 2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, a 2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), a 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate-lyase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA reductase, a adipyl-CoA dehydrogenase, a 2-oxohept-4-ene-1,7-dioate (OHED) formate-lyase, a 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, a 2-oxoheptane-1,7-dioate (2-OHD) formate-lyase, a 2-oxoheptane-1,7-dioate (2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme (see Examples XIX and XXI; steps A-Q of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/D/E of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/F/G/E of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; or a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/J/D/I of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); or a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/C/H/I of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/L/M/N/O/E of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/E of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/Q/O/E of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/J/T/AA of
The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an adipate reductase, an adipate kinase or an adipylphosphate reductase (see Example XXVIII; steps X/Y/Z of
In one embodiment, the invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate oxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanal oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a 2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and XXVI; steps A/B/D/E/F/G/I of
In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a 2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of
In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase, an [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), a 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase, an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a 6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase (aminating), a 6-acetamidohexanamine N-acetyltransferase, a 6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoA transferase, a 6-acetamidohexanoate CoA ligase, a 6-acetamidohexanoyl-CoA oxidoreductase, a [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, a [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, a 6-aminocaproate CoA transferase and a 6-aminocaproate CoA ligase (see Examples XX and XXI; steps A-N of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/B/C of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/L/N/C of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps M/N/C of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); or a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/H of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); or a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a 3-oxo-1-carboxyheptanal aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a 3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, a homolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a 2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a 6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI;
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/AB/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/P/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/P/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/W/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a 6-aminopimeloylphosphonate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/U/X/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a 2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoate aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate decarboxylase, a 6-aminohexanal aminotransferase or a 6-aminohexanal aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of
The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or D/P/G/H of
In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/H of
A non-naturally occurring microbial organism disclosed herein can have, for example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, wherein the non-naturally occurring microbial organism includes at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product, as disclosed herein. Thus, a non-naturally occurring microbial organism can contain at least one exogenous nucleic acid encoding a polypeptide, where the polypeptide is an enzyme or protein that converts the substrates and products of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, such as that shown in
For example, a non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate (see
Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproyl-CoA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to 3-hydroxy-6-aminohexanoyl-CoA; 3-hydroxy-6-aminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA; 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA (
Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED): 2-oxohept-4-ene-1,7-dioate (OHED) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (
Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate to glutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (
Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to 6-aminocaproate; 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal; 2-amino-7-oxoheptanoate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (
Additionally, a non-naturally occurring microbial organism can have an hexamethylenediamine (HMDA) pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate to glutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; -amino-7-oxoheptanoate to homolysine; and homolysine to HMDA (
Additionally, a non-naturally occurring microbial organism can have a levulinic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; and 3-oxoadipate to levulinic acid. It is understood that any of the pathways disclosed herein which produce an intermediate of one pathway can be used to produce that intermediate for another pathway, if desired. For example, as disclosed herein, the alpha-ketoadipate to adipate pathway shown in
In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from any of the substrates or products disclosed herein or shown in any of
While generally described herein as a microbial organism that contains a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme expressed in a sufficient amount to produce an intermediate of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. For example, as disclosed herein, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway are exemplified in
It is understood that any of the pathways disclosed herein, including those as described in the Examples and exemplified in the Figures, including the pathways of
The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes as well as the reactants and products of the reaction.
The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes participating in one or more 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) to achieve 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme activities that, together with one or more endogenous enzymes, produces a desired product such as 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more adipate, 6-aminocaproic acid or caprolactam biosynthetic pathways. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis can be established in a host deficient in a pathway enzyme through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, exogenous expression of all enzymes in the pathway can be included, although it is understood that all enzymes of a pathway can be expressed even if the host contains at least one of the pathway enzymes.
For example, exogenous expression of all enzymes in a pathway for production of adipate can be included in a host organism, such as succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. In particular, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase. Alternatively, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotransadipylase/adipate kinase. In addition, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA:acetyl-CoA transferase. Further, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA hydrolase.
In the case of a 6-aminocaproic acid producing microbial organism, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid can be included in a host organism, such as CoA-dependent aldehyde dehydrogenase and transaminase or CoA-dependent aldehyde dehydrogenase and 6-aminocaproate dehydrogenase. For a caprolactam producing microbial organism, exogenous expression of all enzymes in a pathway for production of caprolactam can be included in a host organism, such as CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase. In another example, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid (6-ACA) can be included in a host organism, such as an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase, or alternatively an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase, or alternatively an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In a further aspect, all of the 6-ACA pathway described above can include a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another example, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid (6-ACA) can be included in a host organism, such as a glutamyl-CoA transferase or glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelate decarboxylase.
In another example, exogenous expression of all enzymes in a pathway for production of hexamethylenediamine can be included in a host organism, such as a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, or alternatively a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, or alternatively a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, or alternatively a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide), or alternatively a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide), or alternatively a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In another example, exogenous expression of all enzymes in a pathway for production of hexamethylenediamine can be included in a host organism, such as a glutamyl-CoA transferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 5-oxopimeloyl-CoA hydrolase or a 5-oxopimeloyl-CoA ligase; a 3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating), or alternatively a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase; a 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).
Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathways. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be included, as disclosed herein.
Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, up to all nucleic acids encoding the above enzymes constituting a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway precursors such as succinyl-CoA and/or acetyl-CoA in the case of adipate synthesis, or adipyl-CoA or adipate in the case of 6-aminocaproic acid or caprolactam synthesis, including the adipate pathway enzymes disclosed herein, or pyruvate and succinic semialdehyde, glutamate, glutaryl-CoA, homolysine or 2-amino-7-oxosubarate in the case of 6-aminocaprioate synthesis, or 6-aminocaproate, glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2-amino-7-oxosubarate in the case of hexamethylenediamine synthesis.
Generally, a host microbial organism is selected such that it produces the precursor of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway.
In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In this specific embodiment it can be useful to increase the synthesis or accumulation of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway product to, for example, drive 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway reactions toward 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzymes. Over expression of the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme or enzymes can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, through overexpression of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, that is, up to all nucleic acids encoding 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway enzymes. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway.
In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
The invention additionally provides a non-naturally occurring microbial organism that includes one or more gene disruptions, such as the gene disruptions disclosed in Example XXX and Tables 14-16, where the organism produces a 6-ACA, adipate and/or HMDA. The disruptions occur in genes encoding an enzyme that couples production of adipate, 6-ACA and/or HMDA to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer increased production of adipate, 6-ACA and/or HMDA onto the non-naturally occurring organism. Thus, the invention provides a non-naturally occurring microbial organism, comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding proteins or enzymes wherein the one or more gene disruptions confer increased production of adipate, 6-ACA and/or HMDA in the organism. As disclosed herein, such an organism contains a pathway for production of adipate, 6-ACA and/or HMDA, in addition to the gene disruptions, such as those exemplified in Example XXX and Tables 14-16.
It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic capability. For example, a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes. In the case of adipate production, at least two exogenous nucleic acids can encode the enzymes such as the combination of succinyl-CoA:acetyl-CoA acyl transferase and 3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyl transferase and 3-hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoA and 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA and adipyl-CoA synthetase, and the like. In the case of caprolactam production, at least two exogenous nucleic acids can encode the enzymes such as the combination of CoA-dependent aldehyde dehydrogenase and transaminase, or CoA-dependent aldehyde dehydrogenase and amidohydrolase, or transaminase and amidohydrolase. In the case of 6-aminocaproic acid production, at least two exogenous nucleic acids can encode the enzymes such as the combination of an 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase and a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, or a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a 3-hydroxyadipyl-CoA dehydratase and a adipyl-CoA dehydrogenase, a glutamyl-CoA transferase and a 6-aminopimeloyl-CoA hydrolase, or a glutaryl-CoA beta-ketothiolase and a 3-aminopimelate 2,3-aminomutase. In the case of hexamethylenediamine production, at least two exogenous nucleic acids can encode the enzymes such as the combination of 6-aminocaproate kinase and [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or a 6-acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase and a 2-amino-7-oxoheptanoate aminotransferase, or a 3-oxopimeloyl-CoA ligase and a homolysine decarboxylase. Thus, it is understood that any combination of two or more enzymes of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention.
Similarly, it is understood that any combination of three or more enzymes of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, in the case of adipate production, the combination of enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase; or succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase; or succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and adipyl-CoA:acetyl-CoA transferase, and so forth, as desired, so long as the combination of enzymes of the desired biosynthetic pathway results in production of the corresponding desired product. In the case of 6-aminocaproic acid production, the at least three exogenous nucleic acids can encode the enzymes such as the combination of an 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, or a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase and a 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, or a 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase and a adipyl-CoA dehydrogenase, or a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA hydrolase and a 2-aminopimelate decarboxylase, or a glutaryl-CoA beta-ketothiolase, a 3-aminating oxidoreductase and a 2-aminopimelate decarboxylase, or a 3-oxoadipyl-CoA thiolase, a 5-carboxy-2-pentenoate reductase and a adipate reductase. In the case of hexamethylenediamine production, at least three exogenous nucleic acids can encode the enzymes such as the combination of 6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase and 6-aminocaproic semialdehyde aminotransferase, or a 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, or 6-aminocaproate N-acetyltransferase, a [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, or a 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase and a homolysine decarboxylase, or a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a 2-oxo-7-aminohept-3-enoate reductase and a homolysine decarboxylase, or a 6-acetamidohexanoate reductase, a 6-acetamidohexanal aminotransferase and a 6-acetamidohexanamine N-acetyltransferase. Similarly, any combination of four or more enzymes of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes of the desired biosynthetic pathway results in production of the corresponding desired product.
In addition to the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid other than use of the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers is through addition of another microbial organism capable of converting an adipate, 6-aminocaproic acid or caprolactam pathway intermediate to 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. One such procedure includes, for example, the fermentation of a microbial organism that produces a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate. The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate can then be used as a substrate for a second microbial organism that converts the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate to 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate can be added directly to another culture of the second organism or the original culture of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate and the second microbial organism converts the intermediate to 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis. In a particular embodiment, the increased production couples biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism, and can obligatorily couple production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism if desired and as disclosed herein.
Sources of encoding nucleic acids for a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Escherichia coli str. K12, Escherichia coli C, Escherichia coli W, Pseudomonas sp, Pseudomonas knackmussii, Pseudomonas sp. Strain B13, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas mendocina, Rhodopseudomonas palustris, Mycobacterium tuberculosis, Vibrio cholera, Heliobacter pylori, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces sp. 2065, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1, Ralstonia eutropha, Ralstonia eutropha H16, Clostridium acetobutylicum, Euglena gracilis, Treponema denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium botulinum A3 str, Clostridium tyrobutyricum, Clostridium pasteurianum, Clostridium thermoaceticum (Moorella thermoaceticum), Moorella thermoacetica Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp. M62/1, Fusobacterium nucleatum, Bos taurus, Zoogloea ramigera, Rhodobacter sphaeroides, Clostridium beijerinckii, Metallosphaera sedula, Thermoanaerobacter species, Thermoanaerobacter brockii, Acinetobacter baylyi, Porphyromonas gingivalis, Leuconostoc mesenteroides, Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sulfolobus solfataricus, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Salmonella typhimurium, Salmonella enterica, Thermotoga maritima, Halobacterium salinarum, Bacillus cereus, Clostridium difficile, Alkaliphilus metalliredigenes, Thermoanaerobacter tengcongensis, Saccharomyces kluyveri, Helicobacter pylori, Corynebacterium glutamicum, Clostridium sacchanopenbutylacetonicum, Pseudomonas chlononaphis, Streptomyces clavuligenus, Campylobacter jejuni, Thermus thenmophilus, Pelotomaculum thermopropionicum, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thenmophilius, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Haloancula marismortui, Pynobaculum aenophilum, Pynobaculum aenophilum str. IM2, Nicotiana tabacum, Menthe piperita, Pinus taeda, Hordeum vulgane, Zea mays, Rhodococcus opacus, Cupriavidus necator, Bradyrhizobium japonicum, Bradyrhizobium japonicum USDA110, Ascarius suum, butyrate-producing bacterium L2-50, Bacillus megaterium, Methanococcus manipaludis, Methanosarcina mazei, Methanosarcina mazei, Methanocarcina barkeri, Methanocaldococcus jannaschii, Caenorhabditis elegans, Leishmania major, Methylomicrobium alcaliphilum 20Z, Chromohalobacten salexigens, Archaeglubus fulgidus, Chlamydomonas reinhardtii, trichomonas vaginalis G3, Trypanosoma brucei, Mycoplana ramose, Micrococcus luteas, Acetobacter pasteunians, Kluyvenomyces lactis, Mesorhizobium loti, Lactococcus lactis, Lysinibacillus sphaericus, Candida boidinii, Candida albicans SC5314, Burkholderia ambifaria AMMD, Ascaris suun, Acinetobacter baumanii, Acinetobacter calcoaceticus, Burkholderia phymatum, Candida albicans, Clostridium subterminale, Cupriavidus taiwanensis, Flavobacterium lutescens, Lachancea kluyveri, Lactobacillus sp. 30a, Leptospira interrogans, Moorella thermoacetica, Myxococcus xanthus, Nicotiana glutinosa, Nocardia iowensis (sp. NRRL 5646), Pseudomonas reinekei MT1, Ralstonia eutropha JMP134, Ralstonia metallidurans, Rhodococcus jostii, Schizosaccharomyces pombe, Selenomonas ruminantium, Streptomyces clavuligenus, Syntrophus aciditrophicus, Vibrio parahaemolyticus, Vibrio vulnificus, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes (see Examples). However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
In some instances, such as when a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway exists in an unrelated species, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. For example, E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metoblic and/or genetic modifications to produce a desired product.
Methods for constructing and testing the expression levels of a non-naturally occurring 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).
Exogenous nucleic acid sequences involved in a pathway for production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
An expression vector or vectors can be constructed to include one or more 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.
Directed evolution is one approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through implementation screening assays that allow for the identification of useful variants. Particularly useful screening methods include sensitive high-throughput assays that allow the automated screening of many enzyme variants (e.g., >104). Iterative rounds of mutagenesis and screening typically are performed to identify an enzyme with optimized properties. The greater the number of variants screened, the higher the probability of identifying an ideally suitable variant. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened.
Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol.Eng 22:1-9 (2005); and Sen et al., Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes.
Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example, selectivity/specificity—for conversion of non-natural substrates; temperature stability—for robust high temperature processing; pH stability—for bioprocessing under lower or higher pH conditions; substrate or product tolerance—so that high product titers can be achieved; binding (Km)—broadens substrate binding to include non-natural substrates; inhibition (Ki)—to remove inhibition by products, substrates, or key intermediates; activity (kcat)—increases enzymatic reaction rates to achieve desired flux; expression levels—increases protein yields and overall pathway flux; oxygen stability—for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity—for operation of an aerobic enzyme in the absence of oxygen.
The following exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Any of these can be used to alter/optimize activity of a decarboxylase enzyme.
EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn2+ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance. This method can generate multiple mutations in a single gene simultaneously, which can be useful. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics.
Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats. Adjusting the Mn2+ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3-4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit.
DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci U.S.A. 91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >1 kbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious, random and neutral mutations that might confer antigenicity.
Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents. Combinations of low-fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra.
In Random Priming Recombination (RPR) random sequence primers are used to generate many short DNA fragments complementary to different segments of the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.
In Heteroduplex Recombination linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair. (Volkov et al, Nucleic Acids Res 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)) The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons.
Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of ssDNA. Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in, and then ligated to give a pool of full-length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.
Recombined Extension on Truncated templates (RETT) entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates. (Lee et al., J. Molec. Catalysis 26:119-129 (2003)) No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps—no direct shuffling. This method can also be more random than StEP due to the absence of pauses.
In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.
Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest. (Ostermeier et al., Proc Natl Acad Sci U.S.A. 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol 17:1205-1209 (1999)) Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured.
Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations. (Lutz et al., Nucleic Acids Res 29:E16 (2001)) Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide more reproducibility, and adjustability.
SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling. (Lutz et al., Proc Natl Acad Sci U.S.A. 98:11248-11253 (2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.
In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed by screening/selection for those retaining usable activity. (Bergquist et al., Biomol.Eng 22:63-72 (2005)) Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.
Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of “universal” bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis. (Wong et al., Biotechnol J 3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)) Using this technique it can be possible to generate a large library of mutants within 2-3 days using simple methods. This technique is non-directed in comparison to the mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or an alternative) to epPCR.
In Synthetic Shuffling, overlapping oligonucleotides are designed to encode “all genetic diversity in targets” and allow a very high diversity for the shuffled progeny. (Ness et al., Nat. Biotechnol 20:1251-1255 (2002)) In this technique, one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny. One can design sequence/codon biases to make more distantly related sequences recombine at rates approaching those observed with more closely related sequences. Additionally, the technique does not require physically possessing the template genes.
Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation. (Muller et al., Nucleic Acids Res 33:e117 (2005)) The gene is reassembled using internal PCR primer extension with proofreading polymerase. The sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage. Other nucleotide analogs, such as 8-oxo-guanine, can be used with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. The chemical cleavage of DNA used in this technique results in very few unshuffled clones.
In Sequence Homology-Independent Protein Recombination (SHIPREC) a linker is used to facilitate fusion between two distantly/unrelated genes. Nuclease treatment is used to generate a range of chimeras between the two genes. These fusions result in libraries of single-crossover hybrids. (Sieber et al., Nat. Biotechnol 19:456-460 (2001)) This produces a limited type of shuffling and a separate process is required for mutagenesis. In addition, since no homology is needed this technique can create a library of chimeras with varying fractions of each of the two unrelated parent genes. SHIPREC was tested with a heme-binding domain of a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme.
In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations. (Kretz et al., Methods Enzymol. 388:3-11 (2004)) Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA. The mutation is typically in the middle of the primer and flanked on each side by ˜20 nucleotides of correct sequence. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). After extension, DpnI is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (i.e., one codon). The technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.
Combinatorial Cassette Mutagenesis (CCM) involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations. (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)) Simultaneous substitutions at two or three sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. This technique has been used to explore the information content of the lambda repressor DNA-binding domain.
Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentially similar to CCM except it is employed as part of a larger program: 1) Use of epPCR at high mutation rate to 2) ID hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space. (Reetz, M. T., S. Wilensek, D. Zha, and K. E. Jaeger, 2001, Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis. Angew. Chem. Int. Ed Engl. 40:3589-3591.) As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51-fold, the enantioselectivity of an enzyme.
In the Mutator Strains technique conditional is mutator plasmids allow increases of 20- to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required. (Selifonova et al., Appl Environ Microbiol 67:3645-3649 (2001)) This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur. In order for effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows for plasmid curing at 41° C. It should be noted that mutator strains have been explored for quite some time (e.g., see Low et al., J. Mol. Biol. 260:359-3680 (1996)). In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.
“Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids.” (Rajpal et al., Proc Natl Acad Sci U.S.A. 102:8466-8471 (2005)) Rather than saturating each site with all possible amino acid changes, a set of nine is chosen to cover the range of amino acid R-group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800-fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This method is a rational approach to minimize the number of random combinations and can increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections.
Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation). Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombinations independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM™, a large range of mutations can be tested for improved activity. The method allows “blending” and “fine tuning” of DNA shuffling, e.g. codon usage can be optimized.
In Silico Protein Design Automation (PDA) is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics. (Hayes et al., Proc Natl Acad Sci U.S.A. 99:15926-15931 (2002)) This technology uses in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position. Structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (1050). The choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely experimental technologies due to exponential increases in numbers.
Iterative Saturation Mutagenesis (ISM) involves: 1) use knowledge of structure/function to choose a likely site for enzyme improvement; 2) saturation mutagenesis at chosen site using Stratagene QuikChange (or other suitable means); 3) screen/select for desired properties; and 4) with improved clone(s), start over at another site and continue repeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem.Int.Ed Engl. 45:7745-7751 (2006)) This is a proven methodology, which assures all possible replacements at a given position are made for screening/selection.
Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques.
The invention additionally provides methods for producing a desired intermediate or product such as adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. For example, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Additionally, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.
Further, a method for producing 6-aminocaproic acid can involve culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway, the pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, under conditions and for a sufficient period of time to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase and transaminase or 6-aminocaproate dehydrogenase. Additionally, a method for producing caprolactam can involve culturing a non-naturally occurring microbial organism having a caprolactam pathway, the pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, under conditions and for a sufficient period of time to produce caprolactam, the caprolactam pathway including CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase.
The invention additionally provides methods for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway described herein under conditions and for a sufficient period of time to produce 6-ACA. In one aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase. In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase. In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In a further aspect, the 6-ACA pathways described above can include a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase.
The invention additionally provides methods for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway described herein under conditions and for a sufficient period of time to produce HMDA. In one aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. In another aspect, the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. In yet another aspect, the HMDA pathway includes a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide).
Also, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase/alpha-ketoadipate kinase or alpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Furthermore, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase.
As disclosed herein, the invention also provides method for producing 6-aminocaproic acid by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; steps A/B/C/D/K/L/M of
In another embodiment, the invention provides a method for producing caprolactam by culturing a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase (see Examples XII and XV; steps K/L of
In yet another embodiment, the invention provides a method for producing caprolactam by culturing a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps A/B/C/D of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway, the microbial organism including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase, a phosphoenolpyruvate (PEP) carboxykinase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a 2-oxohept-4-ene-1,7-dioate (OHED) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, an adipate semialdehyde oxidoreductase (aminating), a 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate (6-OHE) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase, a 2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), a 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a 2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, a 2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), a 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate-lyase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA reductase, a adipyl-CoA dehydrogenase, a 2-oxohept-4-ene-1,7-dioate (OHED) formate-lyase, a 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, a 2-oxoheptane-1,7-dioate (2-OHD) formate-lyase, a 2-oxoheptane-1,7-dioate (2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme (see Examples XIX and XXI; steps A-Q of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway, the microbial organism including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA. In one aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/D/E of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/J/T/AA of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a homolysine 2-monooxygenase (see Examples XXV and XXVI; steps A of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an adipate reductase, an adipate kinase or an adipylphosphate reductase (see Example)(XVIII; steps X/Y/Z of
In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate oxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanal oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a 2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and XXVI; steps A/B/D/E/F/G/I of
In another embodiment of the invention, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a 2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway, the microbial organism including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase, an [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), a 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase, an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a 6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase (aminating), a 6-acetamidohexanamine N-acetyltransferase, a 6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoA transferase, a 6-acetamidohexanoate CoA ligase, a 6-acetamidohexanoyl-CoA oxidoreductase, a [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, a [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, a 6-aminocaproate CoA transferase and a 6-aminocaproate CoA ligase (see Examples XX and XXI; steps A-N of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway, the microbial organism including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA. In one aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/B/C of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A-H of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a 3-oxo-1-carboxyheptanal aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a 3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, a homolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a 2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a 6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI;
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/AB/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/P/Q/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/P/Q/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/W/Q/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a 6-aminopimeloylphosphonate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/U/X/Q/S of
In yet another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of
The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a 2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoate aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate decarboxylase, a 6-aminohexanal aminotransferase or 6-aminohexanal aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of
The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or D/P/G/H of
The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanal aminating oxidoreductase, a 6-aminohexanal aminotransferase, a 2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, a 2-oxo-7-aminoheptanoate decarboxylase, a homolysine decarboxylase, a 2-amino-7-oxosubarate amino acid decarboxylase, a 2-oxo-7-aminoheptanoate aminating oxidoreductase, a 2-oxo-7-aminoheptanoate aminotransferase, a 2-amino-7-oxosubarate aminating oxidoreductase, a 2-amino-7-oxosubarate aminotransferase or a 2,7-diaminosubarate decarboxylase (see Examples XXIV and XXVI; Steps A/B/C/G/H/I/J/K/L/M of
In another embodiment, the invention provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/H of
The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a levulinic acid (LA) pathway including at least one exogenous nucleic acid encoding a LA pathway enzyme expressed in a sufficient amount to produce LA, the LA pathway including a 3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, or a 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of
The invention further provides methods of producing non-naturally microbial organisms having increased production of adipate, 6-ACA and/or HMDA by disruption of one or more genes to confer increased production of adiate, 6-ACA and/or HMDA. Such gene disruptions include those exemplified herein in Example XXX and Tables 14-16.
The invention additionally provides a method for producing adipate, 6-ACA and/or HMDA that includes culturing a non-naturally occurring microbial organism that includes one or more gene disruptions that confer increased production of adiapte, 6-ACA and/or HMDA. The disruptions can occur in genes encoding an enzyme obligatory to coupling adipate, 6-ACA and/or HMDA production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, such that the disruptions confer stable growth-coupled production of adipate, 6-ACA and/or HMDA onto the non-naturally microbial organism.
In some embodiments, the gene disruption can include a complete gene deletion. Methods for gene disruption are well known to those skilled in the art and are described herein (see Example XXX). In some embodiments other methods to disrupt a gene include, for example, frameshifting by omission, addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it can confer to the non-naturally occurring organism from reverting to a phenotype expressing the previously disrupted gene. In particular, the gene disruptions are selected from the gene sets that described in Tables 14-16.
Suitable purification and/or assays to test for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme activities from the exogenous DNA sequences can also be assayed using methods well known in the art.
The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers can be cultured for the biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
For the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719 (Ser. No. 11/891,602), filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.
The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
In addition to renewable feedstocks such as those exemplified above, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.
Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H2 and CO, syngas can also include CO2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO2.
The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO2 and CO2/H2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H2-dependent conversion of CO2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:
2CO2+4H2+nADP+nPi→CH3COOH+2H2O+nATP
Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO2 and H2 mixtures as well for the production of acetyl-CoA and other desired products.
The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase, and these enzymes can also be referred to as methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.
Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, CO2 and/or H2 to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO2 via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the p-toluate, terepathalate, or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability.
Given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid and any of the intermediate metabolites in the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. All that is required is to engineer in one or more of the required enzyme activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid when grown on a carbohydrate and produces and/or secretes any of the intermediate metabolites shown in the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway when grown on a carbohydrate. For example, an adipate producing microbial organisms can initiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA, 3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see
The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme in sufficient amounts to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.
In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic conditions, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers can synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing microbial organisms can produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intracellularly and/or secrete the product into the culture medium.
The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.
As described herein, one exemplary growth condition for achieving biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N2/CO2 mixture or other suitable non-oxygen gas or gases.
The culture conditions described herein can be scaled up and grown continuously for manufacturing of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid will include culturing a non-naturally occurring 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.
Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.
In addition to the above fermentation procedures using the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers of the invention for continuous production of substantial quantities of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired. As described herein, an intermediate in the adipate pathway utilizing 3-oxoadipate, hexa-2-enedioate, can be converted to adipate, for example, by chemical hydrogenation over a platinum catalyst (see Example III).
As described herein, exemplary growth conditions for achieving biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid includes the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described above in the presence of an osmoprotectant. Briefly, an osmoprotectant means a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. For example, as described in Example XXII, Escherichia coli in the presence of varying amounts of 6-aminocaproic acid is suitably grown in the presence of 2 mM glycine betaine. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM.
To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003). OptKnock is a metabolic modeling and simulation program that suggests gene deletion strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.
The concept of growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates. The knockouts suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point should lie within its calculated solution boundary. Plots such as these allow one to visualize how close strains are to their performance limits or, in other words, how much room is available for improvement. The OptKnock framework has already been able to identify promising gene deletion strategies for biochemical overproduction, (Burgard et al., Biotechnol Bioeng, 84(6):647-657 (2003); Pharkya et al., Biotechnol Bioeng, 84(7):887-899 (2003)) and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.
Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that enable an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation method referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patent application serial No. 2009/0047719, filed Aug. 10, 2007.
Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed Jun. 14, 2002, and in International Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components. Analysis methods such as convex analysis, linear programming and the calculation of extreme pathways as described, for example, in Schilling et al., J. Theon. Biol. 203:229-248 (2000); Schilling et al., Biotech. Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Prog. 15:288-295 (1999), can be used to determine such phenotypic capabilities.
As described above, one constraints-based method used in the computational programs applicable to the invention is flux balance analysis. Flux balance analysis is based on flux balancing in a steady state condition and can be performed as described in, for example, Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approaches have been applied to reaction networks to simulate or predict systemic properties of, for example, adipocyte metabolism as described in Fell and Small, J. Biochem. 138:781-786 (1986), acetate secretion from E. coli under ATP maximization conditions as described in Majewski and Domach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeast as described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996). Additionally, this approach can be used to predict or simulate the growth of S. cerevisiae on a variety of single-carbon sources as well as the metabolism of H. influenzae as described in Edwards and Palsson, Proc. Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio. Chem. 274:17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130 (2001).
Once the solution space has been defined, it can be analyzed to determine possible solutions under various conditions. This computational approach is consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.
These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.
Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.
The ability of a cell or organism to obligatory couple growth to the production of a biochemical product can be illustrated in the context of the biochemical production limits of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. The production of a desired biochemical generally is in direct competition with biomass formation for intracellular resources. Under these circumstances, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates. The knockouts suggested by the above metabolic modeling and simulation programs such as OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point will lie within its calculated solution boundary. Plots such as these allow accurate predictions of how close the designed strains are to their performance limits which also indicates how much room is available for improvement.
The OptKnock mathematical framework is exemplified herein for pinpointing gene deletions leading to growth-coupled biochemical production (see Example XXX). The procedure builds upon constraint-based metabolic modeling which narrows the range of possible phenotypes that a cellular system can display through the successive imposition of governing physico-chemical constraints, Price et al., Nat Rev Microbiol, 2: 886-97 (2004). As described above, constraint-based models and simulations are well known in the art and generally invoke the optimization of a particular cellular objective, subject to network stoichiometry, to suggest a likely flux distribution.
Briefly, the maximization of a cellular objective quantified as an aggregate reaction flux for a steady state metabolic network comprising a set N={1, . . . , N} of metabolites and a set M={1, . . . , M} of metabolic reactions is expressed mathematically as follows:
where Sij is the stoichiometric coefficient of metabolite i in reaction j, vj is the flux of reaction j, vsubstrate
The modeling of gene deletions, and thus reaction elimination, first employs the incorporation of binary variables into the constraint-based approach framework, Burgard et al., Biotechnol Bioeng, 74: 364-375 (2001), Burgard et al., Biotechnol Prog, 17: 791-797 (2001). These binary variables,
assume a value of 1if reaction j is active and a value of 0 if it is inactive. The following constraint,
vjmin·yj≦vj≦vjmax·yj,∀jεM
ensures that reaction flux vj is set to zero only if variable yj is equal to zero. Alternatively, when yj is equal to one, vj is free to assume any value between a lower vjmin and an upper vjmax bound. Here, vjmin and vjmax are identified by minimizing and maximizing, respectively, every reaction flux subject to the network constraints described above, Mahadevan et al., Metab Eng, 5: 264-76 (2003).
Optimal gene/reaction knockouts are identified by solving a bilevel optimization problem that chooses the set of active reactions (yj=1) such that an optimal growth solution for the resulting network overproduces the chemical of interest. Schematically, this bilevel optimization problem is illustrated in
where vchemical is the production of the desired target product, for example adipate, 6-ACA and/or HMDA, or other biochemical product, and K is the number of allowable knockouts. Note that setting K equal to zero returns the maximum biomass solution of the complete network, while setting K equal to one identifies the single gene/reaction knockout (yj=0) such that the resulting network involves the maximum overproduction given its maximum biomass yield. The final constraint ensures that the resulting network meets a minimum biomass yield. Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), provide a more detailed description of the model formulation and solution procedure. Problems containing hundreds of binary variables can be solved in the order of minutes to hours using CPLEX 8.0, GAMS: The Solver Manuals. 2003: GAMS Development Corporation, accessed via the GAMS, Brooke et al., GAMS Development Corporation (1998), modeling environment on an IBM RS6000-270 workstation. The OptKnock framework has already been able to identify promising gene deletion strategies for biochemical overproduction, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), Pharkya et al., Biotechnol Bioeng, 84: 887-899 (2003), and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.
The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.
Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.
To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.
The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).
An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.
Given the teachings and guidance provided herein, those skilled in the art will understand that to disrupt an enzymatic reaction the catalytic activity of the one or more enzymes involved in the reaction is to be disrupted. Disruption can occur by a variety of means including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity disruption can occur by a genetic alteration that reduces or destroys the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits in order to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is reduced or eliminated.
Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the growth-coupled product production. Examplary disruptions to confer increased production of adipate, 6-ACA and/or HMDA are described in Example XXX and Tables 14-16.
Employing the methods exemplified above, the methods of the invention allow the construction of cells and organisms that increase production of a desired product, for example, by coupling the production of a desired product to growth of the cell or organism engineered to harbor the identified genetic alterations. As disclosed herein, metabolic alterations have been identified that couple the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism. Microbial organism strains constructed with the identified metabolic alterations produce elevated levels, relative to the absence of the metabolic alterations, of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid during the exponential growth phase. These strains can be beneficially used for the commercial production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in continuous fermentation process without being subjected to the negative selective pressures described previously. Although exemplified herein as metabolic alterations, in particular one or more gene disruptions, that confer growth coupled production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, it is understood that any gene disruption that increases the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be introduced into a host microbial organism, as desired.
Therefore, the methods of the invention provide a set of metabolic modifications that are identified by an in silico method such as OptKnock. The set of metabolic modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. For 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production, metabolic modifications can be selected from the set of metabolic modifications listed in Tables 14-16 (see Example XXX).
Also provided is a method of producing a non-naturally occurring microbial organisms having stable growth-coupled production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The method can include identifying in silico a set of metabolic modifications that increase production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, for example, increase production during exponential growth; genetically modifying an organism to contain the set of metabolic modifications that increase production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, and culturing the genetically modified organism. If desired, culturing can include adaptively evolving the genetically modified organism under conditions requiring production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The methods of the invention are applicable to bacterium, yeast and fungus as well as a variety of other cells and microorganism, as disclosed herein.
Thus, the invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions that confer increased production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In one embodiment, the one or more gene disruptions confer growth-coupled production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, and can, for example, confer stable growth-coupled production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In another embodiment, the one or more gene disruptions can confer obligatory coupling of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production to growth of the microbial organism. Such one or more gene disruptions reduce the activity of the respective one or more encoded enzymes.
The non-naturally occurring microbial organism can have one or more gene disruptions included in a metabolic modification listed in Tables 14-16. As disclosed herein, the one or more gene disruptions can be a deletion. Such non-naturally occurring microbial organisms of the invention include bacteria, yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes, as disclosed herein.
Thus, the invention provides a non-naturally occurring microbial organism, comprising one or more gene disruptions, where the one or more gene disruptions occur in genes encoding proteins or enzymes where the one or more gene disruptions confer increased production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in the organism. The production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be growth-coupled or not growth-coupled. In a particular embodiment, the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be obligatorily coupled to growth of the organism, as disclosed herein.
The invention provides non naturally occurring microbial organisms having genetic alterations such as gene disruptions that increase production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, for example, growth-coupled production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Product production can be, for example, obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell, as disclosed herein. The genetic alterations can increase the production of the desired product or even make the desired product an obligatory product during the growth phase. Sets of metabolic alterations or transformations that result in increased production and elevated levels of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis are exemplified in Tables 14-16 (see Example XXX). Each alteration within a set corresponds to the requisite metabolic reaction that should be functionally disrupted. Functional disruption of all reactions within each set can result in the increased production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid by the engineered strain during the growth phase. The corresponding reactions to the referenced alterations can be found in Tables 14-16 (see Example XXX), and the gene or genes that encode enzymes or proteins that carry out the reactions are set forth in Tables 14-16.
For example, for each strain exemplified in Tables 14-16, the metabolic alterations that can be generated for 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production are shown in each row. These alterations include the functional disruption of the reactions shown in Tables 14-16. Each of these non-naturally occurring alterations result in increased production and an enhanced level of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production, for example, during the exponential growth phase of the microbial organism, compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.
It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.
This example describes an exemplary adipate synthesis pathway via a reverse adipate degradation pathway.
Organisms such as Penicillium chrysogenum have the ability to naturally degrade adipate (Thykaer et al., Metab. Eng. 4:151-158. (2002)). The mechanism is similar to the oxidation of fatty acids (see
A highly efficient pathway for the production of adipate is achieved through genetically altering a microorganism such that similar enzymatic reactions are employed for adipate synthesis from succinyl-CoA and acetyl-CoA (see
The maximum theoretical yields of adipate using this pathway were calculated both in the presence and absence of an external electron acceptor such as oxygen. These calculations show that the pathway can efficiently transform glucose into adipate and CO2 under anaerobic conditions with a 92% molar yield (Table 1). The production of adipate using this pathway does not require the uptake of oxygen as NAD+ can be regenerated in the two hydrogenase steps that form 3-hydroxyadipyl-CoA and adipyl-CoA (see
TABLE 1
The maximum theoretical yields of adipate and the associated
ATP yields per mole of glucose using the reverse degradation
pathway assuming the final step in the pathway is a synthetase,
phosphotransadipylase/kinase, or transferase.
Aerobic
Anaerobic
Adipate Yield
0.92
0.92
Max ATP yield @ max adipate yield
1.55
1.55
Max ATP yield @ max adipate yield
2.47
2.47
PPCK assumed
Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of adipate, one or more exogenous DNA sequence(s) are expressed in a suitable host microorganism. In addition, the microorganisms can have endogenous gene(s) functionally deleted. These modifications allow the production of adipate using renewable feedstock.
Below is described a number of biochemically characterized candidate genes that encode enzymes that catalyze each step of the reverse adipate degradation pathway in a production host. Although described using E. coli as a host organism to engineer the pathway, essentially any suitable host organism can be used. Specifically listed are genes that are native to E. coli as well as genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed.
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
paaJ
16129358
NP_415915.1
Escherichia coli
pcaF
17736947
AAL02407
Pseudomonas
knackmussii (B13)
phaD
3253200
AAC24332.1
Pseudomonas putida
paaE
106636097
ABF82237.1
Pseudomonas fluorescens
Bamb_0447
115350501
YP_772340
Burkholderia
ambifaria AMMD
These exemplary sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (for example, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to generate production hosts.
For example, orthologs of paaJ from Escherichia coli K12 can be found using the following GI numbers and/or GenBank identifiers:
GI#
GenBank Accession #
Organism
152970031
YP_001335140.1
Klebsiella pneumoniae
157371321
YP_001479310.1
Serratia proteamaculans
3253200
AAC24332.1
Pseudomonas putida
Example orthologs of pcaF from Pseudomonas knackmussii can be found using the following GI numbers and/or GenBank identifiers:
GI#
GenBank Accession #
Organism
4530443
AAD22035.1
Streptomyces sp. 2065
24982839
AAN67000.1
Pseudomonas putida
115589162
ABJ15177.1
Pseudomonas aeruginosa
Additional native candidate genes for the ketothiolase step include atoB, which can catalyze the reversible condensation of 2 acetyl-CoA molecules (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), and its homolog yqeF. Non-native gene candidates include phaA (Sato et al., supra, 2007) and bktB (Slater et al., J. Bacteriol. 180:1979-1987 (1998)) from R. eutropha, and the two ketothiolases, thiA and thiB, from Clostridium acetobutylicum (Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541 (2000)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
atoB
16130161
NP_416728.1
Escherichia coli
yqeF
90111494
NP_417321.2
Escherichia coli
phaA
113867452
YP_725941
Ralstonia eutropha
bktB
3046397
AAC38322.1
Ralstonia eutropha
thiA
15896127
NP_349476.1
Clostridium acetobutylicum
thiB
15004782
NP_149242.1
Clostridium acetobutylicum
It is less desirable to use the thiolase-encoding genes fadA and fadB, genes in fatty acid degradation pathway in E. coli, in this exemplary pathway. These genes form a complex that encodes for multiple activities, most of which are not desired in this pathway.
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
paaH
16129356
NP_415913.1
Escherichia coli
phaC
26990000
NP_745425.1
Pseudomonas putida
paaC
106636095
ABF82235.1
Pseudomonas fluorescens
hbd
15895965
NP_349314.1
Clostridium acetobutylicum
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
maoC
16129348
NP_415905.1
Escherichia coli
paaF
16129354
NP_415911.1
Escherichia coli
paaG
16129355
NP_415912.1
Escherichia coli
cr
15895969
NP_349318.1
Clostridium acetobutylicum
paaA
26990002
NP_745427.1
Pseudomonas putida
paaB
26990001
NP_745426.1
Pseudomonas putida
phaA
106636093
ABF82233.1
Pseudomonas fluorescens
phaB
106636094
ABF82234.1
Pseudomonas fluorescens
Alternatively, beta-oxidation genes are candidates for the first three steps in adipate synthesis. Candidate genes for the proposed adipate synthesis pathway also include the native fatty acid oxidation genes of E. coli and their homologs in other organisms. The E. coli genes fadA and fadB encode a multienzyme complex that exhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al., J. Biol. Chem. 265:10424-10429 (1990); Yang et al., J. Biol. Chem. 266:16255 (1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937 (1990)). These activities are mechanistically similar to the first three transformations shown in
Gene
GenBank
name
GI#
Accession #
Organism
fadA
49176430
YP_026272.1
Escherichia coli
fadB
16131692
NP_418288.1
Escherichia coli
fadI
16130275
NP_416844.1
Escherichia coli
fadJ
16130274
NP_416843.1
Escherichia coli
fadR
16129150
NP_415705.1
Escherichia coli
Referring to
One candidate gene for the enoyl-CoA reductase step is the gene product of bcd from C. acetobutylicum (Atsumi et al., supra, 2007; Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA, a reaction similar in mechanism to the desired reduction of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA in the adipate synthesis pathway. Activity of this enzyme can be enhanced by expressing bcd in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli, resulting in an active enzyme (Hoffmeister et al., supra, 2005). This approach is well known to those skilled in the art of expressing eukarytotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci and Martin, FEBS Lett. 581:1561-1566 (2007)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
bcd
15895968
NP_349317.1
Clostridium acetobutylicum
etfA
15895966
NP_349315.1
Clostridium acetobutylicum
etfB
15895967
NP_349316.1
Clostridium acetobutylicum
TER
62287512
Q5EU90.1
Euglena gracilis
TDE0597
42526113
NP_971211.1
Treponema denticola
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
sucC
16128703
NP_415256.1
Escherichia coli
sucD
1786949
AAC73823.1
Escherichia coli
Another option, using phosphotransadipylase/adipate kinase, is catalyzed by the gene products of buk1, buk2, and ptb from C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptb gene encodes an enzyme that can convert butyryl-CoA into butyryl-phosphate, which is then converted to butyrate via either of the buk gene products with the concomitant generation of ATP. The analogous set of transformations, that is, conversion of adipyl-CoA to adipyl-phosphate followed by conversion of adipyl-phosphate to adipate, can be carried out by the buk1, buk2, and ptb gene products. The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
ptb
15896327
NP_349676
Clostridium acetobutylicum
buk1
15896326
NP_349675
Clostridium acetobutylicum
buk2
20137415
Q97II1
Clostridium acetobutylicum
Alternatively, an acetyltransferase capable of transferring the CoA group from adipyl-CoA to acetate can be applied. Similar transformations are catalyzed by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996); Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
cat1
729048
P38946.1
Clostridium kluyveri
cat2
172046066
P38942.2
Clostridium kluyveri
cat3
146349050
EDK35586.1
Clostridium kluyveri
Finally, though not as desirable from an energetic standpoint, the conversion of adipyl-CoA to adipate can also be carried out by an acyl-CoA hydrolase or equivalently a thioesterase. The top E. coli gene candidate is tesB (Naggert et al., J. Biol. Chem. 266:11044-11050 (1991)), which shows high similarity to the human acot8, which is a dicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has also been characterized in the rat liver (Deana, Biochem. Int. 26:767-773 (1992)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
tesB
16128437
NP_414986
Escherichia coli
acot8
3191970
CAA15502
Homo sapiens
acot8
51036669
NP_570112
Rattus norvegicus
Other native candidate genes include tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc et al., J. Bacteriol. 189:7112-7126 (2007)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
tesA
16128478
NP_415027
Escherichia coli
ybgC
16128711
NP_415264
Escherichia coli
paaI
16129357
NP_415914
Escherichia coli
ybdB
16128580
NP_415129
Escherichia coli
The above description provides an exemplary adipate synthesis pathway by way of a reverse adipate degradation pathway.
This example describes the generation of a microbial organism capable of producing adipate using the reverse degradation pathway.
Escherichia coli is used as a target organism to engineer a reverse adipate degradation pathway as shown in
To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in the reverse degradation pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP—415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the reverse degradation pathway.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of reverse degradation pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional adipate synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of adipate. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates or the adipate product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production.
For large-scale production of adipate, the above reverse degradation pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the preparation of an adipate producing microbial organism using a reverse degradation pathway.
This example describes an exemplary adipate synthesis pathway through 3-oxoadipate.
An additional pathway from that described in Examples I and II that uses acetyl-CoA and succinyl-CoA as precursors for adipate formation and passes through the metabolic intermediate, 3-oxoadipate, is shown in
The subsequent steps involve reduction of 3-oxoadipate to 3-hydroxyadipate (conversion of a keto group to hydroxyl group), dehydration of 3-hydroxyadipate to yield hexa-2-enedioate, and reduction of hexa-2-enedioate to form adipate. These steps of the pathway are analogous to the conversion of oxaloacetate into succinate via the reductive TCA cycle (see
The maximum theoretical yield of adipate using this pathway is 0.92 mole per mole glucose consumed, and oxygen is not required for attaining these yields (see Table 2). The associated energetics are identical to those of the reverse adipate pathway. Theoretically, ATP formation of up to 1.55 moles is observed per mole of glucose utilized through this pathway. The ATP yield improves to approximately 2.47 moles if phosphoenolpyruvate kinase (PPCK) is assumed to operate in the direction of ATP generation. Interestingly, the product yield can be increased further to 1 mole adipate per mole of glucose consumed if chemical hydrogenation is used for the last step and a 100% efficiency of catalysis is assumed. In this scenario, up to 1.95 moles of ATP are formed theoretically without assuming the reverse functionality of PPCK.
TABLE 2
The maximum theoretical yields of adipate and the associated ATP
yields per mole of glucose using the 3-oxoadipate pathway.
Final step chemical
Final step enzymatic
hydrogenation
Aerobic
Anaerobic
Aerobic
Anaerobic
Adipate Yield
0.92
0.92
1.00
1.00
Max ATP yield @ max
1.55
1.55
1.95
1.95
adipate yield
Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of adipate, one or more exogenous DNA sequence(s) can be expressed in a host microorganism. In addition, the host microorganism can have endogenous gene(s) functionally deleted. These modifications allow the production of adipate using renewable feedstock.
Described below are a number of biochemically characterized candidate genes capable of encoding enzymes that catalyze each step of the 3-oxoadipate pathway for adipate synthesis. Although this method is described for E. coli, one skilled in the art can apply these teachings to any other suitable host organism. Specifically, listed below are genes that are native to E. coli as well as genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed.
Referring to
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
pcaI
24985644
AAN69545.1
Pseudomonas putida
pcaI
50084858
YP_046368.1
Acinetobacter sp. ADP1
pcaI
21224997
NP_630776.1
Streptomyces coelicolor
The protein sequences of exemplary gene products for subunit B of this complex can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
pcaJ
26990657
NP_746082.1
Pseudomonas putida
pcaJ
21224996
NP_630775.1
Streptomyces coelicolor
pcaJ
141776
AAC37147.1
Acinetobacter sp. ADP1
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
mdh
1789632
AAC76268.1
Escherichia coli
ldhA
16129341
NP_415898.1
Escherichia coli
ldh
113866693
YP_725182.1
Ralstonia eutropha
bdh
177198
AAA58352.1
Homo sapiens
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
acnA
166215073
P25516.3
Escherichia coli
fumB
33112655
P14407.2
Escherichia coli
ilvD
146451
AAA24013.1
Escherichia coli
Other good candidates for carrying out this function are the serine dehydratases. These enzymes catalyze a very similar transformation in the removal of ammonia from serine as required in this dehydration step. The protein sequence for exemplary gene product can be found using the following GI number and/or GenBank identifier:
Gene
GenBank
name
GI#
Accession #
Organism
dsdA
2507445
P00926
Escherichia coli
Non-native gene candidates for this transformation have been identified as well. For example, the multi-subunit L-serine dehydratase from Peptostreptococcus asaccharolyticus was shown to complement an E. coli strain deficient in L-serine dehydratase activity (Hofineister et al., J. Bacteriol. 179:4937-4941 (1997)). Further, a putative 2-(hydroxymethyl)glutarate dehydratase, encoded by the gene hmd in Eubacterium barkeri shows similarity to both α- and β-subunits of [4Fe-4S]-containing bacterial serine dehydratases (Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006)). The protein sequence for exemplary gene product can be found using the following GI number and/or GenBank identifier:
Gene
GenBank
name
GI#
Accession #
Organism
Hmd
86278275
ABC88407.1
Eubacterium barkeri
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
fadH
16130976
NP_417552.1
Escherichia coli
enr
169405742
ACA54153.1
Clostridium botulinum A3 str
enr
2765041
CAA71086.1
Clostridium tyrobutyricum
enr
3402834
CAA76083.1
Clostridium kluyveri
The above description provides an exemplary adipate synthesis pathway by way of an 3-oxoadipate pathway.
This example describes the generation of a microbial organism capable of producing adipate using the 3-oxoadipate pathway.
Escherichia coli is used as a target organism to engineer the 3-oxoadipate pathway as shown in
To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in the 3-oxoadipate pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the paaJ (NP—415915.1), pcaIJ (AAN69545.1 and NP—746082.1), and bdh (AAA58352.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, and 3-oxoadipate reductase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the acnA (P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipate dehydratase and 2-enoate reductase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the 3-oxoadipate pathway.
The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 3-oxoadipate pathway genes for adipate synthesis is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional adipate synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of adipate. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates or the adipate product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production.
For large-scale production of adipate, the 3-oxoadipate pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the preparation of an adipate-producing microbial organism containing a 3-oxidoadipate pathway.
This example describes an adipate synthesis pathway previously described (see Niu et al., Biotechnol. Prog. 18(2): p. 201-11. 2002; Frost et al., U.S. Pat. No. 5,487,987, issued Jan. 30, 1996).
Adipate synthesis via a combined biological and chemical conversion process has been previously described. (Niu et al., Biotechnol. Prog. 18:201-211 (2002)) and is shown in
TABLE 3
The maximum theoretical yields of adipate per mole of glucose
using the using the cis,cis-muconic acid pathway.
Final step chemical
Final step enzymatic
hydrogenation
Aerobic
Anaerobic
Aerobic
Anaerobic
Adipate Yield
0.75
0.00
0.85
0.00
Although this is an exemplary method, there are disadvantages of this method compared to others, such as those described in Examples I-IV. For example, the first limitation of this method is the lower theoretical yields compared to the reverse adipate degradation and 3-oxoadipate pathways. The second limitation is that the ATP yields of this pathway are negligible. A third limitation of this pathway is that it involves a dioxygenase, necessitating a supply of oxygen to the bioreactor and precluding the option of anaerobic fermentation.
The above description provides an exemplary adipate synthesis pathway by way of a cis,cis-muconic acid pathway
This example describes an exemplary adipate synthesis pathway via an alpha-ketoadipate pathway.
Alpha-keto adipate is a known intermediate in lysine biosynthesis in S. cerevisiae, and this information was used to identify an additional pathway for adipic acid biosynthesis (see
The adipate yields associated with this pathway are shown in Table 4. Because of the loss of two CO2 molecules during the conversion of acetyl-CoA to adipate, only 67% of the glucose can be converted into adipate. This is reflected in the molar yields for this pathway under aerobic conditions. The yields are further reduced in the absence of oxygen uptake. Also since the maximum ATP yields under anaerobic conditions are negligible, the engineered organism will have to utilize additional substrate to form energy for cell growth and maintenance under such conditions.
TABLE 4
The maximum theoretical yields of adipate and
the associated ATP yields per mole of glucose
using the using the alpha-ketoadipate pathway.
Final step chemical
Final step enzymatic
hydrogenation
Aerobic
Anaerobic
Aerobic
Anaerobic
Adipate Yield
0.67
0.45
0.67
0.40
Max ATP yield @ max
6.17
0.00
7.50
0.00
adipate yield
The above description provides an exemplary adipate synthesis pathway by way of an alpha-ketoadipate pathway.
This example describes an exemplary adipate synthesis pathway via a lysine degradation pathway.
Two additional pathways for adipate synthesis rely on lysine degradation to form adipate. One pathway starts from alpha-ketoglutarate to form lysine (pathway non-native to E. coli and found in S. cerevisiae), and the other uses aspartate as a starting point for lysine biosynthesis (pathway native to E. coli).
TABLE 5
The maximum theoretical yield of adipate and the accompanying
ATP yield per mole of glucose assuming the lysine biosynthesis
pathway with alpha-ketoglutarate as a starting point.
Aerobic
Anaerobic
Adipate Yield
0.40
0.20
Max ATP yield @ max adipate yield
5.60
0.00
TABLE 6
The maximum theoretical yield of adipate and the accompanying
ATP yield per mole of glucose assuming the lysine biosynthesis
pathway with aspartate as a starting point.
Aerobic
Anaerobic
Adipate Yield
0.50
0.34
Max ATP yield @ max adipate yield
0.50
0.04
The above description provides an exemplary adipate synthesis pathway by way of a lysine degradation pathway.
This example describes an exemplary caprolactam and/or 6-aminocaproic acid synthesis pathway via an adipyl-CoA pathway.
An exemplary pathway for forming caprolactam and/or 6-aminocaproic acid using adipyl-CoA as the precursor is shown in
The final amidohydrolase step is energetically and redox neutral, and thus the product and ATP molar yields associated with 6-aminocaproic acid production are equivalent to those associated with caprolactam production. Thus one can alternatively envision a microorganism and associated fermentation process that forms 6-aminocaproic acid instead of caprolactam followed by an additional unit operation to dehydrate/cyclize 6-aminocaproic acid to caprolactam.
TABLE 7
The maximum theoretical yield of caprolactam and the
accompanying ATP yield per mole of glucose assuming
that the reverse fatty acid degradation pathway is complemented
with the reaction scheme from FIG. 8.
Aerobic
Anaerobic
Caprolactam Yield
0.80
0.80
Max ATP yield @ max Caprolactam yield
0.78
0.78
Max ATP yield @ max Caprolactam yield
1.63
1.63
PPCK assumed
Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of 6-aminocaproic acid or caprolactam, one or more exogenous DNA sequence(s) can be expressed in a host microorganism. In addition, the microorganism can have endogenous gene(s) functionally deleted. These modifications will allow the production of 6-aminocaproate or caprolactam using renewable feedstock.
Below is described a number of biochemically characterized candidate genes capable of encoding enzymes that catalyze each step of the caprolactam formation pathway described in
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
acr1
50086359
YP_047869.1
Acinetobacter calcoaceticus
18857901
BAB85476.1
Acinetobacter sp. Strain M-1
sucD
172046062
P38947.1
Clostridium kluyveri
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
gabT
16130576
NP_417148.1
Escherichia coli
puuE
16129263
NP_415818.1
Escherichia coli
abat
37202121
NP_766549.2
Mus musculus
gabT
70733692
YP_257332.1
Pseudomonas fluorescens
abat
47523600
NP_999428.1
Sus scrofa
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
lysDH
13429872
BAB39707
Geobacillus stearothermophilus
lysDH
15888285
NP_353966
Agrobacterium tumefaciens
lysDH
74026644
AAZ94428
Achromobacter denitrificans
Referring to
Plasmids contained in several strains of Pseudomonas sp. isolated from soil have been shown to confer ability to grow on caprolactam as a sole carbon source (Boronin et al., FEMS Microbiol. Lett. 22:167-170 (1984)); however, associated gene or protein sequences have not been associated with this function to date.
The most closely related candidate enzyme with available sequence information is 6-aminohexanoate-cyclic dimer hydrolase, which has been characterized in Pseudomonas sp. and Flavobacterium sp. The nylB gene product from Pseudomonas sp NK87 was cloned and expressed in E. coli (Kanagawa et al., J. Gen. Microbiol. 139:787-795 (1993)). The substrate specificity of the enzyme was tested in Flavobacterium sp K172 and was shown to react with higher-order oligomers of 6-aminohexanoate but not caprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). The reversibility and ability of 6-aminohexanoate dimer hydrolases in other organisms to react with the desired substrate in the direction of interest can be further tested. The protein sequences for exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:
Gene
GenBank
name
GI#
Accession #
Organism
nylB
148711
AAA24929.1
Pseudomonas sp NK87
nylB
129000
P13397
Flavobacterium sp K172
nylB
119961013
YP_949627.1
Arthrobacter aurescens TC1
The above description provides an exemplary pathway to produce caprolactam and/or 6-aminocaproic acid by way of an adipyl-CoA pathway.
This example describes the generation of a microbial organism capable of producing adipate using the reverse degradation pathway and converting the intracellular adipate to 6-aminocaproate and/or caprolactam.
Escherichia coli is used as a target organism to engineer the necessary genes for adipate, 6-aminocaproate, and/or caprolactam synthesis (see
To generate an E. coli strain engineered to produce 6-aminocaproate and/or caprolactam, nucleic acids encoding the enzymes utilized in the reverse adipate degradation pathway and 6-aminocaproate or caprolactam synthesis pathways are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP—415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the acr1 (YP—047869.1), gabT (NP—417148.1), and nylB (AAA24929.1) genes encoding CoA-dependent aldehyde dehydrogenase, transaminase, and amidohydrolase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproate and/or caprolactam synthesis.
The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproate and caprolactam synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6-aminocaproate and/or caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional pathway for the synthesis of 6-aminocaproate and/or caprolactam are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproate and/or caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6-aminocaproate and/or caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the products. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproate and/or caprolactam producer to further increase production.
For large-scale production of 6-aminocaproate and/or caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes two exemplary adipate synthesis pathways proceeding from alpha-ketoadipate and passing through a 2-hydroxyadipyl-CoA intermediate.
As described in example VI, alpha-ketoadipate is a known intermediate in lysine biosynthesis that can be formed from alpha-ketoglutarate via homocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase. Alpha-ketoadipate can be converted to 2-hydroxyadipyl-CoA by the two routes depicted in
Conversion of alpha-ketoadipate into 2-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977). Alternatively, enzymes capable of reducing alpha-ketoglutarate to 2-hydroxyglutarate may also show activity on alpha-ketoadipate, which is only one carbon atom longer. One such enzyme possessing alpha-ketoglutarate reductase activity is serA of Escherichia coli (Zhao and Winkler, J. Bacteriol. 178(1):232-9 (1996)). Additional exemplary enzymes can be found in Arabidopsis thaliana (Ho, et al., J. Biol. Chem. 274(1):397-402 (1999)) and Haemophilus influenzae.
Gene
GenBank
name
GI#
Accession #
Organism
serA
16130814
NP_417388.1
Escherichia coli
PGDH
18394525
NP_564034
Arabidopsis thaliana
serA
1173427
P43885
Haemophilus influenzae
Referring to
Gene
GenBank
name
GI#
Accession #
Organism
gctA
3122155
Q59111
Acidaminococcus fermentans
gctB
3122156
Q59112
Acidaminococcus fermentans
The dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoA can be carried out by a 2-hydroxyacyl-CoA dehydratase. A 2-hydroxyglutaryl-CoA dehydratase system has been characterized in Acidaminococcus fermentans and requires both the hgdA and hgdB subunits and the activator protein, hgdC, for optimal activity (Dutscho et al., Eur. J. Biochem. 181(3):741-746 (1989); Locher et al. J. Mol. Biol. 307(1):297-308; Muller and Buckel, Eur. J. Biochem. 230(2):698-704 (2001); Schweiger et al. Eur. J. Biochem. 169(2):441-448 (1987)). This enzyme system is similar in mechanism to the lactoyl-CoA dehydratase from Clostridium propionicum (Hofineister and Buckel, Eur. J. Biochem. 206(2):547-552 (1992); Kuchta and Abeles, J. Biol. Chem. 260(24):13181-13189 (1985)). Homologs to hgdA, hgdB, and hgdC exist in several organisms.
Gene
GenBank
name
GI#
Accession #
Organism
hgdA
123112
P11569
Acidaminococcus fermentans
hgdB
123113
P11570
Acidaminococcus fermentans
hgdC
2506909
P11568
Acidaminococcus fermentans
hgdA
225177593
ZP_03731126.1
Clostridium sp. M62/1
hgdB
225177592
ZP_03731125.1
Clostridium sp. M62/1
hgdC
225177594
ZP_03731127.1
Clostridium sp. M62/1
hgdA
19703552
NP_603114.1
Fusobacterium nucleatum
hgdB
19703553
NP_603115.1
Fusobacterium nucleatum
hgdC
19703551
NP_603113.1
Fusobacterium nucleatum
Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out by the enzymes described in Example I.
The above description provides an exemplary adipate synthesis pathway by way of a 2-hydroxyadipyl-CoA pathway.
This example describes the generation of a microbial organism capable of producing adipate using a 2-hydroxyadipyl-CoA pathway.
Escherichia coli is used as a target organism to engineer the necessary genes for adipate synthesis (see
To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in a 2-hydroxyadipyl-CoA to adipate pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the serA (NP—417388.1), gctA (Q59111), and gctB (Q59112) genes encoding the 2-hydroxyadipate dehydrogenase and 2-hydroxyadipyl-CoA:acetyl-CoA transferase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the hgdA (P11569), hgdB (P11570), and hgdC (P11568) genes encoding 2-hydroxyadipyl-CoA dehydratase activity, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Further, the bcd (NP—349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP—415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis.
The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 2-hydroxyadipyl-CoA pathway genes for adipate synthesis is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional adipate synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of adipate. Adaptive evolution also can be used to generate better producers of, for example, the alpha-ketoadipate intermediate or the adipate product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production.
For large-scale production of adipate, the 2-hydroxyadipyl-CoA pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the preparation of an adipate-producing microbial organism containing a 2-hydroxyadipyl-CoA pathway.
This example describes exemplary pathways for production of hexamethylenediamine, caprolactam and 6-aminocaproic acid.
Described below are various pathways leading to the production of caprolactam, hexamethylenediamine (HMDA), or 6-aminocaproate from common central metabolites. The first described pathway entails the activation of 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthase enzyme (
Another option for minimizing or even eliminating the formation of the cyclic imine or caprolactam during the conversion of 6-aminocaproic acid to HMDA entails adding a functional group (for example, acetyl, succinyl) to the amine group of 6-aminocaproic acid to protect it from cyclization. This is analogous to ornithine formation from L-glutamate in Escherichia coli. Specifically, glutamate is first converted to N-acetyl-L-glutamate by N-acetylglutamate synthase. N-Acetyl-L-glutamate is then activated to N-acetylglutamyl-phosphate, which is reduced and transaminated to form N-acetyl-L-ornithine. The acetyl group is then removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase forming L-ornithine. Such a route is necessary because formation of glutamate-5-phosphate from glutamate followed by reduction to glutamate-5-semialdehyde leads to the formation of (S)-1-pyrroline-5-carboxylate, a cyclic imine formed spontaneously from glutamate-5-semialdehyde. In the case of forming HMDA from 6-aminocaproic acid, the steps can involve acetylating 6-aminocaproic acid to acetyl-6-aminocaproic acid, activating the carboxylic acid group with a CoA or phosphate group, reducing, aminating, and deacetylating.
Note that 6-aminocaproate can be formed from various starting molecules. For example, the carbon backbone of 6-aminocaproate can be derived from succinyl-CoA and acetyl-CoA as depicted in
The starting molecule, 4-aminobutyryl-CoA, can be formed from various common central metabolites. For example, glutamate can be decarboxylated to 4-aminobutyrate, which is then activated by a CoA-transferase or synthase to 4-aminobutyryl-CoA. Alternatively, succinate semialdehyde, formed from either the reduction of succinyl-CoA or the decarboxylation of alpha-ketoglutarate, can be transaminated to 4-aminobutyrate prior to activation by a CoA-transferase or synthase to form 4-aminobutyryl-CoA. It is noted that 4-aminobutyryl-CoA and several of the intermediates of the 4-aminobutyryl-CoA to 6-aminocaproyl-CoA pathway may spontaneously cyclize to their corresponding lactams. Thus, adding a protective functional group to the terminal amine group of 4-aminobutyryl-CoA and/or several of the amino-CoA intermediates can be used to minimize the formation of unwanted cyclic byproducts. In this case, the same general set of transformations depicted in
All transformations depicted in
TABLE 8
Enzyme types for conversion of succinyl-CoA, acetyl-
CoA, and/or 4-aminobutyryl-CoA to 6-aminocaproate,
caprolactam, and/or hexamethylenediamine.
Label
Function
1.1.1.a
Oxidoreductase (ketone to hydroxyl or aldehyde to
alcohol)
1.2.1.b
Oxidoreductase (acyl-CoA to aldehyde)
1.3.1.a
Oxidoreductase operating on CH—CH donors
1.4.1.a
Oxidoreductase operating on amino acids
2.3.1.b
Acyltransferase
2.6.1.a
Aminotransferase
2.8.3.a
Coenzyme-A transferase
3.1.2.a
Thiolester hydrolase (CoA specific)
4.2.1.a
Hydro-lyase
6.2.1.a
Acid-thiol ligase
6.3.1.a/6.3.2.a
Amide synthases/peptide synthases
No enzyme
Spontaneous cyclization
required
The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.
1.1.1.a Oxidoreductases. Four transformations depicted in
Exemplary enzymes that can convert 3-oxoacyl-CoA molecules such as 3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA into 3-hydroxyacyl-CoA molecules such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA, respectively, include enzymes whose natural physiological roles are in fatty acid beta-oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71:403-411 (1981)). Furthermore, the gene products encoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze the reverse reaction of step B in
Gene
GenBank
name
GI#
Accession #
Organism
fadB
119811
P21177.2
Escherichia coli
fadJ
3334437
P77399.1
Escherichia coli
paaH
16129356
NP_415913.1
Escherichia coli
phaC
26990000
NP_745425.1
Pseudomonas putida
paaC
106636095
ABF82235.1
Pseudomonas fluorescens
Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807 (1989)). Additional gene candidates include Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al., J. Biol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol.Microbiol 61:297-309 (2006)). The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol. 3:349-357 (1989)) and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al., supra).
Gene
GenBank
name
GI#
Accession #
Organism
hbd
18266893
P52041.2
Clostridium
acetobutylicum
Hbd2
146348271
EDK34807.1
Clostridium kluyveri
Hbd1
146345976
EDK32512.1
Clostridium kluyveri
HSD17B10
3183024
O02691.3
Bos taurus
phbB
130017
P23238.1
Zoogloea ramigera
phaB
146278501
YP_001168660.1
Rhodobacter sphaeroides
A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
hbd
15895965
NP_349314.1
Clostridium
acetobutylicum
hbd
20162442
AAM14586.1
Clostridium beijerinckii
Msed_1423
146304189
YP_001191505
Metallosphaera sedula
Msed_0399
146303184
YP_001190500
Metallosphaera sedula
Msed_0389
146303174
YP_001190490
Metallosphaera sedula
Msed_1993
146304741
YP_001192057
Metallosphaera sedula
Various alcohol dehydrogenases represent good candidates for converting 3-oxoadipate to 3-hydroxyadipate (step H,
Gene
GenBank
name
GI#
Accession #
Organism
mdh
1789632
AAC76268.1
Escherichia coli
ldhA
16129341
NP_415898.1
Escherichia coli
ldh
113866693
YP_725182.1
Ralstonia eutropha
bdh
177198
AAA58352.1
Homo sapiens
adh
60592974
AAA23199.2
Clostridium beijerinckii
adh
113443
P14941.1
Thermoanaerobacter brockii
1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde). The transformations of adipyl-CoA to adipate semialdehyde (Step N,
Gene
GenBank
name
GI#
Accession #
Organism
acr1
50086359
YP_047869.1
Acinetobacter calcoaceticus
acr1
1684886
AAC45217
Acinetobacter baylyi
acr1
18857901
BAB85476.1
Acinetobacter sp. Strain M-1
sucD
172046062
P38947.1
Clostridium kluyveri
sucD
34540484
NP_904963.1
Porphyromonas gingivalis
bphG
425213
BAA03892.1
Pseudomonas sp
adhE
55818563
AAV66076.1
Leuconostoc mesenteroides
An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., supra; Thauer R. K., Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed—0709 in Metallosphaera sedula (Alber et al., supra; Berg et al., supra). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., supra). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to cutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra).
Gene
GenBank
name
GI#
Accession #
Organism
Msed_0709
146303492
YP_001190808.1
Metallosphaera sedula
mcr
15922498
NP_378167.1
Sulfolobus tokodaii
asd-2
15898958
NP_343563.1
Sulfolobus solfataricus
Saci_2370
70608071
YP_256941.1
Sulfolobus
acidocaldarius
Ald
49473535
AAT66436
Clostridium beijerinckii
eutE
687645
AAA80209
Salmonella typhimurium
eutE
2498347
P77445
Escherichia coli
1.3.1.a Oxidoreductase Operating on CH—CH Donors. Referring to
Gene
GenBank
name
GI#
Accession #
Organism
bcd
15895968
NP_349317.1
Clostridium acetobutylicum
etfA
15895966
NP_349315.1
Clostridium acetobutylicum
etfB
15895967
NP_349316.1
Clostridium acetobutylicum
TER
62287512
Q5EU90.1
Euglena gracilis
TDE0597
42526113
NP_971211.1
Treponema denticola
Step J of both
Gene
GenBank
name
GI#
Accession #
Organism
fadH
16130976
NP_417552.1
Escherichia coli
enr
169405742
ACA54153.1
Clostridium botulinum A3 str
enr
2765041
CAA71086.1
Clostridium tyrobutyricum
enr
3402834
CAA76083.1
Clostridium kluyveri
enr
83590886
YP_430895.1
Moorella thermoacetica
1.4.1.a Oxidoreductase Operating on Amino Acids.
Most oxidoreductases operating on amino acids catalyze the oxidative deamination of alpha-amino acids with NAD+ or NADP+ as acceptor, though the reactions are typically reversible. Exemplary oxidoreductases operating on amino acids include glutamate dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh, and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (McPherson et al., Nucleic. Acids Res. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273 (1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles 1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998); Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Stoyan et al., J. Biotechnol 54:77-80 (1997); Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)). The nadX gene from Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808 (2003)).
Gene
GenBank
name
GI#
Accession #
Organism
gdhA
118547
P00370
Escherichia coli
gdh
6226595
P96110.4
Thermotoga maritima
gdhA1
15789827
NP_279651.1
Halobacterium salinarum
ldh
61222614
P0A393
Bacillus cereus
nadX
15644391
NP_229443.1
Thermotoga maritima
The lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes, catalyze the oxidative deamination of the 8-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn nonenzymatically cyclizes to form Δ1-piperideine-6-carboxylate (Misono et al., J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); Misono et al., supra), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate given the structural similarity between adipate semialdehyde and 2-aminoadipate-6-semialdehyde.
Gene
GenBank
name
GI#
Accession #
Organism
lysDH
13429872
BAB39707
Geobacillus stearothermophilus
lysDH
15888285
NP_353966
Agrobacterium tumefaciens
lysDH
74026644
AAZ94428
Achromobacter denitrificans
2.3.1.b Acyl Transferase. Referring to
Gene
GenBank
name
GI#
Accession #
Organism
paaJ
16129358
NP_415915.1
Escherichia coli
pcaF
17736947
AAL02407
Pseudomonas knackmussii (B13)
phaD
3253200
AAC24332.1
Pseudomonas putida
paaE
106636097
ABF82237.1
Pseudomonas fluorescens
2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymes present additional candidates for performing step A in
Gene
GenBank
name
GI#
Accession #
Organism
ortA (α)
126698017
YP_001086914.1
Clostridium
difficile 630
ortB (β)
126698018
YP_001086915.1
Clostridium
difficile 630
Amet_2368
150390132
YP_001320181.1
Alkaliphilus
(α)
metalliredigenes QYF
Amet_2369
150390133
YP_001320182.1
Alkaliphilus
(β)
metalliredigenes QYF
Teth514_1478
167040116
YP_001663101.1
Thermoanaerobacter
(α)
sp. X514
Teth514_1479
167040117
YP_001663102.1
Thermoanaerobacter
(β)
sp. X514
TTE1235
20807687
NP_622858.1
Thermoanaerobacter
(α)
tengcongensis MB4
thrC (β)
20807688
NP_622859.1
Thermoanaerobacter
tengcongensis MB4
2.6.1.a Aminotransferase. Step O of
Gene
GenBank
name
GI#
Accession #
Organism
gabT
16130576
NP_417148.1
Escherichia coli
puuE
16129263
NP_415818.1
Escherichia coli
abat
37202121
NP_766549.2
Mus musculus
gabT
70733692
YP_257332.1
Pseudomonas fluorescens
abat
47523600
NP_999428.1
Sus scrofa
Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to hexamethylenediamine The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K. H., J Biol Chem 239:783-786 (1964)). A putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol 184:3765-3773 (2002)).
Gene
GenBank
name
GI#
Accession #
Organism
ygjG
145698310
NP_417544
Escherichia coli
spuC
9946143
AAG03688
Pseudomonas aeruginosa
Yet additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferases which produce malonate semialdehyde from beta-alanine (WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri was also shown to preferentially use beta-alanine as the amino group donor (Andersen et al., FEBS. J. 274:1804-1817 (2007)). SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem., 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both β-alanine and GABA transamination (Andersen et al., supra). 3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2-methylpropionate. This enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Tamaki et al, Methods Enzymol, 324:376-389 (2000)).
Gene
GenBank
name
GI#
Accession #
Organism
SkyPYD4
98626772
ABF58893.1
Saccharomyces kluyveri
SkUGA1
98626792
ABF58894.1
Saccharomyces kluyveri
UGA1
6321456
NP_011533.1
Saccharomyces cerevisiae
Abat
122065191
P50554.3
Rattus norvegicus
Abat
120968
P80147.2
Sus scrofa
2.8.3.a Coenzyme-A Transferase. CoA transferases catalyze reversible reactions that involve the transfer of a CoA moiety from one molecule to another. For example, step E of
Gene
GenBank
name
GI#
Accession #
Organism
pcaI
24985644
AAN69545.1
Pseudomonas putida
pcaJ
26990657
NP_746082.1
Pseudomonas putida
pcaI
50084858
YP_046368.1
Acinetobacter sp. ADP1
pcaJ
141776
AAC37147.1
Acinetobacter sp. ADP1
pcaI
21224997
NP_630776.1
Streptomyces coelicolor
pcaJ
21224996
NP_630775.1
Streptomyces coelicolor
HPAG1_0676
108563101
YP_627417
Helicobacter pylori
HPAG1_0677
108563102
YP_627418
Helicobacter pylori
ScoA
16080950
NP_391778
Bacillus subtilis
ScoB
16080949
NP_391777
Bacillus subtilis
A 3-oxoacyl-CoA transferase that can utilize acetate as the CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme has also been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583 (1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
atoA
2492994
P76459.1
Escherichia coli K12
atoD
2492990
P76458.1
Escherichia coli K12
actA
62391407
YP_226809.1
Corynebacterium
glutamicum ATCC 13032
cg0592
62389399
YP_224801.1
Corynebacterium
glutamicum ATCC 13032
ctfA
15004866
NP_149326.1
Clostridium acetobutylicum
ctfB
15004867
NP_149327.1
Clostridium acetobutylicum
ctfA
31075384
AAP42564.1
Clostridium
saccharoperbutylacetonicum
ctfB
31075385
AAP42565.1
Clostridium
saccharoperbutylacetonicum
The above enzymes may also exhibit the desired activities on adipyl-CoA and adipate (
Gene
GenBank
name
GI#
Accession #
Organism
cat1
729048
P38946.1
Clostridium kluyveri
cat2
172046066
P38942.2
Clostridium kluyveri
cat3
146349050
EDK35586.1
Clostridium kluyveri
The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).
Gene
GenBank
name
GI#
Accession #
Organism
gctA
559392
CAA57199.1
Acidaminococcus fermentans
gctB
559393
CAA57200.1
Acidaminococcus fermentans
3.1.2.a Thiolester Hydrolase (CoA Specific). Several eukaryotic acetyl-CoA hydrolases have broad substrate specificity and thus represent suitable candidate enzymes for hydrolyzing 3-oxoadipyl-CoA, adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA, or 6-aminocaproyl-CoA (Steps G and M of
Gene
GenBank
name
GI#
Accession #
Organism
acot12
18543355
NP_570103.1
Rattus norvegicus
Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valine degradation (Shimomura et al., J Biol. Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus cereus.
Gene
GenBank
name
GI#
Accession #
Organism
hibch
146324906
Q5XIE6.2
Rattus norvegicus
hibch
146324905
Q6NVY1.2
Homo sapiens
hibch
2506374
P28817.2
Saccharomyces cerevisiae
BC_292
29895975
AP09256
Bacillus cereus
Yet another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of CoA thiolesters (Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)).
Gene
GenBank
name
GI#
Accession #
Organism
tesB
16128437
NP_414986
Escherichia coli
acot8
3191970
CAA15502
Homo sapiens
acot8
51036669
NP_570112
Rattus norvegicus
Other potential E. coli thiolester hydrolases include the gene products of tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); Zhuang et al., FEBS Lett 516:161-163 (2002)), paaI (Song et al., J Biol Chem 281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol 189:7112-7126 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
tesA
16128478
NP_415027
Escherichia coli
ybgC
16128711
NP_415264
Escherichia coli
paaI
16129357
NP_415914
Escherichia coli
ybdB
16128580
NP_415129
Escherichia coli
6.3.1.a/6.3.2.a Amide Synthases/Peptide Synthases. The direct conversion of 6-aminocaproate to caprolactam (Step S,
Gene
GenBank
name
GI#
Accession #
Organism
acsA
60650089
BAD90933
Pseudomonas chlororaphis
puuA
87081870
AAC74379
Escherichia coli
bls
41016784
Q9R8E3
Streptomyces clavuligerus
4.2.1.a Hydrolyase. Most dehydratases catalyze the α, β-elimination of water. This involves activation of the a-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the β-position. Enzymes exhibiting activity on substrates with an electron-withdrawing carboxylate group are excellent candidates for dehydrating 3-hydroxyadipate (
For example, fumarase enzymes naturally catalyze the reversible dehydration of malate to fumarate. E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001);Woods et al., Biochim Biophys Acta 954:14-26 (1988);Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates are found in Campylobacter jejuni (Smith et al., Int. J Biochem.Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology include fum1 from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
fumA
81175318
P0AC33
Escherichia coli
fumB
33112655
P14407
Escherichia coli
fumC
120601
P05042
Escherichia coli
fumC
9789756
O69294
Campylobacter jejuni
fumC
3062847
BAA25700
Thermus thermophilus
fumH
120605
P14408
Rattus norvegicus
fum1
39931311
P93033
Arabidopsis thaliana
fumC
39931596
Q8NRN8
Corynebacterium glutamicum
MmcB
147677691
YP_001211906
Pelotomaculum
thermopropionicum
MmcC
147677692
YP_001211907
Pelotomaculum
thermopropionicum
Two additional dehydratase candidates are 2-(hydroxymethyl)glutarate dehydratase and dimethylmaleate hydratase, enzymes studied for their role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006)). 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate. This enzyme is encoded by hmd in Eubacterium barkeri (Alhapel et al., supra). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing bacterial serine dehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA).
Gene
GenBank
name
GI#
Accession #
Organism
hmd
86278275
ABC88407.1
Eubacterium
barkeri
BACCAP_02294
154498305
ZP_02036683.1
Bacteroides
capillosus
ANACOL_02527
167771169
ZP_02443222.1
Anaerotruncus
colihominis DSM
17241
NtherDRAFT_2368
169192667
ZP_02852366.1
Natranaerobius
thermophilus
JW/NM-WN-LF
Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857 (1984)).
Gene
GenBank
name
GI#
Accession #
Organism
dmdA
86278276
ABC88408
Eubacterium barkeri
dmdB
86278277
ABC88409.1
Eubacterium barkeri
An additional enzyme candidate is 2-methylmalate dehydratase, also called citramalate hydrolyase, a reversible hydrolyase that catalyzes the alpha, beta elimination of water from citramalate to form mesaconate. This enzyme has been purified and characterized in Clostridium tetanomorphum (Wang et al., J Biol. Chem. 244:2516-2526 (1969)). The activity of this enzyme has also been detected in several bacteria in the genera Citrobacter and Morganella in the context of the glutamate degradation VI pathway (Kato et al., Arch. Microbiol 168:457-463 (1997)). Genes encoding this enzyme have not been identified in any organism to date.
Enzymes exhibiting activity on substrates with an electron-withdrawing CoA-thiol ester group adjacent to the α-hydrogen are excellent candidates for dehydrating 3-hydroxyadipyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
paaA
26990002
NP_745427.1
Pseudomonas fluorescens
paaB
26990001
NP_745426.1
Pseudomonas fluorescens
phaA
106636093
ABF82233.1
Pseudomonas putida
phaB
106636094
ABF82234.1
Pseudomonas putida
maoC
16129348
NP_415905.1
Escherichia coli
paaF
16129354
NP_415911.1
Escherichia coli
paaG
16129355
NP_415912.1
Escherichia coli
crt
15895969
NP_349318.1
Clostridium acetobutylicum
crt1
153953091
YP_001393856
Clostridium kluyveri DSM 555
6.2.1.a Acid-Thiol Ligase. Steps F, L, and R of
Gene
GenBank
name
GI#
Accession #
Organism
sucC
16128703
NP_415256.1
Escherichia coli
sucD
1786949
AAC73823.1
Escherichia coli
Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Bower et al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.
Gene
GenBank
name
GI#
Accession #
Organism
phl
77019264
CAJ15517.1
Penicillium chrysogenum
phlB
152002983
ABS19624.1
Penicillium chrysogenum
paaF
22711873
AAC24333.2
Pseudomonas putida
bioW
50812281
NP_390902.2
Bacillus subtilis
AACS
21313520
NP_084486.1
Mus musculus
AACS
31982927
NP_076417.2
Homo sapiens
ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra).
Gene
GenBank
name
GI#
Accession #
Organism
AF1211
11498810
NP_070039.1
Archaeoglobus fulgidus
DSM 4304
scs
55377722
YP_135572.1
Haloarcula marismortui
ATCC 43049
PAE3250
18313937
NP_560604.1
Pyrobaculum aerophilum
str. IM2
Yet another option is to employ a set of enzymes with net ligase or synthetase activity. For example, phosphotransadipylase and adipate kinase enzymes are catalyzed by the gene products of buk1, buk2, and ptb from C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb gene encodes an enzyme that can convert butyryl-CoA into butyryl-phosphate, which is then converted to butyrate via either of the buk gene products with the concomitant generation of ATP.
Gene
GenBank
name
GI#
Accession #
Organism
ptb
15896327
NP_349676
Clostridium acetobutylicum
buk1
15896326
NP_349675
Clostridium acetobutylicum
buk2
20137415
Q97II1
Clostridium acetobutylicum
No enzyme required—Spontaneous cyclization. 6-Aminocaproyl-CoA will cyclize spontaneously to caprolactam, thus eliminating the need for a dedicated enzyme for this step. A similar spontaneous cyclization is observed with 4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi et al., J Biol Chem 256:7642-7651 (1981)).
This example describes the generation of a microbial organism capable of producing 6-aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA.
Escherichia coli is used as a target organism to engineer the 6-aminocaproic acid pathway shown in
To generate an E. coli strain engineered to produce 6-aminocaproic acid, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1), etfAB (NP—349315.1 and NP—349316.1), and acot8 (CAA15502) genes encoding 6-aminohex-2-enoyl-CoA reductase and 6-aminocaproyl-CoA hydrolase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD (NP—904963.1), gabT (NP—417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproic acid synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6-aminocaproic acid. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production.
For large-scale production of 6-aminocaproic acid, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the generation of a microbial organism capable of producing 6-aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA.
Escherichia coli is used as a target organism to engineer the 6-aminocaproic acid pathway shown in
To generate an E. coli strain engineered to produce 6-aminocaproic acid, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), pcaIJ (AAN69545.1 and NP—746082.1), and bdh (AAA58352.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoate reductase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the enr (CAA76083.1) and hmd (ABC88407.1) genes encoding 6-aminohex-2-enoate reductase and 3-hydroxy-6-aminohexanoate dehydratase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD (NP—904963.1), gabT (NP—417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproic acid synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6-aminocaproic acid. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production.
For large-scale production of 6-aminocaproic acid, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the generation of a microbial organism capable of producing caprolactam from acetyl-CoA and succinyl-CoA.
Escherichia coli is used as a target organism to engineer the caprolactam pathway shown in
To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1) and etfAB (NP—349315.1 and NP—349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the acr1 (YP—047869.1), gabT (NP—417148.1), and bioW (NP—390902.2) genes encoding adipyl-CoA reductase (aldehyde forming), 6-aminocaproic acid transaminase, and 6-aminocaproyl-CoA synthase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the caprolactam synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional caprolactam synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the caprolactam producer to further increase production.
For large-scale production of caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the generation of a microbial organism capable of producing hexamethylenediamine from acetyl-CoA and succinyl-CoA.
Escherichia coli is used as a target organism to engineer the hexamethylenediamine pathway shown in
To generate an E. coli strain engineered to produce hexamethylenediamine, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1) and etfAB (NP—349315.1 and NP—349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the acr1 (YP—047869.1), gabT (NP—417148.1), bioW (NP—390902.2), and ygjG (NP—417544) genes encoding adipyl-CoA reductase (aldehyde forming), 6-aminocaproyl-CoA reductase (aldehyde forming), 6-aminocaproic acid transaminase, 6-aminocaproyl-CoA synthase, and hexamethylenediamine transaminase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylenediamine synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the hexamethylenediamine synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce hexamethylenediamine is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional hexamethylenediamine synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of hexamethylenediamine One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of hexamethylenediamine Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the hexamethylenediamine product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the hexamethylenediamine producer to further increase production.
For large-scale production of hexamethylenediamine, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the generation of a microbial organism capable of producing caprolactam from acetyl-CoA and 4-aminobutyryl-CoA.
Escherichia coli is used as a target organism to engineer the caprolactam pathway shown in
To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1) and etfAB (NP—349315.1 and NP—349316.1) genes encoding 6-aminohex-2-enoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD (NP—904963.1), gabT (NP—417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the caprolactam synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional caprolactam synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the caprolactam producer to further increase production.
For large-scale production of caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes the generation of a microbial organism capable of producing hexamethylenediamine from acetyl-CoA and 4-aminobutyryl-CoA.
Escherichia coli is used as a target organism to engineer the hexamethylenediamine pathway shown in Figure XVII that starts from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing hexamethylenediamine E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.
To generate an E. coli strain engineered to produce hexamethylenediamine, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP—415915.1), paaH (NP—415913.1), and maoC (NP—415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP—349317.1), etfAB (NP—349315.1 and NP—349316.1), acr1 (YP—047869.1), and ygjG (NP—417544) genes encoding 6-aminohex-2-enoyl-CoA reductase, 6-aminocaproyl-CoA reductase (aldehyde forming), and hexamethylenediamine transaminase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD (NP—904963.1), gabT (NP—417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylenediamine synthesis.
The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the hexamethylenediamine synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce hexamethylenediamine is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional hexamethylenediamine synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of hexamethylenediamine One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of hexamethylenediamine Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the hexamethylenediamine product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the hexamethylenediamine producer to further increase production.
For large-scale production of hexamethylenediamine, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).
This example describes exemplary pathways for production of 6-aminocaproic acid.
Novel pathways for producing 6-aminocaproic acid (6-ACA) and related products are described herein. These pathways synthesize 6-ACA from succinic semialdehyde and pyruvate, utilizing aldolase and hydratase enzymes from the 4-hydroxyphenylacetic acid degradation pathway. The candidate enzymes, and associated risks of implementation are discussed in Example XXI below.
This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 6-ACA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.
6-aminocaproic acid and derivatives are produced from succinic semialdehyde and pyruvate in a minimum of five enzymatic steps. In the first step of all pathways, pyruvate and succinic semialdehyde are joined by 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase. The product of this reaction, HODH, is then dehydrated by 2-oxohept-4-ene-1,7-dioate (OHED) hydratase to form OHED. In subsequent steps, OHED is transaminated, decarboxylated or reduced as shown in
In one route, the alkene of OHED is reduced by OHED reductase, forming 2-oxoheptane-1,7-dioate (2-OHD) (
In a similar route, the 2-keto group of 2-OHD is transaminated by an aminotransferase or an aminating oxidoreductase (
In an alternate route, OHED is first decarboxylated by OHED decarboxylase (
Yet another route calls for an aminotransferase or aminating oxidoreductase to convert OHED to 2-aminohept-4-ene-1,7-dioate (2-AHE) (
In yet another route, HODH is converted to 3-hydroxyadipyl-CoA by either an HODH dehydrogenase or and HODH formate-lyase (
In a similar route, HODH is first converted to OHED (
In the final route, HODH is converted to 2-OHD via steps B and C, as described previously. A 2-OHD formate-lyase or dehydrogenase converts 2-OHD to adipyl-CoA (
The routes detailed in
This example describes exemplary pathways for production of hexamethylenediamine
Novel pathways for producing hexamethylenediamine (HMDA) and related products are described herein. This pathway synthesizes HMDA from 6-Aminocaproate (6-ACA). These pathways involve activation of the acid group by phosphorylation and/or acylation. Acetylation of the terminal amino group provides protection from spontaneous cyclization of pathway intermediates. The candidate enzymes, and associated risks of implementation are discussed in Example XXI below.
This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze HMDA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.
Several pathways for producing HMDA from 6-aminocaproate are detailed in
In one route, 6-aminocaproate is phosphorylated to 6-AHOP by 6-aminocaproate kinase (
Alternately, 6-AHOP is converted to 6-aminocaproyl-CoA by an acyltransferase (
In yet another route, 6-aminocaproate is first activated to a CoA derivative by a CoA transferase or CoA ligase (
Additional routes proceed from 6-acetamidohexanoate, the acetylated product of 6-aminocaproate N-acetyltransferase. 6-Acetamidohexanoate is converted to 6-acetamidohexanal by different routes (described below). In the final two steps of these routes, 6-acetamidohexanal is first converted to 6-acetamidohexanamine by an aminotransferase or an aminating oxidoreductase (
In one route, 6-acetamidohexanoate is phosphorylated by 6-acetamidohexanoate kinase (
In another route, 6-acetamidohexanoate is activated to 6-acetamidohexanoyl-CoA by a CoA transferase or CoA ligase (
Alternately, 6-acetamidohexanoate is phosphorylated to 6-AAHOP (
This example describes the enzyme classification system for the exemplary pathways described in Examples XIX and XX for production of 6-aminocaproate or hexamethylenediamine
All transformations depicted in
Table 9 shows the enzyme types useful to convert common central metabolic intermediates into 6-aminocaproate and hexamethylenediamine. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.
TABLE 9
LABEL
FUNCTION
1.2.1.b
Oxidoreductase (acyl-CoA to aldehyde)
1.2.1.c
Oxidoreductase (2-ketoacid to acyl-CoA)
1.2.1.d
Oxidoreductase (phosphonic acid to aldehyde)
1.3.1.a
Oxidoreductase (alkene to alkane)
1.4.1.a
Oxidoreductase (ketone or aldehyde to amino)
2.3.1.a
Acyltransferase (transferring CoA to phospho)
2.3.1.c
Acyltransferase (N-acetyltransferase)
2.3.1.d
Acyltransferase (formate C-acyltransferase)
2.6.1.a
Aminotransferase
2.7.2.a
Phosphotransferase (carboxy acceptor)
2.8.3.a
Coenzyme-A transferase
3.5.1.a
Hydrolase (acting on linear amides)
4.1.1.a
Carboxy-lyase
4.1.2.a
Aldehyde-lyase
4.2.1.a
Hydro-lyase
6.2.1.a
Acid-thiol ligase
1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde). The transformations of 6-acetamidohexanoyl-CoA to 6-acetamidohexanal (
Gene
GenBank
name
GI#
Accession #
Organism
acr1
50086359
YP_047869.1
Acinetobacter calcoaceticus
acr1
1684886
AAC45217
Acinetobacter baylyi
acr1
18857901
BAB85476.1
Acinetobacter sp. Strain M-1
sucD
172046062
P38947.1
Clostridium kluyveri
sucD
34540484
NP_904963.1
Porphyromonas gingivalis
bphG
425213
BAA03892.1
Pseudomonas sp
adhE
55818563
AAV66076.1
Leuconostoc mesenteroides
An additional enzyme that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786 (2007); and Thauer, R. K., Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed—0709 in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al., Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WIPO Patent Application WO/2007/141208 Kind Code: A2). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)).
Gene
GenBank
name
GI#
Accession #
Organism
Msed_0709
146303492
YP_001190808.1
Metallosphaera sedula
mcr
15922498
NP_378167.1
Sulfolobus tokodaii
asd-2
15898958
NP_343563.1
Sulfolobus solfataricus
Saci_2370
70608071
YP_256941.1
Sulfolobus
acidocaldarius
Ald
49473535
AAT66436
Clostridium beijerinckii
eutE
687645
AAA80209
Salmonella typhimurium
eutE
2498347
P77445
Escherichia coli
1.2.1.c Oxidoreductase (2-Ketoacid to Acyl-CoA). Several transformations in
Activity of enzymes in the 2-keto-acid dehydrogenase family is normally low or limited under anaerobic conditions in E. coli. Increased production of NADH (or NADPH) could lead to a redox-imbalance, and NADH itself serves as an inhibitor to enzyme function. Engineering efforts have increased the anaerobic activity of the E. coli pyruvate dehydrogenase complex (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effect of NADH can be overcome by engineering an H322Y mutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858 (2008)). Structural studies of individual components and how they work together in complex provide insight into the catalytic mechanisms and architecture of enzymes in this family (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Zhou et al., Proc. Natl. Acad. Sci. U.S. A 98:14802-14807 (2001)). The substrate specificity of the dehydrogenase complexes varies in different organisms, but generally branched-chain keto-acid dehydrogenases have the broadest substrate range.
Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA and is the primary site of control of metabolic flux through the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278 (1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD gene expression is downregulated under anaerobic conditions and during growth on glucose (Park et al., Mol. Microbiol. 15:473-482 (1995)). Although the substrate range of AKGD is narrow, structural studies of the catalytic core of the E2 component pinpoint specific residues responsible for substrate specificity (Knapp et al., J. Mol. Biol. 280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and pdhD (E3, shared domain), is regulated at the transcriptional level and is dependent on the carbon source and growth phase of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296 (1992)). In yeast, the LPD1 gene encoding the E3 component is regulated at the transcriptional level by glucose (Roy and Dawes, J. Gen. Microbiol. 133:925-933 (1987)). The E1 component, encoded by KGD1, is also regulated by glucose and activated by the products of HAP2 and HAP3 (Repetto and Tzagoloff, Mol. Cell. Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited by products NADH and succinyl-CoA, is well-studied in mammalian systems, as impaired function of has been linked to several neurological diseases (Tretter and dam-Vizi, Philos. Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2005)).
GenBank
Gene name
GI#
Accession #
Organism
sucA
16128701
NP_415254.1
Escherichia coli
sucB
16128702
NP_415255.1
Escherichia coli
lpd
16128109
NP_414658.1
Escherichia coli
odhA
51704265
P23129.2
Bacillus subtilis
odhB
129041
P16263.1
Bacillus subtilis
pdhD
118672
P21880.1
Bacillus subtilis
KGD1
6322066
NP_012141.1
Saccharomyces
cerevisiae
KGD2
6320352
NP_010432.1
Saccharomyces
cerevisiae
LPD1
14318501
NP_116635.1
Saccharomyces
cerevisiae
Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as 2-oxoisovalerate dehydrogenase, participates in branched-chain amino acid degradation pathways, converting 2-keto acids derivatives of valine, leucine and isoleucine to their acyl-CoA derivatives and CO2. The complex has been studied in many organisms including Bacillus subtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), Rattus norvegicus (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). In Bacillus subtilis the enzyme is encoded by genes pdhD (E3 component), bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)). In mammals, the complex is regulated by phosphorylation by specific phosphatases and protein kinases. The complex has been studied in rat hepatocites (Chicco et al., J. Biol. Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1 alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3 components of the Pseudomonas putida BCKAD complex have been crystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Mattevi et al., Science. 255:1544-1550 (1992)) and the enzyme complex has been studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putida BCKAD genes is activated by the gene product of bkdR (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)). In some organisms including Rattus norvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int. 31:911-922 (1993)), this complex has been shown to have a broad substrate range that includes linear oxo-acids such as 2-oxobutanoate and alpha-ketoglutarate, in addition to the branched-chain amino acid precursors. The active site of the bovine BCKAD was engineered to favor alternate substrate acetyl-CoA (Meng and Chuang, Biochemistry. 33:12879-12885 (1994)).
GenBank
Gene name
GI#
Accession #
Organism
bfmBB
16079459
NP_390283.1
Bacillus subtilis
bfmBAA
16079461
NP_390285.1
Bacillus subtilis
bfmBAB
16079460
NP_390284.1
Bacillus subtilis
pdhD
118672
P21880.1
Bacillus subtilis
lpdV
118677
P09063.1
Pseudomonas
putida
bkdB
129044
P09062.1
Pseudomonas
putida
bkdA1
26991090
NP_746515.1
Pseudomonas
putida
bkdA2
26991091
NP_746516.1
Pseudomonas
putida
Bckdha
77736548
NP_036914.1
Rattus norvegicus
Bckdhb
158749538
NP_062140.1
Rattus norvegicus
Dbt
158749632
NP_445764.1
Rattus norvegicus
Dld
40786469
NP_955417.1
Rattus norvegicus
The pyruvate dehydrogenase complex, catalyzing the conversion of pyruvate to acetyl-CoA, has also been extensively studied. In the E. coli enzyme, specific residues in the E1 component are responsible for substrate specificity (Bisswanger, J Biol. Chem. 256:815-822 (1981); Bremer, Eur. J Biochem. 8:535-540 (1969); and Gong et al., J Biol. Chem. 275:13645-13653 (2000)). As mentioned previously, enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008)); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., J. Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S. A 98:14802-14807 (2001)) and the E2 catalytic domain from Azotobacter vinelandii are available (Mattevi et al., Science. 255:1544-1550 (1992)). Some mammalian PDH enzymes complexes can react on alternate substrates such as 2-oxobutanoate, although comparative kinetics of Rattus norvegicus PDH and BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton et al., Biochem. J. 234:295-303 (1986)).
GenBank
Gene name
GI#
Accession #
Organism
aceE
16128107
NP_414656.1
Escherichia coli
aceF
16128108
NP_414657.1
Escherichia coli
lpd
16128109
NP_414658.1
Escherichia coli
pdhA
3123238
P21881.1
Bacillus subtilis
pdhB
129068
P21882.1
Bacillus subtilis
pdhC
129054
P21883.2
Bacillus subtilis
pdhD
118672
P21880.1
Bacillus subtilis
aceE
152968699
YP_001333808.1
Klebsiella
pneumonia
aceF
152968700
YP_001333809.1
Klebsiella
pneumonia
lpdA
152968701
YP_001333810.1
Klebsiella
pneumonia
Pdha1
124430510
NP_001004072.2
Rattus
norvegicus
Pdha2
16758900
NP_446446.1
Rattus
norvegicus
Dlat
78365255
NP_112287.1
Rattus
norvegicus
Dld
40786469
NP_955417.1
Rattus
norvegicus
As an alternative to the large multienzyme 2-keto-acid dehydrogenase complexes described above, some anaerobic organisms utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze acylating oxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenase complexes, these enzymes contain iron-sulfur clusters, utilize different cofactors, and use ferredoxin or flavodoxin as electron acceptors in lieu of NAD(P)H. While most enzymes in this family are specific to pyruvate as a substrate (POR) some 2-keto-acid:ferredoxin oxidoreductases have been shown to accept a broad range of 2-ketoacids as substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from the thermoacidophilic archaeon Sulfolobus tokodaii 7, which contains an alpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599 (1996)). A plasmid-based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str. K1 have also been recently cloned into E. coli, characterized, and found to react with a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)). The gene sequences of these OFOR candidates are available, although they do not have GenBank identifiers assigned to date. There is bioinformatic evidence that similar enzymes are present in all archaea, some anaerobic bacteria and amitochondrial eukarya (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). This class of enzyme is also interesting from an energetic standpoint, as reduced ferredoxin could be used to generate NADH by ferredoxin-NAD reductase (Petitdemange et al., Biochim. Biophys. Acta 421:334-337 (1976)). Also, since most of the enzymes are designed to operate under anaerobic conditions, less enzyme engineering may be required relative to enzymes in the 2-keto-acid dehydrogenase complex family for activity in an anaerobic environment.
GenBank
Gene name
GI#
Accession #
Organism
ST2300
15922633
NP_378302.1
Sulfolobus tokodaii 7
1.2.1.d Oxidoreductase (Phosphonic Acid to Aldehyde). The reduction of a phosphonic acid to its corresponding aldehyde is catalyzed by an oxidoreductase in the EC class 1.2.1. Steps B and F in
GenBank
Gene name
GI#
Accession #
Organism
Asd
16131307
NP_417891.1
Escherichia coli
Asd
68249223
YP_248335.1
Haemophilus
influenzae
Asd
1899206
AAB49996
Mycobacterium
tuberculosis
VC2036
15642038
NP_231670
Vibrio cholera
Asd
210135348
YP_002301787.1
Heliobacter pylori
ARG5,6
6320913
NP_010992.1
Saccharomyces
cerevisiae
argC
16078184
NP_389001.1
Bacillus subtilis
1.3.1.a Oxidoreductase (Alkene to Alkane). Several transformations fall into the category of oxidoreductases that reduce an alkene to an alkane (EC 1.3.1.-). For example, Steps C, G, K and N in
Enzymes with enone reductase activity have been identified in prokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur. J. Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolic fraction of Saccharomyces cerevisiae were purified and characterized, and found to accept a variety of alkenals (similar to 6-OHE) and enoyl ketones (similar to OHED) as substrates (Wanner and Tressl, Eur. J. Biochem. 255:271-278 (1998)). Genes encoding these enzymes have not been identified to date. Cell extracts of cyanobacterium Synechococcus sp. PCC7942 reduced a variety enone substrates to their corresponding alkyl ketones (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004)). Genes have not been associated with this activity in this organism. Enone reductases in other organisms can also catalyze this transformation.
A recombinant NADPH-dependent enone reductase from Nicotiana tabacum, encoded by NtRed1, was functionally expressed and characterized in E. coli (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). This reductase was functional on the exocyclic enoyl ketone pulegone (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). An enzyme candidate in S. cerevisiae at the locus YML131W, bears 30% identity to NtRed1 (evalue=1e-26). The amino acid sequence of NtRed1 shares significant homology with 2-alkenal reductase from Arabidopsis thaliana, zeta-crystallin homolog from A. thaliana, pulegone reductase from Menthe piperita and phenylpropenal alkene reductase from Pinus taeda. These enzymes are known to catalyze the reduction of alkenes of α,β-unsaturated ketones and aldehydes.
GenBank
Gene name
GI#
Accession #
Organism
NtRed1
6692816
BAA89423
Nicotiana tabacum
YML131W
45269874
AAS56318.1
Saccharomyces
cerevisiae
AtDBR1
15237888
NP-197199
Arabidopsis
thaliana
P2
886430
CAA89262
Arabidopsis
thaliana
PulR
34559418
AAQ75423
Menthe piperita
PtPPDBR
110816011
ABG91753
Pinus taeda
2-Alkenal reductase catalyzes the reduction of α, β-unsaturated double bonds of aldehydes and ketones. A barley alkenal hydrogenase ALH1 was identified with activity for a range of α,β-unsaturated ketones and aldehydes including trans-2-nonenal, 2-hexenal, traumatin and 1-octene-3-one (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)). The Hordeum vulgare ALH1 cDNA was cloned expressed in E. coli (Hambraeus and Nyberg, J. Agric. Food Chem. 53:8714-8721 (2005)).
GenBank
Gene name
GI#
Accession #
Organism
ALH1
62765876
AAX99161
Hordeum vulgare
ALH1
195652571
ACG45753
Zea mays
2-Enoate reductase enzymes are known to catalyze the NAD(P)H-dependent reduction of a wide variety of α, β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been characterized (Seedorf et al., Proc. Natl. Acad. Sci. U.S. A 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel and Simon, Arch. Microbiol 135:51-57 (1983)). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). The C. thermoaceticum enr gene has also been expressed in a catalytically active form in E. coli (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).
GenBank
Gene name
GI#
Accession #
Organism
enr
169405742
ACA54153.1
Clostridium
botulinum A3 str
enr
2765041
CAA71086.1
Clostridium
tyrobutyricum
enr
3402834
CAA76083.1
Clostridium kluyveri
enr
83590886
YP_430895.1
Moorella thermoacetica
fadH
16130976
NP_417552.1
Escherichia coli
Another candidate enoate reductase is 3-oxoadipate oxidoreductase (maleylacetate reductase), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. The enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); and Kaschabek. and Reineke, J. Bacteriol. 175:6075-6081 (1993)), and the coding gene was cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)). Candidate genes for 3-oxoadipate oxidoreductase include cicE gene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., J. Bacteriol. 180:3503-3508 (1998)), and macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)).
GenBank
Gene name
GI#
Accession #
Organism
clcE
3913241
O30847.1
Pseudomonas
sp. strain B13
macA
7387876
O84992.1
Rhodococcus opacus
macA
5916089
AAD55886
Cupriavidus necator
Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the reduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (
GenBank
Gene name
GI#
Accession #
Organism
bcd
15895968
NP_349317.1
Clostridium acetobutylicum
etfA
15895966
NP_349315.1
Clostridium acetobutylicum
etfB
15895967
NP_349316.1
Clostridium acetobutylicum
TER
62287512
Q5EU90.1
Euglena gracilis
TDE0597
42526113
NP_971211.1
Treponema denticola
Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes in the pim operon, pimC and pimD, bear sequence homology to C. acetobutylicum bcd and are predicted to encode a flavin-containing pimeloyl-CoA dehydrogenase (Harrison and Harwood, Microbiology 151:727-736 (2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison and Harwood, Microbiology 151:727-736 (2005)).
GenBank
Gene name
GI#
Accession #
Organism
pimC
39650632
CAE29155
Rhodopseudomonas palustris
pimD
39650631
CAE29154
Rhodopseudomonas palustris
pimC
27356102
BAC53083
Bradyrhizobium japonicum
pimD
27356101
BAC53082
Bradyrhizobium japonicum
An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascarius suum and is capable of reducing a variety of linear and branched chain substrates including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Two isoforms of the enzyme, encoded by genes acad1 and acad, have been characterized.
GenBank
Gene name
GI#
Accession #
Organism
acad1
2407655
AAC48316.1
Ascarius suum
acad
347404
AAA16096.1
Ascarius suum
1.4.1.a Oxidoreductase (Ketone or Aldehyde to Amino). Oxidoreductases in the EC class 1.4.1 that convert an aldehyde or ketone to its corresponding amine group catalyze several biosynthetic steps in the disclosed pathways. In
Most aminating oxidoreductases catalyze the reversible oxidative deamination of alpha-amino acids with NAD+ or NADP+ as acceptor, and the reactions are typically reversible. Exemplary enzymes include glutamate dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh, and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (Korber et al., J Mol. Biol. 234:1270-1273 (1993); and McPherson et al., Nucleic Acids Res. 11:5257-5266 (1983)), gdh from Thermotoga maritime (Kort et al., Extremophiles. 1:52-60 (1997); Lebbink et al., J Mol. Biol. 280:287-296 (1998); and Lebbink et al., J Mol. Biol. 289:357-369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula, Biotechnol Bioeng 68:557-562 (2000); and Stoyan et al., J Biotechnol. 54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al., J Biol. Chem. 278:8804-8808 (2003)).
GenBank
Gene name
GI#
Accession #
Organism
gdhA
118547
P00370
Escherichia coli
gdh
6226595
P96110.4
Thermotoga maritima
gdhA1
15789827
NP_279651.1
Halobacterium salinarum
ldh
61222614
P0A393
Bacillus cereus
nadX
15644391
NP_229443.1
Thermotoga maritima
Lysine 6-dehydrogenase (deaminating), encoded by lysDH, catalyzes the oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn non-enzymatically cyclizes to form Δ1-piperideine-6-carboxylate (Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol. 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem. 106:76-80 (1989); and Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate given the structural similarity between adipate semialdehyde and 2-aminoadipate-6-semialdehyde.
GenBank
Gene name
GI#
Accession #
Organism
lysDH
13429872
BAB39707
Geobacillus stearothermophilus
lysDH
15888285
NP_353966
Agrobacterium tumefaciens
lysDH
74026644
AAZ94428
Achromobacter denitrificans
2.3.1.a Acyltransferase (Transferring CoA to Phospho). Acyltransferases that exchange a CoA moiety for a phosphate are in the EC class 2.3.1. Transformations in this category include the conversions of 6-AAHOP to 6-acetamidohexanoyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
Pta
16130232
NP_416800.1
Escherichia coli
Ptb
15896327
NP_349676
Clostridium acetobutylicum
Ptb
38425288
AAR19757.1
butyrate-producing bacterium L2-50
Ptb
10046659
CAC07932.1
Bacillus megaterium
2.3.1.c Acyltransferase (N-Acetyltransferase). N-Acetyltransferases transfer an acetyl group to an amine, forming an N-acetyl group. N-Acetylation serves diverse functions in biological systems including transcriptional regulation, nuclear import, chromosome assembly and nucleosome remodeling (Kouzarides, EMBO J. 19:1176-1179 (2000)). N-Acetylation of metabolic intermediates of arginine biosynthetic pathways serves both to protect reactive intermediates from spontaneous cyclization and also to sequester pathway intermediates from competing pathways (Caldovic and Tuchman, Biochem. J 372:279-290 (2003)). Acetylation of 6-ACA (
One candidate enzyme for acetylating 6-ACA is lysine N-acetyltransferase (EC 2.3.1.32), an enzyme which selectively transfers the acetyl moiety from acetyl phosphate to the terminal amino group of L-lysine, beta-L-lysine or L-ornithine. Although this enzyme is not known to acetylate 6-ACA, this substrate is structurally similar to the natural substrate. Lysine N-acetyltransferase has been characterized in Bos taurus (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) and Methanosarcina mazei (Pfluger et al., Appl Environ. Microbiol. 69:6047-6055 (2003)). Methanogenic archaea M. maripaludis, M. acetivorans, M. barkeri and M. jannaschii are also predicted to encode enzymes with this functionality (Pfluger et al., Appl Environ. Microbiol. 69:6047-6055 (2003)).
Gene
GenBank
name
GI#
Accession #
Organism
ablB
21227037
NP_632959.1
Methanosarcina mazei
yodP
44921183
CAF30418
Methanococcus maripaludis
MA3978
20092772
NP_618847.1
Methanosarcina
acetivorans
MJ0635
15668816
NP_247619.1
Methanocaldococcus
jannaschii
Mbar_A0671
73668215
YP_304230.1
Methanosarcina barkeri
Alternately, 6-ACA acetylation can be catalyzed by an enzyme in the GNAT family of N-acetyltransferases. Such enzymes transfer an acetyl group from acetyl-CoA to a primary amine. The enzyme spermidine N-acetyltransferase (SSAT), also known as diamine N-acetyltransferase (EC 2.3.1.57), is capable of acetylating a variety of small molecule substrates. Purified enzymes from Ascaris suum and Onchocerca volvulus exhibit a broad substrate range that includes HMDA (Davids et al., Mol. Biochem. Parasitol. 64:341-344 (1994); and Wittich and Walter, Mol. Biochem. Parasitol. 38:13-17 (1990)), but the associated genes have not been identified to date. Other enzymes with this functionality are found in Bacillus subtilis (Forouhar et al., J Biol. Chem. 280:40328-40336 (2005)) and Homo sapiens (Casero and Pegg, FASEB J 7:653-661 (1993)). A closely related enzyme is thialysine N-acetyltransferase in C. elegans, an enzyme that accepts a range of substrates including lysine, ornithine, thialysine and others (bo-Dalo et al., Biochem. J 384:129-137 (2004)) Amino acid residues involved in substrate binding were identified in the thialysine N-acetyltransferase from Leishmania major (Luersen, K., FEBS Lett. 579:5347-5352 (2005)). An additional candidate is the diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzyme participating in ectoine biosynthesis in Methylomicrobium alcaliphilum (Reshetnikov et al., Arch. Microbiol. 184:286-297 (2006)) C. salexigens (formerly Halomonas elongata) (Canovas et al., Syst. Appl Microbiol 21:487-497 (1998)).
Gene
GenBank
name
GI#
Accession #
Organism
paiA
16080268
NP_391095.1
Bacillus subtilis
SSAT1
114322
P21673
Homo sapiens
D2023.4
17559148
NP_505978.1
Caenorhabditis elegans
LmjF36.2750
68129928
CAJ09234.1
Leishmania major
ectA
68366269
AAY96770.1
Methylomicrobium
alcaliphilum 20Z
ectA
6685422
Q9ZEU8.1
Chromohalobacter
salexigens
An additional enzyme candidate for acetylating 6-ACA (
Gene
GenBank
name
GI#
Accession #
Organism
argJ
16078185
NP_389002.1
Bacillus subtilis
ECM40 (ARG7)
6323707
NP_013778.1
Saccharomyces
cerevisiae
Rv1653
15608791
NP_216169.1
Mycobacterium
tuberculosis
2.3.1.d Acyltransferase (Formate C-Acyltransferase). The acylation of ketoacids HODH, OHED and 2-OHD to their corresponding CoA derivatives (
Gene
GenBank
name
GI#
Accession #
Organism
pflB
16128870
NP_415423.1
Escherichia coli
pflA
16128869
NP_415422.1
Escherichia coli
tdcE
48994926
AAT48170.1
Escherichia coli
pflD
11499044
NP_070278.1
Archaeglubus fulgidus
pfl
2500058
Q46266.1
Clostridium pasteurianum
act
1072362
CAA63749.1
Clostridium pasteurianum
pfl1
159462978
XP_001689719.1
Chlamydomonas reinhardtii
pflA1
159485246
XP_001700657.1
Chlamydomonas reinhardtii
2.6.1.a Aminotransferase. Steps E, H and J of
Gene
GenBank
name
GI#
Accession #
Organism
gabT
16130576
NP_417148.1
Escherichia coli
puuE
16129263
NP_415818.1
Escherichia coli
abat
37202121
NP_766549.2
Mus musculus
gabT
70733692
YP_257332.1
Pseudomonas fluorescens
abat
47523600
NP_999428.1
Sus scrofa
Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to HMDA. The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC. Microbiol. 3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova et al., BMC. Microbiol. 3:2 (2003)). A putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu et al., J. Bacteriol. 184:3765-3773 (2002)).
Gene
GenBank
name
GI#
Accession #
Organism
ygjG
145698310
NP_417544
Escherichia coli
spuC
9946143
AAG03688
Pseudomonas aeruginosa
Additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferases which produce malonic semialdehyde from beta-alanine (WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri was shown to preferentially use beta-alanine as the amino group donor (Andersen and Hansen, Gene 124:105-109 (1993)). SkUGAJ encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGAJ (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both β-alanine and GABA transamination (Andersen and Hansen, Gene 124:105-109 (1993)). 3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2-methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat 1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al., Methods Enzymol. 324:376-389 (2000)).
Gene
GenBank
name
GI#
Accession #
Organism
SkyPYD4
98626772
ABF58893.1
Saccharomyces kluyveri
SkUGA1
98626792
ABF58894.1
Saccharomyces kluyveri
UGA1
6321456
NP_011533.1
Saccharomyces cerevisiae
Abat
122065191
P50554.3
Rattus norvegicus
Abat
120968
P80147.2
Sus scrofa
Steps J and H of
Gene
GenBank
name
GI#
Accession #
Organism
aspC
16128895
NP_415448.1
Escherichia coli
AAT2
1703040
P23542.3
Saccharomyces cerevisiae
ASP5
20532373
P46248.2
Arabidopsis thaliana
Got2
112987
P00507
Rattus norvegicus
avtA
49176374
YP_026231.1
Escherichia coli
serC
16128874
NP_415427.1
Escherichia coli
2.7.2.a Phosphotransferase (Carboxy Acceptor). Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. Steps A and E in
Gene
GenBank
name
GI#
Accession #
Organism
buk1
15896326
NP_349675
Clostridium acetobutylicum
buk2
20137415
Q97II1
Clostridium acetobutylicum
buk2
6685256
Q9X278.1
Thermotoga maritima
lysC
16131850
NP_418448.1
Escherichia coli
ackA
16130231
NP_416799.1
Escherichia coli
proB
16128228
NP_414777.1
Escherichia coli
Acetylglutamate kinase phosphorylates acetylated glutamate during arginine biosynthesis and is a good candidate for phosphorylating 6-acetamidohexanoate (
Gene
GenBank
name
GI#
Accession #
Organism
argB
145698337
NP_418394.3
Escherichia coli
argB
16078186
NP_389003.1
Bacillus subtilis
ARG5,6
6320913
NP_010992.1
Saccharomyces cerevisiae
2.8.3.a Coenzyme-A Transferase. Coenzyme-A (CoA) transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. In Step M of
Gene
GenBank
name
GI#
Accession #
Organism
cat1
729048
P38946.1
Clostridium kluyveri
cat2
172046066
P38942.2
Clostridium kluyveri
cat3
146349050
EDK35586.1
Clostridium kluyveri
TVAG_395550
123975034
XP_001330176
Trichomonas
vaginalis G3
Tb11.02.0290
71754875
XP_828352
Trypanosoma brucei
A CoA transferase that can utilize acetyl-CoA as the CoA donor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek and Frerman, Arch. Biochem. Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies and Schink, Appl Environ. Microbiol. 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli and Overath, Eur. J. Biochem. 29:553-562 (1972)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol. 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol. 56:1576-1583 (1990); and Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
AtoA
2492994
NP_416726
Escherichia coli K12
AtoD
2492990
NP_416725
Escherichia coli K12
actA
62391407
YP_226809.1
Corynebacterium glutamicum
ATCC 13032
cg0592
62389399
YP_224801.1
Corynebacterium glutamicum
ATCC 13032
ctfA
15004866
NP_149326.1
Clostridium acetobutylicum
ctfB
15004867
NP_149327.1
Clostridium acetobutylicum
ctfA
31075384
AAP42564.1
Clostridium
saccharoperbutylacetonicum
ctfB
31075385
AAP42565.1
Clostridium
saccharoperbutylacetonicum
The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).
Gene
GenBank
name
GI#
Accession #
Organism
gctA
559392
CAA57199.1
Acidaminococcus fermentans
gctB
559393
CAA57200.1
Acidaminococcus fermentans
Yet another CoA transferase is the two-unit succinyl-CoA:3:oxoacid-CoA transferase encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)). Similar enzymes based on homology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
pcaI
24985644
AAN69545.1
Pseudomonas putida
pcaJ
26990657
NP_746082.1
Pseudomonas putida
pcaI
50084858
YP_046368.1
Acinetobacter sp. ADP1
pcaJ
141776
AAC37147.1
Acinetobacter sp. ADP1
pcaI
21224997
NP_630776.1
Streptomyces coelicolor
pcaJ
21224996
NP_630775.1
Streptomyces coelicolor
HPAG1_0676
108563101
YP_627417
Helicobacter pylori
HPAG1_0677
108563102
YP_627418
Helicobacter pylori
ScoA
16080950
NP_391778
Bacillus subtilis
ScoB
16080949
NP_391777
Bacillus subtilis
3.5.1.a Hydrolase (Acting on Linear Amides).
Deacetylation of linear acetamides is catalyzed by an amidohydrolase in the 3.5.1 family of enzymes. Such an enzyme is required for the deacetylation of 6-acetamidohexanamine to HMDA (
Acetylpolyamine amidohydrolase (EC 3.5.1.62), is another candidate enzyme that forms the diamines putrescine and cadaverine from their acetylated precursors. The acetylpolyamine deacetylase (AphA) from Mycoplana ramosa has been cloned in E. coli and characterized (Sakurada et al., J. Bacteriol. 178:5781-5786 (1996)) and a crystal structure is available (Fujishiro et al., Biochem. Biophys. Res. Commun. 157:1169-1174 (1988)). This enzyme has also been studied in Micrococcus luteus, but the associated gene has not been identified to date (Suzuki et al., Biochim. Biophys. Acta 882:140-142 (1986)). A protein the histone deacetylase superfamily with high sequence similarity to AphA was identified in the M. luteus genome (evalue=1e-18, 37% identity). The N-acetyl-L-ornithine deacetylase from E. coli is another candidate amidohydrolase (EC 3.5.1.16). The E. coli enzyme, encoded by the argE gene (McGregor et al., J Am. Chem. Soc. 127:14100-14107 (2005); and Meinnel et al., J. Bacteriol. 174:2323-2331 (1992)), removes N-acetyl groups from a variety of substrates including ornithine, lysine, glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry 39:1285-1293 (2000)).
Gene
GenBank
name
GI#
Accession #
Organism
aphA
3023317
Q48935.1
Mycoplana ramose
MlutDRAFT_1143
172071524
EDT57566.1
Micrococcus luteus
argE
16131795
NP_418392.1
Escherichia coli
4.1.1.a Carboxy-Lyase.
Steps D and F in
Gene
GenBank
name
GI#
Accession #
Organism
pdc
118391
P06672.1
Zymomonas mobilus
pdc1
30923172
P06169
Saccharomyces cerevisiae
pdc
20385191
Q8L388
Acetobacter pasteurians
pdc1
52788279
Q12629
Kluyveromyces lactis
Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry 42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).
Gene
GenBank
name
GI#
Accession #
Organism
mdlC
3915757
P20906.2
Pseudomonas putida
mdlC
81539678
Q9HUR2.1
Pseudomonas aeruginosa
dpgB
126202187
ABN80423.1
Pseudomonas stutzeri
ilvB-1
70730840
YP_260581.1
Pseudomonas fluorescens
A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci U S. A 102:10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (˜130 kD) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 1) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene can be identified by testing candidate genes containing this N-terminal sequence for KDC activity.
Gene
GenBank
name
GI#
Accession #
Organism
kgd
160395583
O50463.4
Mycobacterium tuberculosis
kgd
27375563
NP_767092.1
Bradyrhizobium japonicum
kgd
13473636
NP_105204.1
Mesorhizobium loti
A fourth candidate enzyme for catalyzing this step is branched chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988); and Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.
Gene
GenBank
name
GI#
Accession #
Organism
kdcA
44921617
AAS49166.1
Lactococcus lactis
Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the E1 subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J. Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.
Gene
GenBank
name
GI#
Accession #
Organism
BCKDHB
34101272
NP_898871.1
Homo sapiens
BCKDHA
11386135
NP_000700.1
Homo sapiens
BCKDHB
115502434
P21839
Bos taurus
BCKDHA
129030
P11178
Bos taurus
The decarboxylation of 2-AHD to 6-aminocaproate (
Gene
GenBank
name
GI#
Accession #
Organism
panD
67470411
P0A790
Escherichia coli K12
panD
18203593
Q9X4N0
Corynebacterium glutamicum
panD
54041701
P65660.1
Mycobacterium tuberculosis
4.1.2.a Aldehyde-Lyase.
HOHD aldolase, also known as HHED aldolase, catalyzes the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate (HOHD) into pyruvate and succinic semialdehyde (
Gene
GenBank
name
GI#
Accession #
Organism
hpcH
633197
CAA87759.1
Escherichia coli C
hpaI
38112625
AAR11360.1
Escherichia coli W
4.2.1.a Hydro-Lyase.
The enzyme OHED hydratase participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) using magnesium as a cofactor (Burks et al., J. Am. Chem. Soc. 120 (1998)) (
Gene
GenBank
name
GI#
Accession #
Organism
hpcG
556840
CAA57202.1
Escherichia coli C
hpaH
757830
CAA86044.1
Escherichia coli W
hpaH
150958100
ABR80130.1
Klebsiella pneumoniae
Sari_01896
160865156
ABX21779.1
Salmonella enterica
Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
crt
15895969
NP_349318.1
Clostridium acetobutylicum
crt1
153953091
YP_001393856.1
Clostridium kluyveri
Enoyl-CoA hydratases (EC 4.2.1.17) also catalyze the dehydration of 3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J. Bacteriol. 188:8551-8559 (2003); Conrad et al., J. Bacteriol. 118:103-111 (1974); and Roberts et al., Arch. Microbiol. 117:99-108 (1978)). The enoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al., Arch. Microbiol. 117:99-108 (1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci. U.S. A 95:6419-6424 (1998)). The gene product of pimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation (Harrison and Harwood, Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park and Lee, J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., J Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., J. Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)).
Gene
GenBank
name
GI#
Accession #
Organism
ech
26990073
NP_745498.1
Pseudomonas putida
paaA
26990002
NP_745427.1
Pseudomonas putida
paaB
26990001
NP_745426.1
Pseudomonas putida
phaA
106636093
ABF82233.1
Pseudomonas fluorescens
phaB
106636094
ABF82234.1
Pseudomonas fluorescens
pimF
39650635
CAE29158
Rhodopseudomonas palustris
maoC
16129348
NP_415905.1
Escherichia coli
paaF
16129354
NP_415911.1
Escherichia coli
paaG
16129355
NP_415912.1
Escherichia coli
Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Nakahigashi and Inokuchi, Nucleic Acids Res. 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991); and Yang et al., Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol. 47:793-805 (2003)).
Gene
GenBank
name
GI#
Accession #
Organism
fadA
49176430
YP_026272.1
Escherichia coli
fadB
16131692
NP_418288.1
Escherichia coli
fadI
16130275
NP_416844.1
Escherichia coli
fadJ
16130274
NP_416843.1
Escherichia coli
fadR
16129150
NP_415705.1
Escherichia coli
6.2.1.a Acid-Thiol Ligase (Also Called CoA Synthetase).
Steps I and M of
Gene
GenBank
name
GI#
Accession #
Organism
AF1211
11498810
NP_070039.1
Archaeoglobus fulgidus
DSM 4304
AF1983
11499565
NP_070807.1
Archaeoglobus fulgidus
DSM 4304
scs
55377722
YP_135572.1
Haloarcula marismortui
ATCC 43049
PAE3250
18313937
NP_560604.1
Pyrobaculum aerophilum str.
IM2
sucC
16128703
NP_415256.1
Escherichia coli
sucD
1786949
AAC73823.1
Escherichia coli
Another candidate enzyme for this step is 6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)). Other candidates are found in Bacillus subtilis (Bower et al., J. Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690 (1992)).
Gene
GenBank
name
GI#
Accession #
Organism
pauA
15596214
NP_249708.1
Pseudomonas mendocina
bioW
50812281
NP_390902.2
Bacillus subtilis
bioW
115012
P22822.1
Lysinibacillus sphaericus
Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem. J 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J 395:147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun. 360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.
Gene
GenBank
name
GI#
Accession #
Organism
phl
77019264
CAJ15517.1
Penicillium chrysogenum
phlB
152002983
ABS19624.1
Penicillium chrysogenum
paaF
22711873
AAC24333.2
Pseudomonas putida
AACS
21313520
NP_084486.1
Mus musculus
AACS
31982927
NP_076417.2
Homo sapiens
E. coli was assayed for tolerance, metabolic activity and growth during exposure to various concentrations of 6-aminocaproate (6-ACA). Aerobically, cultures were able to grow media with up to 10% 6-ACA, while anaerobic cultures could grow in media with approximately 6% 6-ACA (
At high concentrations (>65 g/L) the osmolarity of 6-ACA is ˜0.5 M which may cause osmotic stress. To determine osmotic stress as the basis for 6-ACA growth inhibition, cultures were grown in various concentrations of 6-ACA with and without the osmoprotectant glycine betaine. As seen in
Several β-ketothiolase enzymes have been shown to break β-ketoadipyl-CoA into acetyl-CoA and succinyl-CoA. For example, the gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol, 184(1): 207-15 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc Natl Acad Sci USA, 95(11), 6419-24 (1998)), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., Arch Microbiol, 188(2), 117-25 (2007)), and paaJ from E. coli (Nogales et al., Microbiology, 153(Pt 2), 357-65 (2007)) catalyze the conversion of 3-oxoadipyl-CoA into succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate or styrene. To confirm that β-ketothiolase enzymes exhibit condensation activity, several thiolases (Table 10; SEQ ID NOS: 3-50, respectively) were cloned into a derivative of pZE13(Lutz et al., Nucleic Acids Res, 29(18), 3873-81 (2001)), which results in the clones having a carboxy-terminal 6×His tag (SEQ ID NO: 2).
TABLE 10
Cloned Thiolases
Species
Enzyme
template
Gene
Length
5′ PRIMER
3′ PRIMER
ORF SEQ
beta-
Ralstonia
bktB
1185
ATGACGCGTGAAGTG
GATACGCTCGAAGAT
atgacgcgtgaagtggtagtggtaagcggtgtccgtaccgcgatcgggacctttggcggcagcctgaagga
ketothiolase
eutropha
GTAGTGGTAAG
GGCGG
tgtggcaccggcggagctgggcgcactggtggtgcgcgaggcgctggcgcgcgcgcaggtgtcgggcgacg
H16
(SEQ ID NO: 3)
(SEQ ID NO: 4)
atgtcggccacgtggtattcggcaacgtgatccagaccgagccgcgcgacatgtatctgggccgcgtcgcg
gccgtcaacggcggggtgacgatcaacgcccccgcgctgaccgtgaaccgcctgtgcggctcgggcctgca
ggccattgtcagcgccgcgcagaccatcctgctgggcgataccgacgtcgccatcggcggcggcgcggaaa
gcatgagccgcgcaccgtacctggcgccggcagcgcgctggggcgcacgcatgggcgacgccggcctggtc
gacatgatgctgggtgcgctgcacgatcccttccatcgcatccacatgggcgtgaccgccgagaatgtcgc
caaggaatacgacatctcgcgcgcgcagcaggacgaggccgcgctggaatcgcaccgccgcgcttcggcag
cgatcaaggccggctacttcaaggaccagatcgtcccggtggtgagcaagggccgcaagggcgacgtgacc
ttcgacaccgacgagcacgtgcgccatgacgccaccatcgacgacatgaccaagctcaggccggtcttcgt
caaggaaaacggcacggtcacggccggcaatgcctcgggcctgaacgacgccgccgccgcggtggtgatga
tggagcgcgccgaagccgagcgccgcggcctgaagccgctggcccgcctggtgtcgtacggccatgccggc
gtggacccgaaggccatgggcatcggcccggtgccggcgacgaagatcgcgctggagcgcgccggcctgca
ggtgtcggacctggacgtgatcgaagccaacgaagcctttgccgcacaggcgtgcgccgtgaccaaggcgc
tcggtctggacccggccaaggttaacccgaacggctcgggcatctcgctgggccacccgatcggcgccacc
ggtgccctgatcacggtgaaggcgctgcatgagctgaaccgcgtgcagggccgctacgcgctggtgacgat
gtgcatcggcggcgggcagggcattgccgccatcttcgagcgtatctga
(SEQ ID NO: 5)
2-Methyl-
Mus
ACA
1215
ATGGAAGTAAGATGC
CAGCTTCTCAATCAG
atggaagtaagatgcctggaacgaagttatgcatccaaacccactttgaatgaagtggttatagtaagtgc
acetoacetyl-
musculus
T1
CTGGAACGAAG
CAGGGC
tataagaactcccattggatccttcctgggcagccttgcctctcagccggccactaaacttggtactgctg
CoA Thiolase
(SEQ ID NO: 6)
(SEQ ID NO: 7)
caattcagggagccattgagaaggcagggattccaaaagaagaagtgaaggaagtctacatgggcaatgtc
(branched
atccaagggggtgaaggacaggcccctaccaggcaagcaacactgggcgcaggtttacctatttccactcc
chain?)
atgcaccacagtaaacaaggtttgtgcttcaggaatgaaagccatcatgatggcctctcaaagtcttatgt
gtggacatcaggatgtgatggtggcaggcgggatggagagcatgtccaatgtcccatacgtaatgagcaga
ggagcaacaccatatggtggggtaaaacttgaagacctgattgtaaaagacgggctaactgatgtctacaa
taaaattcatatgggtaactgtgctgagaatactgcaaagaagatgaatatctcacggcaggaacaggata
cgtacgctctcagctcttacaccagaagtaaagaagcgtgggacgcagggaagtttgccagtgagattact
cccatcaccatctcagtgaaaggtaaaccagatgtggtggtgaaagaagatgaagaatacaagcgtgttga
ctttagtaaagtgccaaagctcaagaccgtgttccagaaagaaaatggcacaataacagctgccaatgcca
gcacactgaacgatggagcagctgctctggttctcatgactgcagaggcagcccagaggctcaatgttaag
ccattggcacgaattgcagcatttgctgatgctgccgtagaccccattgattttccacttgcgcctgcata
tgccgtacctaaggttcttaaatatgcaggactgaaaaaagaagacattgccatgtgggaagtaaatgaag
cattcagtgtggttgtgctagccaacattaaaatgctggagattgacccccaaaaagtaaatatccacgga
ggagctgtttctctgggccatccaattgggatgtctggagcccggattgttgttcatatggctcatgccct
gaagccaggagagttcggtctggctagtatttgcaacggaggaggaggtgcttccgccctgctgattgaga
agctgtag
(SEQ ID NO: 8)
2-Methyl-
Pseudomonas
fadAX
1194
ATGACCCTCGCCAAT
GTACAGGCATTCAAC
atgaccctcgccaatgaccccatcgttatcgtcagcgccgtgcgcacgcccatgggcgggttgcagggcga
acetoacetyl-
putida
GACCC
AGCCATGG
cctcaagagcctgactgcgccgcaactgggcagcgccgccattcgtgctgccgtggaacgggccggcatcg
CoA Thiolase
(KT2440)
(SEQ ID NO: 9)
(SEQ ID NO: 10)
atgccgccggtgtcgagcaggtactgttcggctgcgtgctgccggccggccagggccaggcaccggcacgc
(branched
caggccgcgctgggcgccgggctggacaagcacaccacctgcaccaccctgaacaagatgtgcggctcggg
chain?)
tatgcaagccgcgatcatggcccatgacctgctgctggccggcaccgcagacgtggtagtggcgggtggca
tggaaagcatgaccaacgcgccgtacctgctggacaaagcccgtggcggctaccgcatgggccacggcaag
atcatcgaccacatgttcatggacggtctcgaagacgcctacgacaaaggccgcctgatgggtacctttgc
cgaggactgtgcccaggccaatgccttcagccgcgaggcccaggaccagttcgccatcgcctcgctgaccc
gagcgcaggaagccatcagcagcggccgttttgccgccgagatcgtgccggtggaagtcaccgagggcaag
gaaaagcgcgtcatcaaggatgacgagcagccgcccaaggcgcgtctggacaagattgcgcagctcaaacc
ggcgtttcgtgaaggcggcaccgtgacggcggccaacgccagttcgatttccgacggcgctgcggcgctgg
tactgatgcgccgctccgaggccgacaaacgtggcctcaagccattggccgtcatccacggccacgccgcc
tttgccgacaccccggcgctgttcccgaccgccccgatcggcgcgatcgacaaactgatgaaacgcaccgg
ctggaacctggccgaagtcgacctgttcgagatcaacgaggccttcgccgtggtcaccctggcggccatga
aacacctcgacctgccacacgacaaggtcaatatccacggcggcgcctgcgccctcggtcacccgatcggc
gcttctggcgcacgtattctggtcaccctgttgtcggccttgcgccagaacaatctgcgtcggggtgtggc
ggccatctgcatcggcggtggcgaggccacggccatggctgttgaatgcctgtactga
(SEQ ID NO: 11)
beta-
Caenorhab-
kat-1
1167
ATGAACAAACATGCT
TAATTTCTGGATAAC
atgaacaaacatgctttcatcgtcggagccgcccgtacacctattggatcatttcgttcttctctctcttc
ketothiolase
ditis elegans
TTCATCGTCG
CATTCCACTTGAGC
ggtaactgctccagagctcgcctcggttgccatcaaagcagcattggagcgtggagcagtgaagccgagtt
(SEQ ID NO: 12)
(SEQ ID NO: 13)
caattcaggaggtgttccttggtcaagtctgtcaagcaaatgctggtcaagctcccgctcgtcaagcagct
cttggagccggactcgatctttcggttgctgttaccaccgtcaataaagtgtgctcttctgggctgaaagc
aatcattcttgctgcccagcaaattcaaaccggtcatcaagattttgccattggcggaggaatggagagca
tgtcacaagtaccattttatgttcaaagaggagagatcccatatggtggatttcaagtgattgatggaatc
gtcaaagacggactgaccgatgcttatgataaagttcacatgggaaactgcggagagaagacttcaaaaga
aatgggaattacacgtaaagaccaagacgaatatgctatcaacagctacaaaaagtcagctaaagcatggg
agaatggaaatatcggaccagaagtggtgccagtgaacgtcaaatcaaagaagggagtcacgattgttgat
aaagatgaagagttcacaaaagtcaatttcgacaagttcacctcgctgagaactgttttccagaaagacgg
aactatcactgctgctaatgcttcaacattgaacgacggtgcagctgctgtcattgttgcctcacaggaag
cagtttccgagcaaagcttaaagcctctggcccgaattttggcttatggagatgccgccacgcacccactc
gatttcgctgtagcaccaactttgatgttcccaaaaattcttgaaagagcaggagtgaagcaatcagatgt
tgctcaatgggaagttaatgaagccttctcatgtgttccccttgctttcatcaaaaaactaggagtcgatc
catcccttgtgaacccacatggaggagctgtttcaattggtcaccccatcggaatgtccggagcccgcctc
atcactcatcttgtgcacacactcaaaagtggccaaatcggagttgctgccatttgcaatggaggtggtgg
ctcaagtggaatggttatccagaaattataa
(SEQ ID NO: 14)
beta-
Escherichia
paaJ
1206
ATGCGTGAAGCCTTT
AACACGCTCCAGAAT
atgcgtgaagcctttatttgtgacggaattcgtacgccaattggtcgctacggcggggcattatcaagtgt
ketothiolase
coli
ATTTGTGACG
CATGGCG
tcgggctgatgatctggctgctatccctttgcgggaactgctggtgcgaaacccgcgtctcgatgcggagt
NP_415915.1
(SEQ ID NO: 15)
(SEQ ID NO: 16)
gtatcgatgatgtgatcctcggctgtgctaatcaggcgggagaagataaccgtaacgtagcccggatggcg
actttactggcggggctgccgcagagtgtttccggcacaaccattaaccgcttgtgtggttccgggctgga
cgcactggggtttgccgcacgggcgattaaagcgggcgatggcgatttgctgatcgccggtggcgtggagt
caatgtcacgggcaccgtttgttatgggcaaggcagccagtgcattttctcgtcaggctgagatgttcgat
accactattggctggcgatttgtgaacccgctcatggctcagcaatttggaactgacagcatgccggaaac
ggcagagaatgtagctgaactgttaaaaatctcacgagaagatcaagatagttttgcgctacgcagtcagc
aacgtacggcaaaagcgcaatcctcaggcattctggctgaggagattgttccggttgtgttgaaaaacaag
aaaggtgttgtaacagaaatacaacatgatgagcatctgcgcccggaaacgacgctggaacagttacgtgg
gttaaaagcaccatttcgtgccaatggggtgattaccgcaggcaatgcttccggggtgaatgacggagccg
ctgcgttgattattgccagtgaacagatggcagcagcgcaaggactgacaccgcgggcgcgtatcgtagcc
atggcaaccgccggggtggaaccgcgcctgatggggcttggtccggtgcctgcaactcgccgggtgctgga
acgcgcagggctgagtattcacgatatggacgtgattgaactgaacgaagcgttcgcggcccaggcgttgg
gtgtactacgcgaattggggctgcctgatgatgccccacatgttaaccccaacggaggcgctatcgcctta
ggccatccgttgggaatgagtggtgcccgcctggcactggctgccagccatgagctgcatcggcgtaacgg
tcgttacgcattgtgcaccatgtgcatcggtgtcggtcagggcatcgccatgattctggagcgtgtttga
(SEQ ID NO: 17)
beta-
Pseudomonas
phaD
1221
ATGAATGAACCGACC
GAGGCGCTCGATGAT
atgaatgaaccgacccacgccgatgccttgatcatcgacgccgtgcgcacgcccattggccgctatgccgg
ketothiolase
putida
CACGCC
CATGG
ggccctgagcagcgtgcgcgccgacgacctggcggccatcccgctcaaagccttgatccagcgtcaccccg
AAN68887.1
(KT2440)
(SEQ ID NO: 18)
(SEQ ID NO: 19)
aactggactggaaagccattgatgacgttatcttcggctgtgccaaccaggctggcgaagacaaccgcaac
gtggcccacatggcgagcctgctggccgggctgccactcgaagtaccagggaccacgatcaaccgcctgtg
cggttccggtctggatgccatcggtaatgcggcacgtgccctgcgctgcggtgaagcggggctcatgctgg
ccggtggtgtggagtccatgtcgcgtgcaccgtttgtgatgggtaagtcggagcaggcattcgggcgtgcg
gccgagctgttcgacaccaccatcggctggcgtttcgtcaacccgctgatgaaggccgcctacggcatcga
ttcgatgccggaaacggctgaaaacgtggccgaacagttcggcatctcgcgcgccgaccaggatgcctttg
ccctgcgcagccagcacaaagccgcagcagctcaggcccgcggccgcctggcgcgggaaatcgtgccggtc
gaaatcccgcaacgcaaaggcccagccaaagtggtcgagcatgacgagcacccgcgcggcgacacgaccct
ggagcagctggctcggctcgggacgccgtttcgtgaaggcggcagcgtaacggcgggtaatgcctccggcg
tgaatgacggcgcttgcgccctgctgctggccagcagcgccgcggcccgccgccatgggttgaaggcccgc
ggccgcatcgtcggcatggcggtggccggggttgagcccaggctgatgggcattggtccggtgcctgcgac
ccgcaaggtgctggcgctcaccggcctggcactggctgacctggatgtcatcgaactcaatgaggcctttg
ccgcccaagggctggccgtgttgcgcgagctgggcctggccgacgacgacccgcgagtcaaccgcaacggc
ggcgccatcgccctgggccatcccctgggcatgagcggtgcccggttggtgaccactgccttgcacgagct
tgaagaaacggccggccgctacgccctgtgcaccatgtgcatcggcgtaggccaaggcattgccatgatca
tcgagcgcctctga
(SEQ ID NO: 20)
beta-
Clostridium
thiA
1179
ATGAAAGAAGTTGTA
GCACTTTTCTAGCAA
atgaaagaagttgtaatagctagtgcagtaagaacagcgattggatcttatggaaagtctcttaaggatgt
ketothiolase
aceto-
ATAGCTAGTGCAGTA
TATTGCTGTTCC
accagcagtagatttaggagctacagctataaaggaagcagttaaaaaagcaggaataaaaccagaggatg
NP_349476.1
butylicum
AGAAC
(SEQ ID NO: 22)
ttaatgaagtcattttaggaaatgttcttcaagcaggtttaggacagaatccagcaagacaggcatctttt
ATCC 824
(SEQ ID NO: 21)
aaagcaggattaccagttgaaattccagctatgactattaataaggtttgtggttcaggacttagaacagt
tagcttagcagcacaaattataaaagcaggagatgctgacgtaataatagcaggtggtatggaaaatatgt
ctagagctccttacttagcgaataacgctagatggggatatagaatgggaaacgctaaatttgttgatgaa
atgatcactgacggattgtgggatgcatttaatgattaccacatgggaataacagcagaaaacatagctga
gagatggaacatttcaagagaagaacaagatgagtttgctcttgcatcacaaaaaaaagctgaagaagcta
taaaatcaggtcaatttaaagatgaaatagttcctgtagtaattaaaggcagaaagggagaaactgtagtt
gatacagatgagcaccctagatttggatcaactatagaaggacttgcaaaattaaaacctgccttcaaaaa
agatggaacagttacagctggtaatgcatcaggattaaatgactgtgcagcagtacttgtaatcatgagtg
cagaaaaagctaaagagcttggagtaaaaccacttgctaagatagtttcttatggttcagcaggagttgac
ccagcaataatgggatatggacctttctatgcaacaaaagcagctattgaaaaagcaggttggacagttga
tgaattagatttaatagaatcaaatgaagcttttgcagctcaaagtttagcagtagcaaaagatttaaaat
ttgatatgaataaagtaaatgtaaatggaggagctattgcccttggtcatccaattggagcatcaggtgca
agaatactcgttactcttgtacacgcaatgcaaaaaagagatgcaaaaaaaggcttagcaactttatgtat
aggtggcggacaaggaacagcaatattgctagaaaagtgctag
(SEQ ID NO: 23)
beta-
Clostridium
thiB
1179
ATGAGAGATGTAGTA
GTCTCTTTCAACTAC
atgagagatgtagtaatagtaagtgctgtaagaactgcaataggagcatatggaaaaacattaaaggatgt
ketothiolase
aceto-
ATAGTAAGTGCTGTA
GAGAGCTGTTCCC
acctgcaacagagttaggagctatagtaataaaggaagctgtaagaagagctaatataaatccaaatgaga
NP_149242.1
butylicum
AGAACTG
(SEQ ID NO: 25)
ttaatgaagttatttttggaaatgtacttcaagctggattaggccaaaacccagcaagacaagcagcagta
ATCC 824
(SEQ ID NO: 24)
aaagcaggattacctttagaaacacctgcgtttacaatcaataaggtttgtggttcaggtttaagatctat
aagtttagcagctcaaattataaaagctggagatgctgataccattgtagtaggtggtatggaaaatatgt
ctagatcaccatatttgattaacaatcagagatggggtcaaagaatgggagatagtgaattagttgatgaa
atgataaaggatggtttgtgggatgcatttaatggatatcatatgggagtaactgcagaaaatattgcaga
acaatggaatataacaagagaagagcaagatgaattttcacttatgtcacaacaaaaagctgaaaaagcca
ttaaaaatggagaatttaaggatgaaatagttcctgtattaataaagactaaaaaaggtgaaatagtcttt
gatcaagatgaatttcctagattcggaaacactattgaagcattaagaaaacttaaacctattttcaagga
aaatggtactgttacagcaggtaatgcatccggattaaatgatggagctgcagcactagtaataatgagcg
ctgataaagctaacgctctcggaataaaaccacttgctaagattacttcttacggatcatatggggtagat
ccatcaataatgggatatggagctttttatgcaactaaagctgccttagataaaattaatttaaaacctga
agacttagatttaattgaagctaacgaggcatatgcttctcaaagtatagcagtaactagagatttaaatt
tagatatgagtaaagttaatgttaatggtggagctatagcacttggacatccaataggtgcatctggtgca
cgtattttagtaacattactatacgctatgcaaaaaagagattcaaaaaaaggtcttgctactctatgtat
tggtggaggtcagggaacagctctcgtagttgaaagagactaa
(SEQ ID NO: 26)
3-oxoadipyl-
Candida
POT98
1182
ATGTTCAAGAAATCA
CTCGTTAGCAAACAA
atgttcaagaaatcagctaatgatattgttgttattgcagcaaagagaactccaatcaccaagtcaattaa
CoA thiolase
albicans
GCTAATGATATTGTT
GGCAGCG
aggtgggttgagtagattatttcctgaggaaatattatatcaagtggttaagggtactgtatcagattcac
SC5314
G
(SEQ ID NO: 28)
aagttgatttaaacttgattgatgatgtgttagtcggtacggtcttgcaaactttagggggacagaaagct
(SEQ ID NO: 27)
agtgccttggccattaaaaagattggattcccaattaagaccacggttaatacggtcaatcgtcaatgtgc
tagttctgctcaagcgattacttatcaagcaggtagtttgcgtagtggggagaatcaatttgctattgctg
ctggagtagaaagtatgactcatgattattttcctcatcgtgggattcccacaagaatttctgaatcattt
ttagctgatgcatccgatgaagctaaaaacgtcttgatgccaatggggataaccagtgaaaatgttgccac
taaatatggaatttctcgtaaacaacaagatgagtttgcccttaattctcatttgaaagcagacaaggcta
caaaactgggtcattttgcaaaagaaatcattcctattcaaacaacggatgaaaacaaccaacacgtttca
ataaccaaagatgatggtataaggggaagttcaacaattgaaaagttgggtggcttaaaacctgtgttcaa
ggatgatgggactactactgctggtaattcctcgcaaatttcagatggagggtctgctgtgattttaacta
ctcgtcaaaatgctgagaaatcgggagtaaagccaatagctagatttattggttcgtcagtagctggtgtt
ccttcgggacttatgggaattggtccatcggctgctattcctcaattgttgtcgagattaaatgttgacac
gaaagacattgatatttttgaattgaacgaggcatttgcatcccaactgatttattgtattgaaaaattgg
gtcttgattatgataaagtcaatccatatggtggagctatagccttgggacatccattaggagccactggc
gcaagagttacggcaacgttgcttaatggattaaaagatcagaataaagagttgggtgtcatctcaatgtg
cacatccacaggtcaaggatacgctgccttgtttgctaacgagtag
(SEQ ID NO: 29)
3-oxoadipyl-
Candida
POT1
1227
ATGGATAGATTAAAT
TTCCTTAATCAATAT
atggatagattaaatcaattaagtggtcaattaaaaccaacttcaaaacaatcccttactcaaaagaaccc
CoA thiolase
albicans
CAATTAAGTGGTCAA
GGAGGCAGCAC
agacgatgttgtcatcgttgcagcatacagaactgccatcggtaaaggtttcaaagggtctttcaaatctg
SC5314
TTAAAACC
(SEQ ID NO: 31)
tgcaatctgaattcatcttgactgaattcttgaaagaatttattaaaaagactggagtcgatgcatctttg
(SEQ ID NO: 30)
attgaagatgttgctattggtaacgttttgaaccaagctgctggtgccaccgaacacagaggtgctagttt
ggctgcaggtattccttacactgcagctttccttgccatcaacagattgtgttcctcagggttaatggcca
tttctgacattgccaacaaaatcaaaaccggtgaaatcgaatgtggtcttgctggtggtattgaatccatg
tctaaaaactatggtagtccaaaagttattccaaagattgacccacacttggctgatgacgaacaaatgag
taaatgtttgattccaatgggtatcaccaacgaaaatgttgctaatgaattcaacattccaagagaaaaac
aagatgcctttgctgctaaatcttatagtaaagccgaaaaagccatctcctctggagctttcaaagatgaa
atcttaccaatcagatccattatcagatccccagacggttctgaaaaagaaatcattgtcgataccgacga
aggtccaagaaagggtgttgacgctgcttccttgagcaaattgaaaccagcatttggtggtactaccactg
ccggtaacgcttctcaaatttcagatggtgctgctggtgttttattgatgaagagaagtttggctgaagcc
aaaggttacccaattgttgctaaatacattgcttgttcaactgttggtgttccgccagaaatcatgggtgt
tggtccagcttacgccattccagaagtgttgaagagaactggattgactgtggatgacgttgatgtgtttg
aaatcaacgaagcttttgctgctcaatgtctttactcagctgaacaatgtaatgttccagaagaaaaattg
aacataaacggtggtgccatcgctttaggtcatcctcttggttgtactggtgccagacaatatgccactat
cttgagattgttgaaaccaggtgaaattggtttgacttctatgtgtatcggtagtggtatgggtgctgcct
ccatattgattaaggaatag
(SEQ ID NO: 32)
3-oxoadipyl-
Candida
POT2
1233
ATGTCATCCAAACAA
TTCTCTAACCAAAAC
atgtcatccaaacaacaatacttgaagaagaatcctgacgatgtcgttgtcgttgcagcatacagaactgc
CoA thiolase
albicans
CAATACTTGAAGAAG
AGAAGCAGCACC
tttaaccaaaggtggaagaggtggattcaaagatgttggatctgatttccttttgaaaaaattgactgaag
SC5314
(SEQ ID NO: 33)
(SEQ ID NO: 34)
aatttgttaaaaaaactggtgttgaccctaaaatcattcaagatgctgccattggtaatgtcttgaacaga
agagctggtgatttcgaacatagaggtgcattattatctgctggattaccttattcagttccatttgttgc
ccttaacagacaatgttcatctgggttaatggccatttctcaagtggccaacaagatcaagactggtgaaa
ttgaatgtggtttagctggtggtgttgaaagtatgacaaaaaactatggtccagaagcattgattgctatt
gaccctgcttatgaaaaagacccagaatttgttaaaaacggtattccaatgggtattactaatgaaaatgt
ttgtgccaaattcaatatttcaagagatgttcaagatcaatttgctgctgaatcttatcaaaaagctgaaa
aggcacaaaaagaaggtaaatttgatgatgaaattttaccaattgaagttttccaagaagatgaagatgct
gaagatgaagacgaagatgaagatgaagatgctgaaccaaaagaaaaattggttgttattagtaaagatga
aggtattagaccaggtgttactaaagaaaaattggctaaaattaaaccagctttcaaatctgatggtgtat
cttcagctggtaactcttcacaagtttccgatggtgctgccttggtgttattgatgaaacgttcatttgct
gaaaagaatggattcaaaccattggctaaatacatttcttgtggtgttgctggtgtcccaccagaaattat
gggtattggtccagctgttgccattccaaaagttttgaaacaaactggattatcagtcagtgatattgata
tttatgaaatcaatgaagcatttgccggtcaatgtttgtactcaattgaaagttgtaatattccaagagaa
aaagtcaatcttaatgggggtgctattgccttgggtcaccctcttggttgtactggtgctagacaatacgc
tactattttaagattgttaaaaccaggtgaatttggtgtgacttctatgtgtattggtactggtatgggtg
ctgcttctgttttggttagagaataa
(SEQ ID NO: 35)
beta-
Pseudomonas
pcaF
1206
ATGAGCCGCGAGGTA
GACCCGCTCGATGGC
atgagccgcgaggtattcatctgcgatgccgtgcgcacgccgatcggccgtttcggcggcagtctttccgc
ketoadipyl
aeruginosa
TTCATCTG
CAG
ggtgcgcgccgacgacctcgcggcggtgccgctgaaggccctggtcgagcgcaacccgggggtcgactggt
CoA thiolase
PAO1
(SEQ ID NO: 36)
(SEQ ID NO: 37)
cggcgttggacgaggtgttcctcggctgcgccaaccaggccggcgaggacaaccgtaacgtggcgcgcatg
pcaF
gcgctgctgctggccggtttgccggagagcgtgcccggcgtcaccctcaaccgcctctgcgcctcggggat
ggacgccatcggcacggcgttccgcgccatcgcctgcggcgagatggagctggccatcgccggcggcgtcg
agtcgatgtcgcgcgcgccgtacgtgatgggcaaggccgatagcgccttcggtcgcggccagaagatcgag
gacaccaccatcggctggcgcttcgtcaatccgctgatgaaggagcagtacggcatcgacccgatgccgca
gaccgccgacaacgtcgccgacgactatcgcgtgtcgcgtgccgaccaggatgccttcgccctgcgcagcc
agcagcgcgccggcagggcgcaggaggccggtttcttcgccgaggaaatcgtcccggtgacgattcgcggg
cgcaagggcgacaccctggtcgagcacgacgagcatccgcgtcccgacaccaccctggaggcgctggcccg
gctcaagccggtcaacgggccggagaagaccgtcaccgccggcaacgcgtccggggtcaacgacggcgccg
ccgcgctggtcctggcctccgccgaggcagtggagaagcacggcctgactccgcgcgcgcgggtgctgggc
atggccagcgccggcgtcgccccacggatcatgggcatcggcccggtgccggcggtgcgcaagctgctgcg
gcgcctggacctggcgatcgacgccttcgacgtgatcgaactcaacgaagccttcgccagccagggcctgg
cctgcctgcgcgaactgggcgtggccgacgacagtgagaaggtcaacccgaacggcggtgccatcgccctc
ggccacccgctggggatgagcggtgcgcggctggtcctcaccgcgctccatcaacttgagaagagcggcgg
ccggcgcggcctggcgaccatgtgcgtaggcgtcggccaaggcctggcgctggccatcgagcgggtctga
(SEQ ID NO: 38)
acyl-CoA
Pseudomonas
bkt
1206
ATGCTCGATGCCTAT
TCGGCAGCGCTCGAT
atgctcgatgcctatatctacgccggcctgcgtacgcctttcggccggcatgccggtgcactctcgacggt
thiolase
aeruginosa
ATCTACGCC
CAC
gcgtccggacgacctggccggcctgctgctggcgcgtctcgcggaaacctccgggttcgccgtcgacgacc
POA1
(SEQ ID NO: 39)
(SEQ ID NO: 40)
tggaggatgtgatcctcggttgcaccaaccaggccggcgaagacagccgcaacctggcgcgcaacgcgctg
ctcgcagccggcctgccggcgcggctgcccgggcagacggtcaaccgcttgtgtgccagcggactgtcggc
ggtgatcgacgcggcgcgcgcgatcagttgcggtgagggccggctgtacctggccggcggcgccgaaagca
tgtcccgggcgccgttcgtcatgggcaaggcggagagcgccttcagccgcacgctggaggtcttcgacagc
accatcggcgcgcgcttcgccaaccccaggctggtcgagcgctatggcaacgacagcatgccggagaccgg
cgacaacgtggcccgcgccttcggcatcgcccgcgaagacgccgaccgtttcgccgcttcttcccaggcgc
gctaccaggctgcgctggaggagggctttttcctcggcgagatccttccggtggaggtgcgtgccggacgc
aagggcgagacgcggctggtggagcgcgacgagcatccgcgaccgcaggccgacctggcggccctggcgcg
cttgccggcgttgttcgccggtggggtagtgaccgccggtaatgcgtctgggatcaacgacggggcggcgg
tagtgctgctgggcgatcgcgcgatcggcgagcgcgagggcatccggccgttggcgcggatcctcgccagc
gccagcgtcggcgtcgagccccggttgatgggcatcggcccgcagcaggcgatcctccgcgcgctgcaacg
cgccggcatcgacctggacgaggtcggcctgatcgagatcaacgaagccttcgcgccgcaggtcctggcct
gcctgaagttgctcggcctggactacgaggacccgcgggtcaatccccatggcggcgccattgccctcggc
catccgctcggcgcctccggtgcgcgcctggtgctcaccgccgcccgcgggctgcaacgcatcgagcggcg
ctacgcggtggtcagcctgtgcgtcgggctcggccagggcgtggcgatggtgatcgagcgctgccgatga
(SEQ ID NO: 41)
3-oxoadipyl-
Pseudomonas
pcaF
1203
ATGCACGACGTATTC
AACCCGCTCGATGGC
atgcacgacgtattcatctgtgacgccatccgtaccccgatcggccgcttcggcggcgccctggccagcgt
CoA thiolase
putida
ATCTGTGACG
CAAC
gcgggccgacgacctggccgccgtgccgctgaaggcgctgatcgagcgcaaccctggcgtgcagtgggacc
(KT2440)
(SEQ ID NO: 42)
(SEQ ID NO: 43)
aggtagacgaagtgttcttcggctgcgccaaccaggccggtgaagacaaccgcaacgtggcccgcatggca
ctgctgctggccggcctgccggaaagcatcccgggcgtcaccctgaaccgtctgtgcgcgtcgggcatgga
tgccgtcggcaccgcgttccgcgccatcgccagcggcgagatggagctggtgattgccggtggcgtcgagt
cgatgtcgcgcgccccgttcgtcatgggcaaggctgaaagcgcctattcgcgcaacatgaagctggaagac
accaccattggctggcgtttcatcaacccgctgatgaagagccagtacggtgtggattccatgccggaaac
cgccgacaacgtggccgacgactatcaggtttcgcgtgctgatcaggacgctttcgccctgcgcagccagc
agaaggctgccgctgcgcaggctgccggcttctttgccgaagaaatcgtgccggtgcgtatcgctcacaag
aagggcgaaatcatcgtcgaacgtgacgaacacctgcgcccggaaaccacgctggaggcgctgaccaagct
caaaccggtcaacggcccggacaagacggtcaccgccggcaacgcctcgggcgtgaacgacggtgctgcgg
cgatgatcctggcctcggccgcagcggtgaagaaacacggcctgactccgcgtgcccgcgttctgggcatg
gccagcggcggcgttgcgccacgtgtcatgggcattggcccggtgccggcggtgcgcaaactgaccgagcg
tctggggatagcggtaagtgatttcgacgtgatcgagcttaacgaagcgtttgccagccaaggcctggcgg
tgctgcgtgagctgggtgtggctgacgatgcgccccaggtaaaccctaatggcggtgccattgccctgggc
caccccctgggcatgagcggtgcacgcctggtactgactgcgttgcaccagctggagaagagtggcggtcg
caagggcctggcgaccatgtgtgtgggtgtcggccaaggtctggcgttggccatcgagcgggtttga
(SEQ ID NO: 44)
3-oxoadipyl-
Burkholderia
bkt
1203
ATGACCGACGCCTAC
CACGCGTTCGATCGC
atgaccgacgcctacatctgcgatgcgattcgcacacccatcggccgctacggcggcgccctgaaagacgt
CoA thiolase
ambifaria
ATCTGCG
GATC
tcgtgccgacgatctcggcgcggtgccgctcaaggcgctgatcgaacgcaaccggaacgtcgactggtcgg
AMMD
(SEQ ID NO: 45)
(SEQ ID NO: 46)
cgatcgacgacgtgatctatggctgcgcgaaccaggccggcgaagacaaccgcaacgtcgcgcgcatgtcc
gcgctgctcgcgggcttgccgaccgccgtgccgggcacgacgctgaaccggttatgcggctcgggcatgga
cgccgtcggcacggccgcgcgcgcgatcaaggcgggcgaggcacgcttgatgatcgcgggcggcgtcgaaa
gcatgacgcgcgcgccgttcgtgatgggcaaggccgccagcgcattcgcgcgccaggctgcgattttcgac
acgacgatcggctggcgtttcattaatccgctgatgaaacagcaatacggcgtcgattcgatgcccgagac
ggccgagaacgtcgcggtcgactacaacatcagccgcgccgaccaggatctattcgcgctgcgcagccagc
agaaggccgcgcgtgcgcagcaggacggcacgctcgccgccgaaatcgtccccgtcacgattgcgcagaaa
aaaggcgacgcgctcgtcgtatcgctcgacgagcatccgcgcgaaacatcgctcgaagcgctcgcgaagct
gaagggcgtcgtgcgtcccgacggctcggtcacggccggcaacgcgtcaggcgtcaacgacggcgcatgcg
cactgctgctcgccaacgcggaagccgccgatcaatatgggctgcgccgccgcgcgcgtgtcgtcggcatg
gcgagcgccggcgtcgagccgcgcgtgatgggtatcggcccggcgccggccacgcagaaactgttgcgcca
gctcggcatgacagatcgaccagttcgacgtgatcgagctgaacgaagcgttcgcgtcgcagggtctcgcg
gtgctgcgcatgctcggtgtcgccgacgacgatccgcgcgtgaaccccaacggcggtgcgatcgcgctcgg
ccatccgctcggcgcatcgggtgcgcggctcgtgaccacggcgcttcaccaactcgagcgtacgggcggcc
gctttgcgctctgtacgatgtgcatcggcgtcggccagggcatcgcgatcgcgatcgaacgcgtgtaa
(SEQ ID NO: 47)
beta-
Ascaris
bkt
1242
ATGGCCACCTCAAGA
CAATTTCTCGATGAC
gtgatggccacctcaagacttgtctgcagcaatttaacgaagcaatgctttacgatctcgtcacgtgctgc
ketothiolase
suum
CTTGTCTGC
CATTCCACC
tagccaatttaccgatgtggtattcgtgggtgccgcacgaacaccggtcggatcgtttcgctcttcgcttt
(SEQ ID NO: 48)
(SEQ ID NO: 49)
ccactgttccagccactgtcctcggagctgaggctattaagggtgcacttaaacatgccaatctaaaaccc
tcacaagtgcaagaggtgttctttggctgtgtcgttccatccaactgtggacaagttcctgcccgtcaagc
gacacttggagctggatgcgatccttcgacaatcgttacaactctcaataaattgtgcgcctcgggaatga
agtcgattgcttgtgccgcctcacttttgcaacttggtcttcaagaggttaccgttggtggcggtatggag
agcatgagcttagtgccgtactatcttgaacgtggtgaaactacttatggtggaatgaagctcatcgacgg
tatcccaagagatggtccgactgatgcatatagtaatcaacttatgggtgcatgcgctgataatgtggcta
aacgattcaacatcacccgtgaggaacaggataaattcgctattgaaagctataaacgatctgctgctgca
tgggagagtggagcatgcaaagctgaagtagttcctattgaagtgacaaagggcaagaaaacatacattgt
caacaaggatgaggaatacatcaaagtcaacttcgagaagcttcccaaactgaaacccgccttcttgaaag
acggaaccatcacggctggcaatgcttcaacactgaacgatggtgctgcggcagttgtgatgacgactgtc
gaaggagcgaaaaaatacggtgtgaaaccattggcccgattgctctcatatggtgatgcggcaacaaatcc
agtcgattttgctattgcaccatcaatggttatcccaaaggtacttaaattggctaatctcgagatcaagg
atattgatttgtgggaaatcaacgaggctttcgccgttgttccccttcattcaatgaagacactcggtatc
gatcactcgaaagtgaacattcatggtggtggcgtatctcttggacatcctattggaatgtctggagctcg
aattatcgttcatctgattcatgcgttgaaacctggccagaaaggctgcgctgcaatctgcaatggtggcg
gtggcgctggtggaatggtcatcgagaaattgtaa
(SEQ ID NO: 50)
The genes were expressed in E. coli and the proteins purified using Ni-NTA spin columns and quantified. To assay enzyme activity in vitro, a 5× CoA:DTNB (Ellman's reagent or 5,5′-dithiobis-(2-nitrobenzoic acid)) mixture was prepared. The mixture consisted of 10 mM succinyl-CoA, 5 mM acetyl-CoA, 30 mM DTNB in 100 mM Tris buffer, pH 7.4. Five μL of the CoA:DTNB mixture was added to 0.5 μM purified thiolase enzyme in 100 mM Tris buffer, pH 7.8 in a final volume of 50 μL. The reaction was incubated at 30° C. for 30 minutes, then quenched with 2.5 μL 10% formic acid and samples frozen at −20° C. until ready for analysis by LC/MS. Because many thiolases can condense two acetyl-CoA molecules into acetoaceytl-CoA, production of acetoacetyl-CoA was examined.
This example describes exemplary pathways for production of hexamethylenediamine (HMDA) from glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2-amino-7-oxosubarate through homolysine, the seven-carbon analog of lysine. Homolysine is an attractive precursor to HMDA. Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Homolysine can be formed biocatalytically from the central metabolic precursors glutamate, glutaryl-CoA or pyruvate and 4-aminobutanal. Subsequent decarboxylation of homolysine by an enzyme analogous to lysine decarboxylase yields HMDA.
This example describes additional pathways that proceed from 2-amino-7-oxosubarate, or pyruvate and 4-aminobutanal through the intermediate 6-aminohexanal. 6-Aminohexanal can readily be converted to HMDA by an aminotransferase or an aminating oxidoreductase.
The maximum theoretical yield of HMDA is 0.71 moles per mole glucose utilized (0.46 g/g). The pathways disclosed in
C6H12O6+1.41NH4→0.71C6H18N2+1.76CO2+2.47H2O
Novel pathways for producing hexamethylenediamine (HMDA) and related products are described herein. The candidate enzymes, and associated risks of implementation are discussed in Example XXVI below.
This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze HMDA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.
HMDA can be produced from glutamate via glutaryl-CoA in eight enzymatic steps, shown in
HMDA can also be produced from glutaryl-CoA by several routes. Exemplary routes for HMDA production are shown in
One route entails conversion of 3-oxopimelate to 3-oxo-1-carboxyheptanal. This conversion can be catalyzed by an ATP- and NAD(P)H dependent enzyme with 3-oxopimelate reductase activity (Step C of
In an alternate route, 3-oxopimelate is transaminated to 3-aminopimelate (Step J of
3-Aminopimelate can be converted to 2-aminopimelate by a 2,3-aminomutase enzyme (Step T of
Two routes for producing HMDA from pyruvate and 4-aminobutanal are shown in
Several routes for producing HMDA from 2-amino-7-oxosubarate are shown in
Alternately, the intermediate 2-amino-7-oxoheptanoate is first converted to homolysine by an aminotransferase or aminating oxidoreductase (Step M of
In yet another route, the 2-amino acid group of 2-amino-7-oxosubarate is decarboxylated, yielding 2-oxo-7-aminoheptanoate (Step I of
In yet another route, the 2-oxo group of 2-amino-7-oxosubarate is converted to an amino group, forming 2,7-diaminosubarate (Step K of
Described herein is the generation of a microbial organism that has been engineered to produce HMDA from pyruvate and 4-aminobutanal, as shown in
Escherichia coli is used as a target organism to engineer a HMDA-producing pathway as shown in
An E. coli strain is engineered to produce HMDA from 4-aminobutanal via the route outlined in
The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of HMDA pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA and immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce HMDA through this pathway is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).
Microbial strains engineered to have a functional HMDA synthesis pathway from 4-aminobutanal are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.
After successful demonstration of enhanced HMDA production via the activities of the exogenous enzymes, the genes encoding these enzymes are inserted into the chromosome of a wild type E. coli host using methods known in the art. Such methods include, for example, sequential single crossover (Gay et al., J. Bacteriol. 3:153 (1983)). and Red/ET methods from GeneBridges (Zhang et al., European Patent Application No. 01117 (2001))). Chromosomal insertion provides several advantages over a plasmid-based system, including greater stability and the ability to co-localize expression of pathway genes.
To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of HMDA. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of HMDA. Adaptive evolution also can be used to generate better producers of, for example, the 2-oxo-4-hydroxy-7-aminoheptanoate intermediate or the HMDA product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the HMDA producer to further increase production.
For large-scale production of HMDA, the above HMDA pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng., 775-779 (2005).
Novel pathways for producing 6-aminocaproate (6-ACA) and related products are described herein. The candidate enzymes, and associated risks of implementation are discussed in Example XXVI below.
This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 6-ACA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.
6-ACA can be produced from glutamate as a starting molecule. Glutamate is transformed to 6-aminopimeloyl-CoA as described previously (
6-ACA can also be produced from glutaryl-CoA as a starting molecule. In the disclosed pathway to 6-ACA, similar to the HMDA pathway described above, glutaryl-CoA is first condensed with acetyl-CoA by a beta-ketothiolase to form 3-oxopimeloyl-CoA (Step A of
Homolysine is also an attractive precursor to 6-aminocaproate (6-ACA) production. Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Under aerobic conditions, oxidation of homolysine by a lysine 2-monooxygenase yields 6-aminohexanamide, which is readily hydrolyzed to 6-ACA in dilute acid or basic solution (
6-ACA can also be produced from 2-amino-7-oxosubarate as a starting molecule (
This example describes the enzyme classification system for the exemplary pathways described in Examples XXIV and XXV for production of hexamethylenediamine or 6-aminocaproate.
All transformations depicted in
Table 11 shows the enzyme types useful to convert common central metabolic intermediates into 6-aminocaproate and hexamethylenediamine. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.
TABLE 11
LABEL
FUNCTION
1.1.1.a
Oxidoreductase (oxo to alcohol)
1.13.12.a
Monooxygenase (O2 incorporating)
1.2.1.a
Oxidoreductase (aldehyde to acid)
1.2.1.b
Oxidoreductase (acyl-CoA to aldehyde)
1.2.1.d
Oxidoreductase (phosphonate reductase)
1.2.1.e
Acid reductase
1.3.1.a
Oxidoreductase (alkene to alkane)
1.4.1.a
Oxidoreductase (aminating)
2.3.1.b
Acyltransferase (beta-ketothiolase)
2.6.1.a
Aminotransferase
2.7.2.a
Phosphotransferase (carboxy acceptor)
2.8.3.a
Coenzyme-A transferase
3.1.2.a
CoA hydrolase
4.1.1.a
Carboxy-lyase
4.1.2.a
Aldehyde-lyase
4.2.1.a
Hydro-lyase
5.4.3.a
Aminomutase
6.2.1.a
Acid-thiol ligase
1.1.1.a Oxidoreductase (Oxo to Alcohol)—The reduction of 3-oxo-6-aminopimeloyl-CoA to 3-hydroxy-6-aminopimeloyl-CoA is catalyzed by a 3-oxoacyl-CoA dehydrogenase (
Gene
GenBank
name
GI#
Accession #
Organism
fadB
119811
P21177.2
Escherichia coli
fadJ
3334437
P77399.1
Escherichia coli
paaH
16129356
NP_415913.1
Escherichia coli
phaC
26990000
NP_745425.1
Pseudomonas putida
paaC
106636095
ABF82235.1
Pseudomonas fluorescens
Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J Bacteriol. 171:6800-6807 (1989)). Additional gene candidates include Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al., J Biol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61:297-309 (2006)).) The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol. 3:349-357 (1989)) and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as an alternate substrate (Peoples et al., Mol. Microbiol. 3:349-357 (1989)).
Gene
GenBank
name
GI#
Accession #
Organism
hbd
18266893
P52041.2
Clostridium acetobutylicum
Hbd2
146348271
EDK34807.1
Clostridium kluyveri
Hbd1
146345976
EDK32512.1
Clostridium kluyveri
HSD17B10
3183024
O02691.3
Bos taurus
phaB
77464321
YP_353825.1
Rhodobacter sphaeroides
phbB
130017
P23238.1
Zoogloea ramigera
A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007).
Gene
GenBank
name
GI#
Accession #
Organism
hbd
15895965
NP_349314.1
Clostridium
acetobutylicum
hbd
20162442
AAM14586.1
Clostridium beijerinckii
Msed_1423
146304189
YP_001191505
Metallosphaera sedula
Msed_0399
146303184
YP_001190500
Metallosphaera sedula
Msed_0389
146303174
YP_001190490
Metallosphaera sedula
Msed_1993
146304741
YP_001192057
Metallosphaera sedula
1.13.12.a Monooxygenase (O2 Incorporating)
An O2-incorporating monooxygenase is required to convert homolysine to 6-aminohexanamide (Step A of
Gene
GenBank
name
GI#
Accession #
Organism
davB
193805882
BAG54787.1
Pseudomonas putida
pfl_5670
68347255
AAY94861.1
Pseudomonas
fluorescens
SCO1454
7209214
CAB76876.1
Streptomyces
coelicolor
RHA1_ro03531
110820050
ABG95334.1
Rhodococcus jostii
1.2.1.a Oxidoreductase (Aldehyde to Acid)—Two transformations in
Gene
GenBank
name
GI#
Accession #
Organism
ALDH-2
118504
P05091.2
Homo sapiens
ALDH-2
14192933
NP_115792.1
Rattus norvegicus
astD
3913108
P76217.1
Escherichia coli
1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde)—Reductive de-acylation of 3-oxopimeloyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
acr1
50086359
YP_047869.1
Acinetobacter calcoaceticus
acr1
1684886
AAC45217
Acinetobacter baylyi
acr1
18857901
BAB85476.1
Acinetobacter sp. Strain M-1
sucD
172046062
P38947.1
Clostridium kluyveri
sucD
34540484
NP_904963.1
Porphyromonas gingivalis
bphG
425213
BAA03892.1
Pseudomonas sp
An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg et al., Science. 318:1782-1786 (2007); and Thauer et al., Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed—0709 in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al., Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ. Microbiol. 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to cutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., Appl Environ. Microbiol. 65:4973-4980 (1999)).
Gene
GenBank
name
GI#
Accession #
Organism
Msed_0709
146303492
YP_001190808.1
Metallosphaera sedula
mcr
15922498
NP_378167.1
Sulfolobus tokodaii
asd-2
15898958
NP_343563.1
Sulfolobus solfataricus
Saci_2370
70608071
YP_256941.1
Sulfolobus
acidocaldarius
Ald
49473535
AAT66436
Clostridium beijerinckii
eutE
687645
AAA80209
Salmonella typhimurium
eutE
2498347
P77445
Escherichia coli
1.2.1.d Oxidoreductase (Phosphonate Reductase)—The reduction of a phosphonic acid to its corresponding aldehyde is catalyzed by an oxidoreductase in the EC class 1.2.1. Steps G, N and X in
Gene
GenBank
name
GI#
Accession #
Organism
asd
16131307
NP_417891.1
Escherichia coli
asd
68249223
YP_248335.1
Haemophilus influenzae
asd
1899206
AAB49996
Mycobacterium tuberculosis
VC2036
15642038
NP_231670
Vibrio cholera
asd
210135348
YP_002301787.1
Heliobacter pylori
ARG5,6
6320913
NP_010992.1
Saccharomyces cerevisiae
argC
16078184
NP_389001.1
Bacillus subtilis
Other exemplary phosphonate reductase enzymes include glyceraldehyde 3-phosphate dehydrogenase which converts glyceraldehyde-3-phosphate into D-glycerate 1,3-bisphosphate (e.g., E. coli gapA (Branlant et al., Eur. J. Biochem. 150:61-66 (1985)).23)), N-acetyl-gamma-glutamyl-phosphate reductase which converts N-acetyl-L-glutamate-5-semialdehyde into N-acetyl-L-glutamyl-5-phosphate (e.g., E. coli argC (Parsot et al., Gene. 68:275-283 (1988)), and glutamate-5-semialdehyde dehydrogenase which converts L-glutamate-5-semialdehyde into L-glutamyl-5-phospate (e.g., E. coli proA (Smith et al., J. Bacteriol. 157:545-551 (1984))). Genes encoding glutamate-5-semialdehyde dehydrogenase enzymes from Salmonella typhimurium (Mahan et al., J Bacteriol. 156:1249-1262 (1983)) and Campylobacter jejuni (Louie et al., Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E. coli.
Gene
GenBank
name
GI#
Accession #
Organism
gapA
71159358
P0A9B2.2
Escherichia coli
argC
16131796
NP_418393.1
Escherichia coli
proA
16128229
NP_414778.1
Escherichia coli
proA
16763704
NP_459319.1
Salmonella typhimurium
proA
9087222
P53000.2
Campylobacter jejuni
1.2.1.e Acid Reductase—Several transformations in
Carboxylic acid reductase, found in Nocardia iowensis, catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). This enzyme, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al. “Biocatalytic Reduction of Carboxylic Acids Mechanism and Applications” Chapter 15 in Biocatalysis in the Pharmaceutical and Biotechnology Industries, ed. R. N. Patel, CRC Press LLC, Boca Raton, Fla. (2006)).
Gene
GenBank
name
GI#
Accession #
Organism
car
40796035
AAR91681.1
Nocardia iowensis (sp. NRRL 5646)
npt
114848891
ABI83656.1
Nocardia iowensis (sp. NRRL 5646)
An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified by sequence comparison homology searching. Directed evolution or other enzyme engineering methods may be required to enhance reactivity with the substrates in
Gene
GenBank
name
GI#
Accession #
Organism
LYS2
171867
AAA34747.1
Saccharomyces cerevisiae
LYS5
1708896
P50113.1
Saccharomyces cerevisiae
LYS2
2853226
AAC02241.1
Candida albicans
LYS5
28136195
AAO26020.1
Candida albicans
Lys1p
13124791
P40976.3
Schizosaccharomyces pombe
Lys7p
1723561
Q10474.1
Schizosaccharomyces pombe
Lys2
3282044
CAA74300.1
Penicillium chrysogenum
1.3.1.a Oxidoreductase (Alkene to Alkane)—Three transformations fall into the category of oxidoreductases that reduce an alkene to an alkane (EC 1.3.1.-). The conversion of 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
enr
169405742
ACA54153.1
Clostridium botulinum A3 str
enr
2765041
CAA71086.1
Clostridium tyrobutyricum
enr
3402834
CAA76083.1
Clostridium kluyveri
enr
83590886
YP_430895.1
Moorella thermoacetica
fadH
16130976
NP_417552.1
Escherichia coli
Another candidate 2-enoate reductase is maleylacetate reductase (MAR), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. MAR enzymes naturally participate in aromatic degradation pathways (Camara et al., J. Bacteriol. (2009); Huang et al., Appl Environ. Microbiol. 72:7238-7245 (2006)); Kaschabek et al., J. Bacteriol. 177:320-325 (1995) and Kaschabek et al., J. Bacteriol. 175:6075-6081 (1993)). The enzyme activity was identified and characterized in Pseudomonas sp. strain B 13 (Kaschabek et al., J Bacteriol 177:320-325 (1995); and Kaschabek et al., J Bacteriol 175:6075-6081 (1993)), and the coding gene was cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)). Additional MAR gene candidates include cicE gene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., J Bacteriol 180:3503-3508 (1998)), the macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha (Seibert et al., J. Bacteriol. 175:6745-6754 (1993)) and NCgl1112 in Corynebacterium glutamicum (Huang et al., Appl Environ. Microbiol. 72:7238-7245 (2006)). A MAR in Pseudomonas reinekei MT1, encoded by ccaD, was recently identified and the nucleotide sequence is available under the DBJ/EMBL GenBank accession number EF159980 (Camara et al., J. Bacteriol. (2009)).
Gene
GenBank
name
GI#
Accession #
Organism
clcE
3913241
O30847.1
Pseudomonas sp. strain B13
macA
7387876
O84992.1
Rhodococcus opacus
macA
5916089
AAD55886
Cupriavidus necator
tfdFII
1747424
AAC44727.1
Ralstonia eutropha JMP134
NCgl1112
19552383
NP_600385
Corynebacterium glutamicum
ccaD
134133940
ABO61029.1
Pseudomonas reinekei MT1
Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the reduction of 6-amino-7-carboxyhept-2-enoyl-CoA to 6-aminopimeloyl-CoA (
Gene
GenBank
name
GI#
Accession #
Organism
bcd
15895968
NP_349317.1
Clostridium acetobutylicum
etfA
15895966
NP_349315.1
Clostridium acetobutylicum
etfB
15895967
NP_349316.1
Clostridium acetobutylicum
TER
62287512
Q5EU90.1
Euglena gracilis
TDE0597
42526113
NP_971211.1
Treponema denticola
Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes in the pim operon, pimC and pimD, bear sequence homology to C. acetobutylicum bcd and are predicted to encode a flavin-containing pimeloyl-CoA dehydrogenase (Harrison et al., Microbiology 151:727-736 (2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison et al., Microbiology 151:727-736 (2005)).
Gene
GenBank
name
GI#
Accession #
Organism
pimC
39650632
CAE29155
Rhodopseudomonas palustris
pimD
39650631
CAE29154
Rhodopseudomonas palustris
pimC
27356102
BAC53083
Bradyrhizobium japonicum
pimD
27356101
BAC53082
Bradyrhizobium japonicum
An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascarius suum and is capable of reducing a variety of linear and branched chain substrates including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993))). Two isoforms of the enzyme, encoded by genes acad1 and acad, have been characterized.
Gene
GenBank
name
GI#
Accession #
Organism
acad1
2407655
AAC48316.1
Ascarius suum
acad
347404
AAA16096.1
Ascarius suum
1.4.1.a Oxidoreductase (Aminating)—Several reactions in
In Step D of
Gene
GenBank
name
GI#
Accession #
Organism
gdhA
118547
P00370
Escherichia coli
gdh
6226595
P96110.4
Thermotoga maritima
gdhA1
15789827
NP_279651.1
Halobacterium salinarum
ldh
61222614
P0A393
Bacillus cereus
nadX
15644391
NP_229443.1
Thermotoga maritima
Two reactions entail conversion of 3-oxoacids to 3-amino acids: 3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (
Gene
GenBank
name
GI#
Accession #
Organism
kdd
19713113
AAL93966.1
Fusobacterium nucleatum
mxan_4391
108462082
ABF87267.1
Myxococcus xanthus
pg_1069
34397119
AAQ66183.1
Porphyromonas gingivalis
The conversions of 2-amino-7-oxoheptanoate to homolysine (
Gene
GenBank
name
GI#
Accession #
Organism
lysDH
13429872
BAB39707
Geobacillus stearothermophilus
lysDH
15888285
NP_353966
Agrobacterium tumefaciens
lysDH
74026644
AAZ94428
Achromobacter denitrificans
2.3.1.b Acyltransferase (Beta-Ketothiolase)—In Step A of
Gene
GenBank
name
GI#
Accession #
Organism
bktB
11386745
YP_725948
Ralstonia eutropha
pimB
39650633
CAE29156
Rhodopseudomonas palustris
syn_02642
85860483
YP_462685.1
Syntrophus aciditrophicus
Beta-ketothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl-CoA and propionyl-CoA may also be able to catalyze the formation of 3-oxopimeloyl-CoA. Zoogloea ramigera possesses two ketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a β-oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., U.S. Pat. No. 5,958,745 (1999)). The sequences of these genes or their translated proteins have not been reported, but several candidates in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from R. eutropha. These include:
Gene
GenBank
name
GI#
Accession #
Organism
phaA
113867452
YP_725941.1
Ralstonia eutropha
h16_A1713
113867716
YP_726205.1
Ralstonia eutropha
pcaF
116694155
YP_728366.1
Ralstonia eutropha
h16_B1369
116695312
YP_840888.1
Ralstonia eutropha
h16_A0170
113866201
YP_724690.1
Ralstonia eutropha
h16_A0462
113866491
YP_724980.1
Ralstonia eutropha
h16_A1528
113867539
YP_726028.1
Ralstonia eutropha
h16_B0381
116694334
YP_728545.1
Ralstonia eutropha
h16_B0662
116694613
YP_728824.1
Ralstonia eutropha
h16_B0759
116694710
YP_728921.1
Ralstonia eutropha
h16_B0668
116694619
YP_728830.1
Ralstonia eutropha
h16_A1720
113867723
YP_726212.1
Ralstonia eutropha
h16_A1887
113867867
YP_726356.1
Ralstonia eutropha
phbA
135759
P07097.4
Zoogloea ramigera
bktB
194289475
YP_002005382.1
Cupriavidus taiwanensis
Rmet_1362
94310304
YP_583514.1
Ralstonia metallidurans
Bphy_0975
186475740
YP_001857210.1
Burkholderia phymatum
Additional candidates include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)); and Winzer et al., J. Mol. Microbiol. Biotechnol 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser, et al., J. Biol. Chem. 269:31383-31389 (1994)).
Gene
GenBank
name
GI#
Accession #
Organism
atoB
16130161
NP_416728
Escherichia coli
thlA
15896127
NP_349476.1
Clostridium acetobutylicum
thlB
15004782
NP_149242.1
Clostridium acetobutylicum
ERG 10
6325229
NP_015297
Saccharomyces cerevisiae
Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J Bacteriol 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. U.S. A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di Arch et al., Microbiol 188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PAO1, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida. These enzymes can also be employed for the synthesis of 3-oxopimeloyl-CoA, a compound structurally similar to 3-oxoadipyl-CoA.
Gene
GenBank
name
GI#
Accession #
Organism
paaJ
16129358
NP_415915.1
Escherichia coli
pcaF
17736947
AAL02407
Pseudomonas knackmussii (B13)
phaD
3253200
AAC24332.1
Pseudomonas putida
pcaF
506695
AAA85138.1
Pseudomonas putida
pcaF
141777
AAC37148.1
Acinetobacter calcoaceticus
paaE
106636097
ABF82237.1
Pseudomonas fluorescens
bkt
115360515
YP_777652.1
Burkholderia ambifaria AMMD
bkt
9949744
AAG06977.1
Pseudomonas aeruginosa PAO1
pcaF
9946065
AAG03617.1
Pseudomonas aeruginosa PAO1
A beta-ketothiolase is also required to condense glutamyl-CoA and acetyl-CoA (
2.6.1.a Aminotransferase—Several reactions in
Aminotransferases selective for aldehydes are required for transaminating 2-amino-7-oxoheptanoate (
Gene
GenBank
name
GI#
Accession #
Organism
lat
10336502
BAB13756.1
Flavobacterium lutescens
lat
153343
AAA26777.1
Streptomyces clavuligenus
dat
6685373
P56744.1
Acinetobacter baumanii
Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme was also able to transaminate cadaverine and spermidine (Samsonova et al., Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim et al., J Biol. Chem. 239:783-786 (1964); and Samsonova et al., Microbiol 3:2 (2003)). The spuC gene of Pseudomonas aeruginosa encodes a putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate (Lu et al., J. Bacteriol. 184:3765-3773 (2002)).
Gene
GenBank
name
GI#
Accession #
Organism
ygjG
145698310
NP_417544
Escherichia coli
spuC
9946143
AAG03688
Pseudomonas aeruginosa
The conversion of an aldehyde to a terminal amine can also be catalyzed by gamma-aminobutyrate transaminase (GABA transaminase). This enzyme naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Liu et al., Biochemistry 43:10896-10905 (2004); and Schulz et al., Appl Environ Microbiol 56:1-6 (1990)). The two GABA transaminases in E. coli are encoded by gabT (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with a range of alternate substrates including 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985)); and Scott et al., J. Biol. Chem. 234:932-936 (1959)).
Gene
GenBank
name
GI#
Accession #
Organism
gabT
16130576
NP_417148.1
Escherichia coli
puuE
16129263
NP_415818.1
Escherichia coli
abat
37202121
NP_766549.2
Mus musculus
gabT
70733692
YP_257332.1
Pseudomonas fluorescens
abat
47523600
NP_999428.1
Sus scrofa
Enzymes that transaminate 3-oxoacids are required to convert 3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (
Gene
GenBank
name
GI#
Accession #
Organism
SkyPYD4
98626772
ABF58893.1
Lachancea kluyveri
SkUGA1
98626792
ABF58894.1
Lachancea kluyveri
UGA1
6321456
NP_011533.1
Saccharomyces cerevisiae
Abat
122065191
P50554.3
Rattus norvegicus
Abat
120968
P80147.2
Sus scrofa
Several aminotransferases transaminate the amino groups of 2-oxo acids to form amino acids. Such an enzyme is required for the transamination of 2-oxo-7-aminoheptanoate to homolysine (
Gene
GenBank
name
GI#
Accession #
Organism
lysN
31096548
BAC76939.1
Thermus thermophilus
AadAT-II
46395904
Q8N5Z0.2
Homo sapiens
Another candidate is aspartate aminotransferase, an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84 (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43 (1982)) and ASPS from Arabidopsis thaliana (de la et al., Plant J 46:414-425 (2006); Kwok et al., J Exp. Bot. 55:595-604 (2004) and Wilkie et al., Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that work on other amino-acid substrates may also be able to catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen et al., J. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the transamination of a-ketobutyrate to generate α-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen et al., J. Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam, J. et al., Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et al., FEBS. Lett. 390:179-182 (1996)).
Gene
GenBank
name
GI#
Accession #
Organism
aspC
16128895
NP_415448.1
Escherichia coli
AAT2
1703040
P23542.3
Saccharomyces cerevisiae
ASP5
20532373
P46248.2
Arabidopsis thaliana
Got2
112987
P00507
Rattus norvegicus
avtA
49176374
YP_026231.1
Escherichia coli
serC
16128874
NP_415427.1
Escherichia coli
2.7.2.a Phosphotransferase (Carboxy Acceptor)—Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. Steps F, M and U in
Gene
GenBank
name
GI#
Accession #
Organism
buk1
15896326
NP_349675
Clostridium acetobutylicum
buk2
20137415
Q97II1
Clostridium acetobutylicum
buk2
6685256
Q9X278.1
Thermotoga maritima
lysC
16131850
NP_418448.1
Escherichia coli
ackA
16130231
NP_416799.1
Escherichia coli
proB
16128228
NP_414777.1
Escherichia coli
2.8.3.a Coenzyme-A Transferase—CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Several transformations in
GenBank
Gene name
GI#
Accession #
Organism
cat1
729048
P38946.1
Clostridium kluyveri
cat2
172046066
P38942.2
Clostridium kluyveri
cat3
146349050
EDK35586.1
Clostridium kluyveri
TVAG_395550
123975034
XP_001330176
Trichomonas
vaginalis G3
Tb11.02.0290
71754875
XP_828352
Trypanosoma brucei
The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)), substrates similar in structure to 2,3-dehydroadipyl-CoA. The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).
Gene
GenBank
name
GI#
Accession #
Organism
gctA
559392
CAA57199.1
Acidaminococcus fermentans
gctB
559393
CAA57200.1
Acidaminococcus fermentans
A CoA transferase that can utilize acetyl-CoA as the CoA donor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch. Biochem. Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ. Microbiol. 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli et al., Eur. J Biochem. 29:553-562 (1972)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol. 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol. 56:1576-1583 (1990); and Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem 71:58-68 (2007)).
GenBank
Gene name
GI#
Accession #
Organism
atoA
2492994
P76459.1
Escherichia coli
atoD
2492990
P76458.1
Escherichia coli
actA
62391407
YP_226809.1
Corynebacterium
glutamicum
cg0592
62389399
YP_224801.1
Corynebacterium
glutamicum
ctfA
15004866
NP_149326.1
Clostridium acetobutylicum
ctfB
15004867
NP_149327.1
Clostridium acetobutylicum
ctfA
31075384
AAP42564.1
Clostridium
saccharoperbutylacetonicum
ctfB
31075385
AAP42565.1
Clostridium
saccharoperbutylacetonicum
The de-acylation 3-oxopimeloyl-CoA to 3-oxopimelate (
GenBank
Gene name
GI#
Accession #
Organism
pcaI
24985644
AAN69545.1
Pseudomonas putida
pcaJ
26990657
NP_746082.1
Pseudomonas putida
pcaI
50084858
YP_046368.1
Acinetobacter
sp. ADP1
pcaJ
141776
AAC37147.1
Acinetobacter
sp. ADP1
pcaI
21224997
NP_630776.1
Streptomyces
coelicolor
pcaJ
21224996
NP_630775.1
Streptomyces
coelicolor
HPAG1_0676
108563101
YP_627417
Helicobacter pylori
HPAG1_0677
108563102
YP_627418
Helicobacter pylori
ScoA
16080950
NP_391778
Bacillus subtilis
ScoB
16080949
NP_391777
Bacillus subtilis
3.1.2.a CoA Hydrolase—The hydrolysis of 6-aminopimeloyl-CoA to 6-aminopimelate (
GenBank
Gene name
GI#
Accession #
Organism
acot12
18543355
NP_570103.1
Rattus
norvegicus
ACH1
6319456
NP_009538
Saccharomyces
cerevisiae
Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol. Chem. 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana et al., Biochem. Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolases include the gene products of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); and (Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol. 189:7112-7126 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
tesB
16128437
NP_414986
Escherichia coli
acot8
3191970
CAA15502
Homo sapiens
acot8
51036669
NP_570112
Rattus norvegicus
tesA
16128478
NP_415027
Escherichia coli
ybgC
16128711
NP_415264
Escherichia coli
paaI
16129357
NP_415914
Escherichia coli
ybdB
16128580
NP_415129
Escherichia coli
Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this reaction step but would require certain mutations to change their function.
Gene
GenBank
name
GI#
Accession #
Organism
gctA
559392
CAA57199
Acidaminococcus fermentans
gctB
559393
CAA57200
Acidaminococcus fermentans
4.1.1.a Carboxy-Lyase—The decarboxylation reactions of homolysine to HMDA (
Gene
GenBank
name
GI#
Accession #
Organism
cadA
145458
AAA23536.1
Escherichia coli
ldcC
1786384
AAC73297.1
Escherichia coli
ldc
13124043
O50657.1
Selenomonas
ruminantium
cadA
44886078
AB124819.1
Vibrio
parahaemolyticus
Several ornithine decarboxylase enzymes (EC 4.1.1.17) exhibit activity on lysine and other similar compounds. Such enzymes are found in Nicotiana glutinosa (Lee et al., Biochem. J. 360:657-665 (2001)), Lactobacillus sp. 30a (Guirard et al., J Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). The enzymes from Lactobacillus sp. 30a (Momany et al., J Mol. Biol. 252:643-655 (1995)) and V. vulnificus have been crystallized. The V. vulnificus enzyme efficiently catalyzes lysine decarboxylation and the residues involved in substrate specificity have been elucidated (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). A similar enzyme has been characterized in Trichomonas vaginalis but the gene encoding this enzyme is not known (Yarlett et al., Biochem. J 293 (Pt 2):487-493 (1993)).
GenBank
Gene name
GI#
Accession #
Organism
AF323910.1:1 . . . 1299
12007488
AAG45222.1
Nicotiana
glutinosa
odc1
1169251
P43099.2
Lactobacillus
sp. 30a
VV2_1235
27367615
NP_763142.1
Vibrio
vulnificus
Keto-acid decarboxylase enzymes are required to convert 2-oxo-7-aminoheptanoate to 6-aminohexanal (Step F of
Gene
GenBank
name
GI#
Accession #
Organism
pdc
118391
P06672.1
Zymomonas mobilus
pdc1
30923172
P06169
Saccharomyces cerevisiae
pdc
20385191
AM21208
Acetobacter pasteurians
pdc1
52788279
Q12629
Kluyveromyces lactis
Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry 42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).
Gene
GenBank
name
GI#
Accession #
Organism
mdlC
3915757
P20906.2
Pseudomonas
putida
mdlC
81539678
Q9HUR2.1
Pseudomonas
aeruginosa
dpgB
126202187
ABN80423.1
Pseudomonas
stutzeri
ilvB-1
70730840
YP_260581.1
Pseudomonas
fluorescens
A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci U S. A 102:10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (−130 kD) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 1) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.
Gene
GenBank
name
GI#
Accession #
Organism
kgd
160395583
O50463.4
Mycobacterium
tuberculosis
kgd
27375563
NP_767092.1
Bradyrhizobium
japonicum
kgd
13473636
NP_105204.1
Mesorhizobium loti
A fourth candidate enzyme for catalyzing this reaction is branched chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988); and Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.
Gene
GenBank
name
GI#
Accession #
Organism
kdcA
44921617
AAS49166.1
Lactococcus lactis
Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the E1 subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J. Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.
GenBank
Gene name
GI#
Accession #
Organism
BCKDHB
34101272
NP_898871.1
Homo sapiens
BCKDHA
11386135
NP_000700.1
Homo sapiens
BCKDHB
115502434
P21839
Bos taurus
BCKDHA
129030
P11178
Bos taurus
4.1.2.a The condensation of pyruvate with 4-aminobutanal (
Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme is encoded by hpal (Prieto et al., J Bacteriol. 178:111-120 (1996)).
Gene
GenBank
name
GI#
Accession #
Organism
hpcH
633197
CAA87759.1
Escherichia
coli C
hpaI
38112625
AAR11360.1
Escherichia
coli W
Another pyruvate-utilizing aldehyde lyase is 2-dehydro-3-deoxyglucarate aldolase (DDGA, EC 4.1.2.20), a type II aldolase that participates in the catabolic pathway for D-glucarate/galactarate utilization in E. coli. The natural donor of this enzyme is tartronate semialdehyde, but this enzyme has a broad substrate specificity and has been shown to reversibly condense a wide range of aldehydes with pyruvate (Fish et al., Methods Enzymol. 9:529-534 (1966)). The crystal structure of this enzyme has been determined and a catalytic mechanism proposed (Izard et al., EMBO J. 19:3849-3856 (2000)). Additional candidate DDGA enzymes are found in Leptospira interrogans (118) and Sulfolobus solfataricus (Buchanan et al., Biochem. J. 343 Pt 3:563-570 (1999)). The S. solfataricus enzyme is highly thermostable and was cloned and expressed in E. coli (Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)).
GenBank
Gene name
GI#
Accession #
Organism
garL
1176153
P23522.2
Escherichia coli
LA_1624
24195249
AAN48823.1
Leptospira interrogans
AJ224174.1:1..885
2879782
CAA11866.1
Sulfolobus solfataricus
4.2.1.a Hydro-Lyase—Two reactions in
Gene
GenBank
name
GI#
Accession #
Organism
ech
26990073
NP_745498.1
Pseudomonas putida
paaA
26990002
NP_745427.1
Pseudomonas putida
paaB
26990001
NP_745426.1
Pseudomonas putida
phaA
106636093
ABF82233.1
Pseudomonas fluorescens
phaB
106636094
ABF82234.1
Pseudomonas fluorescens
pimF
39650635
CAE29158
Rhodopseudomonas palustris
maoC
16129348
NP_415905.1
Escherichia coli
paaF
16129354
NP_415911.1
Escherichia coli
paaG
16129355
NP_415912.1
Escherichia coli
3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase, is an enoyl-CoA hydratase that dehydrates 3-hydroxyisobutyryl-CoA to form crotonyl-CoA. Crotonase enzymes are required for n-butanol formation in some organisms, particularly Clostridial species, and also comprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genes encoding crotonase enzymes can be found in C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311 (2008); and Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer et al., FEBS Lett. 21:351-354. 1972)), and Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)) though the sequence of the latter gene is not known.
Gene
name
GI#
GenBank Accession #
Organism
crt
15895969
NP_349318.1
Clostridium acetobutylicum
crt1
153953091
YP_001393856.1
Clostridium kluyveri
Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Nakahigashi et al., Nucleic Acids Res. 18:4937 (1990); Yang et al., J. Bacteriol. 173:7405-7406 (1991) and Yang et al., Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol. 47:793-805 (2003)).
Gene name
GI#
GenBank Accession #
Organism
fadA
49176430
YP_026272.1
Escherichia coli
fadB
16131692
NP_418288.1
Escherichia coli
fadI
16130275
NP_416844.1
Escherichia coli
fadJ
16130274
NP_416843.1
Escherichia coli
fadR
16129150
NP_415705.1
Escherichia coli
2-Oxo-7-aminohept-3-enoate is formed from the dehydration of 2-oxo-4-hydroxy-7-aminoheptanoate (
GenBank
Gene name
GI#
Accession #
Organism
hpcG
556840
CAA57202.1
Escherichia coli C
hpaH
757830
CAA86044.1
Escherichia coli W
hpaH
150958100
ABR80130.1
Klebsiella pneumoniae
Sari_01896
160865156
ABX21779.1
Salmonella enterica
An alternate enzyme candidate for catalyzing this reaction is fumarase, also known as fumarate hydratase (EC 4.2.1.2). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Guest et al., J Gen Microbiol. 131:2971-2984 (1985); Tseng et al., J Bacteriol 183:461-467 (2001) and Woods et al., Biochim Biophys Acta 954:14-26 (1988)). FumC has been shown to dehydrate alternate substrates including tartrate and threo-hydroxyaspartate (Teipel et al., J Biol. Chem. 243:5684-5694 (1968)). A wealth of structural information is available for FumC and researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver et al., D Biol Crystallogr. 61:1395-1401 (2005)). Additional fumarate hydratase enzymes are found in Escherichia coli (Estevez et al., Protein Sci 11:1552-1557 (2002); Hong, et al., Biotechnol. Bioprocess Eng. 9:252-255 (2005)) and Rose et al., Proc Natl Acad Sci US. A 101:3393-3397 (2004)), Corynebacterium glutamicum (Genda et al., Biotechnol Biochem. 70:1102-1109 (2006)), Campylobacter jejuni (Smith et al., Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)), and Rattus norvegicus (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).
Gene
GenBank
name
GI#
Accession #
Organism
fumA
81175318
P0AC33
Escherichia coli K12
fumB
33112655
P14407
Escherichia coli K12
fumC
120601
P05042.1
Escherichia coli K12
fumC
39931596
Q8NRN8.1
Corynebacterium glutamicum
fumC
9789756
O69294.1
Campylobacter jejuni
fumC
75427690
P84127
Thermus thermophilus
fumH
120605
P14408.1
Rattus norvegicus
MmcB
147677691
YP_001211906
Pelotomaculum
thermopropionicum
MmcC
147677692
YP_001211907
Pelotomaculum
thermopropionicum
Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch. Microbiol. 168:457-463 1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms.
GenBank
Gene name
GI#
Accession #
Organism
leuD
3122345
Q58673.1
Methanocaldococcus jannaschii
5.4.3.a Aminomutase—Several reactions in
Gene
name
GI#
GenBank Accession #
Organism
kamA
75423266
Q9XBQ8.1
Clostridium subterminale
kamA
81485301
Q8RHX4
Fusobacterium nucleatum
yodO
4033499
O34676.1
Bacillus subtilis
Other enzymes with 2,3-aminomutase activity include tyrosine 2,3-aminomutase (EC 5.4.3.6) and leucine 2,3-aminomutase (EC 5.4.3.7). Tyrosine 2,3-aminomutase participates in tyrosine biosynthesis, reversibly converting tyrosine to 3-amino-3-(4-hydroxyphenyl)-propionoate by shifting an amine from the 2- to the 3-position. In Streptomyces globisporus the enzyme has also been shown to react with tyrosine derivatives (Christenson et al., Biochemistry 42:12708-12718 (2003)); however, the sequence of this enzyme is not yet available. Leucine 2,3-aminomutase converts L-leucine to beta-leucine during leucine biosynthesis and degradation. A leucine 2,3-aminomutase-specific assay detected enzyme activity in many organisms (Poston et al., Methods Enzymol. 166:130-135 (1988)) but genes encoding the enzyme have not been identified to date.
6.2.1.a Acid-Thiol Ligase—The activation of carboxylic acids to acyl-CoA derivatives is catalyzed by CoA acid-thiol ligases or CoA synthetases in the EC class 6.2.1 (the terms ligase, synthetase, and synthase are used herein interchangeably and refer to the same enzyme class). Such enzymes couple the energetic cost of thioester bond formation to the hydrolysis of ATP into ADP or AMP. Several ADP-forming CoA ligases have been demonstrated to react in the reverse direction, removing the CoA moiety from acyl-CoA molecules and concomitantly forming ATP. Reversible CoA ligases are required to de-acylate 6-aminopimeloyl-CoA (
ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on cyclic compounds phenylacetate and indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., Arch. Microbiol. 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen et al., Arch. Microbiol. 182:277-287 (2004); and Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).
GenBank
Gene name
GI#
Accession #
Organism
AF1211
11498810
NP_070039.1
Archaeoglobus fulgidus
DSM 4304
AF1983
11499565
NP_070807.1
Archaeoglobus fulgidus
DSM 4304
scs
55377722
YP_135572.1
Haloarcula marismortui
PAE3250
18313937
NP_560604.1
Pyrobaculum aerophilum
str. IM2
sucC
16128703
NP_415256.1
Escherichia coli
sucD
1786949
AAC73823.1
Escherichia coli
Another candidate enzyme is the AMP-forming pimeloyl-CoA ligase (EC 6.2.1.14) which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J. 340 (Pt 3):793-801 (1999)). Other pimeloyl-CoA ligase candidates are found in Bacillus subtilis (Bower et al., J Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J. 287 (Pt 3):685-690 (1992)).
GenBank
Gene name
GI#
Accession #
Organism
pauA
15596214
NP_249708.1
Pseudomonas mendocina
bioW
50812281
NP_390902.2
Bacillus subtilis
bioW
115012
P22822.1
Lysinibacillus sphaericus
Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem J 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-et al., Maceiras, J 395:147-155 (2006); and Wang et al., Biophys. Res. Commun. 360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J Biol. Chem. 265:7084-7090 (1990)). Acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.
Gene
name
GI#
GenBank Accession #
Organism
phl
77019264
CAJ15517.1
Penicillium chrysogenum
phlB
152002983
ABS19624.1
Penicillium chrysogenum
paaF
22711873
AAC24333.2
Pseudomonas putida
AACS
21313520
NP_084486.1
Mus musculus
AACS
31982927
NP_076417.2
Homo sapiens
Levulinic acid (LA), also known as 4-oxopentanoic acid and 4-ketovaleric acid, is a precursor to nylon-like polymers, synthetic rubbers and plastics. It is also a precursor of other commodity chemicals such as methyltetrahydrofuran, valerolactone and ethyl levulinate. Other potential applications include use as a fuel extender and a biodegradable herbicide/pesticide. It is traditionally prepared by treating cellulosic biomass with strong acids such as hydrochloric and sulfuric acids. This process has the disadvantages of low LA yield and numerous byproducts. More recently, the Biofine Process was developed which converts cellulosic biomass into LA, formic acid and furfural at a yield of 70% the theoretical maximum (Hayes et al., “The biofine process-production of levulinic acid, furfural and formic acid from lignocellulosic feedstock” p. 139-164. In Biorefineries: Industrial Processes and Products. Wiley, Weinheim, Germany (2006)). Described herein is a process for selectively producing LA from sugar or syngas feedstocks using a microbial organism.
The maximum theoretical yield of LA from glucose is 1.45 moles of LA per mole glucose utilized (0.938 g/g), according to the following equation:
Glucose(C6H12O2)+1.27CO2→1.45LA(C5H8O3)+0.18H2O
LA is produced from the central metabolites succinyl-CoA and acetyl-CoA in three enzymatic steps. In the first step, acetyl-CoA and succinyl-CoA are condensed by a beta-ketothiolase to form 3-oxoadipyl-CoA (Step A of
The decarboxylation of 3-oxoadipate to LA can occur enzymatically or spontaneously. In E. coli, several 3-oxoacids produced during amino acid biosynthesis have been shown to undergo spontaneous decarboxylation (Boylan et al., Biochem. Biophys. Res Commun. 85:190-197 (1978)). An enzyme catalyzing the decarboxylation of 3-oxoadipate to LA has not been demonstrated to our knowledge. An exemplary enzyme candidate catalyzing a similar reaction is acetoacetate decarboxylase (EC 4.1.1.4). The acetoacetate decarboxylase from Clostridium acetobutylicum, encoded by adc, has a broad substrate specificity and has been shown to decarboxylate 3-oxopentanoate, 2-oxo-3-phenylpropionic acid and 2-methyl-3-oxobutyrate (Benner et al., J. Am. Chem. Soc. 103:993-994 (1981) and Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984)). An acetoacetate decarboylase has also been characterized in Clostridium beijerinckii (Ravagnani et al., Mol. Microbiol. 37:1172-1185 (2000)). The acetoacetate decarboxylase from Bacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylate 3-oxopentanoate (Matiasek et al., Curr. Microbiol. 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence of B. polymyxa is not yet available. Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).
GenBank
Gene name
GI#
Accession #
Organism
adc
15004868
NP_149328.1
Clostridium
acetobutylicum
adc
31075386
AAP42566.1
Clostridium
saccharoperbutyl-
acetonicum
cbei_3835
150018652
YP_001310906.1
Clostridium beijerinckii
This example describes gene disruption strategies for production of adipate 6-aminocaproic acid (6-ACA) and hexamethylenediamine (HMDA).
Described below in more detail are sets of enzymatic activities that can be reduced by appropriate gene disruptions or deletions in a production host engineered to contain the adipate, 6-aminocaproic acid (6-ACA) and hexamethylenediamine (HMDA) production pathways, for example, pathways using succinyl CoA and acetyl CoA as precursors.
OptKnock is a bilevel computational framework formulated with the overall objective of developing genetically stable overproducing microorganisms. Specifically, the framework examines the complete network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene disruptions or deletions, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, in the case of a gene deletion, there is negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are completely removed from the genome.
Growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical production will generally result in sub-maximal growth rates. The knockouts suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point should lie within its calculated solution boundary. Plots such as these allow visualization of how close strains are to their performance limits or, in other words, how much room is available for improvement. The OptKnock framework has been used to identify promising gene deletion strategies for biochemical overproduction and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.
Described below are sets of enzyme activities that should be absent, attenuated, or eliminated for creating host organisms that achieve growth-coupled adipate, 6-ACA or HMDA production upon the addition of the biosynthetic pathway that proceeds through succinyl-CoA and acetyl-CoA. To enumerate all potential strategies, an optimization technique, termed integer cuts, has been implemented which entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration.
The OptKnock algorithm was used to identify designs based on a stoichiometric model of Escherichia coli metabolism. Assumptions include (i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic or microaerobic conditions; and (iii) a minimum non-growth associated maintenance requirement of 4 mmol/gdw/hr. Table 12 provides a list of all the reaction stoichiometries and the associated genes known to be associated with the reactions identified for deletion in the strategies. Table 13 provides a list of the metabolite abbreviations, the corresponding names and locations of all the metabolites that participate in the reactions listed in Table 12. The growth-coupled productions designs for adipic acid, 6ACA and HMDA are provided in Tables 14-16. The product formation rates shown in Tables 14-16 are in mmol/gDCW·hr. The basis glucose uptake rate is 10 mmol/gDCW·hr and the biomass formation rate is shown in units of 1/hr. These tables list the reactions that are knocked out in a particular strategy, the anticipated product and biomass yields. Although the designs were identified using a metabolic model of E. coli metabolism, and the gene names listed are specific to E. coli, the method of choosing the metabolic engineering strategies and also the designs themselves are applicable to any HMDA, 6-ACA or adipate-producing organism. Thus the designs are essentially lists of enzymatic transformations whose activity is to be either eliminated, attenuated, or initially absent from a microorganism to provide the growth coupled production of adipate, 6ACA and HMDA.
The key criterion for prioritizing the final selection of designs was the growth-coupled yield of each of the products. To examine this, production cones were constructed for each strategy by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation, as described above. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs were given a lower priority.
The metabolic engineering strategies described below assume that the organism can produce adipate, 6-ACA or HMDA via the succinyl CoA and acetyl-CoA utilizing pathway. The construction of a recombinant host organism capable of producing these products via the pathway is described herein.
Strain Construction: In order to validate the computational predictions proposed in this report, the strains are constructed, evolved, and tested. Escherichia coli K-12 MG1655 housing the succinyl-CoA-acetyl-CoA pathway serves as the strain into which the deletions are introduced. The strains are constructed by incorporating in-frame deletions using homologous recombination via the λ Red recombinase system of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97(12):6640-6645 2000)). The approach involves replacing a chromosomal sequence, that is, the gene targeted for removal, with a selectable antibiotic resistance gene, which itself is later removed. The knockouts are integrated one by one into the recipient strain. No antibiotic resistance markers remain after each deletion, allowing accumulation of multiple mutations in each target strain. The deletion technology completely removes the gene targeted for removal so as to substantially reduce the possibility of the constructed mutants reverting back to the wild-type.
Shake Flask Characterization: As intermediate strains are being constructed, strain performance is quantified by performing shake flask fermentations. Anaerobic conditions are obtained by sealing the flasks with a rubber septum and then sparging the medium with nitrogen. For strains where growth is not observed under strict anaerobic conditions, microaerobic conditions can be applied by covering the flask with foil and poking a small hole for limited aeration. All experiments are performed using M9 minimal medium supplemented with glucose unless otherwise stated. Pre-cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. The products, ethanol, and organic acids are analyzed by GC-MS or HPLC using routine procedures. Triplicate cultures are grown for each strain.
Batch Fermenter Testing: The performance of select strains is tested in anaerobic, pH-controlled batch fermentations. This allows reliable quantification of the growth, glucose uptake, and formation rates of all products, as well as ensure that the accumulation of acidic fermentation products will not limit cell growth. In addition, it allows accurate determination of volumetric productivity and yield of product formation, two of the most important parameters in benchmarking strain performance. Fermentations are carried out in 1-L bioreactors with 600 mL working volume, equipped with temperature and pH control. The reactor is continuously sparged with N2 at approximately 0.5 L/min to ensure that dissolved oxygen (DO) levels remain below detection levels. The culture medium is the same as described above, except that the glucose concentration is increased in accordance with the higher cell density achievable in a fermentation vessel.
Chemostat Testing: Chemostat experiments are conducted to obtain a direct measure of how the switch in fermentation mode from batch to continuous affects product yield and volumetric productivity. The bioreactors described above using batch mode are operated in chemostat mode through continuous supply of medium and removal of spent culture. The inlet flow rate is set to maintain a constant dilution rate of 80% of the maximum growth rate observed for each strain in batch, and the outlet flow is controlled to maintain level. Glucose is the limiting nutrient in the medium, and set to achieve the desired optical density in the vessel.
Adaptive Evolution: The knockout strains are initially expected to exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To allow this adjustment, the strains is adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat. Genet. 36(10):1056-1058 (2004). The OptKnock-generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli (Fong and Palsson, Nat. Genet. 36(10):1056-1058 (2004); Fong et al., J. Bacteriol. 185(21):6400-6408 (2003); Ibarra et al., Nature 420(6912):186-189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions are run for a period of 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained. The growth-coupled biochemical production concept behind the OptKnock approach results in the generation of genetically stable overproducers.
Although described as deletion sets, it is understood, as disclosed herein, that gene sets can be deleted or disrupted so that encoded gene product activities are reduced or eliminated. Thus, the gene deletion sets of Tables 14-16 can be used to delete or disrupt a gene set in a host organism in which an increased production of 6-ACA, adipate and/or HMDA is desired. It is understood that any of the disclosed gene deletion sets can be used to generate knockout strains with disrupted or deleted genes that confer increased production of 6-ACA, adipate and/or HMDA.
TABLE 12
A list of all the reaction stoichiometries and the associated genes known to be
associated with the reactions identified for deletion in the strategies listed in Tables 1 and 2.
Reaction
Abbreviation
Reaction Name
Reaction Stoichiometry
Associated genes
ACKr
Acetate kinase
[c]: ac + atp <==> actp + adp
(b3115 or b2296 or b1849)
ADHEr
Alcohol dehydrogenase
[c]: accoa + (2) h + (2) nadh
(b0356 or b1478 or b1241)
<==> coa + etoh + (2) nad
AKGD
Alpha-ketoglutarate
[c]: akg + coa + nad --> co2 +
(b0116 and b0726 and b0727)
dehydrogenase
nadh + succoa
ALAR
Alanine racemase
[c]: ala-L <==> ala-D
b4053
ASNS2
Asparagine synthetase
[c]: asp-L + atp + nh4 --> amp +
b3744
asn-L + h + ppi
ASPT
L-aspartase
[c]: asp-L --> fum + nh4
b4139
ATPS4r
ATP synthase
adp[c] + (4) h[p] + pi[c] <==>
(((b3736 and b3737 and b3738)
atp[c] + (3) h[c] + h2o[c]
and (b3731 and b3732 and b3733
and b3734 and b3735)) or ((b3736
and b3737 and b3738) and (b3731
and b3732 and b3733 and b3734 and
b3735) and b3739))
CBMK2
Carbamate kinase
[c]: atp + co2 + nh4 --> adp +
(b0521 or b0323 or b2874)
cbp + (2) h
DAAD
D-amino acid
[c]: ala-D + fad + h2o --> fadh2 +
b1189
dehydrogenase
nh4 + pyr
EDA
2-dehydro-3-deoxy-
[c]: 2ddg6p --> g3p + pyr
b1850
phosphogluconate
aldolase
ENO
Enolase
[c]: 2pg <==> h2o + pep
b2779
FBA
Fructose-bis-phosphate
[c]: fdp <==> dhap + g3p
(b2097 or b2925 or b1773)
aldolase
FRD
Fumarate reductase
[c]: fum + mql8 --> mqn8 +
(b4151 and b4152 and b4153 and
succ
b4154)
FUM
Fumarase
[c]: fum + h2o <==> mal-L
(b1612 or b4122 or b1611)
G5SD
Glutamate-5-
[c]: glu5p + h + nadph -->
b0243
semialdehyde
glu5sa + nadp + pi
dehyrogenase
G6PDHy
Glucose-6-phosphate
[c]: g6p + nadp <==> 6pgl + h +
b1852
dehydrogenase
nadph
GLCpts
D-glucose transport via
glc-D[e] + pep[c] --> g6p[c] +
((b2417 and b1101 and b2415 and
PTS mechanism
pyr[c]
b2416) or (b1817 and b1818 and
b1819 and b2415 and b2416) or
(b2417 and b1621 and b2415 and
b2416))
GLU5K
Gluatmate-5-kinase
[c]: atp + glu-L --> adp + glu5p
b0242
GLUDy
Glutamate dehydrogenase
[c]: glu-L + h2o + nadp <==>
b1761
akg + h + nadph + nh4
HEX1
Hexokinase
[c]: atp + glc-D --> adp + g6p + h
b2388
LDH_D
Lactate dehydrogenase
[c]: lac-D + nad <==> h + nadh +
b1380 or b2133
pyr
MDH
Malate dehydrogenase
[c]: mal-L + nad <==> h + nadh +
b3236
oaa
ME1x
Malic enzyme (NAD)
[c]: mal-L + nad --> co2 + nadh +
b1479
pyr
ME2
Malic enzyme (NADP)
[c]: mal-L + nadp --> co2 +
b2463
nadph + pyr
NADH12
NADH dehydrogenase
[c]: h + nadh + ubq8 --> nad +
b1109
(ubiquinone-8)
ubq8h2
NADH6
NADH dehydrogenase
(4.5) h[c] + nadh[c] + ubq8[c] -->
(b2276 and b2277 and b2278 and
(ubiquinone-8 and 3.5
(3.5) h[e] + nad[c] +
b2279 and b2280 and b2281 and
protons)
ubq8h2[c]
b2282 and b2283 and b2284 and
b2285 and b2286 and b2287 and
b2288)
PDH
Pyruvate dehydrogenase
[c]: coa + nad + pyr --> accoa +
((b0114 and b0115 and b0116) or (b0116
co2 + nadh
and b0726 and b0727) or (b0116 and
b2903 and b2904 and b2905))
PFK
Phosphofructokinase
[c]: atp + f6p --> adp + fdp + h
(b3916 or b1723)
PFLi
Pyruvate formate lyase
[c]: coa + pyr --> accoa + for
(((b0902 and b0903) and b2579) or
(b0902 and b0903) or (b0902 and
b3114) or (b3951 and b3952))
PGDH
Phosphogluconate
[c]: 6pgc + nadp --> co2 +
b2029
dehyrogenase
nadph + ru5p-D
PGDHY
Phosphogluconate
[c]: 6pgc --> 2ddg6p + h2o
b1851
dehydratase
PGI
Glucose-6-phosphate
[c]: g6p <==> f6p
b4025
isomerase
PGL
6-
[c]: 6pgl + h2o --> 6pgc + h
b0767
Phosphogluconolactonase
PGM
Phosphoglycerate mutase
[c]: 3pg <==> 2pg
b3612
PPC
Phosphoenolpyruvate
[c]: co2 + h2o + pep --> h + oaa +
b3956
carboxylase
pi
PPCK
Phosphoenolpyruvate
[c]: atp + oaa --> adp + co2 +
b3403
carboxykinase
pep
PRO1z
Proline oxidase
[c]: fad + pro-L --> 1pyr5c +
b1014
fadh2 + h
PTAr
Phosphotransacetylase
[c]: accoa + pi <==> actp + coa
b2297
PYK
Pyruvate kinase
[c]: adp + h + pep --> atp + pyr
(b1854 or b1676)
RPE
Ribulose-5-phosphate-5-
[c]: ru5p-D <==> xu5p-D
(b4301 or b3386)
epimerase
SUCD4
Succinate dehydrogenase
[c]: fadh2 + ubq8 <==> fad +
(b0721 and b0722 and b0723 and
ubq8h2
b0724
SUCOAS
Succinyl-CoA synthetase
[c]: atp + coa + succ <==> adp +
(b0728 and b0729)
pi + succoa
TAL
Transaldoalse
[c]: g3p + s7p <==> e4p + f6p
(b2464 or b0008)
THD2
NADP transhydrogenase
(2) h[e] + nadh[c] + nadp[c] -->
(b1602 and b1603)
(2) h[c] + nad[c] + nadph[c]
THD5
NAD transhydrogenase
[c]: nad + nadph --> nadh +
(b3962 or (b1602 and b1603))
nadp
TKT1
Transketolase
[c]: r5p + xu5p-D <==> g3p +
(b2935 or b2465)
s7p
TKT2
Transketolase
[c]: e4p + xu5p-D <==> f6p +
(b2935 or b2465)
g3p
TPI
Triosephosphate
[c]: dhap <==> g3p
b3919
isomerase
VALTA
Valine transaminase
[c]: akg + val-L <==> 3mob +
b3770
glu-L
TABLE 13
List of the metabolite abbreviations, the corresponding names and
locations of all the metabolites that participate in the reactions
listed in Table 12.
Metabolite
Abbreviation
Compartment
Metabolite Name
13dpg
Cytosol
3-Phospho-D-glyceroyl phosphate
1pyr5c
Cytosol
1-Pyrroline-5-carboxylate
2ddg6p
Cytosol
2-Dehydro-3-deoxy-D-gluconate
6-phosphate
2pg
Cytosol
D-Glycerate 2-phosphate
3mob
Cytosol
3-Methyl-2-oxobutanoate
3pg
Cytosol
3-Phospho-D-glycerate
6pgc
Cytosol
6-Phospho-D-gluconate
6pgl
Cytosol
6-phospho-D-glucono-1,5-lactone
ac
Cytosol
Acetate
accoa
Cytosol
Acetyl-CoA
actp
Cytosol
Acetyl phosphate
adp
Cytosol
Adenosine diphosphate
akg
Cytosol
2-Oxoglutarate
ala-D
Cytosol
D-alanine
ala-L
Cytosol
L-alanine
amp
Cytosol
Adenosine monophosphate
asn-L
Cytosol
L-asparagine
asp-L
Cytosol
L-aspartate
atp
Cytosol
Adenosine triphosphate
cbp
Cytosol
Carbamoyl phosphate
co2
Cytosol
Carbon dioxide
coa
Cytosol
Coenzyme A
dha
Cytosol
Dihydroxyacetone
dhap
Cytosol
Dihydroxyacetone phosphate
e4p
Cytosol
D-Erythrose 4-phosphate
etoh
Cytosol
Ethanol
f6p
Cytosol
D-Fructose 6-phosphate
fad
Cytosol
Flavin adenine dinucleotide
fadh2
Cytosol
Flavin adenine dinucleotide-reduced
fdp
Cytosol
D-Fructose 1,6-bisphosphate
for
Cytosol
Formate
fum
Cytosol
Fumarate
g3p
Cytosol
Glyceraldehyde 3-phosphate
g6p
Cytosol
D-Glucose 6-phosphate
glc-D[e]
Extra-organism
D-Glucose
glu5p
Cytosol
L-glutamate 5-phosphate
glu5sa
Cytosol
L-glutamate 5-semialdehyde
glu-L
Cytosol
L-Glutamate
h
Cytosol
H+
h[e]
Extra-organism
H+
h2o
Cytosol
Water
lac-D
Cytosol
D-Lactate
mal-L
Cytosol
L-Malate
mql-8
Cytosol
Menaquinol-8
mqn-8
Cytosol
Menaquinone-8
nad
Cytosol
Nicotinamide adenine dinucleotide
nadh
Cytosol
Nicotinamide adenine dinucleotide -
reduced
nadp
Cytosol
Nicotinamide adenine dinucleotide
phosphate
nadph
Cytosol
Nicotinamide adenine dinucleotide
phosphate - reduced
nh4
Cytosol
Ammonium
o2
Cytosol
Oxygen
oaa
Cytosol
Oxaloacetate
pep
Cytosol
Phosphoenolpyruvate
pi
Cytosol
Phosphate
ppi
Cytosol
Diphosphate
pyr
Cytosol
Pyruvate
r5p
Cytosol
alpha-D-Ribose 5-phosphate
ru5p-D
Cytosol
D-Ribulose 5-phosphate
s7p
Cytosol
Sedoheptulose 7-phosphate
succ
Cytosol
Succinate
succoa
Cytosol
Succinyl-CoA
ubq8
Cytosol
Ubiquinone-8
ubq8h2
Cytosol
Ubiquinol-8
val-L
Cytosol
L-valine
xu5p-D
Cytosol
D-Xylulose 5-phosphate
TABLE 14
Knockout strain designs for increased production of 6-ACA,
showing yields of 6-ACA and biomass.
New
Design
Num
6ACA.
ID
KO
Yield
Biomass
Rxnlist
1
1
1.72604
0.38773
ATPS4r
2
1
0.83466
0.26712
PGI
3
2
5.04234
0.22255
HEX1, PGI
4
2
4.11897
0.24338
EDA and/or PGDHY, PGI
5
2
3.26272
0.35236
ADHEr, NADH6
6
2
2.8403
0.28275
ADHEr, PPCK
7
2
2.05537
0.33531
GLCpts, NADH6
8
2
2.0373
0.38161
ATPS4r, RPE
9
2
1.89007
0.3845
ATPS4r, TAL
10
2
1.49147
0.26024
PFLi, PGI
11
2
1.11406
0.32382
NADH6, PFLi
12
2
0.95551
0.27773
PFLi, PPCK
13
2
0.89333
0.37885
ADHEr, FUM
14
2
0.41621
0.38953
ADHEr, HEX1
15
2
0.31773
0.36065
ASPT, FUM
16
3
6.93034
0.18126
EDA and/or PGDHY, NADH6, PGI
17
3
6.91845
0.19552
HEX1, PFLi, PGI
18
3
6.14899
0.1758
EDA and/or PGDHY, PFLi, PGI
19
3
5.81422
0.16481
ATPS4r, EDA and/or PGDHY, PGI
20
3
5.71646
0.21908
ADHEr, HEX1, PGI
21
3
4.83364
0.29669
ADHEr, NADH6, PFLi
22
3
4.23803
0.24209
ACKr and/or PTAr, EDA and/or PGDHY, PGI
23
3
4.02855
0.29483
ADHEr, MDH, THD2 and/or GLUDy
24
3
3.77533
0.25553
ADHEr, ASPT, MDH
25
3
3.65248
0.19372
ADHEr, NADH6, PGI
26
3
3.47283
0.25194
ADHEr, NADH6, PPCK
27
3
3.39319
0.29001
ADHEr, MDH, NADH6
28
3
3.35305
0.34906
ADHEr, NADH6, RPE
29
3
3.23462
0.28717
ACKr and/or PTAr, ATPS4r, SUCOAS
30
3
3.0877
0.16431
ADHEr, PGI, PPCK
31
3
2.90019
0.24841
ADHEr, GLCpts, PPCK
32
3
2.89855
0.2815
ADHEr, PPCK, RPE
33
3
2.88617
0.25645
ADHEr, FUM, THD2 and/or GLUDy
34
3
2.72186
0.35068
ADHEr, FUM, HEX1
35
3
2.60615
0.3202
ATPS4r, HEX1, PFLi
36
3
2.54001
0.22798
PFLi, PGDH, PGI
37
3
2.5259
0.22921
PFLi, PGI, TAL
38
3
2.5129
0.23034
PFLi, PGI, RPE
39
3
2.50442
0.16853
ATPS4r, PFLi, PGI
40
3
2.38919
0.18418
GLCpts, NADH6, PGI
41
3
2.30741
0.33343
ATPS4r, GLCpts, NADH6
42
3
2.16995
0.33092
GLCpts, NADH6, RPE
43
3
2.11568
0.333
GLCpts, NADH6, TAL
44
3
2.10576
0.25488
ATPS4r, PPCK, PYK
45
3
1.5933
0.25891
FUM, PFLi, PGI
46
3
1.25154
0.3194
NADH6, PFLi, RPE
47
3
1.23093
0.32359
ACKr and/or PTAr, NADH6, PFLi
48
3
1.18643
0.32149
NADH6, PFLi, TAL
49
3
1.08143
0.27599
PFLi, PPCK, RPE
50
3
1.0527
0.27673
ACKr and/or PTAr, PFLi, PPCK
51
3
1.03356
0.34314
FUM, HEX1, PFLi
52
3
1.02156
0.27682
PFLi, PPCK, TAL
53
3
0.8538
0.24817
MDH, PFLi, THD2 and/or GLUDy
54
3
0.5753
0.38695
ADHEr, HEX1, RPE
55
3
0.49968
0.38818
ADHEr, HEX1, TAL
56
3
0.31402
0.35643
FDH2, FUM, NADH6
57
3
0.23944
0.28074
FUM, PFLi, THD2 and/or GLUDy
58
3
0.23418
0.26581
ASPT, MDH, PFLi
59
3
0.20901
0.23724
ASPT, MDH, PYK
60
4
7.08404
0.12641
EDA and/or PGDHY, NADH6, PFLi, PGI
61
4
7.0245
0.10838
EDA and/or PGDHY, PFLi, PGI, PPCK
62
4
6.92332
0.16482
ADHEr, HEX1, NADH6, PGI
63
4
6.89839
0.18171
ACKr and/or PTAr, ADHEr, NADH6, PGI
64
4
6.58534
0.16513
EDA and/or PGDHY, GLCpts, PFLi, PGI
65
4
6.36357
0.11937
EDA and/or PGDHY, PFLi, PGI, THD2 and/or GLUDy
66
4
6.22082
0.11375
ATPS4r, EDA and/or PGDHY, PFLi, PGI
67
4
5.8701
0.13379
ADHEr, EDA and/or PGDHY, PGI, PPCK
68
4
5.85757
0.23874
ADHEr, MDH, NADH6, THD2 and/or GLUDy
69
4
5.85411
0.19685
ADHEr, HEX1, PGI, PPS
70
4
5.67774
0.13549
EDA and/or PGDHY, PGI, PPCK, PYK
71
4
5.46506
0.26673
EDA and/or PGDHY, MDH, PFLi, THD2 and/or GLUDy
72
4
5.40131
0.26362
ADHEr, MDH, PFLi, THD2 and/or GLUDy
73
4
5.08219
0.13778
ATPS4r, NADH6, PFLi, PGI
74
4
4.88764
0.27849
ADHEr, NADH12, NADH6, PFLi
75
4
4.88489
0.27942
ADHEr, FUM, NADH6, PFLi
76
4
4.81778
0.26037
ADHEr, ATPS4r, MDH, NADH6
77
4
4.73419
0.21859
ADHEr, NADH6, PPCK, THD2 and/or GLUDy
78
4
4.63783
0.29595
ADHEr, ATPS4r, FDH2, NADH6
79
4
4.51525
0.21818
ADHEr, FUM, PPCK, THD2 and/or GLUDy
80
4
4.51525
0.21818
ADHEr, MDH, PPCK, THD2 and/or GLUDy
81
4
4.30547
0.19131
ADHEr, ATPS4r, NADH6, PGI
82
4
4.2733
0.23078
ADHEr, ATPS4r, NADH6, PPCK
83
4
4.16417
0.18771
ATPS4r, NADH6, PGI, RPE
84
4
4.16053
0.18697
ATPS4r, NADH6, PGI, TAL
85
4
4.15658
0.18617
ATPS4r, NADH6, PGDH, PGI
86
4
4.12032
0.32701
ADHEr, ATPS4r, FUM, NADH6
87
4
3.9718
0.23354
ADHEr, ATPS4r, MDH, PPCK
88
4
3.9718
0.23354
ADHEr, ATPS4r, FUM, PPCK
89
4
3.8747
0.21758
ADHEr, ASPT, GLCpts, MDH
90
4
3.84814
0.25342
ADHEr, ASPT, MDH, RPE
91
4
3.83986
0.2047
ADHEr, ASPT, MDH, PYK
92
4
3.75472
0.32987
ADHEr, ATPS4r, HEX1, NADH6
93
4
3.54965
0.29114
ADHEr, ATPS4r, MDH, PGDH
94
4
3.54605
0.21695
ADHEr, GLCpts, NADH6, PPCK
95
4
3.54385
0.218
ADHEr, NADH6, PPCK, PYK
96
4
3.53615
0.25027
ADHEr, NADH6, PPCK, RPE
97
4
3.5018
0.32809
ADHEr, ATPS4r, FUM, HEX1
98
4
3.46904
0.25375
ADHEr, GLCpts, MDH, NADH6
99
4
3.46528
0.28851
ADHEr, MDH, NADH6, RPE
100
4
3.44916
0.13425
ADHEr, PFLi, PGI, PPCK
101
4
3.44555
0.26498
ADHEr, HEX1, NADH6, THD2 and/or GLUDy
102
4
3.43776
0.29402
ADHEr, ATPS4r, MDH, TAL
103
4
3.36596
0.19156
FUM, NADH6, PGI, THD5
104
4
3.36596
0.19156
MDH, NADH6, PGI, THD5
105
4
3.33377
0.2967
ADHEr, ATPS4r, MDH, RPE
106
4
3.33152
0.33978
ADHEr, FUM, NADH6, TAL
107
4
3.32935
0.34088
ADHEr, HEX1, NADH6, TAL
108
4
3.32788
0.32122
ADHEr, FUM, HEX1, NADH6
109
4
3.31278
0.3493
ADHEr, GLU5K, NADH6, TAL
110
4
3.31278
0.3493
ADHEr, G5SD, NADH6, TAL
111
4
3.17484
0.10602
ADHEr, PGI, PPCK, THD2 and/or GLUDy
112
4
3.16614
0.11184
ADHEr, MDH, PGI, THD2 and/or GLUDy
113
4
3.16614
0.11184
ADHEr, FUM, PGI, THD2 and/or GLUDy
114
4
3.11125
0.24826
ADHEr, ATPS4r, PPCK, THD2 and/or GLUDy
115
4
2.96628
0.21051
ADHEr, LDH_D, MDH, THD2 and/or GLUDy
116
4
2.95529
0.24477
ADHEr, PPCK, RPE, THD2 and/or GLUDy
117
4
2.95136
0.24731
ADHEr, GLCpts, PPCK, RPE
118
4
2.94249
0.25305
ADHEr, FUM, RPE, THD2 and/or GLUDy
119
4
2.93765
0.22693
ADHEr, MDH, PPCK, PYK
120
4
2.93765
0.22693
ADHEr, FUM, PPCK, PYK
121
4
2.9332
0.24406
ADHEr, PPCK, TAL, THD2 and/or GLUDy
122
4
2.90913
0.24328
ADHEr, PGDH, PPCK, THD2 and/or GLUDy
123
4
2.90913
0.24328
ADHEr, PGL and/or G6PDHy, PPCK, THD2 and/or GLUDy
124
4
2.90081
0.26381
ADHEr, FUM, PPCK, TAL
125
4
2.90081
0.26381
ADHEr, MDH, PPCK, TAL
126
4
2.86334
0.28161
ATPS4r, GLCpts, NADH6, PFLi
127
4
2.79498
0.34856
ADHEr, FUM, HEX1, RPE
128
4
2.7362
0.31504
ATPS4r, HEX1, PFLi, RPE
129
4
2.70922
0.18179
ATPS4r, GLCpts, NADH6, PGI
130
4
2.67469
0.31748
ATPS4r, HEX1, PFLi, TAL
131
4
2.61943
0.36027
ADHEr, HEX1, PFLi, PPS
132
4
2.60629
0.2178
PFLi, PGDH, PGI, TAL
133
4
2.60314
0.31113
ADHEr, HEX1, MDH, PFLi
134
4
2.56456
0.18551
ASPT, NADH6, PGI, THD5
135
4
2.55949
0.17944
ATPS4r, PFLi, PPCK, PYK
136
4
2.45433
0.3546
ADHEr, ATPS4r, HEX1, THD2 and/or GLUDy
137
4
2.45166
0.03625
GLCpts, NADH6, PPCK, PYK
138
4
2.44258
0.32891
ATPS4r, GLCpts, NADH6, RPE
139
4
2.43939
0.25153
ATPS4r, PPCK, PYK, RPE
140
4
2.37857
0.33105
ATPS4r, GLCpts, NADH6, TAL
141
4
2.30961
0.22691
ATPS4r, NADH6, PPCK, PYK
142
4
2.28135
0.25311
ATPS4r, PPCK, PYK, TAL
143
4
2.18633
0.3609
ADHEr, HEX1, PPS, THD2 and/or GLUDy
144
4
2.11347
0.31979
ADHEr, ATPS4r, HEX1, MDH
145
4
2.05497
0.19746
ASPT, ATPS4r, PGI, THD5
146
4
1.83108
0.25754
MDH, PFLi, PGI, THD2 and/or GLUDy
147
4
1.65594
0.25767
ACKr and/or PTAr, FUM, PFLi, PGI
148
4
1.36673
0.31918
ACKr and/or PTAr, NADH6, PFLi, RPE
149
4
1.30242
0.32127
ACKr and/or PTAr, NADH6, PFLi, TAL
150
4
1.28243
0.32216
ACKr and/or PTAr, GLU5K, NADH6, PFLi
151
4
1.28243
0.32216
ACKr and/or PTAr, G5SD, NADH6, PFLi
152
4
1.26654
0.34191
ACKr and/or PTAr, FUM, HEX1, PFLi
153
4
1.18926
0.34046
FUM, HEX1, PFLi, RPE
154
4
1.17756
0.275
ACKr and/or PTAr, PFLi, PPCK, RPE
155
4
1.1182
0.27583
ACKr and/or PTAr, PFLi, PPCK, TAL
156
4
1.1153
0.34173
FUM, HEX1, PFLi, TAL
157
4
0.95889
0.24471
MDH, PFLi, RPE, THD2 and/or GLUDy
158
4
0.9475
0.3633
ADHEr, FUM, PGL and/or G6PDHy, TAL
159
4
0.9475
0.3633
ADHEr, FUM, PGDH, TAL
160
4
0.90913
0.24635
MDH, PFLi, TAL, THD2 and/or GLUDy
161
4
0.36773
0.36076
HEX1, PFLi, PPS, THD2 and/or GLUDy
162
4
0.3476
0.2132
ADHEr, MDH, RPE, THD2 and/or GLUDy
163
4
0.32915
0.27571
FUM, PFLi, RPE, THD2 and/or GLUDy
164
5
6.98742
0.06748
ADHEr, EDA and/or PGDHY, NADH6, PGI, THD2 and/or
GLUDy
165
5
6.97749
0.0848
ACKr and/or PTAr, ATPS4r, HEX1, PFLi, PGI
166
5
6.70882
0.01277
EDA and/or PGDHY, FUM, PFLi, PGI, THD2 and/or GLUDy
167
5
6.70882
0.01277
EDA and/or PGDHY, MDH, PFLi, PGI, THD2 and/or GLUDy
168
5
6.69134
0.13239
ADHEr, ASPT, ATPS4r, GLCpts, MDH
169
5
6.55123
0.09841
ADHEr, ASPT, MDH, PGL and/or G6PDHy, PYK
170
5
6.55123
0.09841
ADHEr, ASPT, EDA and/or PGDHY, MDH, PYK
171
5
6.3052
0.17034
ADHEr, ASPT, ATPS4r, MDH, PGL and/or G6PDHy
172
5
6.20871
0.17434
EDA and/or PGDHY, MDH, PFLi, PGI, THD5
173
5
6.12639
0.08557
ADHEr, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or
GLUDy
174
5
6.03386
0.19348
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH
175
5
5.98628
0.0693
ATPS4r, EDA and/or PGDHY, PGI, PPCK, PYK
176
5
5.98165
0.07186
ADHEr, ATPS4r, EDA and/or PGDHY, PGI, PPCK
177
5
5.93527
0.09761
ADHEr, EDA and/or PGDHY, GLCpts, PGI, PPCK
178
5
5.87444
0.0434
ADHEr, EDA and/or PGDHY, MDH, PGI, THD2 and/or GLUDy
179
5
5.87444
0.0434
ADHEr, EDA and/or PGDHY, FUM, PGI, THD2 and/or GLUDy
180
5
5.82336
0.16439
ACKr and/or PTAr, ADHEr, ASPT, MDH, PYK
181
5
5.75418
0.21654
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6
182
5
5.65488
0.26204
ACKr and/or PTAr, ADHEr, MDH, PFLi, THD2 and/or GLUDy
183
5
5.59555
0.20952
ADHEr, ATPS4r, GLCpts, MDH, NADH6
184
5
5.30614
0.21123
ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy
185
5
5.21058
0.214
ADHEr, ASPT, ATPS4r, LDH_D, MDH
186
5
5.20451
0.17174
ADHEr, LDH_D, NADH6, PFLi, PPCK
187
5
5.1776
0.13724
ADHEr, NADH12, NADH6, PFLi, PGI
188
5
5.11744
0.13758
ADHEr, MDH, NADH6, PFLi, PGI
189
5
5.11744
0.13758
ADHEr, FUM, NADH6, PFLi, PGI
190
5
5.11714
0.20117
ADHEr, LDH_D, MDH, PFLi, PPCK
191
5
5.11714
0.20117
ADHEr, FUM, LDH_D, PFLi, PPCK
192
5
5.10437
0.12227
ADHEr, FUM, PFLi, PGI, PPCK
193
5
5.10437
0.12227
ADHEr, MDH, PFLi, PGI, PPCK
194
5
5.09877
0.17589
ADHEr, ATPS4r, GLCpts, NADH6, PPCK
195
5
5.0606
0.22022
ADHEr, LDH_D, MDH, NADH6, PFLi
196
5
5.02693
0.21085
ADHEr, ATPS4r, NADH6, PGDH, PPCK
197
5
5.02693
0.21085
ADHEr, ATPS4r, NADH6, PGL and/or G6PDHy, PPCK
198
5
5.00057
0.21154
ADHEr, ATPS4r, NADH6, PPCK, TAL
199
5
4.97638
0.21218
ADHEr, ATPS4r, NADH6, PPCK, RPE
200
5
4.88781
0.27844
ADHEr, HEX1, LDH_D, NADH6, PFLi
201
5
4.88512
0.29281
ADHEr, GLU5K, NADH6, PFLi, RPE
202
5
4.88512
0.29281
ADHEr, G5SD, NADH6, PFLi, RPE
203
5
4.885
0.29286
ADHEr, ASNS2, NADH6, PFLi, RPE
204
5
4.85776
0.29446
ADHEr, ATPS4r, FDH2, NADH6, RPE
205
5
4.83644
0.29517
ADHEr, ATPS4r, FDH2, NADH6, TAL
206
5
4.7175
0.34071
ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy
207
5
4.66386
0.18074
ADHEr, ATPS4r, GLCpts, MDH, PPCK
208
5
4.66386
0.18074
ADHEr, ATPS4r, FUM, GLCpts, PPCK
209
5
4.63095
0.20189
ADHEr, ATPS4r, GLCpts, PFLi, PPCK
210
5
4.49707
0.25782
ADHEr, ATPS4r, EDA and/or PGDHY, MDH, THD2 and/or
GLUDy
211
5
4.4958
0.1778
ADHEr, ASPT, MDH, NADH6, PYK
212
5
4.41977
0.25646
ADHEr, ATPS4r, GLCpts, MDH, THD2 and/or GLUDy
213
5
4.29965
0.18828
ADHEr, ASPT, ATPS4r, MDH, PYK
214
5
4.29026
0.24034
ACKr and/or PTAr, EDA and/or PGDHY, GLU5K, GLYCL, PGI
215
5
4.29026
0.24034
ACKr and/or PTAr, EDA and/or PGDHY, G5SD, GLYCL, PGI
216
5
4.28287
0.11665
ACKr and/or PTAr, ATPS4r, PPCK, PYK, SUCOAS
217
5
4.24299
0.26303
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, NADH6
218
5
4.18509
0.32432
ADHEr, ATPS4r, HEX1, NADH6, PGL and/or G6PDHy
219
5
4.18509
0.32432
ADHEr, ATPS4r, HEX1, NADH6, PGDH
220
5
4.15013
0.1124
ADHEr, ASPT, MDH, PYK, THD2 and/or GLUDy
221
5
4.14582
0.32483
ADHEr, ATPS4r, HEX1, NADH6, TAL
222
5
4.1099
0.32529
ADHEr, ATPS4r, HEX1, NADH6, RPE
223
5
4.04473
0.12883
ADHEr, PFLi, PGDH, PGI, PPCK
224
5
4.03844
0.12934
ADHEr, PFLi, PGI, PPCK, TAL
225
5
4.03266
0.12981
ADHEr, PFLi, PGI, PPCK, RPE
226
5
4.02531
0.1111
ADHEr, GLCpts, PFLi, PGI, PPCK
227
5
3.95205
0.10694
ACKr and/or PTAr, ATPS4r, PGDH, PGI, SUCOAS
228
5
3.94203
0.1079
ACKr and/or PTAr, ATPS4r, PGI, SUCOAS, TAL
229
5
3.9367
0.21579
ADHEr, ASPT, GLCpts, MDH, RPE
230
5
3.93273
0.1088
ACKr and/or PTAr, ATPS4r, PGI, RPE, SUCOAS
231
5
3.88257
0.21457
ADHEr, ASPT, LDH_D, MDH, PPCK
232
5
3.84571
0.22865
ADHEr, ASPT, LDH_D, MDH, NADH6
233
5
3.76049
0.30843
ADHEr, ATPS4r, FUM, HEX1, PFLi
234
5
3.74674
0.21738
ADHEr, ATPS4r, LDH_D, PFLi, PPCK
235
5
3.71345
0.27754
ADHEr, ATPS4r, MDH, PGDH, TAL
236
5
3.69299
0.14673
ADHEr, NADH6, PPCK, PYK, THD2 and/or GLUDy
237
5
3.64625
0.1409
ACKr and/or PTAr, ADHEr, FUM, PGI, PPCK
238
5
3.64625
0.1409
ACKr and/or PTAr, ADHEr, MDH, PGI, PPCK
239
5
3.60057
0.21551
ADHEr, GLCpts, NADH6, PPCK, RPE
240
5
3.59735
0.21725
ADHEr, NADH6, PPCK, PYK, RPE
241
5
3.59622
0.19297
ADHEr, GLCpts, MDH, NADH6, PPCK
242
5
3.59622
0.19297
ADHEr, FUM, GLCpts, NADH6, PPCK
243
5
3.59594
0.19311
ADHEr, MDH, NADH6, PPCK, PYK
244
5
3.59594
0.19311
ADHEr, FUM, NADH6, PPCK, PYK
245
5
3.59157
0.1952
ADHEr, FUM, GLCpts, NADH6, THD2 and/or GLUDy
246
5
3.57086
0.18962
ATPS4r, FUM, NADH6, PGI, THD5
247
5
3.57086
0.18962
ATPS4r, MDH, NADH6, PGI, THD5
248
5
3.55204
0.22769
ADHEr, FUM, NADH6, PPCK, TAL
249
5
3.55204
0.22769
ADHEr, MDH, NADH6, PPCK, TAL
250
5
3.53212
0.25244
ADHEr, GLCpts, MDH, NADH6, RPE
251
5
3.52326
0.22785
ADHEr, FUM, GLU5K, NADH6, PPCK
252
5
3.52326
0.22785
ADHEr, G5SD, MDH, NADH6, PPCK
253
5
3.52326
0.22785
ADHEr, GLU5K, MDH, NADH6, PPCK
254
5
3.52326
0.22785
ADHEr, FUM, G5SD, NADH6, PPCK
255
5
3.52319
0.22788
ADHEr, ASNS2, FUM, NADH6, PPCK
256
5
3.52319
0.22788
ADHEr, ASNS2, MDH, NADH6, PPCK
257
5
3.51328
0.26261
ADHEr, HEX1, NADH6, RPE, THD2 and/or GLUDy
258
5
3.40958
0.31856
ADHEr, FUM, HEX1, NADH6, RPE
259
5
3.19551
0.34972
ADHEr, ATPS4r, HEX1, PPS, THD2 and/or GLUDy
260
5
3.04993
0.16253
ADHEr, FUM, LDH_D, PPCK, THD2 and/or GLUDy
261
5
3.04993
0.16253
ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy
262
5
3.02516
0.17674
ADHEr, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
263
5
2.95797
0.22895
ADHEr, GLCpts, MDH, PPCK, TAL
264
5
2.95797
0.22895
ADHEr, FUM, GLCpts, PPCK, TAL
265
5
2.94768
0.30664
ADHEr, ATPS4r, HEX1, MDH, PFLi
266
5
2.93267
0.03242
ATPS4r, GLCpts, NADH6, PPCK, PYK
267
5
2.93156
0.27787
ATPS4r, GLCpts, NADH6, PFLi, RPE
268
5
2.92149
0.26881
ADHEr, ATPS4r, MDH, PGL and/or G6PDHy, PPS
269
5
2.76831
0.35787
ADHEr, HEX1, PFLi, PPS, RPE
270
5
2.73201
0.30945
ADHEr, HEX1, MDH, PFLi, RPE
271
5
2.69754
0.35901
ADHEr, HEX1, PFLi, PPS, TAL
272
5
2.69256
0.22448
ATPS4r, NADH6, PPCK, PYK, RPE
273
5
2.67071
0.31025
ADHEr, HEX1, MDH, PFLi, TAL
274
5
2.63273
0.17703
ATPS4r, PFLi, PPCK, PYK, RPE
275
5
2.61965
0.35455
ADHEr, ATPS4r, HEX1, RPE, THD2 and/or GLUDy
276
5
2.5479
0.2938
ADHEr, ATPS4r, GLUDy, HEX1, THD2 and/or GLUDy
277
5
2.5408
0.35457
ADHEr, ATPS4r, HEX1, TAL, THD2 and/or GLUDy
278
5
2.51093
0.22563
ATPS4r, NADH6, PPCK, PYK, TAL
279
5
2.50239
0.09117
GLCpts, PFLi, PGI, PPCK, PYK
280
5
1.97671
0.25583
ACKr and/or PTAr, MDH, PFLi, PGI, THD2 and/or GLUDy
281
5
1.41987
0.33925
ACKr and/or PTAr, FUM, HEX1, PFLi, RPE
282
5
1.41694
0.31779
ACKr and/or PTAr, GLU5K, NADH6, PFLi, RPE
283
5
1.41694
0.31779
ACKr and/or PTAr, G5SD, NADH6, PFLi, RPE
284
5
1.35397
0.34014
ACKr and/or PTAr, FUM, GLU5K, HEX1, PFLi
285
5
1.35397
0.34014
ACKr and/or PTAr, FUM, G5SD, HEX1, PFLi
286
5
1.35324
0.31986
ACKr and/or PTAr, GLU5K, NADH6, PFLi, TAL
287
5
1.35324
0.31986
ACKr and/or PTAr, G5SD, NADH6, PFLi, TAL
288
5
1.34703
0.34051
ACKr and/or PTAr, FUM, HEX1, PFLi, TAL
289
5
1.16644
0.24173
ADHEr, FRD and/or SUCD4, LDH_D, PPS, THD2 and/or GLUDy
290
5
0.935
0.24579
ACKr and/or PTAr, ACS, MDH, PFLi, THD2 and/or GLUDy
291
5
0.5376
0.11141
ADHEr, FRD and/or SUCD4, GLUDy, LDH_D, THD2 and/or
GLUDy
292
5
0.44145
0.36033
HEX1, PFLi, PPS, RPE, THD2 and/or GLUDy
293
5
0.41407
0.27855
ACKr and/or PTAr, ACS, FUM, PFLi, THD2 and/or GLUDy
294
5
0.34153
0.21403
ACKr and/or PTAr, ADHEr, MDH, TAL, THD2 and/or GLUDy
295
5
0.29506
0.27092
FUM, PFLi, PGDH, TAL, THD2 and/or GLUDy
296
5
0.29506
0.27092
FUM, PFLi, PGL and/or G6PDHy, TAL, THD2 and/or GLUDy
297
5
0.29233
0.27766
FUM, GLYCL, PFLi, TAL, THD2 and/or GLUDy
298
2
1.72604
0.38773
ADHEr, ATPS4r
299
2
0.83466
0.26712
ADHEr, PGI
300
3
4.11897
0.24338
ADHEr, EDA and/or PGDHY, PGI
301
3
2.0373
0.38161
ADHEr, ATPS4r, RPE
302
3
1.89007
0.3845
ADHEr, ATPS4r, TAL
303
3
1.49147
0.26024
ADHEr, PFLi, PGI
304
4
6.93034
0.18126
ADHEr, EDA and/or PGDHY, NADH6, PGI
305
4
6.91845
0.19552
ADHEr, HEX1, PFLi, PGI
306
4
6.14899
0.1758
ADHEr, EDA and/or PGDHY, PFLi, PGI
307
4
5.81422
0.16481
ADHEr, ATPS4r, EDA and/or PGDHY, PGI
308
4
4.23803
0.24209
ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, PGI
309
4
3.23462
0.28717
ACKr and/or PTAr, ADHEr, ATPS4r, SUCOAS
310
4
2.60615
0.3202
ADHEr, ATPS4r, HEX1, PFLi
311
4
2.54001
0.22798
ADHEr, PFLi, PGDH, PGI
312
4
2.5259
0.22921
ADHEr, PFLi, PGI, TAL
313
4
2.5129
0.23034
ADHEr, PFLi, PGI, RPE
314
4
2.50442
0.16853
ADHEr, ATPS4r, PFLi, PGI
315
4
1.5933
0.25891
ADHEr, FUM, PFLi, PGI
316
5
7.08404
0.12641
ADHEr, EDA and/or PGDHY, NADH6, PFLi, PGI
317
5
7.0245
0.10838
ADHEr, EDA and/or PGDHY, PFLi, PGI, PPCK
318
5
6.58534
0.16513
ADHEr, EDA and/or PGDHY, GLCpts, PFLi, PGI
319
5
6.36357
0.11937
ADHEr, EDA and/or PGDHY, PFLi, PGI, THD2 and/or GLUDy
320
5
6.22082
0.11375
ADHEr, ATPS4r, EDA and/or PGDHY, PFLi, PGI
321
5
5.08219
0.13778
ADHEr, ATPS4r, NADH6, PFLi, PGI
322
5
2.7362
0.31504
ADHEr, ATPS4r, HEX1, PFLi, RPE
323
5
2.67469
0.31748
ADHEr, ATPS4r, HEX1, PFLi, TAL
324
5
2.60629
0.2178
ADHEr, PFLi, PGDH, PGI, TAL
325
5
2.05497
0.19746
ADHEr, ASPT, ATPS4r, PGI, THD5
326
5
1.65594
0.25767
ACKr and/or PTAr, ADHEr, FUM, PFLi, PGI
327
6
7.82999
0.02101
ADHEr, ASPT, LDH_D, MDH, PFLi, THD2 and/or GLUDy
328
6
7.21161
0.05631
ADHEr, EDA and/or PGDHY, HEX1, NADH6, PGI, THD2 and/or
GLUDy
329
6
7.16447
0.10326
ADHEr, ASPT, LDH_D, MDH, PFLi, PYK
330
6
6.97749
0.0848
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, PFLi, PGI
331
6
6.89088
0.16538
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy
332
6
6.86543
0.14022
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PFLi
333
6
6.72704
0.08076
ADHEr, ASPT, EDA and/or PGDHY, MDH, NADH6, PYK
334
6
6.72704
0.08076
ADHEr, ASPT, MDH, NADH6, PGL and/or G6PDHy, PYK
335
6
6.67303
0.15616
ADHEr, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
336
6
6.67303
0.15616
ADHEr, FUM, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
337
6
6.62564
0.17535
ADHEr, LDH_D, MDH, NADH6, PFLi, THD2 and/or GLUDy
338
6
6.44535
0.24832
ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/or GLUDy
339
6
6.44535
0.24832
ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy
340
6
6.3515
0.04992
ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, THD2
and/or GLUDy
341
6
6.3052
0.17034
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, MDH, PGDH
342
6
6.25221
0.15227
ACKr and/or PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH
343
6
6.22508
0.18792
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi
344
6
6.20871
0.17434
ADHEr, EDA and/or PGDHY, MDH, PFLi, PGI, THD5
345
6
6.19852
0.09326
ADHEr, ASPT, FUM, LDH_D, MDH, THD2 and/or GLUDy
346
6
6.19408
0.16324
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PYK
347
6
6.19255
0.17194
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, MDH, TAL
348
6
6.18674
0.18098
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, NADH6
349
6
6.18358
0.11862
ADHEr, ATPS4r, GLCpts, MDH, NADH6, PFLi
350
6
6.12313
0.19075
ACKr and/or PTAr, ADHEr, FDH2, LDH_D, MDH, NADH6
351
6
6.09846
0.03275
ADHEr, ASPT, LDH_D, MALS, MDH, THD2 and/or GLUDy
352
6
6.09846
0.03275
ADHEr, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy
353
6
6.08794
0.17343
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, MDH, RPE
354
6
6.01307
0.17456
ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6
355
6
6.00837
0.14517
ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy
356
6
5.99004
0.18339
ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH6
357
6
5.9797
0.19373
ACKr and/or PTAr, ACS, ADHEr, HEX1, PGI, PPS
358
6
5.97334
0.11266
ADHEr, GLCpts, MDH, NADH6, PFLi, PGI
359
6
5.96643
0.16387
ACKr and/or PTAr, ADHEr, ASPT, MDH, PYK, RPE
360
6
5.8983
0.16412
ACKr and/or PTAr, ADHEr, ASPT, MDH, PYK, TAL
361
6
5.89777
0.06803
ADHEr, ATPS4r, EDA and/or PGDHY, HEX1, PGI, THD2 and/or
GLUDy
362
6
5.81808
0.13048
ADHEr, ATPS4r, MDH, NADH6, PFLi, PGI
363
6
5.77313
0.13132
ADHEr, ATPS4r, NADH12, NADH6, PFLi, PGI
364
6
5.75832
0.26044
ACKr and/or PTAr, ADHEr, GLU5K, MDH, PFLi, THD2 and/or
GLUDy
365
6
5.75832
0.26044
ACKr and/or PTAr, ADHEr, G5SD, MDH, PFLi, THD2 and/or
GLUDy
366
6
5.75556
0.13165
ADHEr, ATPS4r, FUM, NADH6, PFLi, PGI
367
6
5.68405
0.15589
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK
368
6
5.64518
0.25081
ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy
369
6
5.61474
0.1274
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPS, THD2 and/or
GLUDy
370
6
5.57706
0.11542
ADHEr, ATPS4r, GLCpts, NADH6, PFLi, THD2 and/or GLUDy
371
6
5.55724
0.20645
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6
372
6
5.39028
0.01098
ADHEr, ASPT, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
373
6
5.36336
0.12039
ADHEr, ASPT, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy
374
6
5.35114
0.12824
ADHEr, NADH12, NADH6, PFLi, PGI, RPE
375
6
5.34992
0.12867
ADHEr, FUM, NADH6, PFLi, PGI, RPE
376
6
5.34421
0.12775
ADHEr, NADH12, NADH6, PFLi, PGI, TAL
377
6
5.34298
0.12817
ADHEr, FUM, NADH6, PFLi, PGI, TAL
378
6
5.33668
0.12721
ADHEr, NADH12, NADH6, PFLi, PGDH, PGI
379
6
5.33544
0.12763
ADHEr, FUM, NADH6, PFLi, PGDH, PGI
380
6
5.3133
0.13509
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK
381
6
5.30121
0.14588
ADHEr, ATPS4r, GLCpts, NADH6, PPCK, RPE
382
6
5.29298
0.14544
ADHEr, ATPS4r, GLCpts, NADH6, PPCK, TAL
383
6
5.28823
0.16642
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PPCK
384
6
5.28401
0.14496
ADHEr, ATPS4r, GLCpts, NADH6, PGL and/or G6PDHy, PPCK
385
6
5.28401
0.14496
ADHEr, ATPS4r, GLCpts, NADH6, PGDH, PPCK
386
6
5.28388
0.145
ADHEr, LDH_D, NADH6, PFLi, PPCK, PYK
387
6
5.23479
0.26578
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi
388
6
5.22487
0.16488
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK
389
6
5.22487
0.16488
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK
390
6
5.18966
0.19912
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK
391
6
5.18966
0.19912
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK
392
6
5.17671
0.18111
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi
393
6
5.17275
0.18244
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi
394
6
5.16547
0.14992
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, MDH, PFLi
395
6
5.14231
0.02949
ADHEr, FRD and/or SUCD4, GLUDy, HEX1, LDH_D, THD2
and/or GLUDy
396
6
5.1335
0.31969
ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy
397
6
5.12241
0.20902
ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE
398
6
5.11384
0.20727
ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL
399
6
5.1046
0.2054
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH
400
6
5.00987
0.10652
ADHEr, ATPS4r, LDH_D, MDH, NADH6, THD2 and/or GLUDy
401
6
4.99324
0.24292
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi
402
6
4.98859
0.24448
ADHEr, FUM, HEX1, LDH_D, NADH6, PFLi
403
6
4.91516
0.18237
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK
404
6
4.91516
0.18237
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK
405
6
4.90997
0.20313
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, THD2 and/or GLUDy
406
6
4.86469
0.20078
ADHEr, ATPS4r, GLCpts, PFLi, PPCK, RPE
407
6
4.79988
0.22937
ACKr and/or PTAr, ADHEr, FUM, MDH, NADH6, PYK
408
6
4.75998
0.22327
ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy
409
6
4.75998
0.22327
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/or
GLUDy
410
6
4.75352
0.20131
ADHEr, ATPS4r, GLCpts, PFLi, PPCK, TAL
411
6
4.72575
0.27672
ADHEr, ATPS4r, FDH2, HEX1, LDH_D, NADH6
412
6
4.68468
0.22968
ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy
413
6
4.67203
0.24577
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, THD2
and/or GLUDy
414
6
4.6646
0.15914
ADHEr, ATPS4r, FUM, GLCpts, NADH6, THD2 and/or GLUDy
415
6
4.6568
0.25634
ADHEr, ATPS4r, ICL, MDH, PGL and/or G6PDHy, PPS
416
6
4.6568
0.25634
ADHEr, ATPS4r, FUM, MDH, PGL and/or G6PDHy, PPS
417
6
4.6568
0.25634
ADHEr, ATPS4r, MALS, MDH, PGL and/or G6PDHy, PPS
418
6
4.64861
0.22305
ADHEr, HEX1, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
419
6
4.61213
0.23585
ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy
420
6
4.54731
0.14834
ADHEr, EDA and/or PGDHY, GLUDy, LDH_D, MDH, THD2
and/or GLUDy
421
6
4.45035
0.18822
ADHEr, ASPT, ATPS4r, MDH, PYK, RPE
422
6
4.44107
0.285
ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH6
423
6
4.37848
0.18825
ADHEr, ASPT, ATPS4r, MDH, PYK, TAL
424
6
4.35711
0.22671
ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6
425
6
4.30745
0.05232
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
426
6
4.29111
0.05856
ADHEr, ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy
427
6
4.29026
0.24034
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, G5SD, GLYCL, PGI
428
6
4.29026
0.24034
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, GLU5K, GLYCL, PGI
429
6
4.19017
0.18892
ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy
430
6
4.14405
0.19951
ADHEr, ATPS4r, FUM, LDH_D, PGDH, PPCK
431
6
4.14405
0.19951
ADHEr, ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK
432
6
4.14405
0.19951
ADHEr, ATPS4r, LDH_D, MDH, PGDH, PPCK
433
6
4.13843
0.19251
ADHEr, ATPS4r, EDA and/or PGDHY, MDH, PPCK, RPE
434
6
4.11865
0.20026
ADHEr, ATPS4r, LDH_D, MDH, PPCK, TAL
435
6
4.11865
0.20026
ADHEr, ATPS4r, FUM, LDH_D, PPCK, TAL
436
6
4.10258
0.11665
ACKr and/or PTAr, ADHEr, ATPS4r, PPCK, PYK, SUCOAS
437
6
4.09791
0.11017
ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2 and/or GLUDy
438
6
4.09791
0.11017
ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2 and/or GLUDy
439
6
4.09533
0.20095
ADHEr, ATPS4r, LDH_D, MDH, PPCK, RPE
440
6
4.09533
0.20095
ADHEr, ATPS4r, FUM, LDH_D, PPCK, RPE
441
6
4.08336
0.13789
ADHEr, ASPT, LDH_D, MDH, PPCK, THD2 and/or GLUDy
442
6
4.08234
0.13828
ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
443
6
4.0683
0.31169
ADHEr, FUM, HEX1, LDH_D, PPS, THD2 and/or GLUDy
444
6
4.05789
0.14762
ADHEr, ASPT, LDH_D, MDH, NADH6, THD2 and/or GLUDy
445
6
4.04139
0.17129
ADHEr, ASPT, LDH_D, MDH, RPE, THD2 and/or GLUDy
446
6
3.97871
0.17786
ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK
447
6
3.95205
0.10694
ACKr and/or PTAr, ADHEr, ATPS4r, PGDH, PGI, SUCOAS
448
6
3.95184
0.18812
ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK
449
6
3.94624
0.19026
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6
450
6
3.94455
0.21245
ADHEr, ASPT, LDH_D, MDH, PPCK, RPE
451
6
3.94203
0.1079
ACKr and/or PTAr, ADHEr, ATPS4r, PGI, SUCOAS, TAL
452
6
3.93874
0.25675
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, THD2 and/or
GLUDy
453
6
3.93273
0.1088
ACKr and/or PTAr, ADHEr, ATPS4r, PGI, RPE, SUCOAS
454
6
3.91186
0.22634
ADHEr, ASPT, LDH_D, MDH, NADH6, RPE
455
6
3.90038
0.04761
ADHEr, GLCpts, NADH6, PGI, PPCK, THD2 and/or GLUDy
456
6
3.8947
0.05032
ADHEr, FUM, GLCpts, NADH6, PGI, THD2 and/or GLUDy
457
6
3.8947
0.05032
ADHEr, GLCpts, MDH, NADH6, PGI, THD2 and/or GLUDy
458
6
3.84046
0.12564
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, THD2
and/or GLUDy
459
6
3.81228
0.08972
ADHEr, EDA and/or PGDHY, MDH, NADH6, PYK, THD2 and/or
GLUDy
460
6
3.81228
0.08972
ADHEr, MDH, NADH6, PGL and/or G6PDHy, PYK, THD2 and/or
GLUDy
461
6
3.76251
0.07312
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK, THD2
and/or GLUDy
462
6
3.70324
0.1395
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK, PYK
463
6
3.70023
0.1556
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK, PYK
464
6
3.70023
0.1556
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PPCK, PYK
465
6
3.67651
0.1546
ADHEr, MDH, NADH6, PGL and/or G6PDHy, PPCK, PYK
466
6
3.67651
0.1546
ADHEr, EDA and/or PGDHY, MDH, NADH6, PPCK, PYK
467
6
3.652
0.16631
ADHEr, LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy
468
6
3.64497
0.19156
ADHEr, FUM, GLCpts, NADH6, RPE, THD2 and/or GLUDy
469
6
3.62254
0.1804
ADHEr, LDH_D, NADH12, NADH6, PPCK, THD2 and/or
GLUDy
470
6
3.5494
0.21535
ADHEr, FUM, LDH_D, NADH12, NADH6, THD2 and/or GLUDy
471
6
3.41696
0.27865
ADHEr, DAAD, FDH2, NADH12, NADH6, PRO1z
472
6
3.41696
0.27865
ADHEr, ALAR, FDH2, NADH12, NADH6, PRO1z
473
6
3.40228
0.13474
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, THD2
and/or GLUDy
474
6
3.27381
0.03414
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PPCK, THD2 and/or
GLUDy
475
6
3.22868
0.19148
ADHEr, FRD and/or SUCD4, LDH_D, PPS, SUCOAS, THD2
and/or GLUDy
476
6
3.11905
0.12289
ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/or GLUDy
477
6
3.11905
0.12289
ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy
478
6
3.10777
0.12936
ADHEr, FUM, GLCpts, LDH_D, PPCK, THD2 and/or GLUDy
479
6
3.10777
0.12936
ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2 and/or GLUDy
480
6
2.9427
0.20328
ADHEr, FRD and/or SUCD4, LDH_D, MALS, PPS, THD2 and/or
GLUDy
481
6
2.9427
0.20328
ADHEr, FRD and/or SUCD4, ICL, LDH_D, PPS, THD2 and/or
GLUDy
482
6
2.80361
0.3231
ADHEr, FUM, HEX1, LDH_D, TAL, THD2 and/or GLUDy
483
6
2.79375
0.29126
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, THD2 and/or
GLUDy
484
6
2.68652
0.29222
ADHEr, ATPS4r, GLUDy, HEX1, RPE, THD2 and/or GLUDy
485
6
2.62059
0.29297
ADHEr, ATPS4r, GLUDy, HEX1, TAL, THD2 and/or GLUDy
486
6
2.46956
0.30762
ADHEr, FUM, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy
487
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, FUM, MDH, PGL and/or
G6PDHy, SUCOAS
488
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, ICL, MDH, PGL and/or
G6PDHy, SUCOAS
489
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, FUM, MDH, PGDH, SUCOAS
490
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, MALS, MDH, PGL and/or
G6PDHy, SUCOAS
491
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, ICL, MDH, PGDH, SUCOAS
492
6
0.97386
0.28207
ACKr and/or PTAr, ADHEr, MALS, MDH, PGDH, SUCOAS
493
6
0.90686
0.28301
ACKr and/or PTAr, ADHEr, MALS, MDH, SUCOAS, TAL
494
6
0.90686
0.28301
ACKr and/or PTAr, ADHEr, ICL, MDH, SUCOAS, TAL
495
6
0.84537
0.28387
ACKr and/or PTAr, ADHEr, ICL, MDH, RPE, SUCOAS
496
6
0.84537
0.28387
ACKr and/or PTAr, ADHEr, MALS, MDH, RPE, SUCOAS
497
3
3.26272
0.35236
ADHEr, LDH_D, NADH6
498
3
2.8403
0.28275
ADHEr, LDH_D, PPCK
499
3
1.72604
0.38773
ADHEr, ATPS4r, LDH_D
500
3
0.89333
0.37885
ADHEr, FUM, LDH_D
501
3
0.83466
0.26712
ADHEr, LDH_D, PGI
502
3
0.41621
0.38953
ADHEr, HEX1, LDH_D
503
4
5.71646
0.21908
ADHEr, HEX1, LDH_D, PGI
504
4
4.83364
0.29669
ADHEr, LDH_D, NADH6, PFLi
505
4
4.11897
0.24338
ADHEr, EDA and/or PGDHY, LDH_D, PGI
506
4
3.77533
0.25553
ADHEr, ASPT, LDH_D, MDH
507
4
3.65248
0.19372
ADHEr, LDH_D, NADH6, PGI
508
4
3.47283
0.25194
ADHEr, LDH_D, NADH6, PPCK
509
4
3.39319
0.29001
ADHEr, LDH_D, MDH, NADH6
510
4
3.35305
0.34906
ADHEr, LDH_D, NADH6, RPE
511
4
3.0877
0.16431
ADHEr, LDH_D, PGI, PPCK
512
4
2.90019
0.24841
ADHEr, GLCpts, LDH_D, PPCK
513
4
2.89855
0.2815
ADHEr, LDH_D, PPCK, RPE
514
4
2.88617
0.25645
ADHEr, FUM, LDH_D, THD2 and/or GLUDy
515
4
2.72186
0.35068
ADHEr, FUM, HEX1, LDH_D
516
4
2.0373
0.38161
ADHEr, ATPS4r, LDH_D, RPE
517
4
1.89007
0.3845
ADHEr, ATPS4r, LDH_D, TAL
518
4
1.49147
0.26024
ADHEr, LDH_D, PFLi, PGI
519
4
0.5753
0.38695
ADHEr, HEX1, LDH_D, RPE
520
4
0.49968
0.38818
ADHEr, HEX1, LDH_D, TAL
521
5
6.93034
0.18126
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI
522
5
6.92332
0.16482
ADHEr, HEX1, LDH_D, NADH6, PGI
523
5
6.91845
0.19552
ADHEr, HEX1, LDH_D, PFLi, PGI
524
5
6.89839
0.18171
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PGI
525
5
6.14899
0.1758
ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI
526
5
5.8701
0.13379
ADHEr, EDA and/or PGDHY, LDH_D, PGI, PPCK
527
5
5.85757
0.23874
ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy
528
5
5.85411
0.19685
ADHEr, HEX1, LDH_D, PGI, PPS
529
5
5.81422
0.16481
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI
530
5
5.40131
0.26362
ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy
531
5
4.88764
0.27849
ADHEr, LDH_D, NADH12, NADH6, PFLi
532
5
4.88489
0.27942
ADHEr, FUM, LDH_D, NADH6, PFLi
533
5
4.81778
0.26037
ADHEr, ATPS4r, LDH_D, MDH, NADH6
534
5
4.73419
0.21859
ADHEr, LDH_D, NADH6, PPCK, THD2 and/or GLUDy
535
5
4.63783
0.29595
ADHEr, ATPS4r, FDH2, LDH_D, NADH6
536
5
4.30547
0.19131
ADHEr, ATPS4r, LDH_D, NADH6, PGI
537
5
4.2733
0.23078
ADHEr, ATPS4r, LDH_D, NADH6, PPCK
538
5
4.23803
0.24209
ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D, PGI
539
5
4.12032
0.32701
ADHEr, ATPS4r, FUM, LDH_D, NADH6
540
5
3.9718
0.23354
ADHEr, ATPS4r, LDH_D, MDH, PPCK
541
5
3.9718
0.23354
ADHEr, ATPS4r, FUM, LDH_D, PPCK
542
5
3.8747
0.21758
ADHEr, ASPT, GLCpts, LDH_D, MDH
543
5
3.84814
0.25342
ADHEr, ASPT, LDH_D, MDH, RPE
544
5
3.83986
0.2047
ADHEr, ASPT, LDH_D, MDH, PYK
545
5
3.75472
0.32987
ADHEr, ATPS4r, HEX1, LDH_D, NADH6
546
5
3.54965
0.29114
ADHEr, ATPS4r, LDH_D, MDH, PGDH
547
5
3.54605
0.21695
ADHEr, GLCpts, LDH_D, NADH6, PPCK
548
5
3.54385
0.218
ADHEr, LDH_D, NADH6, PPCK, PYK
549
5
3.53615
0.25027
ADHEr, LDH_D, NADH6, PPCK, RPE
550
5
3.5018
0.32809
ADHEr, ATPS4r, FUM, HEX1, LDH_D
551
5
3.46904
0.25375
ADHEr, GLCpts, LDH_D, MDH, NADH6
552
5
3.46528
0.28851
ADHEr, LDH_D, MDH, NADH6, RPE
553
5
3.44916
0.13425
ADHEr, LDH_D, PFLi, PGI, PPCK
554
5
3.44555
0.26498
ADHEr, HEX1, LDH_D, NADH6, THD2 and/or GLUDy
555
5
3.43776
0.29402
ADHEr, ATPS4r, LDH_D, MDH, TAL
556
5
3.33377
0.2967
ADHEr, ATPS4r, LDH_D, MDH, RPE
557
5
3.33152
0.33978
ADHEr, FUM, LDH_D, NADH6, TAL
558
5
3.32935
0.34088
ADHEr, HEX1, LDH_D, NADH6, TAL
559
5
3.32788
0.32122
ADHEr, FUM, HEX1, LDH_D, NADH6
560
5
3.31278
0.3493
ADHEr, G5SD, LDH_D, NADH6, TAL
561
5
3.31278
0.3493
ADHEr, GLU5K, LDH_D, NADH6, TAL
562
5
3.23462
0.28717
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, SUCOAS
563
5
3.17484
0.10602
ADHEr, LDH_D, PGI, PPCK, THD2 and/or GLUDy
564
5
3.16614
0.11184
ADHEr, LDH_D, MDH, PGI, THD2 and/or GLUDy
565
5
3.16614
0.11184
ADHEr, FUM, LDH_D, PGI, THD2 and/or GLUDy
566
5
3.11125
0.24826
ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or GLUDy
567
5
2.95529
0.24477
ADHEr, LDH_D, PPCK, RPE, THD2 and/or GLUDy
568
5
2.95136
0.24731
ADHEr, GLCpts, LDH_D, PPCK, RPE
569
5
2.94249
0.25305
ADHEr, FUM, LDH_D, RPE, THD2 and/or GLUDy
570
5
2.93765
0.22693
ADHEr, FUM, LDH_D, PPCK, PYK
571
5
2.93765
0.22693
ADHEr, LDH_D, MDH, PPCK, PYK
572
5
2.9332
0.24406
ADHEr, LDH_D, PPCK, TAL, THD2 and/or GLUDy
573
5
2.90913
0.24328
ADHEr, LDH_D, PGDH, PPCK, THD2 and/or GLUDy
574
5
2.90913
0.24328
ADHEr, LDH_D, PGL and/or G6PDHy, PPCK, THD2 and/or
GLUDy
575
5
2.90081
0.26381
ADHEr, LDH_D, MDH, PPCK, TAL
576
5
2.90081
0.26381
ADHEr, FUM, LDH_D, PPCK, TAL
577
5
2.79498
0.34856
ADHEr, FUM, HEX1, LDH_D, RPE
578
5
2.61943
0.36027
ADHEr, HEX1, LDH_D, PFLi, PPS
579
5
2.60615
0.3202
ADHEr, ATPS4r, HEX1, LDH_D, PFLi
580
5
2.60314
0.31113
ADHEr, HEX1, LDH_D, MDH, PFLi
581
5
2.54001
0.22798
ADHEr, LDH_D, PFLi, PGDH, PGI
582
5
2.5259
0.22921
ADHEr, LDH_D, PFLi, PGI, TAL
583
5
2.5129
0.23034
ADHEr, LDH_D, PFLi, PGI, RPE
584
5
2.50442
0.16853
ADHEr, ATPS4r, LDH_D, PFLi, PGI
585
5
2.45433
0.3546
ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy
586
5
2.18633
0.3609
ADHEr, HEX1, LDH_D, PPS, THD2 and/or GLUDy
587
5
2.11347
0.31979
ADHEr, ATPS4r, HEX1, LDH_D, MDH
588
5
1.5933
0.25891
ADHEr, FUM, LDH_D, PFLi, PGI
589
5
0.9475
0.3633
ADHEr, FUM, LDH_D, PGDH, TAL
590
5
0.9475
0.3633
ADHEr, FUM, LDH_D, PGL and/or G6PDHy, TAL
591
6
7.08404
0.12641
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PFLi, PGI
592
6
7.0245
0.10838
ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI, PPCK
593
6
6.98742
0.06748
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI, THD2
and/or GLUDy
594
6
6.69134
0.13239
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH
595
6
6.58534
0.16513
ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, PFLi, PGI
596
6
6.55123
0.09841
ADHEr, ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK
597
6
6.55123
0.09841
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PYK
598
6
6.36357
0.11937
ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI, THD2 and/or
GLUDy
599
6
6.22082
0.11375
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PFLi, PGI
600
6
5.98165
0.07186
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI, PPCK
601
6
5.93527
0.09761
ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, PGI, PPCK
602
6
5.87444
0.0434
ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI, THD2 and/or
GLUDy
603
6
5.87444
0.0434
ADHEr, EDA and/or PGDHY, LDH_D, MDH, PGI, THD2 and/or
GLUDy
604
6
5.65488
0.26204
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
605
6
5.59555
0.20952
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6
606
6
5.1776
0.13724
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI
607
6
5.11744
0.13758
ADHEr, FUM, LDH_D, NADH6, PFLi, PGI
608
6
5.11744
0.13758
ADHEr, LDH_D, MDH, NADH6, PFLi, PGI
609
6
5.09877
0.17589
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK
610
6
5.08219
0.13778
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGI
611
6
5.02693
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGDH, PPCK
612
6
5.02693
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGL and/or G6PDHy, PPCK
613
6
5.00057
0.21154
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, TAL
614
6
4.97638
0.21218
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, RPE
615
6
4.88512
0.29281
ADHEr, GLU5K, LDH_D, NADH6, PFLi, RPE
616
6
4.88512
0.29281
ADHEr, G5SD, LDH_D, NADH6, PFLi, RPE
617
6
4.885
0.29286
ADHEr, ASNS2, LDH_D, NADH6, PFLi, RPE
618
6
4.85776
0.29446
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, RPE
619
6
4.83644
0.29517
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, TAL
620
6
4.66386
0.18074
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PPCK
621
6
4.66386
0.18074
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PPCK
622
6
4.63095
0.20189
ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK
623
6
4.49707
0.25782
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, THD2
and/or GLUDy
624
6
4.4958
0.1778
ADHEr, ASPT, LDH_D, MDH, NADH6, PYK
625
6
4.41977
0.25646
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
626
6
4.18509
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGL and/or G6PDHy
627
6
4.18509
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGDH
628
6
4.15013
0.1124
ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy
629
6
4.14582
0.32483
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, TAL
630
6
4.1099
0.32529
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, RPE
631
6
4.04473
0.12883
ADHEr, LDH_D, PFLi, PGDH, PGI, PPCK
632
6
4.03844
0.12934
ADHEr, LDH_D, PFLi, PGI, PPCK, TAL
633
6
4.03266
0.12981
ADHEr, LDH_D, PFLi, PGI, PPCK, RPE
634
6
4.02531
0.1111
ADHEr, GLCpts, LDH_D, PFLi, PGI, PPCK
635
6
3.9367
0.21579
ADHEr, ASPT, GLCpts, LDH_D, MDH, RPE
636
6
3.76049
0.30843
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi
637
6
3.71345
0.27754
ADHEr, ATPS4r, LDH_D, MDH, PGDH, TAL
638
6
3.69299
0.14673
ADHEr, LDH_D, NADH6, PPCK, PYK, THD2 and/or GLUDy
639
6
3.64625
0.1409
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PGI, PPCK
640
6
3.64625
0.1409
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PGI, PPCK
641
6
3.60057
0.21551
ADHEr, GLCpts, LDH_D, NADH6, PPCK, RPE
642
6
3.59735
0.21725
ADHEr, LDH_D, NADH6, PPCK, PYK, RPE
643
6
3.59622
0.19297
ADHEr, FUM, GLCpts, LDH_D, NADH6, PPCK
644
6
3.59622
0.19297
ADHEr, GLCpts, LDH_D, MDH, NADH6, PPCK
645
6
3.59594
0.19311
ADHEr, FUM, LDH_D, NADH6, PPCK, PYK
646
6
3.59594
0.19311
ADHEr, LDH_D, MDH, NADH6, PPCK, PYK
647
6
3.59157
0.1952
ADHEr, FUM, GLCpts, LDH_D, NADH6, THD2 and/or GLUDy
648
6
3.55204
0.22769
ADHEr, LDH_D, MDH, NADH6, PPCK, TAL
649
6
3.55204
0.22769
ADHEr, FUM, LDH_D, NADH6, PPCK, TAL
650
6
3.53212
0.25244
ADHEr, GLCpts, LDH_D, MDH, NADH6, RPE
651
6
3.52326
0.22785
ADHEr, GLU5K, LDH_D, MDH, NADH6, PPCK
652
6
3.52326
0.22785
ADHEr, G5SD, LDH_D, MDH, NADH6, PPCK
653
6
3.52326
0.22785
ADHEr, FUM, GLU5K, LDH_D, NADH6, PPCK
654
6
3.52326
0.22785
ADHEr, FUM, G5SD, LDH_D, NADH6, PPCK
655
6
3.52319
0.22788
ADHEr, ASNS2, FUM, LDH_D, NADH6, PPCK
656
6
3.52319
0.22788
ADHEr, ASNS2, LDH_D, MDH, NADH6, PPCK
657
6
3.51328
0.26261
ADHEr, HEX1, LDH_D, NADH6, RPE, THD2 and/or GLUDy
658
6
3.40958
0.31856
ADHEr, FUM, HEX1, LDH_D, NADH6, RPE
659
6
3.19551
0.34972
ADHEr, ATPS4r, HEX1, LDH_D, PPS, THD2 and/or GLUDy
660
6
2.95797
0.22895
ADHEr, FUM, GLCpts, LDH_D, PPCK, TAL
661
6
2.95797
0.22895
ADHEr, GLCpts, LDH_D, MDH, PPCK, TAL
662
6
2.94768
0.30664
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi
663
6
2.92149
0.26881
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPS
664
6
2.76831
0.35787
ADHEr, HEX1, LDH_D, PFLi, PPS, RPE
665
6
2.7362
0.31504
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, RPE
666
6
2.73201
0.30945
ADHEr, HEX1, LDH_D, MDH, PFLi, RPE
667
6
2.69754
0.35901
ADHEr, HEX1, LDH_D, PFLi, PPS, TAL
668
6
2.67469
0.31748
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, TAL
669
6
2.67071
0.31025
ADHEr, HEX1, LDH_D, MDH, PFLi, TAL
670
6
2.61965
0.35455
ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2 and/or GLUDy
671
6
2.60629
0.2178
ADHEr, LDH_D, PFLi, PGDH, PGI, TAL
672
6
2.5479
0.2938
ADHEr, ATPS4r, GLUDy, HEX1, LDH_D, THD2 and/or GLUDy
673
6
2.5408
0.35457
ADHEr, ATPS4r, HEX1, LDH_D, TAL, THD2 and/or GLUDy
674
6
2.05497
0.19746
ADHEr, ASPT, ATPS4r, LDH_D, PGI, THD5
675
6
1.65594
0.25767
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFLi, PGI
676
7
7.90077
0.01226
ADHEr, ASPT, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
677
7
7.84104
0.01965
ADHEr, ASPT, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
678
7
7.7052
0.03644
ADHEr, ASPT, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy
679
7
7.67191
0.04055
ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
680
7
7.61518
0.06956
ADHEr, ATPS4r, LDH_D, MDH, NADH12, PFLi, THD2 and/or
GLUDy
681
7
7.57942
0.07603
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, PGDH
682
7
7.57942
0.07603
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PGDH
683
7
7.57606
0.07664
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, TAL
684
7
7.57606
0.07664
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, TAL
685
7
7.57295
0.0772
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, RPE
686
7
7.57295
0.0772
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, RPE
687
7
7.55163
0.08105
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
688
7
7.38115
0.07649
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, PFLi
689
7
7.293
0.07152
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, NADH6
690
7
7.21161
0.05631
ADHEr, EDA and/or
PGDHY, HEX1, LDH_D, NADH6, PGI, THD2 and/or GLUDy
691
7
7.10953
0.03056
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PYK, THD2
and/or GLUDy
692
7
7.04629
0.01861
ADHEr, ARGt4, ASPT, LDH_D, MALS, MDH, THD2 and/or
GLUDy
693
7
7.04629
0.01861
ADHEr, ARGt4, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy
694
7
7.01619
0.17329
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH
695
7
7.00623
0.10284
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, PGI
696
7
7.00542
0.17519
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL
697
7
6.99744
0.05155
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, NADH6, THD2
and/or GLUDy
698
7
6.99744
0.05155
ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, NADH6, THD2
and/or GLUDy
699
7
6.99744
0.05155
ACKr and/or
PTAr, ADHEr, LDH_D, MALS, MDH, NADH6, THD2 and/or
GLUDy
700
7
6.99618
0.02608
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
701
7
6.99539
0.17696
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE
702
7
6.99098
0.13845
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PFLi, PGL and/or
G6PDHy
703
7
6.99098
0.13845
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PFLi
704
7
6.97749
0.0848
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGI
705
7
6.95495
0.04834
ACKr and/or
PTAr, ADHEr, GLUDy, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
706
7
6.94643
0.04549
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
707
7
6.94643
0.04549
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
708
7
6.94035
0.16409
ACKr and/or
PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGDH
709
7
6.93216
0.19304
ADHEr, HEX1, LDH_D, MDH, PFLi, PPS, THD2 and/or GLUDy
710
7
6.9218
0.16465
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, TAL
711
7
6.90477
0.16517
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, RPE
712
7
6.89223
0.07039
ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
713
7
6.89088
0.16538
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PGDH
714
7
6.88301
0.07117
ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
715
7
6.88301
0.07117
ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
716
7
6.86164
0.16622
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, TAL
717
7
6.83471
0.167
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, RPE
718
7
6.81823
0.05671
ADHEr, ATPS4r, LDH_D, NADH12, PFLi, PPCK, THD2 and/or
GLUDy
719
7
6.75858
0.12153
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
720
7
6.75858
0.12153
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
721
7
6.74354
0.0559
ADHEr, GLUDy, HEX1, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
722
7
6.72704
0.08076
ADHEr, ASPT, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PYK
723
7
6.72704
0.08076
ADHEr, ASPT, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PYK
724
7
6.72478
0.09245
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2
and/or GLUDy
725
7
6.71779
0.1313
ADHEr, FBP, LDH_D, MDH, PFLi, PGDH, THD2 and/or GLUDy
726
7
6.71371
0.1397
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
727
7
6.69882
0.08259
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PGDH, THD2 and/or
GLUDy
728
7
6.69814
0.08084
ADHEr, LDH_D, MDH, NADH6, PFLi, PGDH, THD2 and/or
GLUDy
729
7
6.68517
0.13281
ADHEr, FBP, LDH_D, MDH, PFLi, TAL, THD2 and/or GLUDy
730
7
6.67827
0.0833
ADHEr, ATPS4r, LDH_D, MDH, PFLi, TAL, THD2 and/or
GLUDy
731
7
6.67803
0.08153
ADHEr, LDH_D, MDH, NADH6, PFLi, TAL, THD2 and/or
GLUDy
732
7
6.66553
0.13149
ADHEr, LDH_D, MDH, PFLi, PGDH, PGI, THD2 and/or GLUDy
733
7
6.65922
0.08397
ADHEr, ATPS4r, LDH_D, MDH, PFLi, RPE, THD2 and/or GLUDy
734
7
6.65477
0.13422
ADHEr, FBP, LDH_D, MDH, PFLi, RPE, THD2 and/or GLUDy
735
7
6.65444
0.13292
ADHEr, LDH_D, MDH, PFLi, PGI, TAL, THD2 and/or GLUDy
736
7
6.64411
0.13426
ADHEr, LDH_D, MDH, PFLi, PGI, RPE, THD2 and/or GLUDy
737
7
6.54706
0.05865
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, RPE
738
7
6.50546
0.0488
ADHEr, ASPT, EDA and/or PGDHY, FUM, LDH_D, MDH, THD2
and/or GLUDy
739
7
6.4951
0.09302
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, RPE, THD2
and/or GLUDy
740
7
6.48152
0.09261
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, TAL, THD2
and/or GLUDy
741
7
6.4815
0.14937
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, RPE, THD2
and/or GLUDy
742
7
6.4814
0.1488
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, TAL, THD2
and/or GLUDy
743
7
6.48129
0.14819
ACKr and/or
PTAr, ADHEr, LDH_D, MDH, NADH6, PGDH, THD2 and/or
GLUDy
744
7
6.46677
0.09217
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PGDH, PPCK, THD2 and/or
GLUDy
745
7
6.46677
0.09217
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGL and/or
G6PDHy, PPCK, THD2 and/or GLUDy
746
7
6.40404
0.13985
ACKr and/or
PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6
747
7
6.39358
0.0544
ADHEr, ASPT, FUM, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
748
7
6.36333
0.16503
ACKr and/or
PTAr, ADHEr, ASPT, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
749
7
6.35537
0.14906
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, NADH6, PYK
750
7
6.34075
0.15004
ACKr and/or
PTAr, ADHEr, FDH2, GLCpts, LDH_D, MDH, NADH6
751
7
6.33847
0.16728
ACKr and/or
PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6
752
7
6.30136
0.19463
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
753
7
6.30082
0.1583
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, PYK
754
7
6.23552
0.18763
ACKr and/or
PTAr, ADHEr, FDH2, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
755
7
6.23519
0.15548
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH, PGDH, THD2
and/or GLUDy
756
7
6.22748
0.15693
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH, TAL, THD2
and/or GLUDy
757
7
6.22033
0.15828
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH, RPE, THD2
and/or GLUDy
758
7
6.2176
0.1588
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PGDH, THD2
and/or GLUDy
759
7
6.21321
0.15963
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, TAL, THD2
and/or GLUDy
760
7
6.20917
0.16039
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, RPE, THD2
and/or GLUDy
761
7
6.20871
0.17434
ADHEr, EDA and/or PGDHY, LDH_D, MDH, PFLi, PGI, THD5
762
7
6.18575
0.17904
ACKr and/or
PTAr, ADHEr, FDH2, LDH_D, MDH, NADH12, NADH6
763
7
6.17771
0.01562
ADHEr, ASPT, ICL, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
764
7
6.17771
0.01562
ADHEr, ASPT, LDH_D, MALS, MDH, NADH6, THD2 and/or
GLUDy
765
7
6.10255
0.12296
ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6, PPCK
766
7
6.08364
0.10963
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
767
7
6.05909
0.217
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, HEX1, LDH_D, THD2
and/or GLUDy
768
7
5.99909
0.09659
ADHEr, FUM, GLCpts, LDH_D, PFLi, PGI, PPCK
769
7
5.99909
0.09659
ADHEr, GLCpts, LDH_D, MDH, PFLi, PGI, PPCK
770
7
5.9797
0.19373
ACKr and/or PTAr, ACS, ADHEr, HEX1, LDH_D, PGI, PPS
771
7
5.97334
0.11266
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, PGI
772
7
5.94122
0.14646
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, PPCK, PYK
773
7
5.94122
0.14646
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFLi, PPCK, PYK
774
7
5.9395
0.02821
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
775
7
5.89777
0.06803
ADHEr, ATPS4r, EDA and/or
PGDHY, HEX1, LDH_D, PGI, THD2 and/or GLUDy
776
7
5.86803
0.06511
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
777
7
5.84763
0.01509
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGI, THD2
and/or GLUDy
778
7
5.80727
0.03994
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
779
7
5.80665
0.23182
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2
and/or GLUDy
780
7
5.77313
0.13132
ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PGI
781
7
5.77052
0.23124
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, TAL, THD2
and/or GLUDy
782
7
5.75832
0.26044
ACKr and/or PTAr, ADHEr, G5SD, LDH_D, MDH, PFLi, THD2
and/or GLUDy
783
7
5.75832
0.26044
ACKr and/or PTAr, ADHEr, GLU5K, LDH_D, MDH, PFLi, THD2
and/or GLUDy
784
7
5.75556
0.13165
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PGI
785
7
5.75413
0.21628
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, NADH6, THD2
and/or GLUDy
786
7
5.75142
0.15
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PFLi, PPCK
787
7
5.75142
0.15
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PFLi, PPCK
788
7
5.73569
0.11619
ACKr and/or PTAr, ADHEr, FUM, LDH_D, NADH6, PPCK, THD2
and/or GLUDy
789
7
5.73112
0.23062
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGDH, THD2 and/or
GLUDy
790
7
5.73112
0.23062
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGL and/or
G6PDHy, THD2 and/or GLUDy
791
7
5.7037
0.1229
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6, PPCK
792
7
5.7037
0.1229
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PPCK
793
7
5.67082
0.04313
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGI, THD2 and/or
GLUDy
794
7
5.67082
0.04313
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
795
7
5.6689
0.01529
ADHEr, LDH_D, MDH, NADH6, PFLi, PYK, THD2 and/or
GLUDy
796
7
5.57706
0.11542
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PFLi, THD2 and/or
GLUDy
797
7
5.57479
0.20482
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, MDH, THD2 and/or
GLUDy
798
7
5.56128
0.20483
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH12, THD2 and/or
GLUDy
799
7
5.53687
0.15179
ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK, THD2 and/or
GLUDy
800
7
5.48032
0.07882
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, THD2 and/or
GLUDy
801
7
5.48032
0.07882
ADHEr, ATPS4r, FDH2, FUM, LDH_D, NADH6, THD2 and/or
GLUDy
802
7
5.4574
0.07896
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
803
7
5.43688
0.14109
ADHEr, ASPT, FUM, HEX1, LDH_D, PPS, THD2 and/or GLUDy
804
7
5.42926
0.03599
ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, PPCK, THD2
and/or GLUDy
805
7
5.41288
0.10644
ADHEr, FUM, LDH_D, PFLi, PGI, PPCK, RPE
806
7
5.41288
0.10644
ADHEr, LDH_D, MDH, PFLi, PGI, PPCK, RPE
807
7
5.40727
0.10598
ADHEr, FUM, LDH_D, PFLi, PGI, PPCK, TAL
808
7
5.40727
0.10598
ADHEr, LDH_D, MDH, PFLi, PGI, PPCK, TAL
809
7
5.40541
0.10406
ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK, PYK
810
7
5.40329
0.10477
ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, PYK
811
7
5.40329
0.10477
ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, PYK
812
7
5.40117
0.10549
ADHEr, LDH_D, MDH, PFLi, PGDH, PGI, PPCK
813
7
5.40117
0.10549
ADHEr, FUM, LDH_D, PFLi, PGDH, PGI, PPCK
814
7
5.38947
0.11011
ACKr and/or PTAr, ADHEr, FUM, LDH_D, NADH6, PPCK, PYK
815
7
5.37417
0.11441
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2
and/or GLUDy
816
7
5.37043
0.12143
ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, RPE
817
7
5.37015
0.12013
ADHEr, ASPT, EDA and/or
PGDHY, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
818
7
5.36414
0.12087
ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, TAL
819
7
5.3573
0.12027
ADHEr, LDH_D, MDH, NADH6, PFLi, PGDH, PGI
820
7
5.3512
0.12232
ADHEr, LDH_D, NADH12, NADH6, PFLi, PRO1z, THD2 and/or
GLUDy
821
7
5.35114
0.12824
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, RPE
822
7
5.34992
0.12867
ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, RPE
823
7
5.34421
0.12775
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, TAL
824
7
5.34298
0.12817
ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, TAL
825
7
5.33668
0.12721
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGDH, PGI
826
7
5.33544
0.12763
ADHEr, FUM, LDH_D, NADH6, PFLi, PGDH, PGI
827
7
5.32853
0.04869
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or
G6PDHy, PPCK, THD2 and/or GLUDy
828
7
5.32586
0.13717
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, RPE
829
7
5.31986
0.13616
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, TAL
830
7
5.31768
0.14006
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, RPE
831
7
5.31768
0.14006
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, RPE
832
7
5.31338
0.13506
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGL and/or
G6PDHy, PPCK
833
7
5.31338
0.13506
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGDH, PPCK
834
7
5.31161
0.139
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, TAL
835
7
5.31161
0.139
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, TAL
836
7
5.30507
0.13786
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGL and/or
G6PDHy, PPCK
837
7
5.30507
0.13786
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, PPCK
838
7
5.30507
0.13786
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGDH, PPCK
839
7
5.30121
0.14588
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK, RPE
840
7
5.29298
0.14544
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK, TAL
841
7
5.29144
0.14245
ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PFLi
842
7
5.28753
0.08933
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, THD2 and/or
GLUDy
843
7
5.28422
0.14489
ADHEr, ATPS4r, FDH2, LDH_D, NADH12, NADH6, THD2
and/or GLUDy
844
7
5.28422
0.14489
ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
845
7
5.28401
0.14496
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PGDH, PPCK
846
7
5.28401
0.14496
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PGL and/or
G6PDHy, PPCK
847
7
5.27588
0.18393
ADHEr, FUM, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy
848
7
5.27588
0.18393
ADHEr, FUM, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2
and/or GLUDy
849
7
5.2736
0.14846
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
850
7
5.25759
0.18586
ADHEr, FUM, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy
851
7
5.24329
0.06953
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, THD2 and/or
GLUDy
852
7
5.24057
0.18767
ADHEr, FUM, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy
853
7
5.23194
0.07004
ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL, THD2 and/or
GLUDy
854
7
5.22143
0.07051
ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE, THD2 and/or
GLUDy
855
7
5.1096
0.03265
ADHEr, ASPT, FUM, GLUDy, HEX1, LDH_D, THD2 and/or
GLUDy
856
7
5.07833
0.23187
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH6, THD2 and/or
GLUDy
857
7
4.9126
0.20186
ADHEr, HEX1, LDH_D, PGDH, PPS, TAL, THD2 and/or GLUDy
858
7
4.9126
0.20186
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, TAL, THD2
and/or GLUDy
859
7
4.89537
0.08136
ACKr and/or
PTAr, ADHEr, ATPS4r, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
860
7
4.86469
0.20078
ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK, RPE
861
7
4.86188
0.1035
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, PYK, THD2
and/or GLUDy
862
7
4.85917
0.04186
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
863
7
4.85917
0.04186
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PPCK, THD2 and/or
GLUDy
864
7
4.84043
0.16101
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, PYK
865
7
4.75352
0.20131
ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK, TAL
866
7
4.6646
0.15914
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, THD2 and/or
GLUDy
867
7
4.6568
0.25634
ADHEr, ATPS4r, LDH_D, MALS, MDH, PGL and/or
G6PDHy, PPS
868
7
4.6568
0.25634
ADHEr, ATPS4r, ICL, LDH_D, MDH, PGL and/or G6PDHy, PPS
869
7
4.6568
0.25634
ADHEr, ATPS4r, FUM, LDH_D, MDH, PGL and/or G6PDHy, PPS
870
7
4.60984
0.1687
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, RPE, THD2
and/or GLUDy
871
7
4.60771
0.13834
ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, THD2 and/or
GLUDy
872
7
4.5647
0.0741
ADHEr, ATPS4r, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
873
7
4.51839
0.16783
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, TAL, THD2
and/or GLUDy
874
7
4.48284
0.18237
ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, PPCK
875
7
4.46608
0.18828
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH12, NADH6
876
7
4.43524
0.31394
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGDH, PPS
877
7
4.43524
0.31394
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGL and/or G6PDHy, PPS
878
7
4.43072
0.22439
ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, RPE
879
7
4.41919
0.16689
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, PGDH, THD2 and/or
GLUDy
880
7
4.4053
0.01495
ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2
and/or GLUDy
881
7
4.39394
0.31457
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PPS, TAL
882
7
4.37728
0.02565
ADHEr, ASPT, LDH_D, MDH, PGI, PPCK, THD2 and/or GLUDy
883
7
4.37061
0.04989
ADHEr, ATPS4r, LDH_D, NADH6, PGI, PPCK, THD2 and/or
GLUDy
884
7
4.36653
0.05265
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
885
7
4.36374
0.03082
ADHEr, ASPT, LDH_D, MDH, NADH6, PGI, THD2 and/or
GLUDy
886
7
4.36277
0.03119
ADHEr, ASPT, GLCpts, LDH_D, MDH, PGI, THD2 and/or
GLUDy
887
7
4.35613
0.31514
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PPS, RPE
888
7
4.29026
0.24034
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, GLU5K, GLYCL, LDH_D, PGI
889
7
4.29026
0.24034
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, G5SD, GLYCL, LDH_D, PGI
890
7
4.25283
0.07318
ADHEr, ASPT, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy
891
7
4.17894
0.31889
ADHEr, HEX1, LDH_D, NADH12, NADH6, PPS, THD2 and/or
GLUDy
892
7
4.17527
0.08598
ADHEr, ATPS4r, FRD and/or
SUCD4, HEX1, LDH_D, MALS, THD2 and/or GLUDy
893
7
4.17527
0.08598
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2
and/or GLUDy
894
7
4.17215
0.10398
ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
895
7
4.15042
0.11229
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
896
7
4.14938
0.11268
ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK, THD2 and/or
GLUDy
897
7
4.141
0.08314
ADHEr, ATPS4r, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
898
7
4.10258
0.11665
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PPCK, PYK, SUCOAS
899
7
4.08743
0.09886
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PPCK, SUCOAS, THD2 and/or
GLUDy
900
7
4.04907
0.15099
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, PPCK
901
7
4.03008
0.1761
ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK, RPE
902
7
4.01398
0.06136
ADHEr, ATPS4r, LDH_D, PFLi, PGI, PPCK, THD2 and/or GLUDy
903
7
4.00698
0.18592
ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK, RPE
904
7
4.00128
0.18834
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, RPE
905
7
3.95205
0.10694
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PGDH, PGI, SUCOAS
906
7
3.94203
0.1079
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PGI, SUCOAS, TAL
907
7
3.93273
0.1088
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGI, RPE, SUCOAS
908
7
3.90038
0.04761
ADHEr, GLCpts, LDH_D, NADH6, PGI, PPCK, THD2 and/or
GLUDy
909
7
3.8947
0.05032
ADHEr, FUM, GLCpts, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
910
7
3.8947
0.05032
ADHEr, GLCpts, LDH_D, MDH, NADH6, PGI, THD2 and/or
GLUDy
911
7
3.88125
0.03656
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or
GLUDy
912
7
3.86624
0.19846
ACKr and/or
PTAr, ADHEr, CITL, LDH_D, NADH12, NADH6, THD2 and/or
GLUDy
913
7
3.82356
0.31976
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PPS, THD2 and/or
GLUDy
914
7
3.81228
0.08972
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PYK, THD2 and/or GLUDy
915
7
3.81228
0.08972
ADHEr, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PYK, THD2 and/or GLUDy
916
7
3.77115
0.10937
ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK, THD2 and/or
GLUDy
917
7
3.7291
0.12947
ADHEr, LDH_D, MDH, NADH12, NADH6, PYK, THD2 and/or
GLUDy
918
7
3.7245
0.13167
ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, THD2 and/or
GLUDy
919
7
3.68994
0.14818
ADHEr, GLCpts, LDH_D, NADH12, NADH6, PPCK, THD2 and/or
GLUDy
920
7
3.67651
0.1546
ADHEr, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PPCK, PYK
921
7
3.67651
0.1546
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PPCK, PYK
922
7
3.64497
0.19156
ADHEr, FUM, GLCpts, LDH_D, NADH6, RPE, THD2 and/or
GLUDy
923
7
3.60739
0.21183
ADHEr, FUM, LDH_D, NADH12, NADH6, RPE, THD2 and/or
GLUDy
924
7
3.41696
0.27865
ADHEr, ALAR, FDH2, LDH_D, NADH12, NADH6, PRO1z
925
7
3.41696
0.27865
ADHEr, DAAD, FDH2, LDH_D, NADH12, NADH6, PRO1z
926
7
3.40396
0.28486
ADHEr, FUM, HEX1, LDH_D, NADH12, NADH6, THD2 and/or
GLUDy
927
7
3.25909
0.04258
ADHEr, GLCpts, LDH_D, MDH, PGI, PPCK, THD2 and/or
GLUDy
928
7
3.25909
0.04258
ADHEr, FUM, GLCpts, LDH_D, PGI, PPCK, THD2 and/or GLUDy
929
7
3.2372
0.06431
ADHEr, FBP, LDH_D, MDH, PGI, PPCK, THD2 and/or GLUDy
930
7
3.2372
0.06431
ADHEr, FBP, FUM, LDH_D, PGI, PPCK, THD2 and/or GLUDy
931
7
3.23399
0.06432
ADHEr, FUM, LDH_D, PGI, PPCK, RPE, THD2 and/or GLUDy
932
7
3.23399
0.06432
ADHEr, LDH_D, MDH, PGI, PPCK, RPE, THD2 and/or GLUDy
933
7
3.22783
0.06435
ADHEr, FUM, LDH_D, PGI, PPCK, TAL, THD2 and/or GLUDy
934
7
3.22783
0.06435
ADHEr, LDH_D, MDH, PGI, PPCK, TAL, THD2 and/or GLUDy
935
7
3.01859
0.19992
ADHEr, ASPT, FUM, LDH_D, PPS, SUCOAS, THD2 and/or
GLUDy
936
7
3.00174
0.19757
ADHEr, FRD and/or SUCD4, LDH_D, MALS, PPS, RPE, THD2
and/or GLUDy
937
7
3.00174
0.19757
ADHEr, FRD and/or SUCD4, ICL, LDH_D, PPS, RPE, THD2
and/or GLUDy
938
7
2.96091
0.22716
ADHEr, FUM, GLUDy, HEX1, LDH_D, TAL, THD2 and/or
GLUDy
939
7
2.93716
0.22721
ADHEr, FUM, GLU5K, GLUDy, HEX1, LDH_D, THD2 and/or
GLUDy
940
7
2.93716
0.22721
ADHEr, FUM, G5SD, GLUDy, HEX1, LDH_D, THD2 and/or
GLUDy
941
7
2.93711
0.22724
ADHEr, ASNS2, FUM, GLUDy, HEX1, LDH_D, THD2 and/or
GLUDy
942
7
2.7731
0.32129
ADHEr, ASNS2, FUM, GLU5K, HEX1, LDH_D, THD2 and/or
GLUDy
943
7
2.7731
0.32129
ADHEr, ASNS2, FUM, G5SD, HEX1, LDH_D, THD2 and/or
GLUDy
944
7
2.68652
0.29222
ADHEr, ATPS4r, GLUDy, HEX1, LDH_D, RPE, THD2 and/or
GLUDy
945
7
2.62059
0.29297
ADHEr, ATPS4r, GLUDy, HEX1, LDH_D, TAL, THD2 and/or
GLUDy
946
7
2.35991
0.29396
ACKr and/or PTAr, ADHEr, FUM, LDH_D, RPE, SUCOAS, THD2
and/or GLUDy
947
7
2.35766
0.29368
ACKr and/or PTAr, ADHEr, FUM, LDH_D, SUCOAS, TAL, THD2
and/or GLUDy
948
7
2.3552
0.29337
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PGL and/or
G6PDHy, SUCOAS, THD2 and/or GLUDy
949
7
2.3552
0.29337
ACKr and/or
PTAr, ADHEr, FUM, LDH_D, PGDH, SUCOAS, THD2 and/or
GLUDy
950
7
1.91133
0.2766
ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, PGL and/or
G6PDHy, SUCOAS
951
7
1.91133
0.2766
ACKr and/or
PTAr, ADHEr, FUM, LDH_D, MDH, PGDH, SUCOAS
952
7
1.91133
0.2766
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, PGDH, SUCOAS
953
7
1.91133
0.2766
ACKr and/or PTAr, ADHEr, LDH_D, MALS, MDH, PGL and/or
G6PDHy, SUCOAS
954
7
1.91133
0.2766
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, PGL and/or
G6PDHy, SUCOAS
955
7
1.91133
0.2766
ACKr and/or
PTAr, ADHEr, LDH_D, MALS, MDH, PGDH, SUCOAS
956
7
1.86676
0.2774
ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, SUCOAS, TAL
957
7
1.86676
0.2774
ACKr and/or
PTAr, ADHEr, LDH_D, MALS, MDH, SUCOAS, TAL
958
7
1.86676
0.2774
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, SUCOAS, TAL
959
7
1.8259
0.27813
ACKr and/or
PTAr, ADHEr, LDH_D, MALS, MDH, RPE, SUCOAS
960
7
1.8259
0.27813
ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, RPE, SUCOAS
961
7
1.8259
0.27813
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, RPE, SUCOAS
962
7
1.11652
0.25888
ADHEr, ASPT, FUM, LDH_D, MALS, PPS, THD2 and/or GLUDy
963
7
1.11652
0.25888
ADHEr, ASPT, FUM, ICL, LDH_D, PPS, THD2 and/or GLUDy
964
5
7.09275
0.11213
ADHEr, ASPT, LDH_D, MDH, PFLi
965
5
6.00696
0.20464
ADHEr, ATPS4r, LDH_D, NADH6, PFLi
966
5
5.40138
0.11184
ADHEr, LDH_D, NADH6, PFLi, PGI
967
5
2.59719
0.29318
ADHEr, FUM, HEX1, LDH_D, PFLi
968
5
2.57488
0.22587
ADHEr, HEX1, LDH_D, PFLi, PPCK
969
5
0.16844
0.19119
ADHEr, ASPT, FUM, LDH_D, PFLi
970
6
7.39516
0.07475
ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi
971
6
7.007
0.09285
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PFLi, PGI
972
6
6.90414
0.13454
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PFLi
973
6
6.57261
0.1606
ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi
974
6
6.00781
0.09114
ADHEr, LDH_D, MDH, PFLi, PGI, PPCK
975
6
6.00781
0.09114
ADHEr, FUM, LDH_D, PFLi, PGI, PPCK
976
6
5.39072
0.10901
ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK
977
6
5.38861
0.10972
ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK
978
6
5.38861
0.10972
ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK
979
6
5.06553
0.21856
ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi
980
6
5.00212
0.2515
ADHEr, HEX1, LDH_D, NADH6, PFLi, RPE
981
6
4.98367
0.25221
ADHEr, HEX1, LDH_D, NADH6, PFLi, TAL
982
6
4.96609
0.25206
ADHEr, GLU5K, HEX1, LDH_D, NADH6, PFLi
983
6
4.96609
0.25206
ADHEr, G5SD, HEX1, LDH_D, NADH6, PFLi
984
6
4.96599
0.2521
ADHEr, ASNS2, HEX1, LDH_D, NADH6, PFLi
985
6
4.95562
0.24186
ADHEr, EDA and/or PGDHY, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
986
6
2.71835
0.29123
ADHEr, FUM, HEX1, LDH_D, PFLi, RPE
987
6
2.66849
0.22473
ADHEr, HEX1, LDH_D, PFLi, PPCK, RPE
988
6
2.66076
0.29216
ADHEr, FUM, HEX1, LDH_D, PFLi, TAL
989
6
1.85699
0.28717
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PFLi, SUCOAS
990
6
1.63724
0.03081
ADHEr, FUM, LDH_D, PFLi, PGI, THD2 and/or GLUDy
991
6
1.63724
0.03081
ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy
992
6
1.38263
0.33268
ADHEr, ATPS4r, LDH_D, NADH12, PFLi, THD2 and/or GLUDy
993
6
0.25855
0.07515
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PFLi
994
7
7.86642
0.01651
ADHEr, ASPT, LDH_D, MDH, NADH6, PFLi, PYK
995
7
7.71509
0.0515
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGI, PPS
996
7
7.70416
0.03656
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PFLi, PPCK
997
7
7.48515
0.04452
ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, PFLi, PGI, PPCK
998
7
7.44181
0.09832
ACKr and/or
PTAr, ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi
999
7
7.06656
0.04513
ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi
1000
7
7.00811
0.04002
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PPCK
1001
7
7.00811
0.04002
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PPCK
1002
7
7.00532
0.07752
ADHEr, ATPS4r, FUM, LDH_D, NADH12, NADH6, PFLi
1003
7
6.99105
0.04159
ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PPCK
1004
7
6.96695
0.09776
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PFLi
1005
7
6.69345
0.1479
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
1006
7
6.49322
0.12467
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PPS
1007
7
6.43565
0.1244
ACKr and/or
PTAr, ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi
1008
7
6.21891
0.18801
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGDH, PPS
1009
7
6.21891
0.18801
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGL and/or
G6PDHy, PPS
1010
7
6.21459
0.18835
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPS, TAL
1011
7
6.21064
0.18866
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPS, RPE
1012
7
6.16665
0.08397
ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, PGI
1013
7
6.13458
0.01447
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, PPCK
1014
7
6.13051
0.01457
ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, PPCK
1015
7
6.13051
0.01457
ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, PPCK
1016
7
5.49961
0.07232
ADHEr, GLCpts, LDH_D, NADH12, NADH6, PFLi, PPCK
1017
7
5.49821
0.07279
ADHEr, FUM, GLCpts, LDH_D, NADH6, PFLi, PPCK
1018
7
5.49821
0.07279
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, PPCK
1019
7
5.22113
0.16614
ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2 and/or
GLUDy
1020
7
5.17418
0.18196
ADHEr, FUM, HEX1, LDH_D, NADH12, NADH6, PFLi
1021
7
5.15097
0.18978
ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
1022
7
4.01417
0.12149
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, THD2 and/or
GLUDy
1023
7
3.75439
0.27459
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PPS
1024
7
3.75439
0.27459
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, PPS
1025
7
2.64048
0.21814
ADHEr, HEX1, LDH_D, PFLi, PGL and/or G6PDHy, PPCK, TAL
1026
7
2.64048
0.21814
ADHEr, HEX1, LDH_D, PFLi, PGDH, PPCK, TAL
1027
7
2.62716
0.22497
ADHEr, GLYCL, HEX1, LDH_D, PFLi, PPCK, TAL
1028
7
1.5697
0.24425
ACKr and/or PTAr, ACS, ADHEr, LDH_D, MDH, PFLi, THD2
and/or GLUDy
1029
7
1.09053
0.33566
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGDH, PPS
1030
7
1.09053
0.33566
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGL and/or G6PDHy, PPS
1031
7
0.78572
0.33663
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPS, TAL
1032
7
0.50621
0.33751
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPS, RPE
1033
8
7.88716
0.0204
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi
1034
8
7.88653
0.02051
ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi, POX
1035
8
7.88026
0.02165
ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PFLi, THD2
and/or GLUDy
1036
8
7.84104
0.02874
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, NADH6, PFLi, PGI, PPS
1037
8
7.80925
0.03448
ADHEr, ATPS4r, FUM, LDH_D, NADH12, NADH6, PFLi, PGI
1038
8
7.80925
0.03448
ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi, PGI
1039
8
7.67439
0.05735
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PFLi
1040
8
7.42051
0.09374
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PFLi, PPS
1041
8
7.32674
0.0933
ACKr and/or
PTAr, ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PFLi
1042
8
6.93593
0.04641
ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, PPCK, THD2
and/or GLUDy
1043
8
6.58791
0.09796
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi, PGL and/or
G6PDHy, PPS
1044
8
6.58791
0.09796
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi, PGDH, PPS
1045
8
6.5802
0.09979
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi, PPS, TAL
1046
8
6.57291
0.10152
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi, PPS, RPE
1047
8
5.75656
0.15131
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi, PPS, THD2
and/or GLUDy
1048
8
5.74602
0.16073
ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, PPS, THD2
and/or GLUDy
1049
8
5.67321
0.16148
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2
and/or GLUDy
1050
8
5.60169
0.03793
ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, PPCK, PYK
1051
8
5.60169
0.03793
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, PPCK, PYK
1052
8
5.56692
0.04965
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PFLi, PPCK, PYK
1053
8
5.56692
0.04965
ADHEr, LDH_D, MDH, NADH6, PFLi, PGL and/or
G6PDHy, PPCK, PYK
1054
8
5.50925
0.07241
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PPCK, RPE, THD2 and/or
GLUDy
1055
8
5.50597
0.07191
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PPCK, TAL, THD2 and/or
GLUDy
1056
8
5.50242
0.07138
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGL and/or
G6PDHy, PPCK, THD2 and/or GLUDy
1057
8
5.50242
0.07138
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGDH, PPCK, THD2
and/or GLUDy
1058
8
5.44507
0.0907
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi, PRO1z, THD2
and/or GLUDy
1059
8
5.37306
0.11496
ADHEr, DAAD, LDH_D, MDH, NADH12, NADH6, PFLi, PRO1z
1060
8
5.37306
0.11496
ADHEr, ALAR, LDH_D, MDH, NADH12, NADH6, PFLi, PRO1z
1061
8
5.33606
0.12742
ADHEr, FUM, LDH_D, MDH, NADH12, NADH6, PFLi, PYK
1062
8
5.17764
0.18079
ADHEr, DAAD, HEX1, LDH_D, NADH12, NADH6, PFLi, PRO1z
1063
8
5.17764
0.18079
ADHEr, ALAR, HEX1, LDH_D, NADH12, NADH6, PFLi, PRO1z
1064
8
5.17492
0.2398
ACKr and/or PTAr, ACS, ADHEr, EDA and/or
PGDHY, LDH_D, MDH, PFLi, THD2 and/or GLUDy
1065
8
4.65995
0.20434
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, PPS, RPE
1066
8
4.6123
0.20163
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, PPS, TAL
1067
8
4.56149
0.19874
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, PGDH, PPS
1068
8
4.13777
0.23628
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PPS, RPE
1069
8
4.06785
0.23403
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PPS, TAL
1070
8
4.04062
0.10178
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PDH, PFLi, PPS
1071
8
4.04062
0.10178
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PDH, PFLi, PPS
1072
8
3.9927
0.23161
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PGL and/or
G6PDHy, PPS
1073
8
3.9927
0.23161
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PGDH, PPS
1074
8
2.03637
0.11796
ACKr and/or
PTAr, ADHEr, FBP, LDH_D, MDH, PFLi, PGDH, THD2 and/or
GLUDy
1075
8
1.98516
0.11932
ACKr and/or
PTAr, ADHEr, FBP, LDH_D, MDH, PFLi, TAL, THD2 and/or
GLUDy
1076
8
1.93743
0.12058
ACKr and/or
PTAr, ADHEr, FBP, LDH_D, MDH, PFLi, RPE, THD2 and/or
GLUDy
1077
8
1.76983
0.24401
ACKr and/or
PTAr, ACS, ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or
GLUDy
1078
8
1.5107
0.31956
ADHEr, ATPS4r, LDH_D, NADH12, PFLi, PGL and/or
G6PDHy, TAL, THD2 and/or GLUDy
1079
8
1.5107
0.31956
ADHEr, ATPS4r, LDH_D, NADH12, PFLi, PGDH, TAL, THD2
and/or GLUDy
1080
8
1.45644
0.3201
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGDH, PPS, TAL
1081
8
1.45644
0.3201
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGL and/or
G6PDHy, PPS, TAL
1082
3
0.165681142
0.248770484
ADHEr, LDH_D, PFK and/or FBA and/or TPI
1083
3
2.792461067
0.310192487
ADHEr, FRD and/or SUCD4, LDH_D
1084
4
0.245091981
0.278197481
ADHEr, ASPT, FUM, LDH_D
1085
4
1.163607813
0.384018912
ADHEr, ATPS4r, CBMK2, LDH_D
1086
4
2.27265597
0.332308612
ADHEr, ATPS4r, LDH_D, NADH6
1087
4
1.260046787
0.384504476
ADHEr, ATPS4r, LDH_D, TKT1
1088
4
1.454891201
0.378757536
ADHEr, ATPS4r, LDH_D, TKT2
1089
4
2.042634808
0.153142196
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI
1090
4
2.041701304
0.149789093
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK
1091
4
0.270967943
0.244721556
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT2
1092
4
2.907366868
0.244293538
ADHEr, FUM, LDH_D, PPCK
1093
4
0.363337262
0.340044232
ADHEr, HEX1, LDH_D, MDH
1094
4
2.907366868
0.244293538
ADHEr, LDH_D, MDH, PPCK
1095
4
2.085082479
0.31290916
ADHEr, LDH_D, ME2, NADH6
1096
4
2.881545955
0.259101938
ADHEr, FRD and/or SUCD4, LDH_D, ME2
1097
4
3.647199605
0.168610416
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI
1098
4
2.774595569
0.320438424
ADHEr, FDH2, LDH_D, NADH6
1099
4
3.335904914
0.317384404
ADHEr, FUM, LDH_D, NADH6
1100
4
3.331989713
0.319255557
ADHEr, HEX1, LDH_D, NADH6
1101
4
2.224731454
0.327457036
ADHEr, LDH_D, NADH6, TKT2
1102
4
0.23734264
0.246014656
ADHEr, LDH_D, PFK and/or FBA and/or TPI, RPE
1103
4
0.454138927
0.26931886
ADHEr, LDH_D, PPCK, TKT2
1104
4
0.413317212
0.38682004
ADHEr, HEX1, LDH_D, PPS
1105
4
2.860294682
0.306273023
ADHEr, FRD and/or SUCD4, LDH_D, RPE
1106
4
2.808840722
0.300798688
ADHEr, FRD and/or SUCD4, FUM, LDH_D
1107
4
2.817574813
0.295789651
ADHEr, FRD and/or SUCD4, HEX1, LDH_D
1108
4
2.916137696
0.239263432
ADHEr, FRD and/or SUCD4, LDH_D, MDH
1109
4
2.973865652
0.206156212
ADHEr, FRD and/or SUCD4, LDH_D, PPCK
1110
4
2.892050239
0.30443817
ADHEr, FRD and/or SUCD4, LDH_D, TKT2
1111
4
0.203360099
0.247321495
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TAL
1112
4
2.117903686
0.331070152
ADHEr, LDH_D, NADH6, TAL
1113
4
0.34884992
0.271004016
ADHEr, LDH_D, PPCK, TAL
1114
4
2.828154021
0.308130129
ADHEr, FRD and/or SUCD4, LDH_D, TAL
1115
4
0.203360099
0.247321495
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT1
1116
4
2.117903686
0.331070152
ADHEr, LDH_D, NADH6, TKT1
1117
4
0.34884992
0.271004016
ADHEr, LDH_D, PPCK, TKT1
1118
4
2.828154021
0.308130129
ADHEr, FRD and/or SUCD4, LDH_D, TKT1
1119
5
3.948360602
0.222825522
ADHEr, ASPT, LDH_D, MDH, TKT2
1120
5
3.966967048
0.182343827
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH
1121
5
3.752898719
0.126622688
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, ME2
1122
5
4.408716092
0.258053531
ADHEr, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy
1123
5
6.415221004
0.039824926
ADHEr, FADH4, LDH_D, MDH, THD2 and/or GLUDy
1124
5
2.983060356
0.241650921
ADHEr, FUM, LDH_D, PPCK, TKT2
1125
5
2.997718029
0.231538695
ADHEr, FUM, LDH_D, THD2 and/or GLUDy, TKT2
1126
5
2.937396425
0.227071464
ADHEr, FUM, HEX1, LDH_D, THD2 and/or GLUDy
1127
5
3.001461104
0.190330107
ADHEr, FRD and/or SUCD4, FUM, LDH_D, THD2 and/or
GLUDy
1128
5
2.935886887
0.22793719
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, THD2 and/or
GLUDy
1129
5
3.081083028
0.144666606
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, THD2 and/or
GLUDy
1130
5
2.988449413
0.197792365
ADHEr, FRD and/or SUCD4, LDH_D, PRO1z, THD2 and/or
GLUDy
1131
5
2.948350239
0.234817401
ADHEr, FUM, LDH_D, TAL, THD2 and/or GLUDy
1132
5
3.101419157
0.133003752
ADHEr, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
1133
5
2.948350239
0.234817401
ADHEr, FUM, LDH_D, THD2 and/or GLUDy, TKT1
1134
5
2.983060356
0.241650921
ADHEr, LDH_D, MDH, PPCK, TKT2
1135
5
2.916137696
0.239263432
ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2
1136
5
3.711787388
0.165696592
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT2
1137
5
3.457829994
0.311699463
ADHEr, FUM, LDH_D, NADH6, TKT2
1138
5
3.451407023
0.315392099
ADHEr, HEX1, LDH_D, NADH6, TKT2
1139
5
3.543757983
0.262298527
ADHEr, LDH_D, MDH, NADH6, TKT2
1140
5
3.445461549
0.265025086
ADHEr, FUM, LDH_D, ME2, NADH6
1141
5
3.609567252
0.224464061
ADHEr, LDH_D, NADH6, PPCK, TKT2
1142
5
3.691174432
0.166626529
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, RPE
1143
5
3.065706296
0.153485225
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PFK and/or FBA
and/or TPI
1144
5
3.071383178
0.15022951
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PFK and/or FBA
and/or TPI
1145
5
5.367175665
0.178044995
ADHEr, CBMK2, HEX1, LDH_D, PGI
1146
5
5.888703714
0.106589745
ADHEr, EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, PGI
1147
5
3.110919215
0.148780301
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PGI
1148
5
2.958795095
0.242498072
ADHEr, FUM, LDH_D, PPCK, RPE
1149
5
2.958795095
0.242498072
ADHEr, LDH_D, MDH, PPCK, RPE
1150
5
2.936051582
0.257223558
ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE
1151
5
3.418935049
0.313512999
ADHEr, FUM, LDH_D, NADH6, RPE
1152
5
3.413158878
0.316629525
ADHEr, HEX1, LDH_D, NADH6, RPE
1153
5
2.967063614
0.237144547
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE
1154
5
3.018085814
0.204109778
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, RPE
1155
5
3.009552774
0.185689501
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PPCK
1156
5
3.009552774
0.185689501
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PPCK
1157
5
2.991038247
0.23614703
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT2
1158
5
2.961772661
0.256337155
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT2
1159
5
3.106778446
0.151550201
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PGI
1160
5
3.04583421
0.164881948
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK
1161
5
3.038870487
0.203147899
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT2
1162
5
3.886386663
0.225053566
ADHEr, ASPT, LDH_D, MDH, TAL
1163
5
3.670328799
0.167566962
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TAL
1164
5
3.480501908
0.264053132
ADHEr, LDH_D, MDH, NADH6, TAL
1165
5
3.554829627
0.226273464
ADHEr, LDH_D, NADH6, PPCK, TAL
1166
5
2.942885577
0.238150528
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL
1167
5
2.99710338
0.20508081
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TAL
1168
5
3.964901837
0.267840742
ADHEr, HEX1, LDH_D, MDH, THD2 and/or GLUDy
1169
5
3.00074032
0.229453637
ADHEr, LDH_D, MDH, THD2 and/or GLUDy, TKT2
1170
5
4.557091208
0.201359081
ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or
GLUDy
1171
5
2.935925119
0.227915263
ADHEr, LDH_D, MDH, PGDH, THD2 and/or GLUDy
1172
5
2.97971305
0.22895456
ADHEr, LDH_D, MDH, RPE, THD2 and/or GLUDy
1173
5
2.958777052
0.228457649
ADHEr, LDH_D, MDH, TAL, THD2 and/or GLUDy
1174
5
3.886386663
0.225053566
ADHEr, ASPT, LDH_D, MDH, TKT1
1175
5
2.934359102
0.243351183
ADHEr, FUM, LDH_D, PPCK, TKT1
1176
5
2.934359102
0.243351183
ADHEr, LDH_D, MDH, PPCK, TKT1
1177
5
3.670328799
0.167566962
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT1
1178
5
3.379584856
0.31534776
ADHEr, FUM, LDH_D, NADH6, TKT1
1179
5
3.374609422
0.317876699
ADHEr, HEX1, LDH_D, NADH6, TKT1
1180
5
3.480501908
0.264053132
ADHEr, LDH_D, MDH, NADH6, TKT1
1181
5
3.554829627
0.226273464
ADHEr, LDH_D, NADH6, PPCK, TKT1
1182
5
2.942885577
0.238150528
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT1
1183
5
2.99710338
0.20508081
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT1
1184
5
2.958777052
0.228457649
ADHEr, LDH_D, MDH, THD2 and/or GLUDy, TKT1
TABLE 15
Knockout strain designs for increased production of adipate,
showing yields of adipate and biomass.
New
Design
Num
Adipate.
ID
KO
Yield
Biomass
Rxnlist
1
3
5.47908
0.34054
ADHEr, LDH_D, NADH6
2
3
4.26684
0.28031
ADHEr, LDH_D, PPCK
3
3
2.76166
0.38773
ADHEr, ATPS4r, LDH_D
4
3
1.54669
0.25966
ADHEr, LDH_D, PGI
5
3
1.33726
0.37808
ADHEr, FUM, LDH_D
6
3
0.83242
0.38953
ADHEr, HEX1, LDH_D
7
4
7.52636
0.16586
ADHEr, HEX1, LDH_D, PGI
8
4
6.84269
0.27849
ADHEr, LDH_D, NADH6, PFLi
9
4
6.5702
0.20631
ADHEr, ASPT, LDH_D, MDH
10
4
6.16994
0.16614
ADHEr, LDH_D, NADH6, PGI
11
4
6.0882
0.30766
ADHEr, LDH_D, MDH, THD2 and/or GLUDy
12
4
5.87407
0.22728
ADHEr, LDH_D, NADH6, PPCK
13
4
5.68406
0.28176
ADHEr, LDH_D, MDH, NADH6
14
4
5.62555
0.33704
ADHEr, LDH_D, NADH6, RPE
15
4
5.60596
0.30416
ADHEr, LDH_D, NADH6, THD2 and/or GLUDy
16
4
5.55606
0.3387
ADHEr, LDH_D, NADH6, TAL
17
4
5.54557
0.32148
ADHEr, FUM, LDH_D, NADH6
18
4
5.16028
0.24175
ADHEr, LDH_D, PFLi, PPCK
19
4
4.91511
0.27067
ADHEr, LDH_D, PPCK, THD2 and/or GLUDy
20
4
4.3568
0.24592
ADHEr, GLCpts, LDH_D, PPCK
21
4
4.35358
0.27902
ADHEr, LDH_D, PPCK, RPE
22
4
4.34095
0.25198
ADHEr, FUM, LDH_D, PPCK
23
4
4.34095
0.25198
ADHEr, LDH_D, MDH, PPCK
24
4
4.28493
0.2734
ADHEr, FUM, LDH_D, THD2 and/or GLUDy
25
4
4.0889
0.34834
ADHEr, FUM, HEX1, LDH_D
26
4
3.25969
0.38161
ADHEr, ATPS4r, LDH_D, RPE
27
4
3.18007
0.37054
ADHEr, HEX1, LDH_D, THD2 and/or GLUDy
28
4
3.11658
0.34524
ADHEr, ATPS4r, LDH_D, THD2 and/or GLUDy
29
4
3.02411
0.3845
ADHEr, ATPS4r, LDH_D, TAL
30
4
1.1506
0.38695
ADHEr, HEX1, LDH_D, RPE
31
4
0.99936
0.38818
ADHEr, HEX1, LDH_D, TAL
32
5
7.63281
0.14897
ADHEr, HEX1, LDH_D, PGI, THD2 and/or GLUDy
33
5
7.60368
0.23979
ADHEr, ATPS4r, LDH_D, MDH, NADH6
34
5
7.25714
0.17876
ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy
35
5
7.21061
0.18996
ADHEr, LDH_D, MDH, PFLi, PPCK
36
5
7.21061
0.18996
ADHEr, FUM, LDH_D, PFLi, PPCK
37
5
7.11676
0.21254
ADHEr, LDH_D, MDH, NADH6, PFLi
38
5
7.10568
0.21521
ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy
39
5
6.9706
0.24771
ADHEr, LDH_D, NADH6, PFLi, THD2 and/or GLUDy
40
5
6.90675
0.27577
ADHEr, LDH_D, NADH6, PFLi, RPE
41
5
6.83498
0.1681
ADHEr, ASPT, GLCpts, LDH_D, MDH
42
5
6.80758
0.29402
ADHEr, ATPS4r, LDH_D, MDH, TAL
43
5
6.79015
0.29114
ADHEr, ATPS4r, LDH_D, MDH, PGDH
44
5
6.76743
0.17785
ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy
45
5
6.76201
0.23293
ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy
46
5
6.66991
0.23493
ADHEr, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
47
5
6.66754
0.2967
ADHEr, ATPS4r, LDH_D, MDH, RPE
48
5
6.57746
0.21333
ADHEr, LDH_D, NADH6, PPCK, THD2 and/or GLUDy
49
5
6.34963
0.21785
ADHEr, ATPS4r, LDH_D, NADH6, PPCK
50
5
6.225
0.14772
ADHEr, LDH_D, NADH6, PGI, THD2 and/or GLUDy
51
5
6.18982
0.20058
ADHEr, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
52
5
6.18254
0.21218
ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy
53
5
6.18254
0.21218
ADHEr, FUM, LDH_D, PPCK, THD2 and/or GLUDy
54
5
5.99792
0.19177
ADHEr, GLCpts, LDH_D, NADH6, PPCK
55
5
5.97051
0.22537
ADHEr, LDH_D, NADH6, PPCK, RPE
56
5
5.92744
0.32733
ADHEr, ATPS4r, HEX1, LDH_D, NADH6
57
5
5.92472
0.22627
ADHEr, LDH_D, NADH6, PPCK, TAL
58
5
5.81113
0.24533
ADHEr, GLCpts, LDH_D, MDH, NADH6
59
5
5.80132
0.28014
ADHEr, LDH_D, MDH, NADH6, RPE
60
5
5.74556
0.28091
ADHEr, LDH_D, MDH, NADH6, TAL
61
5
5.74235
0.20973
ADHEr, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy
62
5
5.73575
0.30136
ADHEr, LDH_D, NADH6, RPE, THD2 and/or GLUDy
63
5
5.72421
0.22208
ADHEr, ATPS4r, LDH_D, MDH, PPCK
64
5
5.72421
0.22208
ADHEr, ATPS4r, FUM, LDH_D, PPCK
65
5
5.68542
0.31766
ADHEr, FUM, LDH_D, NADH6, RPE
66
5
5.67414
0.30269
ADHEr, LDH_D, NADH6, TAL, THD2 and/or GLUDy
67
5
5.62871
0.29764
ADHEr, FUM, HEX1, LDH_D, NADH6
68
5
5.61913
0.31947
ADHEr, FUM, LDH_D, NADH6, TAL
69
5
5.56433
0.06187
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI
70
5
5.53618
0.32417
ADHEr, HEX1, LDH_D, NADH6, THD2 and/or GLUDy
71
5
5.36114
0.24084
ADHEr, LDH_D, PFLi, PPCK, RPE
72
5
5.35732
0.32317
ADHEr, ATPS4r, FUM, HEX1, LDH_D
73
5
5.29193
0.25262
ADHEr, ATPS4r, FUM, LDH_D, THD2 and/or GLUDy
74
5
5.26552
0.24127
ADHEr, LDH_D, PFLi, PPCK, TAL
75
5
5.23886
0.36027
ADHEr, HEX1, LDH_D, PFLi, PPS
76
5
5.2123
0.3202
ADHEr, ATPS4r, HEX1, LDH_D, PFLi
77
5
5.20628
0.31113
ADHEr, FUM, HEX1, LDH_D, PFLi
78
5
5.20628
0.31113
ADHEr, HEX1, LDH_D, MDH, PFLi
79
5
5.00885
0.16853
ADHEr, ATPS4r, LDH_D, PFLi, PGI
80
5
4.73325
0.11896
ADHEr, LDH_D, PGI, PPCK, THD2 and/or GLUDy
81
5
4.71871
0.12544
ADHEr, LDH_D, MDH, PGI, THD2 and/or GLUDy
82
5
4.71871
0.12544
ADHEr, FUM, LDH_D, PGI, THD2 and/or GLUDy
83
5
4.46543
0.20438
ADHEr, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
84
5
4.45303
0.23609
ADHEr, LDH_D, MDH, RPE, THD2 and/or GLUDy
85
5
4.44799
0.21105
ADHEr, LDH_D, MDH, PYK, THD2 and/or GLUDy
86
5
4.43313
0.21674
ADHEr, GLCpts, LDH_D, MDH, PPCK
87
5
4.43313
0.21674
ADHEr, FUM, GLCpts, LDH_D, PPCK
88
5
4.43289
0.24479
ADHEr, GLCpts, LDH_D, PPCK, RPE
89
5
4.42239
0.22084
ADHEr, GLCpts, LDH_D, PPCK, THD2 and/or GLUDy
90
5
4.4202
0.25027
ADHEr, FUM, LDH_D, PPCK, RPE
91
5
4.4202
0.25027
ADHEr, LDH_D, MDH, PPCK, RPE
92
5
4.41922
0.25069
ADHEr, LDH_D, PPCK, RPE, THD2 and/or GLUDy
93
5
4.41348
0.22425
ADHEr, LDH_D, MDH, PPCK, PYK
94
5
4.41348
0.22425
ADHEr, FUM, LDH_D, PPCK, PYK
95
5
4.3742
0.27012
ADHEr, FUM, LDH_D, RPE, THD2 and/or GLUDy
96
5
4.34566
0.25018
ADHEr, FUM, GLCpts, LDH_D, THD2 and/or GLUDy
97
5
4.22695
0.31979
ADHEr, ATPS4r, HEX1, LDH_D, MDH
98
5
4.21297
0.36989
ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy
99
5
4.19799
0.34618
ADHEr, FUM, HEX1, LDH_D, RPE
100
5
4.15986
0.32121
ADHEr, FUM, HEX1, LDH_D, THD2 and/or GLUDy
101
5
4.14613
0.34721
ADHEr, FUM, HEX1, LDH_D, TAL
102
5
4.14548
0.36885
ADHEr, HEX1, LDH_D, RPE, THD2 and/or GLUDy
103
5
4.09366
0.36854
ADHEr, HEX1, LDH_D, TAL, THD2 and/or GLUDy
104
5
4.03694
0.36821
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, THD2 and/or
GLUDy
105
5
4.03694
0.36821
ADHEr, HEX1, LDH_D, PGDH, THD2 and/or GLUDy
106
5
3.55579
0.34037
ADHEr, ATPS4r, LDH_D, RPE, THD2 and/or GLUDy
107
5
3.35244
0.11579
ADHEr, FRD and/or SUCD4, LDH_D, PPS, THD2 and/or
GLUDy
108
5
3.34785
0.34268
ADHEr, ATPS4r, LDH_D, TAL, THD2 and/or GLUDy
109
5
2.86477
0.08801
ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI
110
5
1.41816
0.36252
ADHEr, FUM, LDH_D, PGL and/or G6PDHy, TAL
111
5
1.41816
0.36252
ADHEr, FUM, LDH_D, PGDH, TAL
112
6
8.40974
0.12863
ADHEr, GLUDy, LDH_D, MDH, PFLi, THD2 and/or GLUDy
113
6
8.3656
0.13555
ADHEr, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
114
6
8.3656
0.13555
ADHEr, FUM, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
115
6
8.30583
0.11621
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, THD2
and/or GLUDy
116
6
8.24494
0.15445
ADHEr, LDH_D, MDH, NADH6, PFLi, THD2 and/or GLUDy
117
6
7.99084
0.19426
ADHEr, ATPS4r, LDH_D, MDH, PFLi, THD2 and/or GLUDy
118
6
7.9508
0.20053
ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy
119
6
7.92831
0.12763
ADHEr, ATPS4r, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
120
6
7.91305
0.13531
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK
121
6
7.91305
0.13531
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK
122
6
7.87012
0.02274
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK, THD2
and/or GLUDy
123
6
7.7587
0.21695
ADHEr, LDH_D, MDH, NADH12, NADH6, THD2 and/or
GLUDy
124
6
7.75003
0.03607
ADHEr, ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy
125
6
7.74149
0.21948
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6
126
6
7.7368
0.0672
ADHEr, LDH_D, PFLi, PGI, PPCK, THD2 and/or GLUDy
127
6
7.72197
0.07098
ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy
128
6
7.72197
0.07098
ADHEr, FUM, LDH_D, PFLi, PGI, THD2 and/or GLUDy
129
6
7.67273
0.08355
ADHEr, LDH_D, NADH6, PFLi, PGI, THD2 and/or GLUDy
130
6
7.64843
0.19881
ADHEr, GLCpts, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
131
6
7.61824
0.14232
ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy
132
6
7.60825
0.2542
ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/or GLUDy
133
6
7.5467
0.16899
ADHEr, ATPS4r, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
134
6
7.48349
0.12429
ADHEr, FUM, LDH_D, NADH6, PFLi, THD2 and/or GLUDy
135
6
7.4685
0.07894
ADHEr, ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK
136
6
7.4685
0.07894
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PYK
137
6
7.44298
0.08037
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, THD2
and/or GLUDy
138
6
7.40415
0.13677
ADHEr, LDH_D, NADH6, PFLi, PPCK, PYK
139
6
7.4
0.14438
ADHEr, GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy
140
6
7.36241
0.15343
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK
141
6
7.36241
0.15343
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK
142
6
7.27527
0.1744
ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or
GLUDy
143
6
7.27448
0.17459
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi
144
6
7.24864
0.17893
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, THD2
and/or GLUDy
145
6
7.24278
0.26146
ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy
146
6
7.20895
0.28496
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, THD2 and/or GLUDy
147
6
7.18819
0.12101
ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy
148
6
7.17088
0.20914
ADHEr, LDH_D, MDH, PFLi, RPE, THD2 and/or GLUDy
149
6
7.15564
0.2082
ADHEr, LDH_D, MDH, PFLi, TAL, THD2 and/or GLUDy
150
6
7.1458
0.12325
ADHEr, ASPT, LDH_D, MDH, PPCK, THD2 and/or GLUDy
151
6
7.13909
0.20717
ADHEr, LDH_D, MDH, PFLi, PGDH, THD2 and/or GLUDy
152
6
7.12504
0.22071
ADHEr, LDH_D, PFLi, PPCK, RPE, THD2 and/or GLUDy
153
6
7.10894
0.21971
ADHEr, LDH_D, PFLi, PPCK, TAL, THD2 and/or GLUDy
154
6
7.09146
0.21863
ADHEr, LDH_D, PFLi, PGL and/or G6PDHy, PPCK, THD2
and/or GLUDy
155
6
7.09146
0.21863
ADHEr, LDH_D, PFLi, PGDH, PPCK, THD2 and/or GLUDy
156
6
7.0863
0.31638
ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy
157
6
7.06893
0.22405
ADHEr, LDH_D, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
158
6
7.054
0.1365
ADHEr, ASPT, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
159
6
7.02653
0.24555
ADHEr, LDH_D, NADH6, PFLi, RPE, THD2 and/or GLUDy
160
6
6.99568
0.14491
ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
161
6
6.92353
0.16046
ADHEr, ASPT, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
162
6
6.90243
0.27063
ADHEr, HEX1, LDH_D, NADH6, PFLi, TAL
163
6
6.8314
0.18819
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPCK
164
6
6.79251
0.30763
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi
165
6
6.70257
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGL and/or
G6PDHy, PPCK
166
6
6.70257
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGDH, PPCK
167
6
6.66743
0.21154
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, TAL
168
6
6.63518
0.21218
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, RPE
169
6
6.48093
0.19172
ADHEr, ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK
170
6
6.48093
0.19172
ADHEr, ATPS4r, FUM, LDH_D, PGDH, PPCK
171
6
6.44865
0.19659
ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK
172
6
6.44275
0.19249
ADHEr, ATPS4r, FUM, LDH_D, PPCK, TAL
173
6
6.40767
0.1932
ADHEr, ATPS4r, FUM, LDH_D, PPCK, RPE
174
6
6.33204
0.06396
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK, THD2
and/or GLUDy
175
6
6.28774
0.02127
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI, THD2
and/or GLUDy
176
6
6.27764
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGL and/or
G6PDHy
177
6
6.27764
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGDH
178
6
6.21873
0.32483
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, TAL
179
6
6.16484
0.32529
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, RPE
180
6
6.12301
0.15589
ADHEr, LDH_D, MDH, NADH6, PYK, THD2 and/or GLUDy
181
6
6.09151
0.3086
ADHEr, FUM, HEX1, LDH_D, PPS, THD2 and/or GLUDy
182
6
6.08785
0.16598
ADHEr, FUM, LDH_D, NADH6, PPCK, PYK
183
6
6.08785
0.16598
ADHEr, LDH_D, MDH, NADH6, PPCK, PYK
184
6
6.07929
0.19015
ADHEr, GLCpts, LDH_D, NADH6, PPCK, RPE
185
6
6.0681
0.17164
ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or
GLUDy
186
6
6.04302
0.20189
ADHEr, LDH_D, NADH6, PPCK, RPE, THD2 and/or GLUDy
187
6
6.01911
0.18569
ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK
188
6
6.01911
0.18569
ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK
189
6
5.98595
0.1952
ADHEr, FUM, GLCpts, LDH_D, NADH6, THD2 and/or
GLUDy
190
6
5.93003
0.21123
ADHEr, FUM, LDH_D, NADH12, NADH6, THD2 and/or
GLUDy
191
6
5.91322
0.24391
ADHEr, GLCpts, LDH_D, MDH, NADH6, RPE
192
6
5.89537
0.30664
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi
193
6
5.86467
0.24459
ADHEr, GLCpts, LDH_D, MDH, NADH6, TAL
194
6
5.8616
0.12855
ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
195
6
5.8616
0.12855
ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2 and/or GLUDy
196
6
5.75652
0.29464
ADHEr, FUM, HEX1, LDH_D, NADH6, RPE
197
6
5.71417
0.25845
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPS
198
6
5.69588
0.29606
ADHEr, FUM, HEX1, LDH_D, NADH6, TAL
199
6
5.53662
0.35787
ADHEr, HEX1, LDH_D, PFLi, PPS, RPE
200
6
5.4724
0.31504
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, RPE
201
6
5.46402
0.30945
ADHEr, FUM, HEX1, LDH_D, PFLi, RPE
202
6
5.46402
0.30945
ADHEr, HEX1, LDH_D, MDH, PFLi, RPE
203
6
5.39509
0.35901
ADHEr, HEX1, LDH_D, PFLi, PPS, TAL
204
6
5.34939
0.31748
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, TAL
205
6
5.34143
0.31025
ADHEr, HEX1, LDH_D, MDH, PFLi, TAL
206
6
5.34143
0.31025
ADHEr, FUM, HEX1, LDH_D, PFLi, TAL
207
6
5.07203
0.20521
ADHEr, LDH_D, MDH, PFLi, PGDH, PGI
208
6
5.07203
0.20521
ADHEr, FUM, LDH_D, PFLi, PGDH, PGI
209
6
5.04668
0.20656
ADHEr, FUM, LDH_D, PFLi, PGI, TAL
210
6
5.04668
0.20656
ADHEr, LDH_D, MDH, PFLi, PGI, TAL
211
6
5.02327
0.2078
ADHEr, LDH_D, MDH, PFLi, PGI, RPE
212
6
5.02327
0.2078
ADHEr, FUM, LDH_D, PFLi, PGI, RPE
213
6
4.96889
0.09918
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, THD2
and/or GLUDy
214
6
4.92469
0.33322
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2
and/or GLUDy
215
6
4.92469
0.33322
ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy
216
6
4.8531
0.05616
ADHEr, LDH_D, MDH, PGI, PPCK, THD2 and/or GLUDy
217
6
4.8531
0.05616
ADHEr, FUM, LDH_D, PGI, PPCK, THD2 and/or GLUDy
218
6
4.80931
0.34014
ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy
219
6
4.69982
0.3467
ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy
220
6
4.69121
0.11806
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, THD2
and/or GLUDy
221
6
4.64152
0.13706
ADHEr, FUM, GLCpts, LDH_D, PPCK, THD2 and/or GLUDy
222
6
4.64152
0.13706
ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2 and/or GLUDy
223
6
4.6299
0.1415
ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy
224
6
4.6299
0.1415
ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/or GLUDy
225
6
4.60999
0.16834
ADHEr, LDH_D, MDH, PPCK, RPE, THD2 and/or GLUDy
226
6
4.60999
0.16834
ADHEr, FUM, LDH_D, PPCK, RPE, THD2 and/or GLUDy
227
6
4.52953
0.20307
ADHEr, GLCpts, LDH_D, MDH, RPE, THD2 and/or GLUDy
228
6
4.5125
0.21043
ADHEr, LDH_D, MDH, PYK, RPE, THD2 and/or GLUDy
229
6
4.50129
0.21526
ADHEr, GLCpts, LDH_D, MDH, PPCK, RPE
230
6
4.50129
0.21526
ADHEr, FUM, GLCpts, LDH_D, PPCK, RPE
231
6
4.49048
0.21993
ADHEr, GLCpts, LDH_D, PPCK, RPE, THD2 and/or GLUDy
232
6
4.48211
0.22354
ADHEr, FUM, LDH_D, PPCK, PYK, RPE
233
6
4.48211
0.22354
ADHEr, LDH_D, MDH, PPCK, PYK, RPE
234
6
4.42882
0.24654
ADHEr, FUM, GLCpts, LDH_D, RPE, THD2 and/or GLUDy
235
6
4.36373
0.24327
ADHEr, FUM, GLUDy, HEX1, LDH_D, THD2 and/or GLUDy
236
6
4.35757
0.26123
ADHEr, FUM, HEX1, LDH_D, TAL, THD2 and/or GLUDy
237
6
4.31718
0.27765
ADHEr, ASNS2, GLU5K, LDH_D, PPCK, TAL
238
6
4.31718
0.27765
ADHEr, ASNS2, G5SD, LDH_D, PPCK, TAL
239
6
4.2573
0.32058
ADHEr, FUM, HEX1, LDH_D, RPE, THD2 and/or GLUDy
240
6
4.22017
0.33524
ADHEr, GLUDy, HEX1, LDH_D, RPE, THD2 and/or GLUDy
241
6
4.17617
0.33499
ADHEr, GLUDy, HEX1, LDH_D, TAL, THD2 and/or GLUDy
242
6
4.12454
0.33472
ADHEr, GLUDy, HEX1, LDH_D, PGDH, THD2 and/or
GLUDy
243
6
4.12454
0.33472
ADHEr, GLUDy, HEX1, LDH_D, PGL and/or
G6PDHy, THD2 and/or GLUDy
244
6
3.51205
0.21436
ADHEr, LDH_D, PFLi, PGI, PPS, THD2 and/or GLUDy
245
6
3.23372
0.26787
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, THD2
and/or GLUDy
246
6
2.95584
0.24016
ADHEr, FUM, LDH_D, PFLi, PGI, PPS
247
6
2.95584
0.24016
ADHEr, LDH_D, MDH, PFLi, PGI, PPS
248
7
9.10608
0.01953
ADHEr, GLUDy, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
249
7
9.10608
0.01953
ADHEr, FUM, GLUDy, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
250
7
9.00855
0.03482
ADHEr, GLUDy, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
251
7
8.9957
0.03683
ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
252
7
8.9957
0.03683
ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
253
7
8.98751
0.03811
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGI, THD2 and/or
GLUDy
254
7
8.98549
0.03843
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, THD2
and/or GLUDy
255
7
8.78817
0.06934
ADHEr, ATPS4r, GLUDy, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
256
7
8.78695
0.06953
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, THD2
and/or GLUDy
257
7
8.78371
0.07004
ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL, THD2 and/or
GLUDy
258
7
8.78071
0.07051
ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE, THD2 and/or
GLUDy
259
7
8.7615
0.07352
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
260
7
8.7615
0.07352
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
261
7
8.5647
0.10435
ADHEr, GLUDy, HEX1, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
262
7
8.54703
0.10712
ADHEr, GLCpts, GLUDy, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
263
7
8.53424
0.10913
ADHEr, HEX1, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
264
7
8.53424
0.10913
ADHEr, FUM, HEX1, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
265
7
8.50919
0.10634
ACKr and/or
PTAr, ADHEr, LDH_D, MDH, NADH12, NADH6, THD2
and/or GLUDy
266
7
8.50798
0.11324
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK, THD2 and/or
GLUDy
267
7
8.50798
0.11324
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK, THD2 and/or
GLUDy
268
7
8.43629
0.12447
ADHEr, HEX1, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
269
7
8.38405
0.13266
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
270
7
8.37888
0.03978
ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK, THD2
and/or GLUDy
271
7
8.29239
0.14702
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
272
7
8.27109
0.10802
ADHEr, ATPS4r, GLUDy, LDH_D, MDH, NADH6, THD2
and/or GLUDy
273
7
8.25238
0.15328
ADHEr, ATPS4r, LDH_D, MDH, NADH12, PFLi, THD2
and/or GLUDy
274
7
8.24277
0.01559
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
275
7
8.13676
0.03579
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6, THD2
and/or GLUDy
276
7
8.112
0.11376
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, THD2
and/or GLUDy
277
7
8.112
0.11376
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, THD2
and/or GLUDy
278
7
8.02815
0.13154
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
279
7
8.00984
0.13929
ADHEr, ATPS4r, LDH_D, NADH12, PFLi, PPCK, THD2
and/or GLUDy
280
7
8.0064
0.13951
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PFLi, PPCK
281
7
8.0064
0.13951
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PFLi, PPCK
282
7
7.98007
0.18432
ADHEr, HEX1, LDH_D, MDH, NADH12, NADH6, THD2
and/or GLUDy
283
7
7.97463
0.15783
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, THD2 and/or GLUDy
284
7
7.90344
0.19562
ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, THD2
and/or GLUDy
285
7
7.89808
0.01471
ADHEr, ASPT, GLCpts, LDH_D, MDH, PGI, THD2 and/or
GLUDy
286
7
7.89024
0.02802
ADHEr, ATPS4r, LDH_D, PFLi, PGI, PPCK, THD2 and/or
GLUDy
287
7
7.88378
0.02967
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PGI, THD2 and/or
GLUDy
288
7
7.88242
0.11605
ADHEr, GLUDy, LDH_D, MDH, NADH12, NADH6, THD2
and/or GLUDy
289
7
7.87634
0.01837
ADHEr, ASPT, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PYK
290
7
7.87634
0.01837
ADHEr, ASPT, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PYK
291
7
7.84906
0.21647
ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2
and/or GLUDy
292
7
7.84582
0.02225
ACKr and/or
PTAr, ADHEr, ASPT, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
293
7
7.81872
0.2081
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PPS
294
7
7.7627
0.0571
ADHEr, LDH_D, MDH, PFLi, PPCK, PYK, THD2 and/or
GLUDy
295
7
7.7627
0.0571
ADHEr, FUM, LDH_D, PFLi, PPCK, PYK, THD2 and/or
GLUDy
296
7
7.76232
0.02662
ADHEr, ATPS4r, LDH_D, NADH6, PGI, PPCK, THD2 and/or
GLUDy
297
7
7.74907
0.0281
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
298
7
7.74444
0.06699
ACKr and/or
PTAr, ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6
299
7
7.71368
0.10674
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
300
7
7.69617
0.04384
ACKr and/or
PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
301
7
7.69209
0.0741
ADHEr, LDH_D, MDH, NADH6, PFLi, PYK, THD2 and/or
GLUDy
302
7
7.61698
0.05527
ADHEr, ASPT, LDH_D, MDH, PPCK, PYK, THD2 and/or
GLUDy
303
7
7.58851
0.09902
ADHEr, FUM, HEX1, LDH_D, NADH6, PFLi, THD2 and/or
GLUDy
304
7
7.55893
0.07788
ACKr and/or
PTAr, ADHEr, GLUDy, LDH_D, MDH, RPE, THD2 and/or
GLUDy
305
7
7.55773
0.07778
ACKr and/or
PTAr, ADHEr, GLUDy, LDH_D, MDH, TAL, THD2 and/or
GLUDy
306
7
7.55641
0.07767
ACKr and/or
PTAr, ADHEr, GLUDy, LDH_D, MDH, PGDH, THD2 and/or
GLUDy
307
7
7.55604
0.07774
ADHEr, GLUDy, LDH_D, MDH, NADH6, PPCK, THD2
and/or GLUDy
308
7
7.52827
0.18666
ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH6, PPS
309
7
7.5043
0.12303
ACKr and/or
PTAr, ADHEr, HEX1, LDH_D, MDH, PGDH, THD2 and/or
GLUDy
310
7
7.50321
0.07168
ACKr and/or
PTAr, ADHEr, ASPT, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
311
7
7.50183
0.1239
ACKr and/or
PTAr, ADHEr, HEX1, LDH_D, MDH, TAL, THD2 and/or
GLUDy
312
7
7.49954
0.12472
ACKr and/or
PTAr, ADHEr, HEX1, LDH_D, MDH, RPE, THD2 and/or
GLUDy
313
7
7.44132
0.08061
ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK, THD2 and/or
GLUDy
314
7
7.43036
0.11712
ADHEr, EDA and/or
PGDHY, GLUDy, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
315
7
7.38132
0.14888
ADHEr, LDH_D, NADH12, NADH6, PFLi, PRO1z, THD2
and/or GLUDy
316
7
7.37291
0.17014
ACKr and/or
PTAr, ADHEr, ATPS4r, FUM, HEX1, LDH_D, THD2 and/or
GLUDy
317
7
7.37059
0.09082
ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
318
7
7.3166
0.16445
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi, THD2
and/or GLUDy
319
7
7.2905
0.10237
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, THD2
and/or GLUDy
320
7
7.24864
0.17893
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PGDH, THD2 and/or GLUDy
321
7
7.2288
0.11128
ADHEr, ASPT, GLUDy, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
322
7
7.22725
0.1115
ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, THD2
and/or GLUDy
323
7
7.21589
0.1796
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, TAL, THD2 and/or GLUDy
324
7
7.21263
0.18991
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PPCK, TAL
325
7
7.20753
0.1999
ADHEr, GLUDy, LDH_D, PFLi, PPCK, RPE, THD2 and/or
GLUDy
326
7
7.20507
0.13919
ADHEr, FUM, HEX1, LDH_D, PGDH, PPS, THD2 and/or
GLUDy
327
7
7.20507
0.13919
ADHEr, FUM, HEX1, LDH_D, PGL and/or
G6PDHy, PPS, THD2 and/or GLUDy
328
7
7.20053
0.0557
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PPCK, THD2 and/or
GLUDy
329
7
7.20053
0.0557
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
330
7
7.19596
0.14141
ADHEr, FUM, HEX1, LDH_D, PPS, TAL, THD2 and/or
GLUDy
331
7
7.19261
0.19908
ADHEr, GLUDy, LDH_D, PFLi, PPCK, TAL, THD2 and/or
GLUDy
332
7
7.18739
0.14349
ADHEr, FUM, HEX1, LDH_D, PPS, RPE, THD2 and/or
GLUDy
333
7
7.18582
0.18022
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, RPE, THD2 and/or GLUDy
334
7
7.18081
0.1906
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PPCK, RPE
335
7
7.17639
0.19819
ADHEr, GLUDy, LDH_D, PFLi, PGDH, PPCK, THD2 and/or
GLUDy
336
7
7.17639
0.19819
ADHEr, GLUDy, LDH_D, PFLi, PGL and/or
G6PDHy, PPCK, THD2 and/or GLUDy
337
7
7.15844
0.21228
ADHEr, FUM, GLU5K, LDH_D, PFLi, RPE, THD2 and/or
GLUDy
338
7
7.15844
0.21228
ADHEr, FUM, G5SD, LDH_D, PFLi, RPE, THD2 and/or
GLUDy
339
7
7.15832
0.21231
ADHEr, ASNS2, FUM, LDH_D, PFLi, RPE, THD2 and/or
GLUDy
340
7
7.15023
0.12261
ADHEr, ASPT, GLUDy, LDH_D, MDH, NADH6, THD2
and/or GLUDy
341
7
7.14754
0.30973
ADHEr, GLUDy, HEX1, LDH_D, PFLi, PPS, THD2 and/or
GLUDy
342
7
7.12499
0.05298
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2 and/or
GLUDy
343
7
7.12287
0.13074
ADHEr, ASPT, GLCpts, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
344
7
7.12019
0.22193
ADHEr, LDH_D, NADH12, NADH6, PFLi, RPE, THD2 and/or
GLUDy
345
7
7.11351
0.16642
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, MDH, THD2 and/or
GLUDy
346
7
6.91657
0.2887
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PGDH, PPS
347
7
6.89939
0.23701
ADHEr, ATPS4r, FUM, LDH_D, MDH, PGL and/or
G6PDHy, PPS
348
7
6.89939
0.23701
ADHEr, ATPS4r, LDH_D, MALS, MDH, PGL and/or
G6PDHy, PPS
349
7
6.89939
0.23701
ADHEr, ATPS4r, ICL, LDH_D, MDH, PGL and/or
G6PDHy, PPS
350
7
6.84379
0.18963
ADHEr, ATPS4r, EDA and/or
PGDHY, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
351
7
6.75658
0.30629
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PPS, RPE
352
7
6.62545
0.08375
ACKr and/or
PTAr, ADHEr, ATPS4r, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
353
7
6.61503
0.30219
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PPS, TAL
354
7
6.5251
0.02706
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or
GLUDy
355
7
6.50064
0.04761
ADHEr, GLCpts, LDH_D, NADH6, PGI, PPCK, THD2 and/or
GLUDy
356
7
6.49117
0.05032
ADHEr, FUM, GLCpts, LDH_D, NADH6, PGI, THD2 and/or
GLUDy
357
7
6.49117
0.05032
ADHEr, GLCpts, LDH_D, MDH, NADH6, PGI, THD2 and/or
GLUDy
358
7
6.46411
0.29781
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGL and/or
G6PDHy, PPS
359
7
6.46411
0.29781
ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGDH, PPS
360
7
6.37646
0.08322
ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK, THD2
and/or GLUDy
361
7
6.37646
0.08322
ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK, THD2
and/or GLUDy
362
7
6.33505
0.15513
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGDH, PGI
363
7
6.30836
0.31643
ADHEr, HEX1, LDH_D, MALS, PGL and/or
G6PDHy, PPS, THD2 and/or GLUDy
364
7
6.30836
0.31643
ADHEr, HEX1, ICL, LDH_D, PGL and/or
G6PDHy, PPS, THD2 and/or GLUDy
365
7
6.30836
0.31643
ADHEr, HEX1, LDH_D, MALS, PGDH, PPS, THD2 and/or
GLUDy
366
7
6.30836
0.31643
ADHEr, HEX1, ICL, LDH_D, PGDH, PPS, THD2 and/or
GLUDy
367
7
6.29139
0.03885
ACKr and/or
PTAr, ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2 and/or
GLUDy
368
7
6.27848
0.32143
ADHEr, HEX1, LDH_D, NADH12, NADH6, PPS, THD2
and/or GLUDy
369
7
6.27504
0.1561
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGI, TAL
370
7
6.19234
0.13601
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PYK, THD2 and/or
GLUDy
371
7
6.19234
0.13601
ADHEr, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PYK, THD2 and/or GLUDy
372
7
6.18835
0.15484
ADHEr, LDH_D, MDH, NADH6, PYK, RPE, THD2 and/or
GLUDy
373
7
6.16073
0.14508
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PPCK, PYK
374
7
6.16073
0.14508
ADHEr, LDH_D, MDH, NADH6, PGL and/or
G6PDHy, PPCK, PYK
375
7
6.15763
0.16479
ADHEr, FUM, LDH_D, NADH6, PPCK, PYK, RPE
376
7
6.15763
0.16479
ADHEr, LDH_D, MDH, NADH6, PPCK, PYK, RPE
377
7
6.14691
0.14904
ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PPCK
378
7
6.14691
0.14904
ADHEr, FUM, GLCpts, LDH_D, NADH12, NADH6, PPCK
379
7
6.14046
0.17035
ADHEr, GLCpts, LDH_D, NADH6, PPCK, RPE, THD2 and/or
GLUDy
380
7
6.13285
0.17281
ADHEr, LDH_D, MDH, NADH12, NADH6, RPE, THD2
and/or GLUDy
381
7
6.09969
0.18355
ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK, RPE
382
7
6.09969
0.18355
ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK, RPE
383
7
6.07495
0.19156
ADHEr, FUM, GLCpts, LDH_D, NADH6, RPE, THD2 and/or
GLUDy
384
7
6.02503
0.20772
ADHEr, FUM, LDH_D, NADH12, NADH6, RPE, THD2 and/or
GLUDy
385
7
5.9952
0.19254
ADHEr, FUM, HEX1, LDH_D, NADH12, NADH6, THD2
and/or GLUDy
386
7
5.9868
0.32178
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PPS, THD2 and/or
GLUDy
387
7
5.98012
0.20938
ADHEr, FUM, LDH_D, NADH12, NADH6, TAL, THD2
and/or GLUDy
388
7
5.94711
0.3261
ADHEr, HEX1, ICL, LDH_D, PPS, TAL, THD2 and/or GLUDy
389
7
5.94711
0.3261
ADHEr, HEX1, LDH_D, MALS, PPS, TAL, THD2 and/or
GLUDy
390
7
5.93766
0.22464
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH12, THD2
and/or GLUDy
391
7
5.84213
0.21172
ACKr and/or
PTAr, ADHEr, HEX1, LDH_D, NADH12, NADH6, THD2
and/or GLUDy
392
7
5.77732
0.09217
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PGDH, PPCK, THD2 and/or
GLUDy
393
7
5.77732
0.09217
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGL and/or
G6PDHy, PPCK, THD2 and/or GLUDy
394
7
5.7407
0.18622
ACKr and/or
PTAr, ADHEr, GLUDy, HEX1, LDH_D, NADH6, THD2
and/or GLUDy
395
7
5.72608
0.09261
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PPCK, TAL, THD2 and/or
GLUDy
396
7
5.67891
0.09302
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, PPCK, RPE, THD2 and/or
GLUDy
397
7
5.62569
0.20722
ACKr and/or
PTAr, ADHEr, HEX1, LDH_D, NADH6, TAL, THD2 and/or
GLUDy
398
7
5.60804
0.32285
ADHEr, GLU5K, HEX1, LDH_D, NADH6, TAL, THD2 and/or
GLUDy
399
7
5.60804
0.32285
ADHEr, G5SD, HEX1, LDH_D, NADH6, TAL, THD2 and/or
GLUDy
400
7
5.60791
0.32289
ADHEr, ASNS2, HEX1, LDH_D, NADH6, TAL, THD2 and/or
GLUDy
401
7
5.59783
0.33545
ADHEr, HEX1, ICL, LDH_D, PPS, RPE, THD2 and/or GLUDy
402
7
5.59783
0.33545
ADHEr, HEX1, LDH_D, MALS, PPS, RPE, THD2 and/or
GLUDy
403
7
5.58104
0.23062
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGL
and/or G6PDHy, THD2 and/or GLUDy
404
7
5.58104
0.23062
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGDH, THD2 and/or
GLUDy
405
7
5.35846
0.23124
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, TAL, THD2 and/or
GLUDy
406
7
5.27565
0.07793
ADHEr, EDA and/or
PGDHY, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy
407
7
5.19178
0.1965
ADHEr, FUM, LDH_D, PFLi, PGDH, PGI, TAL
408
7
5.19178
0.1965
ADHEr, LDH_D, MDH, PFLi, PGDH, PGI, TAL
409
7
5.16412
0.3061
ADHEr, HEX1, LDH_D, PGL and/or
G6PDHy, PPS, TAL, THD2 and/or GLUDy
410
7
5.16412
0.3061
ADHEr, HEX1, LDH_D, PGDH, PPS, TAL, THD2 and/or
GLUDy
411
7
5.15646
0.16031
ADHEr, ATPS4r, LDH_D, PFLi, PGDH, PGI, TAL
412
7
5.14711
0.23182
ACKr and/or
PTAr, ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2 and/or
GLUDy
413
7
5.06593
0.18784
ADHEr, LDH_D, PFLi, PGDH, PGI, PPS, THD2 and/or
GLUDy
414
7
5.06354
0.16677
ADHEr, ATPS4r, GLYCL, LDH_D, PFLi, PGDH, PGI
415
7
5.04267
0.18882
ADHEr, LDH_D, PFLi, PGI, PPS, TAL, THD2 and/or GLUDy
416
7
5.02125
0.18972
ADHEr, LDH_D, PFLi, PGI, PPS, RPE, THD2 and/or GLUDy
417
7
4.56087
0.18955
ACKr and/or
PTAr, ADHEr, FUM, HEX1, LDH_D, RPE, THD2 and/or
GLUDy
418
7
4.53442
0.18932
ACKr and/or
PTAr, ADHEr, FUM, HEX1, LDH_D, TAL, THD2 and/or
GLUDy
419
7
4.50549
0.18907
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, PGL
and/or G6PDHy, THD2 and/or GLUDy
420
7
4.50549
0.18907
ACKr and/or
PTAr, ADHEr, FUM, HEX1, LDH_D, PGDH, THD2 and/or
GLUDy
421
7
4.43945
0.24195
ADHEr, FUM, GLUDy, HEX1, LDH_D, RPE, THD2 and/or
GLUDy
422
7
4.33649
0.2698
ADHEr, ASNS2, FUM, G5SD, LDH_D, TAL, THD2 and/or
GLUDy
423
7
4.33649
0.2698
ADHEr, ASNS2, FUM, GLU5K, LDH_D, TAL, THD2 and/or
GLUDy
424
7
3.82299
0.23376
ACKr and/or
PTAr, ADHEr, CITL, HEX1, LDH_D, PPS, THD2 and/or
GLUDy
425
7
3.40957
0.10927
ADHEr, FRD and/or
SUCD4, LDH_D, PGDH, PPS, TAL, THD2 and/or GLUDy
426
7
3.40957
0.10927
ADHEr, FRD and/or SUCD4, LDH_D, PGL and/or
G6PDHy, PPS, TAL, THD2 and/or GLUDy
427
7
3.30702
0.26731
ACKr and/or
PTAr, ADHEr, ATPS4r, FUM, LDH_D, TAL, THD2 and/or
GLUDy
428
7
1.7546
0.28013
ACKr and/or PTAr, ADHEr, CITL, FUM, LDH_D, PPS, THD2
and/or GLUDy
429
7
0.1401
0.25687
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, MDH, RPE, SUCOAS
430
7
0.13962
0.25599
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, MDH, SUCOAS, TAL
431
7
0.13909
0.25503
ACKr and/or
PTAr, ADHEr, GLCpts, LDH_D, MDH, PGDH, SUCOAS
432
7
0.13909
0.25503
ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, PGL
and/or G6PDHy, SUCOAS
433
3
0.331362284
0.248770484
ADHEr, LDH_D, PFK and/or FBA and/or TPI
434
3
4.1886916
0.310192487
ADHEr, FRD and/or SUCD4, LDH_D
435
4
2.773932742
0.386261
ADHEr, ASNS2, ATPS4r, LDH_D
436
4
2.792658751
0.384018912
ADHEr, ATPS4r, CBMK2, LDH_D
437
4
5.490131382
0.33737393
ADHEr, CBMK2, LDH_D, NADH6
438
4
6.078666008
0.168610416
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI
439
4
0.474685279
0.246014656
ADHEr, LDH_D, PFK and/or FBA and/or TPI, RPE
440
4
0.406720198
0.247321495
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TAL
441
4
0.406720198
0.247321495
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT1
442
4
0.541935885
0.244721556
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT2
443
4
4.227003016
0.295544651
ADHEr, FRD and/or SUCD4, FUM, LDH_D
444
4
2.774411941
0.386203624
ADHEr, ATPS4r, G5SD, LDH_D
445
4
2.774411941
0.386203624
ADHEr, ATPS4r, GLU5K, LDH_D
446
4
2.552977098
0.152232967
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI
447
4
5.500068328
0.33452449
ADHEr, HEX1, LDH_D, NADH6
448
4
4.226362219
0.295789651
ADHEr, FRD and/or SUCD4, HEX1, LDH_D
449
4
0.999358709
0.388175844
ADHEr, HEX1, LDH_D, TKT1
450
4
1.300899111
0.38573732
ADHEr, HEX1, LDH_D, TKT2
451
4
4.374206544
0.239263432
ADHEr, FRD and/or SUCD4, LDH_D, MDH
452
4
4.29486095
0.269600115
ADHEr, FRD and/or SUCD4, LDH_D, ME2
453
4
4.488943189
0.195395474
ADHEr, FRD and/or SUCD4, LDH_D, PPCK
454
4
4.312306765
0.279635371
ADHEr, LDH_D, PPCK, TAL
455
4
4.312306765
0.279635371
ADHEr, LDH_D, PPCK, TKT1
456
4
4.394666553
0.278410255
ADHEr, LDH_D, PPCK, TKT2
457
4
4.290442023
0.306273023
ADHEr, FRD and/or SUCD4, LDH_D, RPE
458
4
4.242231032
0.308130129
ADHEr, FRD and/or SUCD4, LDH_D, TAL
459
4
3.02411229
0.384504476
ADHEr, ATPS4r, LDH_D, TKT1
460
4
5.556064858
0.338701395
ADHEr, LDH_D, NADH6, TKT1
461
4
4.242231032
0.308130129
ADHEr, FRD and/or SUCD4, LDH_D, TKT1
462
4
3.491738883
0.378757536
ADHEr, ATPS4r, LDH_D, TKT2
463
4
5.694356782
0.335394947
ADHEr, LDH_D, NADH6, TKT2
464
4
4.338075359
0.30443817
ADHEr, FRD and/or SUCD4, LDH_D, TKT2
465
5
3.269904021
0.380186443
ADHEr, ASNS2, ATPS4r, LDH_D, RPE
466
5
3.035292503
0.383059918
ADHEr, ASNS2, ATPS4r, LDH_D, TAL
467
5
2.786583612
0.384746295
ADHEr, ASNS2, ATPS4r, GLU5K, LDH_D
468
5
3.035292503
0.383059918
ADHEr, ASNS2, ATPS4r, LDH_D, TKT1
469
5
4.891772757
0.345085032
ADHEr, ATPS4r, LDH_D, ME2, THD2 and/or GLUDy
470
5
4.906959994
0.170872463
ADHEr, ATPS4r, LDH_D, PFK and/or FBA and/or TPI, PFLi
471
5
2.80469336
0.382577993
ADHEr, ASNS2, ATPS4r, CBMK2, LDH_D
472
5
4.199370617
0.306109514
ADHEr, ASNS2, CBMK2, FRD and/or SUCD4, LDH_D
473
5
3.285503322
0.378014123
ADHEr, ATPS4r, CBMK2, LDH_D, RPE
474
5
3.052359629
0.380854732
ADHEr, ATPS4r, CBMK2, LDH_D, TAL
475
5
2.805163465
0.382521707
ADHEr, ATPS4r, CBMK2, G5SD, LDH_D
476
5
4.199487305
0.3060649
ADHEr, CBMK2, FRD and/or SUCD4, G5SD, LDH_D
477
5
2.805163465
0.382521707
ADHEr, ATPS4r, CBMK2, GLU5K, LDH_D
478
5
4.199487305
0.3060649
ADHEr, CBMK2, FRD and/or SUCD4, GLU5K, LDH_D
479
5
5.566341358
0.335567361
ADHEr, CBMK2, LDH_D, NADH6, TAL
480
5
6.853869476
0.27580388
ADHEr, CBMK2, LDH_D, NADH6, PFLi
481
5
3.052359629
0.380854732
ADHEr, ATPS4r, CBMK2, LDH_D, TKT1
482
5
5.566341358
0.335567361
ADHEr, CBMK2, LDH_D, NADH6, TKT1
483
5
3.515194954
0.375215574
ADHEr, ATPS4r, CBMK2, LDH_D, TKT2
484
5
4.886253722
0.208900511
ADHEr, LDH_D, MDH, PFK and/or FBA and/or TPI, PFLi
485
5
4.621960711
0.144538077
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PFK and/or
FBA and/or TPI
486
5
6.151957387
0.166626529
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, RPE
487
5
6.117214665
0.167566962
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TAL
488
5
0.436376298
0.238196669
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PGDH, TAL
489
5
6.117214665
0.167566962
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or
TPI, TKT1
490
5
0.436376298
0.238196669
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PGDH, TKT1
491
5
6.186312313
0.165696592
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or
TPI, TKT2
492
5
5.556162772
0.318439293
ADHEr, CBMK2, FUM, LDH_D, NADH6
493
5
4.886253722
0.208900511
ADHEr, FUM, LDH_D, PFK and/or FBA and/or TPI, PFLi
494
5
4.613740943
0.14768079
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PFK and/or FBA
and/or TPI
495
5
6.088195155
0.307660476
ADHEr, FUM, LDH_D, ME2, THD2 and/or GLUDy
496
5
5.684064939
0.281763084
ADHEr, FUM, LDH_D, ME2, NADH6
497
5
4.673184878
0.145745102
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PGI
498
5
4.572772914
0.163344346
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PPCK
499
5
4.382533025
0.251079405
ADHEr, FUM, LDH_D, PPCK, TAL
500
5
4.382533025
0.251079405
ADHEr, FUM, LDH_D, PPCK, TKT1
501
5
4.457618973
0.24945663
ADHEr, FUM, LDH_D, PPCK, TKT2
502
5
4.374206544
0.239263432
ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2
503
5
4.324805294
0.29144049
ADHEr, FRD and/or SUCD4, FUM, LDH_D, RPE
504
5
4.278495915
0.29338381
ADHEr, FRD and/or SUCD4, FUM, LDH_D, TAL
505
5
5.619129051
0.319468644
ADHEr, FUM, LDH_D, NADH6, TKT1
506
5
1.418161594
0.362515745
ADHEr, FUM, LDH_D, PGDH, TKT1
507
5
1.418161594
0.362515745
ADHEr, FUM, LDH_D, PGL and/or G6PDHy, TKT1
508
5
4.278495915
0.29338381
ADHEr, FRD and/or SUCD4, FUM, LDH_D, TKT1
509
5
5.750964716
0.315868237
ADHEr, FUM, LDH_D, NADH6, TKT2
510
5
4.370505222
0.289522745
ADHEr, FRD and/or SUCD4, FUM, LDH_D, TKT2
511
5
2.786583612
0.384746295
ADHEr, ASNS2, ATPS4r, G5SD, LDH_D
512
5
3.270303172
0.380130859
ADHEr, ATPS4r, G5SD, LDH_D, RPE
513
5
3.035729231
0.38300349
ADHEr, ATPS4r, G5SD, LDH_D, TAL
514
5
3.035729231
0.38300349
ADHEr, ATPS4r, G5SD, LDH_D, TKT1
515
5
3.270303172
0.380130859
ADHEr, ATPS4r, GLU5K, LDH_D, RPE
516
5
3.035729231
0.38300349
ADHEr, ATPS4r, GLU5K, LDH_D, TAL
517
5
4.101998016
0.149473222
ADHEr, ATPS4r, HEX1, LDH_D, PFK and/or FBA and/or
TPI
518
5
5.510369128
0.331570715
ADHEr, CBMK2, HEX1, LDH_D, NADH6
519
5
3.359019108
0.150796918
ADHEr, HEX1, LDH_D, MDH, PFK and/or FBA and/or TPI
520
5
4.301255739
0.267155156
ADHEr, FRD and/or SUCD4, FUM, HEX1, LDH_D
521
5
4.146125845
0.347209192
ADHEr, FUM, HEX1, LDH_D, TKT1
522
5
4.24954345
0.345156537
ADHEr, FUM, HEX1, LDH_D, TKT2
523
5
5.64062493
0.332159859
ADHEr, HEX1, LDH_D, NADH6, RPE
524
5
5.573829893
0.333283574
ADHEr, HEX1, LDH_D, NADH6, TAL
525
5
6.871954799
0.271451843
ADHEr, HEX1, LDH_D, NADH6, PFLi
526
5
1.026318237
0.371585169
ADHEr, HEX1, LDH_D, PGDH, TAL
527
5
1.026318237
0.371585169
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, TAL
528
5
4.321580803
0.292832307
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, RPE
529
5
4.27640137
0.294235511
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TAL
530
5
4.292100506
0.270655532
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, THD2 and/or
GLUDy
531
5
5.573829893
0.333283574
ADHEr, HEX1, LDH_D, NADH6, TKT1
532
5
1.026318237
0.371585169
ADHEr, HEX1, LDH_D, PGDH, TKT1
533
5
1.026318237
0.371585169
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, TKT1
534
5
4.27640137
0.294235511
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TKT1
535
5
4.093661719
0.368542577
ADHEr, HEX1, LDH_D, THD2 and/or GLUDy, TKT1
536
5
5.706971061
0.331043695
ADHEr, HEX1, LDH_D, NADH6, TKT2
537
5
4.366331362
0.291442423
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TKT2
538
5
4.197378033
0.36914891
ADHEr, HEX1, LDH_D, THD2 and/or GLUDy, TKT2
539
5
6.584217406
0.204283888
ADHEr, ASPT, CBMK2, LDH_D, MDH
540
5
4.679174494
0.143073998
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PGI
541
5
4.572772914
0.163344346
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PPCK
542
5
4.382533025
0.251079405
ADHEr, LDH_D, MDH, PPCK, TAL
543
5
4.382533025
0.251079405
ADHEr, LDH_D, MDH, PPCK, TKT1
544
5
4.457618973
0.24945663
ADHEr, LDH_D, MDH, PPCK, TKT2
545
5
4.450595421
0.237144547
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE
546
5
4.414328365
0.238150528
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL
547
5
6.807575806
0.294019202
ADHEr, ATPS4r, LDH_D, MDH, TKT1
548
5
5.745562276
0.280910173
ADHEr, LDH_D, MDH, NADH6, TKT1
549
5
4.414328365
0.238150528
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT1
550
5
6.455745341
0.299423269
ADHEr, ATPS4r, LDH_D, MDH, TKT2
551
5
5.856779801
0.27936769
ADHEr, LDH_D, MDH, NADH6, TKT2
552
5
4.486557371
0.23614703
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT2
553
5
6.254106519
0.315300181
ADHEr, LDH_D, ME2, NADH6, THD2 and/or GLUDy
554
5
4.339242812
0.268682419
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL
555
5
6.224794308
0.229694348
ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or
GLUDy
556
5
6.876353164
0.277060587
ADHEr, LDH_D, NADH6, PFLi, TAL
557
5
7.122630133
0.211129547
ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi
558
5
4.216706856
0.262624991
ADHEr, FRD and/or SUCD4, LDH_D, PFLi, THD2 and/or
GLUDy
559
5
5.563197905
0.034931763
ADHEr, EDA and/or PGDHY, FRD and/or
SUCD4, LDH_D, PGI
560
5
5.66839105
0.263566425
ADHEr, LDH_D, ME2, PGL and/or G6PDHy, THD2 and/or
GLUDy
561
5
4.552158784
0.193305823
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, RPE
562
5
4.522175259
0.194296957
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TAL
563
5
5.924716044
0.226273464
ADHEr, LDH_D, NADH6, PPCK, TKT1
564
5
5.265518204
0.241270472
ADHEr, LDH_D, PFLi, PPCK, TKT1
565
5
4.522175259
0.194296957
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT1
566
5
6.01594542
0.224464061
ADHEr, LDH_D, NADH6, PPCK, TKT2
567
5
5.456414614
0.240407987
ADHEr, LDH_D, PFLi, PPCK, TKT2
568
5
4.581837961
0.192324751
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT2
569
5
3.352437372
0.115790546
ADHEr, FADH4, LDH_D, PPS, THD2 and/or GLUDy
570
5
4.568751314
0.164881948
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK
571
5
4.379456454
0.26785091
ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE
572
5
3.035729231
0.38300349
ADHEr, ATPS4r, GLU5K, LDH_D, TKT1
573
5
6.876353164
0.277060587
ADHEr, LDH_D, NADH6, PFLi, TKT1
574
5
4.339242812
0.268682419
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT1
575
5
4.419421961
0.267024532
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT2
576
5
6.185324416
0.166037364
ADHEr, LDH_D, NADH6, PGI, TKT2
577
5
6.936871903
0.274483161
ADHEr, LDH_D, NADH6, PFLi, TKT2
578
6
7.614634412
0.238178822
ADHEr, ATPS4r, CBMK2, LDH_D, MDH, NADH6
579
6
7.603679301
0.239793337
ADHEr, ATPS4r, FUM, LDH_D, ME2, NADH6
580
6
7.269154045
0.188693079
ADHEr, FUM, LDH_D, PFLi, PPCK, TKT2
581
6
7.539172655
0.163831584
ADHEr, ASNS2, CBMK2, HEX1, LDH_D, PGI
582
6
7.539312732
0.163809352
ADHEr, CBMK2, G5SD, HEX1, LDH_D, PGI
583
6
7.539312732
0.163809352
ADHEr, CBMK2, GLU5K, HEX1, LDH_D, PGI
584
6
7.497875069
0.097945448
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MDH, THD2
and/or GLUDy
585
6
7.316598461
0.164453176
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PFLi, THD2
and/or GLUDy
586
6
7.269154045
0.188693079
ADHEr, LDH_D, MDH, PFLi, PPCK, TKT2
587
6
7.389036066
0.088156259
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH, THD2
and/or GLUDy
588
6
8.258765764
0.152284822
ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi, THD2
and/or GLUDy
589
6
8.999143093
0.036289126
ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PPCK, THD2
and/or GLUDy
TABLE 16
Knockout strain designs for increased production of HMDA,
showing yields of HMDA and biomass.
New
Design
Num
HMDA.
ID
KO
Yield
Biomass
Rxnlist
1
3
2.32302
0.35737
ADHEr, LDH_D, NADH6
2
3
2.12864
0.28397
ADHEr, LDH_D, PPCK
3
3
1.2553
0.38773
ADHEr, ATPS4r, LDH_D
4
3
1.11254
0.26898
ADHEr, FRD and/or SUCD4, LDH_D
5
3
0.67068
0.37924
ADHEr, FUM, LDH_D
6
3
0.57155
0.26988
ADHEr, LDH_D, PGI
7
3
0.27747
0.38953
ADHEr, HEX1, LDH_D
8
4
4.46145
0.20591
ADHEr, FRD and/or SUCD4, LDH_D, MDH
9
4
4.38802
0.23398
ADHEr, FRD and/or SUCD4, HEX1, LDH_D
10
4
4.03852
0.23697
ADHEr, HEX1, LDH_D, PGI
11
4
3.73656
0.30662
ADHEr, LDH_D, NADH6, PFLi
12
4
3.05653
0.20692
ACKr and/or PTAr, ADHEr, LDH_D, PPCK
13
4
2.59406
0.20532
ADHEr, LDH_D, NADH6, PGI
14
4
2.58406
0.27187
ADHEr, ASPT, LDH_D, MDH
15
4
2.46512
0.26229
ADHEr, LDH_D, NADH6, PPCK
16
4
2.41847
0.29351
ADHEr, LDH_D, MDH, NADH6
17
4
2.38828
0.35417
ADHEr, LDH_D, NADH6, RPE
18
4
2.29878
0.17947
ADHEr, LDH_D, PGI, PPCK
19
4
2.17588
0.24785
ADHEr, FUM, LDH_D, THD2 and/or GLUDy
20
4
2.14745
0.26084
ADHEr, EDA and/or PGDHY, LDH_D, PGI
21
4
2.03987
0.35185
ADHEr, FUM, HEX1, LDH_D
22
4
1.48168
0.38161
ADHEr, ATPS4r, LDH_D, RPE
23
4
1.47082
0.28924
ADHEr, LDH_D, MDH, THD2 and/or GLUDy
24
4
1.3746
0.3845
ADHEr, ATPS4r, LDH_D, TAL
25
4
1.26025
0.2616
ADHEr, FRD and/or SUCD4, LDH_D, SUCOAS
26
4
0.939
0.26603
ADHEr, LDH_D, PFLi, PGI
27
4
0.76149
0.2867
ADHEr, ASPT, FUM, LDH_D
28
4
0.38353
0.38695
ADHEr, HEX1, LDH_D, RPE
29
4
0.33312
0.38818
ADHEr, HEX1, LDH_D, TAL
30
5
5.53962
0.16718
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, MDH
31
5
5.40224
0.22385
ADHEr, HEX1, LDH_D, PFLi, PGI
32
5
5.13405
0.1802
ADHEr, ASPT, LDH_D, MDH, PFLi
33
5
5.0304
0.08351
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PPCK
34
5
4.96585
0.07704
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, THD2 and/or
GLUDy
35
5
4.88127
0.20295
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI
36
5
4.78114
0.2037
ADHEr, HEX1, LDH_D, NADH6, PGI
37
5
4.75518
0.09361
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK
38
5
4.61203
0.20383
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PGI
39
5
4.60391
0.15144
ADHEr, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or GLUDy
40
5
4.56119
0.16777
ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH
41
5
4.52834
0.20359
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE
42
5
4.51845
0.20259
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS
43
5
4.46585
0.23056
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, RPE
44
5
4.45522
0.22919
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS
45
5
4.44183
0.21341
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, THD2 and/or
GLUDy
46
5
4.44155
0.21352
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D
47
5
4.42059
0.20565
ADHEr, ASPT, LDH_D, MDH, SUCOAS
48
5
4.36907
0.15859
ADHEr, EDA and/or PGDHY, LDH_D, PGI, PPCK
49
5
4.36067
0.16481
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI
50
5
4.33827
0.24026
ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy
51
5
4.24585
0.21569
ADHEr, ASPT, FUM, LDH_D, MDH
52
5
4.24585
0.21569
ADHEr, ASPT, ICL, LDH_D, MDH
53
5
4.24585
0.21569
ADHEr, ASPT, LDH_D, MALS, MDH
54
5
4.21843
0.08488
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, THD2 and/or
GLUDy
55
5
4.18265
0.21782
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH
56
5
4.13301
0.24796
ADHEr, ASPT, FUM, HEX1, LDH_D
57
5
4.03737
0.23515
ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D, PGI
58
5
4.02056
0.22023
ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy
59
5
4.0188
0.18437
ADHEr, LDH_D, NADH6, PFLi, PPCK
60
5
3.96576
0.20734
ADHEr, FUM, LDH_D, PFLi, PPCK
61
5
3.96576
0.20734
ADHEr, LDH_D, MDH, PFLi, PPCK
62
5
3.96266
0.23773
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6
63
5
3.92623
0.22446
ADHEr, LDH_D, MDH, NADH6, PFLi
64
5
3.81332
0.27196
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D
65
5
3.81332
0.27196
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH
66
5
3.79273
0.28229
ADHEr, HEX1, LDH_D, NADH6, PFLi
67
5
3.77686
0.28031
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, NADH6
68
5
3.74717
0.3358
ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy
69
5
3.69789
0.22154
ADHEr, LDH_D, NADH6, PPCK, THD2 and/or GLUDy
70
5
3.69693
0.23186
ADHEr, ASPT, ATPS4r, LDH_D, MDH
71
5
3.57925
0.1851
ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK
72
5
3.55622
0.22164
ADHEr, FUM, LDH_D, PPCK, THD2 and/or GLUDy
73
5
3.55622
0.22164
ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy
74
5
3.51036
0.26322
ADHEr, ATPS4r, LDH_D, MDH, NADH6
75
5
3.44912
0.18587
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK
76
5
3.44912
0.18587
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PPCK
77
5
3.23914
0.31877
ADHEr, ATPS4r, FDH2, LDH_D, NADH6
78
5
3.23669
0.2512
ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI
79
5
3.22027
0.23733
ADHEr, ATPS4r, LDH_D, NADH6, PPCK
80
5
3.21813
0.22145
ADHEr, ATPS4r, LDH_D, PFLi, PPCK
81
5
3.20482
0.28717
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, SUCOAS
82
5
3.19987
0.14763
ADHEr, LDH_D, PFLi, PGI, PPCK
83
5
3.04087
0.23963
ADHEr, ATPS4r, FUM, LDH_D, PPCK
84
5
3.04087
0.23963
ADHEr, ATPS4r, LDH_D, MDH, PPCK
85
5
3.01019
0.28849
ADHEr, HEX1, LDH_D, MDH, THD2 and/or GLUDy
86
5
2.94161
0.20506
ADHEr, ATPS4r, LDH_D, NADH6, PGI
87
5
2.88011
0.33864
ADHEr, ATPS4r, FUM, LDH_D, NADH6
88
5
2.85293
0.25411
ADHEr, ASPT, LDH_D, MDH, NADH6
89
5
2.75034
0.26847
ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy
90
5
2.74759
0.33105
ADHEr, ATPS4r, HEX1, LDH_D, NADH6
91
5
2.67997
0.17637
ADHEr, ATPS4r, LDH_D, PGI, PPCK
92
5
2.67853
0.21976
ADHEr, ASPT, LDH_D, MDH, PYK
93
5
2.65374
0.15874
ADHEr, LDH_D, NADH6, PGI, PPCK
94
5
2.65207
0.23435
ADHEr, ASPT, GLCpts, LDH_D, MDH
95
5
2.64298
0.23937
ADHEr, ASPT, LDH_D, MDH, PPCK
96
5
2.63715
0.26997
ADHEr, ASPT, LDH_D, MDH, RPE
97
5
2.60095
0.33049
ADHEr, ATPS4r, FUM, HEX1, LDH_D
98
5
2.52584
0.22167
ADHEr, LDH_D, NADH6, PPCK, PYK
99
5
2.5171
0.22752
ADHEr, GLCpts, LDH_D, NADH6, PPCK
100
5
2.48801
0.24981
ADHEr, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy
101
5
2.47253
0.25734
ADHEr, GLCpts, LDH_D, MDH, NADH6
102
5
2.47049
0.29207
ADHEr, LDH_D, MDH, NADH6, RPE
103
5
2.38845
0.0995
ADHEr, LDH_D, PGI, PPCK, THD2 and/or GLUDy
104
5
2.3823
0.10498
ADHEr, FUM, LDH_D, PGI, THD2 and/or GLUDy
105
5
2.3823
0.10498
ADHEr, LDH_D, MDH, PGI, THD2 and/or GLUDy
106
5
2.37356
0.34412
ADHEr, HEX1, LDH_D, NADH6, TAL
107
5
2.36643
0.29114
ADHEr, ATPS4r, LDH_D, MDH, PGDH
108
5
2.33103
0.23832
ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or GLUDy
109
5
2.32447
0.13422
ADHEr, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
110
5
2.29184
0.29402
ADHEr, ATPS4r, LDH_D, MDH, TAL
111
5
2.282
0.28884
ACKr and/or PTAr, ADHEr, LDH_D, MDH, THD2 and/or GLUDy
112
5
2.22251
0.2967
ADHEr, ATPS4r, LDH_D, MDH, RPE
113
5
2.22197
0.24001
ADHEr, LDH_D, MDH, RPE, THD2 and/or GLUDy
114
5
2.21206
0.24857
ADHEr, GLCpts, LDH_D, PPCK, RPE
115
5
2.20561
0.23941
ADHEr, LDH_D, MDH, TAL, THD2 and/or GLUDy
116
5
2.20149
0.22826
ADHEr, FUM, LDH_D, PPCK, PYK
117
5
2.20149
0.22826
ADHEr, LDH_D, MDH, PPCK, PYK
118
5
2.19372
0.24909
ADHEr, GLCpts, LDH_D, PPCK, TAL
119
5
2.19132
0.23604
ADHEr, GLCpts, LDH_D, MDH, PPCK
120
5
2.19132
0.23604
ADHEr, FUM, GLCpts, LDH_D, PPCK
121
5
2.18792
0.26941
ADHEr, FUM, LDH_D, PPCK, RPE
122
5
2.18792
0.26941
ADHEr, LDH_D, MDH, PPCK, RPE
123
5
2.18777
0.23875
ADHEr, LDH_D, MDH, PGDH, THD2 and/or GLUDy
124
5
2.09486
0.34975
ADHEr, FUM, HEX1, LDH_D, RPE
125
5
1.89618
0.34765
ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy
126
5
1.74629
0.36027
ADHEr, HEX1, LDH_D, PFLi, PPS
127
5
1.73743
0.3202
ADHEr, ATPS4r, HEX1, LDH_D, PFLi
128
5
1.73543
0.31113
ADHEr, HEX1, LDH_D, MDH, PFLi
129
5
1.69405
0.23402
ADHEr, LDH_D, PFLi, PGDH, PGI
130
5
1.68439
0.23524
ADHEr, LDH_D, PFLi, PGI, TAL
131
5
1.67549
0.23637
ADHEr, LDH_D, PFLi, PGI, RPE
132
5
1.66962
0.16853
ADHEr, ATPS4r, LDH_D, PFLi, PGI
133
5
1.66579
0.35584
ADHEr, HEX1, LDH_D, PPS, THD2 and/or GLUDy
134
5
1.44831
0.25358
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, LDH_D, SUCOAS
135
5
1.40898
0.31979
ADHEr, ATPS4r, HEX1, LDH_D, MDH
136
5
1.33078
0.25859
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, SUCOAS
137
5
1.28594
0.26157
ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, ICL, LDH_D
138
5
1.28594
0.26157
ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MALS
139
5
1.15083
0.26676
ADHEr, ASPT, FUM, LDH_D, SUCOAS
140
5
1.08556
0.26021
ACKr and/or PTAr, ADHEr, LDH_D, PFLi, PGI
141
5
1.08281
0.26394
ADHEr, FUM, LDH_D, PFLi, PGI
142
5
0.81325
0.32358
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PPS
143
5
0.73764
0.26814
ADHEr, LDH_D, MDH, PGI, THD5
144
5
0.73764
0.26814
ADHEr, FUM, LDH_D, PGI, THD5
145
6
6.49204
0.11612
ADHEr, ASPT, LDH_D, MDH, PFLi, THD2 and/or GLUDy
146
6
6.18999
0.07445
ADHEr, ATPS4r, FRD and/or SUCD4, GLCpts, LDH_D, MDH
147
6
6.1414
0.1578
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGL and/or G6PDHy
148
6
5.80857
0.1099
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PYK
149
6
5.62948
0.09192
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGI
150
6
5.60131
0.13875
ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI, PPCK
151
6
5.59693
0.15008
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MDH, THD2 and/or
GLUDy
152
6
5.56181
0.13846
ADHEr, EDA and/or PGDHY, FRD and/or
SUCD4, LDH_D, MDH, THD2 and/or GLUDy
153
6
5.52668
0.15619
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PFLi, PGI
154
6
5.52416
0.10076
ADHEr, ASPT, LDH_D, MDH, PFLi, PGI
155
6
5.44943
0.16764
ADHEr, ASPT, ATPS4r, LDH_D, MDH, SUCOAS
156
6
5.43906
0.17387
ADHEr, ASPT, ATPS4r, FUM, LDH_D, MDH
157
6
5.43906
0.17387
ADHEr, ASPT, ATPS4r, ICL, LDH_D, MDH
158
6
5.43906
0.17387
ADHEr, ASPT, ATPS4r, LDH_D, MALS, MDH
159
6
5.4269
0.11474
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PGI
160
6
5.39974
0.15728
ADHEr, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
161
6
5.39974
0.15728
ADHEr, FUM, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
162
6
5.36516
0.17457
ADHEr, LDH_D, MDH, NADH6, PFLi, THD2 and/or GLUDy
163
6
5.29594
0.12992
ADHEr, ASPT, LDH_D, MDH, PFLi, PYK
164
6
5.28218
0.07163
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PGL and/or
G6PDHy, PPCK
165
6
5.28218
0.07163
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PGDH, PPCK
166
6
5.27982
0.10569
ADHEr, ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK
167
6
5.27077
0.07217
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PPCK, TAL
168
6
5.26177
0.14053
ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi
169
6
5.26021
0.07267
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PPCK, RPE
170
6
5.22996
0.24218
ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy
171
6
5.22996
0.24218
ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/or GLUDy
172
6
5.10795
0.18114
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy
173
6
5.07418
0.20379
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS
174
6
5.05205
0.12049
ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI, PPCK
175
6
5.02318
0.17043
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFLi, PPCK
176
6
5.02318
0.17043
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, PPCK
177
6
4.96023
0.0152
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PYK, THD2 and/or
GLUDy
178
6
4.96019
0.14703
ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy
179
6
4.95227
0.18511
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, LDH_D, NADH6, PGI
180
6
4.94767
0.18905
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PFLi
181
6
4.94078
0.08876
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK
182
6
4.88378
0.08315
ADHEr, ASPT, ATPS4r, FUM, LDH_D, THD2 and/or GLUDy
183
6
4.88138
0.0499
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, THD2
and/or GLUDy
184
6
4.8705
0.04951
ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, PPCK, THD2 and/or
GLUDy
185
6
4.86653
0.05103
ADHEr, FRD and/or SUCD4, LDH_D, MALS, MDH, THD2 and/or
GLUDy
186
6
4.86653
0.05103
ADHEr, FRD and/or SUCD4, ICL, LDH_D, MDH, THD2 and/or
GLUDy
187
6
4.85555
0.05127
ADHEr, ASPT, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
188
6
4.81179
0.07918
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, SUCOAS, THD2
and/or GLUDy
189
6
4.80834
0.05481
ADHEr, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy
190
6
4.80834
0.05481
ADHEr, ASPT, LDH_D, MALS, MDH, THD2 and/or GLUDy
191
6
4.80546
0.08184
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS, THD2
and/or GLUDy
192
6
4.79254
0.05156
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, LDH_D, MDH, THD2 and/or GLUDy
193
6
4.7879
0.08109
ADHEr, FRD and/or SUCD4, LDH_D, MALS, PPCK, THD2 and/or
GLUDy
194
6
4.7879
0.08109
ADHEr, FRD and/or SUCD4, ICL, LDH_D, PPCK, THD2 and/or
GLUDy
195
6
4.7863
0.15759
ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI
196
6
4.78022
0.08403
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MALS, THD2 and/or
GLUDy
197
6
4.78022
0.08403
ADHEr, FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2 and/or
GLUDy
198
6
4.75362
0.1717
ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS
199
6
4.74482
0.14204
ADHEr, FUM, LDH_D, PFLi, PGI, PPCK
200
6
4.74482
0.14204
ADHEr, LDH_D, MDH, PFLi, PGI, PPCK
201
6
4.71665
0.15772
ADHEr, FUM, LDH_D, NADH6, PFLi, PGI
202
6
4.71665
0.15772
ADHEr, LDH_D, MDH, NADH6, PFLi, PGI
203
6
4.67938
0.12258
ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
204
6
4.67056
0.08187
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, LDH_D, PPCK, THD2 and/or GLUDy
205
6
4.65509
0.08572
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, HEX1, LDH_D, THD2 and/or GLUDy
206
6
4.65177
0.09959
ADHEr, ASPT, FUM, LDH_D, PPCK, PYK
207
6
4.64856
0.19942
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi
208
6
4.64527
0.14923
ADHEr, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or GLUDy
209
6
4.62501
0.24401
ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy
210
6
4.61569
0.16588
ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, RPE
211
6
4.58364
0.20034
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE, SUCOAS
212
6
4.54149
0.17531
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH, PYK
213
6
4.53277
0.1423
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or
GLUDy
214
6
4.53052
0.2259
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, RPE, SUCOAS
215
6
4.52789
0.16756
ADHEr, ASPT, GLCpts, LDH_D, MDH, SUCOAS
216
6
4.52733
0.16694
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH
217
6
4.50564
0.17546
ADHEr, ASPT, LDH_D, MDH, PYK, SUCOAS
218
6
4.48785
0.20333
ADHEr, ASPT, LDH_D, MDH, RPE, SUCOAS
219
6
4.45673
0.0937
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI, PPCK
220
6
4.44964
0.1574
ADHEr, ASPT, FUM, LDH_D, MDH, THD2 and/or GLUDy
221
6
4.38551
0.17574
ADHEr, ASPT, GLCpts, LDH_D, MALS, MDH
222
6
4.38551
0.17574
ADHEr, ASPT, FUM, GLCpts, LDH_D, MDH
223
6
4.38551
0.17574
ADHEr, ASPT, GLCpts, ICL, LDH_D, MDH
224
6
4.3659
0.18135
ADHEr, ASPT, LDH_D, MALS, MDH, PYK
225
6
4.3659
0.18135
ADHEr, ASPT, FUM, LDH_D, MDH, PYK
226
6
4.3659
0.18135
ADHEr, ASPT, ICL, LDH_D, MDH, PYK
227
6
4.35016
0.23064
ADHEr, FUM, HEX1, LDH_D, NADH6, SUCOAS
228
6
4.34833
0.1713
ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK
229
6
4.34286
0.23323
ADHEr, ASPT, FUM, HEX1, LDH_D, SUCOAS
230
6
4.33401
0.17749
ACKr and/or PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH
231
6
4.31842
0.21314
ADHEr, ASPT, ICL, LDH_D, MDH, RPE
232
6
4.31842
0.21314
ADHEr, ASPT, LDH_D, MALS, MDH, RPE
233
6
4.31842
0.21314
ADHEr, ASPT, FUM, LDH_D, MDH, RPE
234
6
4.31517
0.18251
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PYK
235
6
4.27405
0.08431
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, RPE, THD2 and/or
GLUDy
236
6
4.25521
0.17069
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK, PYK
237
6
4.25521
0.17069
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PPCK, PYK
238
6
4.23738
0.22036
ADHEr, FUM, HEX1, LDH_D, PGI, THD5
239
6
4.23738
0.22036
ADHEr, HEX1, LDH_D, MDH, PGI, THD5
240
6
4.21937
0.24412
ADHEr, ASPT, FUM, HEX1, LDH_D, RPE
241
6
4.20848
0.22638
ADHEr, ASPT, FUM, HEX1, LDH_D, THD2 and/or GLUDy
242
6
4.1949
0.21515
ACKr and/or PTAr, ADHEr, FDH2, LDH_D, MDH, NADH6
243
6
4.193
0.22615
ADHEr, ASPT, ATPS4r, FUM, HEX1, LDH_D
244
6
4.18464
0.18833
ADHEr, HEX1, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy
245
6
4.17022
0.12602
ADHEr, GLCpts, LDH_D, PFLi, PGI, PPCK
246
6
4.15452
0.23423
ACKr and/or PTAr, ACS, ADHEr, EDA and/or
PGDHY, LDH_D, PGI
247
6
4.13958
0.1401
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PGI
248
6
4.12806
0.09037
ADHEr, ASPT, FUM, LDH_D, PPCK, THD2 and/or GLUDy
249
6
4.10281
0.14798
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK
250
6
4.09944
0.14944
ADHEr, LDH_D, NADH6, PFLi, PPCK, PYK
251
6
4.09149
0.20644
ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6
252
6
4.08305
0.24974
ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, NADH6
253
6
4.07707
0.21283
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6
254
6
4.07083
0.2483
ACKr and/or PTAr, ADHEr, FDH2, HEX1, LDH_D, NADH6
255
6
4.0547
0.15755
ADHEr, LDH_D, NADH6, PFLi, PGDH, PGI
256
6
4.04976
0.14332
ADHEr, LDH_D, PFLi, PGDH, PGI, PPCK
257
6
4.04925
0.17118
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK
258
6
4.04925
0.17118
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK
259
6
4.04921
0.20443
ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK
260
6
4.04921
0.20443
ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK
261
6
4.04701
0.15816
ADHEr, LDH_D, NADH6, PFLi, PGI, TAL
262
6
4.04276
0.14386
ADHEr, LDH_D, PFLi, PGI, PPCK, TAL
263
6
4.03993
0.15872
ADHEr, LDH_D, NADH6, PFLi, PGI, RPE
264
6
4.03632
0.14436
ADHEr, LDH_D, PFLi, PGI, PPCK, RPE
265
6
4.01325
0.18677
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi
266
6
3.99577
0.19434
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi
267
6
3.97712
0.21218
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, RPE
268
6
3.96781
0.21154
ADHEr, ATPS4r, LDH_D, NADH6, PPCK, TAL
269
6
3.95767
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGL and/or G6PDHy, PPCK
270
6
3.95767
0.21085
ADHEr, ATPS4r, LDH_D, NADH6, PGDH, PPCK
271
6
3.95117
0.22396
ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE
272
6
3.94143
0.22325
ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL
273
6
3.93082
0.22248
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH
274
6
3.92007
0.30764
ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy
275
6
3.90761
0.02965
ADHEr, EDA and/or PGDHY, LDH_D, MDH, PGI, THD2 and/or
GLUDy
276
6
3.90761
0.02965
ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI, THD2 and/or
GLUDy
277
6
3.88276
0.27815
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi
278
6
3.87622
0.18142
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGI
279
6
3.85591
0.25493
ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi
280
6
3.85214
0.25656
ADHEr, FUM, HEX1, LDH_D, NADH6, PFLi
281
6
3.80756
0.28917
ADHEr, LDH_D, NADH12, NADH6, PFLi, RPE
282
6
3.80403
0.29078
ADHEr, GLCpts, LDH_D, NADH6, PFLi, RPE
283
6
3.80376
0.2909
ADHEr, FUM, LDH_D, NADH6, PFLi, RPE
284
6
3.79095
0.29004
ADHEr, LDH_D, NADH12, NADH6, PFLi, TAL
285
6
3.78678
0.29189
ADHEr, FUM, LDH_D, NADH6, PFLi, TAL
286
6
3.74504
0.18693
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK
287
6
3.66618
0.23348
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6
288
6
3.65047
0.29574
ADHEr, ATPS4r, FDH2, LDH_D, NADH12, NADH6
289
6
3.64636
0.29624
ADHEr, ATPS4r, FDH2, GLCpts, LDH_D, NADH6
290
6
3.61821
0.2978
ADHEr, ATPS4r, FDH2, FUM, LDH_D, NADH6
291
6
3.59369
0.11801
ADHEr, EDA and/or PGDHY, GLUDy, LDH_D, MDH, THD2
and/or GLUDy
292
6
3.56749
0.21671
ADHEr, HEX1, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
293
6
3.56467
0.20357
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK
294
6
3.56467
0.20357
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK
295
6
3.54915
0.24121
ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, THD2
and/or GLUDy
296
6
3.54182
0.17698
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, PGI, THD5
297
6
3.52871
0.18935
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PPCK
298
6
3.52871
0.18935
ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PPCK
299
6
3.48585
0.20551
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK
300
6
3.46875
0.14666
ACKr and/or PTAr, ADHEr, LDH_D, PFLi, PGI, PPCK
301
6
3.39774
0.24244
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
302
6
3.34501
0.09944
ACKr and/or PTAr, ADHEr, ATPS4r, FRD and/or
SUCD4, LDH_D, PGI
303
6
3.21571
0.21219
ADHEr, LDH_D, MDH, NADH12, NADH6, SUCOAS
304
6
3.13882
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGL and/or G6PDHy
305
6
3.13882
0.32432
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGDH
306
6
3.10937
0.32483
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, TAL
307
6
3.08568
0.30099
ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH6
308
6
3.08242
0.32529
ADHEr, ATPS4r, HEX1, LDH_D, NADH6, RPE
309
6
3.05396
0.31324
ADHEr, FUM, HEX1, LDH_D, PPS, THD2 and/or GLUDy
310
6
3.04084
0.1442
ADHEr, ATPS4r, GLCpts, LDH_D, PGI, PPCK
311
6
3.0331
0.26033
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PGDH
312
6
2.95572
0.06686
ADHEr, ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy
313
6
2.95209
0.20166
ADHEr, ASPT, LDH_D, MDH, NADH6, PYK
314
6
2.93749
0.26369
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, TAL
315
6
2.93137
0.23554
ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI, THD5
316
6
2.93119
0.19434
ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy
317
6
2.92428
0.21637
ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6
318
6
2.90707
0.25203
ADHEr, ASPT, LDH_D, MDH, NADH6, RPE
319
6
2.88032
0.10845
ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy
320
6
2.85872
0.13782
ADHEr, ASPT, GLCpts, LDH_D, MDH, PGI
321
6
2.83622
0.13278
ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
322
6
2.83037
0.25344
ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or
GLUDy
323
6
2.73678
0.18763
ADHEr, ASPT, LDH_D, MDH, PPCK, PYK
324
6
2.73413
0.26033
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PGL and/or
G6PDHy
325
6
2.70842
0.20327
ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK
326
6
2.68773
0.20429
ADHEr, FUM, LDH_D, NADH6, PGI, THD5
327
6
2.68773
0.20429
ADHEr, LDH_D, MDH, NADH6, PGI, THD5
328
6
2.6871
0.26683
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, RPE
329
6
2.64071
0.07956
ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, THD2 and/or
GLUDy
330
6
2.61399
0.16269
ADHEr, LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy
331
6
2.60256
0.17034
ADHEr, LDH_D, NADH12, NADH6, PPCK, THD2 and/or GLUDy
332
6
2.56541
0.1952
ADHEr, FUM, GLCpts, LDH_D, NADH6, THD2 and/or GLUDy
333
6
2.53808
0.13335
ADHEr, ATPS4r, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
334
6
2.53559
0.33664
ADHEr, ATPS4r, HEX1, LDH_D, PPS, THD2 and/or GLUDy
335
6
2.53316
0.24472
ADHEr, LDH_D, MDH, NADH6, PPCK, RPE
336
6
2.53316
0.24472
ADHEr, FUM, LDH_D, NADH6, PPCK, RPE
337
6
2.53266
0.21711
ADHEr, FUM, LDH_D, NADH12, NADH6, THD2 and/or GLUDy
338
6
2.50897
0.31991
ADHEr, HEX1, LDH_D, NADH6, PPS, THD2 and/or GLUDy
339
6
2.47564
0.27754
ADHEr, ATPS4r, LDH_D, MDH, PGDH, TAL
340
6
2.37424
0.3231
ADHEr, GLU5K, HEX1, LDH_D, NADH6, THD2 and/or GLUDy
341
6
2.37424
0.3231
ADHEr, G5SD, HEX1, LDH_D, NADH6, THD2 and/or GLUDy
342
6
2.37418
0.32314
ADHEr, ASNS2, HEX1, LDH_D, NADH6, THD2 and/or GLUDy
343
6
2.35165
0.11344
ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/or GLUDy
344
6
2.35165
0.11344
ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy
345
6
2.28934
0.18186
ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy
346
6
2.28203
0.17727
ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy
347
6
2.27443
0.17249
ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy
348
6
2.27443
0.17249
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/or
GLUDy
349
6
2.23539
0.22843
ADHEr, HEX1, LDH_D, PPCK, RPE, THD2 and/or GLUDy
350
6
2.2276
0.23515
ADHEr, FUM, HEX1, LDH_D, RPE, THD2 and/or GLUDy
351
6
2.22058
0.22724
ADHEr, HEX1, LDH_D, PPCK, TAL, THD2 and/or GLUDy
352
6
2.20452
0.22594
ADHEr, HEX1, LDH_D, PGDH, PPCK, THD2 and/or GLUDy
353
6
2.20452
0.22594
ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPCK, THD2 and/or
GLUDy
354
6
2.10135
0.3242
ADHEr, FUM, HEX1, LDH_D, TAL, THD2 and/or GLUDy
355
6
2.02123
0.34714
ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2 and/or GLUDy
356
6
1.98114
0.30095
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, THD2 and/or
GLUDy
357
6
1.96512
0.30664
ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi
358
6
1.96163
0.34738
ADHEr, ATPS4r, HEX1, LDH_D, TAL, THD2 and/or GLUDy
359
6
1.94766
0.26881
ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPS
360
6
1.84554
0.35787
ADHEr, HEX1, LDH_D, PFLi, PPS, RPE
361
6
1.84094
0.27637
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, FUM, LDH_D
362
6
1.82413
0.31504
ADHEr, ATPS4r, HEX1, LDH_D, PFLi, RPE
363
6
1.82134
0.30945
ADHEr, HEX1, LDH_D, MDH, PFLi, RPE
364
6
1.79836
0.35901
ADHEr, HEX1, LDH_D, PFLi, PPS, TAL
365
6
1.74423
0.2066
ADHEr, ASPT, ATPS4r, LDH_D, PGI, THD5
366
6
1.63108
0.27824
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PGDH, SUCOAS
367
6
1.63108
0.27824
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PGL and/or
G6PDHy, SUCOAS
368
6
1.59283
0.279
ACKr and/or PTAr, ADHEr, LDH_D, MDH, SUCOAS, TAL
369
6
1.55776
0.2797
ACKr and/or PTAr, ADHEr, LDH_D, MDH, RPE, SUCOAS
370
6
1.35784
0.33828
ADHEr, FUM, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy
371
6
1.3281
0.25919
ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, SUCOAS
372
6
1.25004
0.31142
ACKr and/or PTAr, ADHEr, FUM, LDH_D, SUCOAS, THD2 and/or
GLUDy
373
6
1.16323
0.25946
ADHEr, FRD and/or SUCD4, GLYCL, LDH_D, PGL and/or
G6PDHy, TAL
374
6
1.16323
0.25946
ADHEr, FRD and/or SUCD4, GLYCL, LDH_D, PGDH, TAL
375
6
0.94735
0.32316
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PPS, RPE
376
6
0.88343
0.32336
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PPS, TAL
377
6
0.83542
0.27562
ADHEr, ASPT, FUM, LDH_D, PGDH, TAL
378
6
0.83542
0.27562
ADHEr, ASPT, FUM, LDH_D, PGL and/or G6PDHy, TAL
379
6
0.81371
0.28445
ACKr and/or PTAr, ADHEr, ASPT, FUM, ICL, LDH_D
380
6
0.81371
0.28445
ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, MALS
381
6
0.40293
0.37998
ACKr and/or PTAr, ADHEr, CITL, ICL, LDH_D, SUCOAS
382
6
0.40293
0.37998
ACKr and/or PTAr, ADHEr, CITL, LDH_D, MALS, SUCOAS
383
7
6.87929
0.03662
ADHEr, ASPT, LDH_D, MDH, PFLi, PGL and/or G6PDHy, PYK
384
7
6.87929
0.03662
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PFLi, PYK
385
7
6.82182
0.04856
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PFLi, THD2
and/or GLUDy
386
7
6.7498
0.06331
ADHEr, ASPT, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
387
7
6.70416
0.07266
ADHEr, ASPT, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
388
7
6.67113
0.07943
ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy
389
7
6.65401
0.08294
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, PGL and/or
G6PDHy
390
7
6.45147
0.12443
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PGL and/or
G6PDHy
391
7
6.44861
0.08805
ADHEr, ASPT, ATPS4r, LDH_D, MALS, MDH, PGL and/or
G6PDHy
392
7
6.44861
0.08805
ADHEr, ASPT, ATPS4r, ICL, LDH_D, MDH, PGL and/or G6PDHy
393
7
6.44861
0.08805
ADHEr, ASPT, ATPS4r, FUM, LDH_D, MDH, PGL and/or G6PDHy
394
7
6.15823
0.07915
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MALS, MDH
395
7
6.15823
0.07915
ADHEr, ASPT, ATPS4r, GLCpts, ICL, LDH_D, MDH
396
7
6.15823
0.07915
ADHEr, ASPT, ATPS4r, FUM, GLCpts, LDH_D, MDH
397
7
6.14605
0.07491
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, SUCOAS
398
7
6.1414
0.1578
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, PGDH
399
7
6.09037
0.16006
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, TAL
400
7
6.08683
0.10358
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, THD2 and/or
GLUDy
401
7
6.0464
0.06183
ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi, PGI
402
7
6.04253
0.16218
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, RPE
403
7
5.86291
0.1062
ADHEr, ASPT, EDA and/or PGDHY, ICL, LDH_D, MDH, THD2
and/or GLUDy
404
7
5.86291
0.1062
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MALS, MDH, THD2
and/or GLUDy
405
7
5.79637
0.08575
ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, NADH6, PGI
406
7
5.74642
0.10456
ADHEr, EDA and/or PGDHY, FRD and/or
SUCD4, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
407
7
5.71711
0.12348
ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6, PFLi
408
7
5.71588
0.18172
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PGL and/or
G6PDHy, PPS
409
7
5.71588
0.18172
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PGDH, PPS
410
7
5.70575
0.12457
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, PFLi
411
7
5.69395
0.07981
ADHEr, ATPS4r, EDA and/or PGDHY, FRD and/or
SUCD4, LDH_D, MDH, PPCK
412
7
5.68826
0.10159
ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, PFLi, PGI, PPCK
413
7
5.6857
0.18276
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS, TAL
414
7
5.67373
0.1078
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, LDH_D, PFLi, PGI, PPCK
415
7
5.67231
0.09516
ACKr and/or PTAr, ADHEr, ATPS4r, EDA and/or PGDHY, FRD
and/or SUCD4, LDH_D, MDH
416
7
5.65788
0.18372
ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS, RPE
417
7
5.65108
0.11748
ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, NADH6, PFLi, PGI
418
7
5.64055
0.12198
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, LDH_D, NADH6, PFLi, PGI
419
7
5.63038
0.12633
ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, PGI
420
7
5.63037
0.14151
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPS, SUCOAS, THD2
and/or GLUDy
421
7
5.61946
0.14377
ADHEr, ATPS4r, EDA and/or PGDHY, FRD and/or
SUCD4, LDH_D, MDH, SUCOAS
422
7
5.61946
0.14377
ADHEr, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, NADH6, SUCOAS
423
7
5.60713
0.15264
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MALS, PPS, THD2
and/or GLUDy
424
7
5.60713
0.15264
ADHEr, FRD and/or SUCD4, HEX1, ICL, LDH_D, PPS, THD2
and/or GLUDy
425
7
5.60017
0.14365
ADHEr, ATPS4r, FRD and/or
SUCD4, LDH_D, MDH, PGDH, SUCOAS
426
7
5.59518
0.14138
ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, PGI
427
7
5.59117
0.14212
ADHEr, ATPS4r, FRD and/or SUCD4, FUM, LDH_D, MDH, PGDH
428
7
5.58992
0.13977
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, MDH, PGDH, TAL
429
7
5.57521
0.1433
ADHEr, HEX1, LDH_D, MDH, PPS, SUCOAS, THD2 and/or
GLUDy
430
7
5.57521
0.1433
ADHEr, FUM, HEX1, LDH_D, PPS, SUCOAS, THD2 and/or
GLUDy
431
7
5.56382
0.059
ADHEr, HEX1, LDH_D, PFLi, PGI, PPS, THD2 and/or GLUDy
432
7
5.55714
0.14423
ADHEr, ASPT, EDA and/or PGDHY, FUM, LDH_D, MDH, THD2
and/or GLUDy
433
7
5.5551
0.14719
ADHEr, HEX1, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or
GLUDy
434
7
5.54832
0.1485
ADHEr, ASPT, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
435
7
5.54749
0.08705
ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
436
7
5.54
0.14449
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, SUCOAS
437
7
5.53962
0.15019
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, SUCOAS
438
7
5.53831
0.08799
ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
439
7
5.53831
0.08799
ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy
440
7
5.53405
0.05732
ACKr and/or PTAr, ADHEr, ASPT, EDA and/or
PGDHY, LDH_D, MDH, PGI
441
7
5.52463
0.06237
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PGI
442
7
5.51713
0.16489
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, SUCOAS
443
7
5.51587
0.14532
ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS, TAL
444
7
5.51034
0.14586
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PGDH
445
7
5.51034
0.14586
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, PGDH
446
7
5.5014
0.07482
ADHEr, ASPT, EDA and/or PGDHY, GLCpts, LDH_D, MDH, PGI
447
7
5.49363
0.14609
ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE, SUCOAS
448
7
5.49315
0.18499
ACKr and/or PTAr, ADHEr, EDA and/or
PGDHY, HEX1, LDH_D, PFLi, PGI
449
7
5.48581
0.14671
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, TAL
450
7
5.48581
0.14671
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, TAL
451
7
5.47686
0.19195
ADHEr, EDA and/or PGDHY, LDH_D, MDH, NADH6, PGI, THD5
452
7
5.47123
0.12153
ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy
453
7
5.47123
0.12153
ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK, THD2 and/or GLUDy
454
7
5.46318
0.14749
ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, RPE
455
7
5.46318
0.14749
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, RPE
456
7
5.43967
0.13732
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, THD2 and/or
GLUDy
457
7
5.41442
0.15603
ADHEr, ASPT, FUM, HEX1, LDH_D, MDH, THD2 and/or GLUDy
458
7
5.41442
0.15603
ADHEr, ASPT, HEX1, LDH_D, MALS, MDH, THD2 and/or
GLUDy
459
7
5.41442
0.15603
ADHEr, ASPT, HEX1, ICL, LDH_D, MDH, THD2 and/or GLUDy
460
7
5.32088
0.07204
ADHEr, FRD and/or
SUCD4, HEX1, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy
461
7
5.29376
0.07385
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MALS, PPCK, THD2
and/or GLUDy
462
7
5.29376
0.07385
ADHEr, FRD and/or SUCD4, HEX1, ICL, LDH_D, PPCK, THD2
and/or GLUDy
463
7
5.29342
0.0731
ADHEr, HEX1, LDH_D, MDH, PPCK, SUCOAS, THD2 and/or
GLUDy
464
7
5.29342
0.0731
ADHEr, FUM, HEX1, LDH_D, PPCK, SUCOAS, THD2 and/or
GLUDy
465
7
5.20741
0.07603
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PGDH, PPCK
466
7
5.20741
0.07603
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PGL and/or
G6PDHy, PPCK
467
7
5.20289
0.16585
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PFLi, RPE
468
7
5.19469
0.07664
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK, TAL
469
7
5.18818
0.16709
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PFLi, TAL
470
7
5.18292
0.0772
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK, RPE
471
7
5.18092
0.10975
ADHEr, FUM, GLCpts, LDH_D, PFLi, PGI, PPCK
472
7
5.18092
0.10975
ADHEr, GLCpts, LDH_D, MDH, PFLi, PGI, PPCK
473
7
5.18025
0.13169
ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK
474
7
5.18025
0.13169
ACKr and/or PTAr, ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK
475
7
5.17526
0.14527
ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi
476
7
5.15999
0.12483
ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, PGI
477
7
5.15742
0.1471
ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6, PFLi
478
7
5.13283
0.14064
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, PGI, PPCK
479
7
5.13283
0.14064
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFLi, PGI, PPCK
480
7
5.11128
0.1487
ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi
481
7
5.10917
0.0835
ADHEr, EDA and/or
PGDHY, GLCpts, LDH_D, NADH6, PGI, PPCK
482
7
5.10795
0.18114
ADHEr, ASPT, ATPS4r, EDA and/or
PGDHY, LDH_D, MDH, PGDH
483
7
5.08422
0.18199
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, TAL
484
7
5.08162
0.15602
ACKr and/or PTAr, ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI
485
7
5.08154
0.15604
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PFLi, PGI
486
7
5.07957
0.15652
ACKr and/or PTAr, ADHEr, FUM, LDH_D, NADH6, PFLi, PGI
487
7
5.06238
0.18278
ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, RPE
488
7
5.04933
0.08188
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, SUCOAS
489
7
5.04086
0.10883
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGDH, PGI
490
7
5.03782
0.10979
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGI, TAL
491
7
5.03499
0.11069
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGI, RPE
492
7
5.0347
0.123
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS
493
7
5.02325
0.08242
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, SUCOAS
494
7
5.02325
0.08242
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, SUCOAS
495
7
5.02233
0.1115
ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK, THD2 and/or
GLUDy
496
7
5.00367
0.01301
ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or
GLUDy
497
7
5.00099
0.08371
ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK, SUCOAS
498
7
4.9785
0.07565
ADHEr, ATPS4r, FDH2, LDH_D, NADH6, SUCOAS, THD2 and/or
GLUDy
499
7
4.97218
0.04755
ADHEr, EDA and/or PGDHY, HEX1, LDH_D, NADH6, PGI, THD2
and/or GLUDy
500
7
4.96921
0.01295
ADHEr, FRD and/or
SUCD4, GLUDy, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
501
7
4.96716
0.01165
ADHEr, GLUDy, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
502
7
4.96611
0.01426
ADHEr, FRD and/or
SUCD4, GLCpts, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
503
7
4.96543
0.01322
ADHEr, FRD and/or SUCD4, GLUDy, ICL, LDH_D, MDH, THD2
and/or GLUDy
504
7
4.96543
0.01322
ADHEr, FRD and/or
SUCD4, GLUDy, LDH_D, MALS, MDH, THD2 and/or GLUDy
505
7
4.96335
0.01401
ADHEr, AKGD, FRD and/or SUCD4, ICL, LDH_D, PPCK, THD2
and/or GLUDy
506
7
4.96335
0.01401
ADHEr, AKGD, FRD and/or SUCD4, LDH_D, MALS, PPCK, THD2
and/or GLUDy
507
7
4.96309
0.0131
ADHEr, GLUDy, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or
GLUDy
508
7
4.96271
0.01323
ADHEr, ASPT, GLUDy, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
509
7
4.96187
0.01458
ADHEr, FRD and/or
SUCD4, GLCpts, LDH_D, MALS, MDH, THD2 and/or GLUDy
510
7
4.96187
0.01458
ADHEr, FRD and/or SUCD4, GLCpts, ICL, LDH_D, MDH, THD2
and/or GLUDy
511
7
4.95873
0.01465
ADHEr, ASPT, GLCpts, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
512
7
4.95686
0.01531
ADHEr, ASPT, LDH_D, MDH, PYK, SUCOAS, THD2 and/or
GLUDy
513
7
4.95085
0.01406
ADHEr, ASPT, GLUDy, LDH_D, MALS, MDH, THD2 and/or
GLUDy
514
7
4.95085
0.01406
ADHEr, ASPT, GLUDy, ICL, LDH_D, MDH, THD2 and/or GLUDy
515
7
4.94529
0.0136
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, GLUDy, LDH_D, MDH, THD2 and/or GLUDy
516
7
4.94524
0.01566
ADHEr, ASPT, GLCpts, LDH_D, MALS, MDH, THD2 and/or
GLUDy
517
7
4.94524
0.01566
ADHEr, ASPT, GLCpts, ICL, LDH_D, MDH, THD2 and/or GLUDy
518
7
4.94393
0.07737
ADHEr, ATPS4r, FUM, LDH_D, NADH6, SUCOAS, THD2 and/or
GLUDy
519
7
4.94353
0.01615
ADHEr, ASPT, FUM, LDH_D, MDH, PYK, THD2 and/or GLUDy
520
7
4.94353
0.01615
ADHEr, ASPT, ICL, LDH_D, MDH, PYK, THD2 and/or GLUDy
521
7
4.94353
0.01615
ADHEr, ASPT, LDH_D, MALS, MDH, PYK, THD2 and/or GLUDy
522
7
4.94073
0.01473
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, GLCpts, LDH_D, MDH, THD2 and/or GLUDy
523
7
4.9406
0.01618
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PYK, THD2
and/or GLUDy
524
7
4.93746
0.13888
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, NADH6
525
7
4.93038
0.19331
ACKr and/or
PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH
526
7
4.92604
0.19438
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL
527
7
4.92485
0.04913
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, NADH6, PGI, THD2
and/or GLUDy
528
7
4.92203
0.19537
ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE
529
7
4.89572
0.18301
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGDH
530
7
4.88586
0.18375
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, TAL
531
7
4.8768
0.18443
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, RPE
532
7
4.87463
0.19612
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGI, THD5
533
7
4.87463
0.19612
ACKr and/or PTAr, ADHEr, FUM, LDH_D, NADH6, PGI, THD5
534
7
4.85806
0.05038
ADHEr, LDH_D, MDH, NADH12, NADH6, SUCOAS, THD2 and/or
GLUDy
535
7
4.85603
0.21579
ADHEr, ASPT, ATPS4r, FUM, HEX1, LDH_D, PPS
536
7
4.84975
0.05712
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PGL and/or
G6PDHy, PPCK, PYK
537
7
4.84975
0.05712
ADHEr, EDA and/or PGDHY, FRD and/or
SUCD4, LDH_D, MDH, PPCK, PYK
538
7
4.81569
0.05271
ADHEr, ASPT, FUM, GLCpts, LDH_D, PPCK, THD2 and/or
GLUDy
539
7
4.81323
0.1198
ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PFLi, PPCK
540
7
4.81194
0.09049
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK, RPE, SUCOAS
541
7
4.81179
0.07918
ADHEr, LDH_D, NADH12, NADH6, PPCK, SUCOAS, THD2
and/or GLUDy
542
7
4.79812
0.05498
ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, NADH6, THD2
and/or GLUDy
543
7
4.79812
0.05498
ACKr and/or PTAr, ADHEr, LDH_D, MALS, MDH, NADH6, THD2
and/or GLUDy
544
7
4.79812
0.05498
ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, NADH6, THD2
and/or GLUDy
545
7
4.79409
0.12192
ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PPCK
546
7
4.79101
0.05495
ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, MDH, THD2
and/or GLUDy
547
7
4.78296
0.12315
ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PPCK
548
7
4.78296
0.12315
ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PPCK
549
7
4.77588
0.08569
ADHEr, AKGD, FRD and/or SUCD4, LDH_D, MALS, MDH, THD2
and/or GLUDy
550
7
4.77588
0.08569
ADHEr, AKGD, FRD and/or SUCD4, ICL, LDH_D, MDH, THD2
and/or GLUDy
551
7
4.77561
0.07964
ADHEr, FUM, LDH_D, NADH6, PPCK, SUCOAS, THD2 and/or
GLUDy
552
7
4.77561
0.07964
ADHEr, LDH_D, MDH, NADH6, PPCK, SUCOAS, THD2 and/or
GLUDy
553
7
4.77329
0.08047
ADHEr, ASPT, LDH_D, MDH, PPCK, SUCOAS, THD2 and/or
GLUDy
554
7
4.77329
0.08047
ADHEr, ASPT, FUM, LDH_D, PPCK, SUCOAS, THD2 and/or
GLUDy
555
7
4.7705
0.16052
ADHEr, ATPS4r, FDH2, GLCpts, LDH_D, MDH, NADH6
556
7
4.76518
0.08334
ADHEr, FUM, HEX1, LDH_D, NADH6, SUCOAS, THD2 and/or
GLUDy
557
7
4.76374
0.0222
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
558
7
4.76264
0.08425
ADHEr, ASPT, FUM, HEX1, LDH_D, SUCOAS, THD2 and/or
GLUDy
559
7
4.74028
0.09218
ADHEr, FUM, LDH_D, NADH6, PPCK, PYK, SUCOAS
560
7
4.74028
0.09218
ADHEr, LDH_D, MDH, NADH6, PPCK, PYK, SUCOAS
561
7
4.73717
0.09329
ADHEr, ASPT, LDH_D, MDH, PPCK, PYK, SUCOAS
562
7
4.73717
0.09329
ADHEr, ASPT, FUM, LDH_D, PPCK, PYK, SUCOAS
563
7
4.71257
0.12092
ADHEr, GLCpts, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or
GLUDy
564
7
4.69803
0.08636
ADHEr, ASPT, FUM, LDH_D, MALS, PPCK, THD2 and/or GLUDy
565
7
4.69803
0.08636
ADHEr, ASPT, FUM, ICL, LDH_D, PPCK, THD2 and/or GLUDy
566
7
4.69803
0.08636
ADHEr, ASPT, LDH_D, MALS, MDH, PPCK, THD2 and/or GLUDy
567
7
4.69803
0.08636
ADHEr, ASPT, ICL, LDH_D, MDH, PPCK, THD2 and/or GLUDy
568
7
4.68998
0.14917
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE, SUCOAS, THD2
and/or GLUDy
569
7
4.68111
0.0912
ADHEr, ASPT, FUM, HEX1, LDH_D, MALS, THD2 and/or GLUDy
570
7
4.68111
0.0912
ADHEr, ASPT, FUM, HEX1, ICL, LDH_D, THD2 and/or GLUDy
571
7
4.68063
0.05195
ACKr and/or
PTAr, ADHEr, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or
GLUDy
572
7
4.67974
0.09159
ADHEr, AKGD, ASPT, LDH_D, MALS, MDH, THD2 and/or
GLUDy
573
7
4.67974
0.09159
ADHEr, AKGD, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy
574
7
4.6782
0.13538
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
575
7
4.67079
0.08656
ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, PPCK, THD2
and/or GLUDy
576
7
4.66866
0.1492
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, TAL, THD2
and/or GLUDy
577
7
4.66526
0.1329
ADHEr, HEX1, LDH_D, MDH, RPE, SUCOAS, THD2 and/or
GLUDy
578
7
4.65177
0.09959
ADHEr, ASPT, LDH_D, MALS, MDH, PPCK, PYK
579
7
4.65177
0.09959
ADHEr, ASPT, ICL, LDH_D, MDH, PPCK, PYK
580
7
4.65148
0.09163
ACKr and/or PTAr, ADHEr, ASPT, FUM, HEX1, LDH_D, THD2
and/or GLUDy
581
7
4.64795
0.13132
ADHEr, ATPS4r, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or
GLUDy
582
7
4.64717
0.13258
ADHEr, HEX1, LDH_D, MDH, SUCOAS, TAL, THD2 and/or
GLUDy
583
7
4.63555
0.16411
ADHEr, FRD and/or
SUCD4, GLCpts, LDH_D, MDH, SUCOAS, TAL
584
7
4.62743
0.13223
ADHEr, HEX1, LDH_D, MDH, PGDH, SUCOAS, THD2 and/or
GLUDy
585
7
4.62722
0.10661
ADHEr, AKGD, ASPT, ICL, LDH_D, MDH, PYK
586
7
4.62722
0.10661
ADHEr, AKGD, ASPT, LDH_D, MALS, MDH, PYK
587
7
4.61442
0.12597
ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2
and/or GLUDy
588
7
4.60692
0.10476
ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGI
589
7
4.60239
0.15202
ADHEr, AKGD, FRD and/or SUCD4, HEX1, LDH_D, MALS, THD2
and/or GLUDy
590
7
4.60239
0.15202
ADHEr, AKGD, FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2
and/or GLUDy
591
7
4.59777
0.03574
ACKr and/or
PTAr, ADHEr, GLUDy, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
592
7
4.59693
0.17398
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH, PYK, RPE
593
7
4.59408
0.12607
ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, PPCK
594
7
4.588
0.17333
ADHEr, ASPT, LDH_D, MDH, NADH6, PYK, SUCOAS
595
7
4.58269
0.16568
ADHEr, ASPT, GLCpts, LDH_D, MDH, RPE, SUCOAS
596
7
4.5614
0.17413
ADHEr, ASPT, LDH_D, MDH, PYK, RPE, SUCOAS
597
7
4.55514
0.12723
ADHEr, ASPT, FUM, GLCpts, LDH_D, MDH, THD2 and/or
GLUDy
598
7
4.54823
0.19006
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
599
7
4.54674
0.19069
ACKr and/or PTAr, ADHEr, ATPS4r, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS
600
7
4.53866
0.20774
ADHEr, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, TAL, THD2 and/or GLUDy
601
7
4.53699
0.1728
ADHEr, FRD and/or SUCD4, FUM, LDH_D, MDH, PYK, TAL
602
7
4.53586
0.209
ADHEr, ATPS4r, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, TAL
603
7
4.51849
0.13149
ADHEr, ATPS4r, FDH2, GLCpts, LDH_D, NADH6, PPCK
604
7
4.51845
0.20259
ADHEr, FDH2, LDH_D, MDH, NADH12, NADH6, SUCOAS
605
7
4.50909
0.20652
ADHEr, ATPS4r, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
606
7
4.50549
0.20804
ADHEr, FRD and/or
SUCD4, GLU5K, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
607
7
4.50549
0.20804
ADHEr, FRD and/or
SUCD4, G5SD, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
608
7
4.50543
0.20807
ADHEr, ASNS2, FRD and/or
SUCD4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
609
7
4.50378
0.20876
ADHEr, HEX1, LDH_D, NADH12, NADH6, SUCOAS, THD2
and/or GLUDy
610
7
4.50198
0.20952
ADHEr, ATPS4r, FDH2, HEX1, LDH_D, NADH6, SUCOAS
611
3
0.669528208
0.263200019
ADHEr, FADH4, LDH_D
612
3
0.110808857
0.249569497
ADHEr, LDH_D, PFK and/or FBA and/or TPI
613
4
0.93088625
0.384018912
ADHEr, ATPS4r, CBMK2, LDH_D
614
4
1.163912961
0.378757536
ADHEr, ATPS4r, LDH_D, TKT2
615
4
4.552254757
0.171189177
ADHEr, FADH4, LDH_D, MDH
616
4
0.710935568
0.257359864
ADHEr, FADH4, LDH_D, RPE
617
4
0.702400877
0.260125701
ADHEr, FADH4, LDH_D, TAL
618
4
0.719290675
0.254652225
ADHEr, FADH4, LDH_D, TKT2
619
4
2.283994469
0.165173433
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK
620
4
0.18159615
0.246009686
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT2
621
4
4.555303308
0.170023606
ADHEr, FRD and/or SUCD4, LDH_D, ME2
622
4
0.710935568
0.257359864
ADHEr, FRD and/or SUCD4, LDH_D, RPE
623
4
0.702400877
0.260125701
ADHEr, FRD and/or SUCD4, LDH_D, TAL
624
4
0.753832099
0.262897433
ADHEr, FRD and/or SUCD4, LDH_D, THD2 and/or GLUDy
625
4
0.719290675
0.254652225
ADHEr, FRD and/or SUCD4, LDH_D, TKT2
626
4
2.165485624
0.255793845
ADHEr, FUM, LDH_D, PPCK
627
4
2.279039418
0.168962421
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI
628
4
0.24281895
0.340878264
ADHEr, HEX1, LDH_D, MDH
629
4
2.165485624
0.255793845
ADHEr, LDH_D, MDH, PPCK
630
4
4.555303308
0.170023606
ADHEr, FADH4, LDH_D, ME2
631
4
2.575639756
0.188350402
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI
632
4
2.359364668
0.333057496
ADHEr, FUM, LDH_D, NADH6
633
4
2.365271913
0.329105029
ADHEr, HEX1, LDH_D, NADH6
634
4
2.381170653
0.338701395
ADHEr, LDH_D, NADH6, TAL
635
4
2.440438621
0.335394947
ADHEr, LDH_D, NADH6, TKT2
636
4
0.303924042
0.270354967
ADHEr, LDH_D, PPCK, TKT2
637
4
0.276184314
0.387717801
ADHEr, HEX1, LDH_D, PPS
638
4
0.158957458
0.24714816
ADHEr, LDH_D, PFK and/or FBA and/or TPI, RPE
639
4
0.268683115
0.271077634
ADHEr, LDH_D, PPCK, RPE
640
4
2.268133298
0.177302028
ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or TPI
641
4
2.303972218
0.174839451
ADHEr, FRD and/or SUCD4, LDH_D, PGI
642
4
2.202777355
0.227277897
ADHEr, FRD and/or SUCD4, LDH_D, PPCK
643
4
0.136108259
0.248297219
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TAL
644
4
0.233253283
0.271804175
ADHEr, LDH_D, PPCK, TAL
645
4
0.753832099
0.262897433
ADHEr, FADH4, LDH_D, THD2 and/or GLUDy
646
4
1.00803743
0.384504476
ADHEr, ATPS4r, LDH_D, TKT1
647
4
0.702400877
0.260125701
ADHEr, FADH4, LDH_D, TKT1
648
4
0.136108259
0.248297219
ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT1
649
4
0.702400877
0.260125701
ADHEr, FRD and/or SUCD4, LDH_D, TKT1
650
4
2.381170653
0.338701395
ADHEr, LDH_D, NADH6, TKT1
651
4
0.233253283
0.271804175
ADHEr, LDH_D, PPCK, TKT1
652
5
0.506864978
0.275350379
ADHEr, ASPT, FUM, LDH_D, TKT2
653
5
2.69303784
0.249064654
ADHEr, ASPT, LDH_D, MDH, TKT2
654
5
4.367851715
0.180794605
ADHEr, ASPT, FUM, LDH_D, ME2
655
5
0.41729748
0.278421057
ADHEr, ASPT, FUM, LDH_D, RPE
656
5
0.325709758
0.281560994
ADHEr, ASPT, FUM, LDH_D, TAL
657
5
2.645877173
0.251090969
ADHEr, ASPT, LDH_D, MDH, TAL
658
5
0.746129455
0.280922235
ADHEr, ASPT, FUM, LDH_D, THD2 and/or GLUDy
659
5
1.038232852
0.250484522
ADHEr, ATPS4r, FADH4, LDH_D, SUCOAS
660
5
1.091156432
0.260562519
ADHEr, ATPS4r, FADH4, LDH_D, THD2 and/or GLUDy
661
5
3.468709118
0.171072336
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, ME2
662
5
1.635653331
0.170872463
ADHEr, ATPS4r, LDH_D, PFK and/or FBA and/or TPI, PFLi
663
5
0.971889592
0.254878539
ADHEr, ATPS4r, LDH_D, PPCK, PYK
664
5
2.773151559
0.15594541
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PGI
665
5
0.794188757
0.220436537
ADHEr, FADH4, HEX1, LDH_D, TKT2
666
5
4.635305085
0.167733685
ADHEr, FADH4, LDH_D, MDH, TKT2
667
5
0.726213349
0.222710178
ADHEr, FADH4, HEX1, LDH_D, RPE
668
5
4.60885325
0.168834272
ADHEr, FADH4, LDH_D, MDH, RPE
669
5
4.600497528
0.168070055
ADHEr, FADH4, LDH_D, MDH, SUCOAS
670
5
4.582051994
0.169949398
ADHEr, FADH4, LDH_D, MDH, TAL
671
5
1.218736747
0.252980412
ADHEr, FADH4, LDH_D, SUCOAS, THD2 and/or GLUDy
672
5
2.323324205
0.162516565
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK, TKT2
673
5
0.794188757
0.220436537
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TKT2
674
5
1.080064187
0.261131964
ADHEr, FRD and/or SUCD4, ICL, LDH_D, THD2 and/or GLUDy
675
5
1.080064187
0.261131964
ADHEr, FRD and/or SUCD4, LDH_D, MALS, THD2 and/or
GLUDy
676
5
4.635305085
0.167733685
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT2
677
5
4.611480051
0.167700442
ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE
678
5
4.603168381
0.166946432
ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS
679
5
4.584877196
0.168800591
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL
680
5
4.692124349
0.117711987
ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or GLUDy
681
5
4.637738386
0.16661454
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT2
682
5
4.582051994
0.169949398
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL
683
5
1.218736747
0.252980412
ADHEr, FRD and/or SUCD4, LDH_D, SUCOAS, THD2 and/or
GLUDy
684
5
2.22459069
0.253337321
ADHEr, FUM, LDH_D, PPCK, TKT2
685
5
2.084553651
0.317680271
ADHEr, FRD and/or SUCD4, FUM, GLU5K, LDH_D
686
5
2.319043241
0.166454441
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, TKT2
687
5
0.379133409
0.337257295
ADHEr, FUM, HEX1, LDH_D, TKT2
688
5
0.379133409
0.337257295
ADHEr, HEX1, LDH_D, MDH, TKT2
689
5
1.080064187
0.261131964
ADHEr, FADH4, ICL, LDH_D, THD2 and/or GLUDy
690
5
1.080064187
0.261131964
ADHEr, FADH4, LDH_D, MALS, THD2 and/or GLUDy
691
5
2.22459069
0.253337321
ADHEr, LDH_D, MDH, PPCK, TKT2
692
5
4.611480051
0.167700442
ADHEr, FADH4, LDH_D, ME2, RPE
693
5
4.603168381
0.166946432
ADHEr, FADH4, LDH_D, ME2, SUCOAS
694
5
4.584877196
0.168800591
ADHEr, FADH4, LDH_D, ME2, TAL
695
5
4.692124349
0.117711987
ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy
696
5
4.637738386
0.16661454
ADHEr, FADH4, LDH_D, ME2, TKT2
697
5
2.871064894
0.268281645
ACKr and/or PTAr, ADHEr, LDH_D, ME2, NADH6
698
5
2.165924808
0.255458013
ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2
699
5
4.328684855
0.179395311
ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, ME2
700
5
1.421860815
0.279607649
ADHEr, FUM, LDH_D, ME2, THD2 and/or GLUDy
701
5
2.626697246
0.185479991
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT2
702
5
2.515320971
0.228708985
ADHEr, FUM, LDH_D, NADH6, PPCK
703
5
2.3870222
0.31455217
ADHEr, FUM, HEX1, LDH_D, NADH6
704
5
2.452754421
0.325482265
ADHEr, HEX1, LDH_D, NADH6, TKT2
705
5
2.515320971
0.228708985
ADHEr, LDH_D, MDH, NADH6, PPCK
706
5
2.517581576
0.273304492
ADHEr, LDH_D, MDH, NADH6, TKT2
707
5
2.44490357
0.275824433
ADHEr, FUM, LDH_D, ME2, NADH6
708
5
2.6103794
0.186397367
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, RPE
709
5
2.555986853
0.242393036
ADHEr, LDH_D, NADH6, PPCK, TKT2
710
5
2.421064631
0.329399782
ADHEr, FUM, LDH_D, NADH6, RPE
711
5
2.424713593
0.326643472
ADHEr, HEX1, LDH_D, NADH6, RPE
712
5
2.535155189
0.243219368
ADHEr, LDH_D, NADH6, PPCK, RPE
713
5
2.593899337
0.187323863
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TAL
714
5
2.391804455
0.331134392
ADHEr, FUM, LDH_D, NADH6, TAL
715
5
2.470792173
0.274926806
ADHEr, LDH_D, MDH, NADH6, TAL
716
5
2.514181006
0.244051353
ADHEr, LDH_D, NADH6, PPCK, TAL
717
5
0.318612958
0.180159999
ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFK and/or FBA
and/or TPI
718
5
2.298067641
0.164222738
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK, TAL
719
5
1.630847318
0.197351873
ADHEr, LDH_D, MDH, PFK and/or FBA and/or TPI, PFLi
720
5
4.068156517
0.116307981
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PFLi, PPCK
721
5
1.630847318
0.197351873
ADHEr, FUM, LDH_D, PFK and/or FBA and/or TPI, PFLi
722
5
1.732749142
0.29901565
ADHEr, FUM, HEX1, LDH_D, PFLi
723
5
1.717432965
0.229711682
ADHEr, HEX1, LDH_D, PFLi, PPCK
724
5
3.998783556
0.193038233
ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi
725
5
4.075902556
0.129526478
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, PFLi
726
5
3.837085522
0.275765851
ADHEr, LDH_D, NADH6, PFLi, RPE
727
5
3.820196202
0.277060587
ADHEr, LDH_D, NADH6, PFLi, TAL
728
5
3.853817724
0.274483161
ADHEr, LDH_D, NADH6, PFLi, TKT2
729
5
3.839632897
0.261974412
ADHEr, FRD and/or SUCD4, LDH_D, PFLi, THD2 and/or GLUDy
730
5
4.393030474
0.140852085
ADHEr, EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, PGI
731
5
0.430495607
0.382946425
ADHEr, HEX1, LDH_D, PPS, TKT2
732
5
0.381078242
0.384474433
ADHEr, HEX1, LDH_D, PPS, RPE
733
5
0.331264934
0.386014683
ADHEr, HEX1, LDH_D, PPS, TAL
734
5
0.331264934
0.386014683
ADHEr, HEX1, LDH_D, PPS, TKT1
735
5
0.318612958
0.180159999
ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFK and/or FBA
and/or TPI
736
5
2.310761865
0.163365197
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK, RPE
737
5
2.306254619
0.167256205
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, RPE
738
5
0.335428873
0.338418234
ADHEr, HEX1, LDH_D, MDH, RPE
739
5
2.674115155
0.222196435
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH
740
5
2.310408468
0.174397201
ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or
TPI, TKT2
741
5
2.084492611
0.317726946
ADHEr, ASNS2, FRD and/or SUCD4, FUM, LDH_D
742
5
2.086878053
0.315902866
ADHEr, CBMK2, FRD and/or SUCD4, FUM, LDH_D
743
5
2.084553651
0.317680271
ADHEr, FRD and/or SUCD4, FUM, G5SD, LDH_D
744
5
2.221671127
0.212830356
ADHEr, FRD and/or SUCD4, FUM, LDH_D, PPCK
745
5
2.110766092
0.297636347
ADHEr, FRD and/or SUCD4, FUM, HEX1, LDH_D
746
5
2.221671127
0.212830356
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PPCK
747
5
2.29690811
0.175324843
ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or
TPI, RPE
748
5
2.283263365
0.176262405
ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or
TPI, TAL
749
5
2.799273371
0.150483141
ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, PGI
750
5
2.255979533
0.224464061
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT2
751
5
4.330026614
0.179036754
ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MDH
752
5
2.238942691
0.225365131
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, RPE
753
5
2.119938959
0.309086971
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TAL
754
5
2.221768517
0.226273464
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TAL
755
5
2.186545813
0.254918542
ADHEr, FUM, LDH_D, PPCK, TAL
756
5
2.293342202
0.16806573
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, TAL
757
5
0.29142241
0.339587193
ADHEr, FUM, HEX1, LDH_D, TAL
758
5
0.29142241
0.339587193
ADHEr, HEX1, LDH_D, MDH, TAL
759
5
2.186545813
0.254918542
ADHEr, LDH_D, MDH, PPCK, TAL
760
5
3.603753429
0.189374217
ADHEr, FADH4, HEX1, LDH_D, THD2 and/or GLUDy
761
5
4.688321717
0.11916587
ADHEr, FADH4, LDH_D, MDH, THD2 and/or GLUDy
762
5
3.879826068
0.057433556
ADHEr, FADH4, LDH_D, PPCK, THD2 and/or GLUDy
763
5
2.092923946
0.31127974
ADHEr, FUM, HEX1, LDH_D, THD2 and/or GLUDy
764
5
1.181362879
0.252361787
ADHEr, HEX1, LDH_D, PPCK, THD2 and/or GLUDy
765
5
0.325709758
0.281560994
ADHEr, ASPT, FUM, LDH_D, TKT1
766
5
2.645877173
0.251090969
ADHEr, ASPT, LDH_D, MDH, TKT1
767
5
4.582051994
0.169949398
ADHEr, FADH4, LDH_D, MDH, TKT1
768
5
2.298067641
0.164222738
ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK, TKT1
769
5
4.582051994
0.169949398
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT1
770
5
4.584877196
0.168800591
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT1
771
5
2.186545813
0.254918542
ADHEr, FUM, LDH_D, PPCK, TKT1
772
5
2.293342202
0.16806573
ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, TKT1
773
5
0.29142241
0.339587193
ADHEr, FUM, HEX1, LDH_D, TKT1
774
5
0.29142241
0.339587193
ADHEr, HEX1, LDH_D, MDH, TKT1
775
5
2.186545813
0.254918542
ADHEr, LDH_D, MDH, PPCK, TKT1
776
5
4.584877196
0.168800591
ADHEr, FADH4, LDH_D, ME2, TKT1
777
5
2.593899337
0.187323863
ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT1
778
5
2.391804455
0.331134392
ADHEr, FUM, LDH_D, NADH6, TKT1
779
5
2.396471968
0.327812994
ADHEr, HEX1, LDH_D, NADH6, TKT1
780
5
2.470792173
0.274926806
ADHEr, LDH_D, MDH, NADH6, TKT1
781
5
2.514181006
0.244051353
ADHEr, LDH_D, NADH6, PPCK, TKT1
782
5
3.820196202
0.277060587
ADHEr, LDH_D, NADH6, PFLi, TKT1
783
5
2.283263365
0.176262405
ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or
TPI, TKT1
784
5
2.119938959
0.309086971
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TKT1
785
5
2.221768517
0.226273464
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT1
786
6
4.63815309
0.138347146
ADHEr, ASPT, FADH4, LDH_D, MDH, PYK
787
6
4.459389393
0.176944819
ADHEr, ASPT, FUM, LDH_D, MDH, TKT2
788
6
4.380192095
0.177265253
ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MALS, MDH
789
6
4.40071793
0.179412355
ADHEr, ASPT, LDH_D, MALS, MDH, TAL
790
6
4.60130813
0.167781976
ADHEr, ASPT, LDH_D, MDH, SUCOAS, TKT2
791
6
4.459389393
0.176944819
ADHEr, ASPT, FUM, LDH_D, ME2, TKT2
792
6
4.430256792
0.178170044
ADHEr, ASPT, FUM, LDH_D, ME2, RPE
793
6
4.40071793
0.179412355
ADHEr, ASPT, FUM, LDH_D, ME2, TAL
794
6
4.563806878
0.124751372
ADHEr, ASPT, FUM, LDH_D, ME2, THD2 and/or GLUDy
795
6
4.380192095
0.177265253
ADHEr, ASPT, FRD and/or SUCD4, FUM, LDH_D, MDH
796
6
4.380192095
0.177265253
ADHEr, ASPT, FRD and/or SUCD4, FUM, LDH_D, ME2
797
6
4.40071793
0.179412355
ADHEr, ASPT, FUM, LDH_D, MDH, TAL
798
6
4.547590285
0.169998972
ADHEr, ASPT, LDH_D, MDH, SUCOAS, TAL
799
6
4.32598859
0.179624623
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, THD2 and/or
GLUDy
800
6
4.503409516
0.124027245
ADHEr, ASPT, ATPS4r, LDH_D, MDH, TKT2
801
6
4.55209485
0.121669936
ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGDH
802
6
4.519337222
0.123256037
ADHEr, ASPT, ATPS4r, LDH_D, MDH, RPE
803
6
4.535068073
0.12249436
ADHEr, ASPT, ATPS4r, LDH_D, MDH, TAL
804
6
5.189612621
0.143792027
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, ME2, PFLi
805
6
4.321178853
0.120168187
ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, MDH, PGI
806
6
4.555303308
0.170023606
ADHEr, DAAD, FRD and/or SUCD4, LDH_D, ME2, PRO1z
807
6
4.552254757
0.171189177
ADHEr, DAAD, FRD and/or SUCD4, LDH_D, MDH, PRO1z
808
6
4.611166872
0.137118286
ADHEr, FADH4, FUM, LDH_D, MDH, PYK
809
6
4.681108251
0.164738111
ADHEr, FADH4, LDH_D, MDH, SUCOAS, TKT2
810
6
4.655427003
0.165799614
ADHEr, FADH4, LDH_D, MDH, RPE, SUCOAS
811
6
4.62941265
0.166874885
ADHEr, FADH4, LDH_D, MDH, SUCOAS, TAL
812
6
4.57471951
0.178914826
ADHEr, FADH4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy
813
6
4.72140048
0.117206382
ADHEr, FADH4, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy
814
6
5.77503303
0.099302315
ADHEr, EDA and/or PGDHY, FADH4, LDH_D, MDH, THD2
and/or GLUDy
815
6
4.802404267
0.049107371
ACKr and/or PTAr, ADHEr, FADH4, LDH_D, PPCK, THD2 and/or
GLUDy
816
6
4.611166872
0.137118286
ADHEr, FRD and/or SUCD4, FUM, LDH_D, MDH, PYK
817
6
4.681108251
0.164738111
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, TKT2
818
6
4.640728933
0.137362308
ADHEr, FRD and/or SUCD4, LDH_D, MDH, ME2, PYK
819
6
4.657699713
0.164706045
ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE, SUCOAS
820
6
4.683198197
0.163658455
ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS, TKT2
821
6
4.63187269
0.165767133
ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS, TAL
822
6
5.443882794
0.111598427
ADHEr, FRD and/or SUCD4, HEX1, LDH_D, ME2, THD2 and/or
GLUDy
823
6
4.714403301
0.10919394
ACKr and/or PTAr, ADHEr, FRD and/or
SUCD4, LDH_D, ME2, THD2 and/or GLUDy
824
6
4.727966085
0.117420509
ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE, THD2 and/or
GLUDy
825
6
4.724744313
0.115799637
ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS, THD2
and/or GLUDy
826
6
4.710892193
0.11755936
ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL, THD2 and/or
GLUDy
827
6
4.744999691
0.117281986
ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or
GLUDy, TKT2
828
6
4.62941265
0.166874885
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, TAL
829
6
4.74186178
0.118725202
ADHEr, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or
GLUDy, TKT2
830
6
4.512318137
0.052959973
ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK, THD2 and/or
GLUDy
831
6
4.724614564
0.118867157
ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE, THD2 and/or
GLUDy
832
6
4.707326054
0.119009453
ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL, THD2 and/or
GLUDy
833
6
4.459389393
0.176944819
ADHEr, ASPT, ICL, LDH_D, MDH, TKT2
834
6
4.380192095
0.177265253
ADHEr, ASPT, FRD and/or SUCD4, ICL, LDH_D, MDH
835
6
4.40071793
0.179412355
ADHEr, ASPT, ICL, LDH_D, MDH, TAL
836
6
4.874048363
0.048155873
ADHEr, FADH4, ICL, LDH_D, PPCK, THD2 and/or GLUDy
837
6
4.520685593
0.183259208
ADHEr, FADH4, HEX1, ICL, LDH_D, THD2 and/or GLUDy
838
6
4.459389393
0.176944819
ADHEr, ASPT, LDH_D, MALS, MDH, TKT2
839
6
4.520685593
0.183259208
ADHEr, FADH4, HEX1, LDH_D, MALS, THD2 and/or GLUDy
840
6
4.874048363
0.048155873
ADHEr, FADH4, LDH_D, MALS, PPCK, THD2 and/or GLUDy
841
6
4.517532821
0.171239478
ADHEr, ASPT, FUM, LDH_D, ME2, SUCOAS
842
6
4.640728933
0.137362308
ADHEr, FADH4, LDH_D, MDH, ME2, PYK
843
6
4.657699713
0.164706045
ADHEr, FADH4, LDH_D, ME2, RPE, SUCOAS
844
6
4.683198197
0.163658455
ADHEr, FADH4, LDH_D, ME2, SUCOAS, TKT2
845
6
4.63187269
0.165767133
ADHEr, FADH4, LDH_D, ME2, SUCOAS, TAL
846
6
4.724744313
0.115799637
ADHEr, FADH4, LDH_D, ME2, SUCOAS, THD2 and/or GLUDy
847
6
4.744999691
0.117281986
ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy, TKT2
848
6
4.526462669
0.168070055
ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2, SUCOAS
849
6
5.443882794
0.111598427
ADHEr, FADH4, HEX1, LDH_D, ME2, THD2 and/or GLUDy
850
6
4.714403301
0.10919394
ACKr and/or PTAr, ADHEr, FADH4, LDH_D, ME2, THD2 and/or
GLUDy
851
6
4.727966085
0.117420509
ADHEr, FADH4, LDH_D, ME2, RPE, THD2 and/or GLUDy
852
6
4.710892193
0.11755936
ADHEr, FADH4, LDH_D, ME2, TAL, THD2 and/or GLUDy
853
6
4.33071542
0.180983121
ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, NADH6
854
6
4.313819364
0.203396482
ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, THD2 and/or
GLUDy
855
6
4.836109146
0.120863498
ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi, PGI
856
6
5.044438059
0.165184738
ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PFLi, PGI
857
6
5.168015107
0.134224131
ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi, THD2 and/or
GLUDy
858
6
5.320455827
0.059323173
ADHEr, FRD and/or SUCD4, LDH_D, MDH, PFLi, THD2 and/or
GLUDy
859
6
4.74186178
0.118725202
ADHEr, FADH4, LDH_D, MDH, THD2 and/or GLUDy, TKT2
860
6
4.512318137
0.052959973
ADHEr, FADH4, LDH_D, PPCK, PYK, THD2 and/or GLUDy
861
6
4.724614564
0.118867157
ADHEr, FADH4, LDH_D, MDH, RPE, THD2 and/or GLUDy
862
6
4.888461627
0.046924032
ADHEr, FADH4, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy
863
6
4.707326054
0.119009453
ADHEr, FADH4, LDH_D, MDH, TAL, THD2 and/or GLUDy
864
6
4.40071793
0.179412355
ADHEr, ASPT, FUM, LDH_D, MDH, TKT1
865
6
4.547590285
0.169998972
ADHEr, ASPT, LDH_D, MDH, SUCOAS, TKT1
866
6
4.535068073
0.12249436
ADHEr, ASPT, ATPS4r, LDH_D, MDH, TKT1
867
6
4.62941265
0.166874885
ADHEr, FADH4, LDH_D, MDH, SUCOAS, TKT1
868
6
4.63187269
0.165767133
ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS, TKT1
869
6
4.710892193
0.11755936
ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or
GLUDy, TKT1
870
6
4.62941265
0.166874885
ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, TKT1
871
6
4.707326054
0.119009453
ADHEr, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or
GLUDy, TKT1
872
6
4.40071793
0.179412355
ADHEr, ASPT, ICL, LDH_D, MDH, TKT1
873
6
4.40071793
0.179412355
ADHEr, ASPT, LDH_D, MALS, MDH, TKT1
874
6
4.40071793
0.179412355
ADHEr, ASPT, FUM, LDH_D, ME2, TKT1
875
6
4.63187269
0.165767133
ADHEr, FADH4, LDH_D, ME2, SUCOAS, TKT1
876
6
4.710892193
0.11755936
ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy, TKT1
877
6
4.707326054
0.119009453
ADHEr, FADH4, LDH_D, MDH, THD2 and/or GLUDy, TKT1
Minimal Gene Deletion Sets for Enhanced Production of 6-ACA. Described above are strain design strategies for improving 6-aminocaproate (6-ACA) production in microorganisms that possess the 6-ACA pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for 6-ACA production described in Table 14, a minimum set of deletions that is required for growth-coupled 6-ACA production has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible.
Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required for preventing the formation of competing byproducts, ethanol and lactate. Therefore, the minimal deletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include strains lacking at least one of the following activities in addition to ADHEr and LDH_D: malate dehydrogenase (MDH), aspartase (ASPT), NAD(P) transhydrogenase (THD2), and glutamate dehydrogenase (GLUDy). Such additional deletions result in a much tighter coupling of production with cell growth.
An additional minimal set of deletions includes phosphoglucoisomerase (PGI). This design focuses on generating reducing equivalents via the pentose phosphate pathway. Additional beneficial deletions include any of: acetaldehyde dehydrogenase (ADHEr), hexokinase (HEX1), 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA) and phosphogluconate dehydratase (PGDHy).
Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. For example, succinate dehydrogenase that oxidizes succinate to fumarate and is known to be active only under aerobic conditions can assume significant activity even under anaerobic conditions, and therefore such activity can be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled 6-ACA production strains.
Minimal Gene Deletion Sets for Enhanced Production of Adipate. Described above are strain design strategies for improving adipate production in microorganisms that possess the adipate pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for adipate production described in Table 15, a minimum set of deletions that is required for growth-coupled adipate production in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.
Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required for preventing the formation of competing byproducts, ethanol and lactate. Therefore, the minimal deletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include a strain lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D): fumarase (FUM), phosphoglucose isomerase (PGI), PEP carboxykinase (PPCK), hexokinase (HEX1), malate dehydrogenase (MDH), and NADH dehydrogenase (NADH6).
Additional deletions have been identified by the OptKnock framework for improving the growth-coupled formation of adipate. These include one or more of the following: malic enzyme (ME2), aspartate transaminase (ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), transhydrogenase (THD2), and glutamate dehydrogenase (GLUDy), and PTS system of glucose uptake (GLCpts). Further improvements in yields can be achieved by additional deletions in any of the following enzymes: ATP synthase (ATPS4r), phosphogluconate dehydratase (PGDHY), 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA), 6-phosphogluconolactonase (PGL), glucose 6-phosphate dehydrogenase (G6PDHY), and phosphogluconate dehydrogenase (PGDH).
Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled adipate production strains.
Minimal Gene Deletion Sets for Enhanced Production of HMDA. Described above are strain design strategies for improving hexamethylene diamine (HMDA) production in microorganisms that possess the HMDA pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for HMDA production described in Table 16, a minimum set of deletions that is required for growth-coupled HMDA production in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.
Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required for preventing the formation of competing byproducts, ethanol and lactate. Therefore, the minimal deletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include a strain lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D): fumarate reductase (FRD2), fumarase (FUM), phosphoglucose isomerase (PGI), or PEP carboxykinase (PPCK).
Additional deletions have been identified by the OptKnock framework for improving the growth-coupled formation of HMDA. These include one or more of the following: hexokinase (HEX1), malic enzyme (ME2), malate dehydrogenase (MDH), aspartate transaminase (ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), and pyruvate kinase (PYK). The HMDA yields can be further improved by further deleting one or more of the following enzymes: transhydrogenase (THD2), glutamate dehydrogenase (GLUDy), ATP synthase (ATPS4r), GLCpts (PTS system of glucose uptake), PGDHY (phosphogluconate dehydratase) and EDA (2-dehydro-3-deoxy-phosphogluconate aldolase).
Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. For example, succinate dehydrogenase that oxidizes succinate to fumarate and is known to be active only under aerobic conditions can assume significant activity even under anaerobic conditions, and therefore such activity can be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled HMDA production strains.
Optknock Strain Design for Growth-Coupled Production of Adipic Acid. Described below is a further exemplification of a deletion strategy for engineering a strain to synthesize adipate using a succinyl-CoA pathway. All high-priority growth-coupled designs for adipate synthesis build upon a strain lacking acetylaldehyde-CoA dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) activities to prevent the formation of fermentation byprouducts, as discussed above. The further deletion of malate dehydrogenase (MDH) also reduces byproduct production.
Designs 2-4 build on Design 1 as a base design. Design 2 entails the removal of phosphoenolpyruvate carboxykinase (PPCK). This design improves the adipate yield to 3.6 g/g at the maximum biomass yield. The additional deletion of pyruvate formate lyase (PFLi) activity in Design 3 further improves yield by preventing secretion of formate as a byproduct. The predicted adipate yield of this design is 5.8 g/g. Design 4 features the deletion of NAD(P) transhydrogenase (THD2) in addition to ADHEr, LDH_D, MDH, PPCK and PFLi. This results in an adipate yield of 6.8 g/g at a growth rate of 0.117 1/hr. Design 4 serves to tightly couple adipate production to cell growth while achieving 91% of the theoretical maximum yield.
This Example describes the biosynthetic production of adipate semialdehyde from adipate and of 6-aminocaproate semialdehyde from 6-aminocaproate.
The transformation from adipate to adipate semialdehyde (
TABLE 17
CAR genes used this example
Protein
GenBank ID
GI Number
Organism
720
AAR91681.1
40796035
Nocardia iowensis
889
YP_887275.1
118473501
Mycobacterium smegmatis str.
MC2 155
890
YP_889972.1
118469671
Mycobacterium smegmatis str.
MC2 155
891
NP_959974.1
41407138
Mycobacterium avium subsp.
paratuberculosis K-10
892
YP_001850422.1
183982131
Mycobacterium marinum M
Cell pellets were lysed by addition of 500 μl B-PER with 0.5 μl lysozyme and benzonase. CAR activity was measured by addition of 2 μl of crude lysate to the assay solution of 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, 10% (v/v) glycerol, 1 mM ATP, 0.5 mM NADPH and 20 mM Adipate or 50 mM 6-Aminocaproate in a 96 well format microplate with a total volume of 250 μl. The oxidation of NADPH to NADP+ was monitored at an absorbance of 340 nm for 30 minutes at room temperature. The rate of NADPH depletion was used to calculate activity of the various CAR proteins. Total protein concentration of each lysate was determined by Bradford, and the activity was normalized to the total protein concentration (Units/mg).
Transformation from Adipate to Adipate Semialdehyde (
Furthermore, a reaction was setup consisting of 500 μl of 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, 10% (v/v) glycerol, 5 mM ATP, 3 mM NADPH and 20 mM Adipate. The reaction was incubated at room temperature for 30 minutes and stopped by addition of 1% formic acid. The sample was then centrifuged and the supernatant was analyzed by LC-MS. Low mM level of adipate semialdehyde was detected that confirmed the transformation from adipate to adipate semialdehyde.
Transformation from 6-Aminocaproate to 6-Aminocaproate Semialdehyde. As shown in
Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.
Burk, Mark J., Burgard, Anthony P., Osterhout, Robin E., Pharkya, Priti
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