Microorganisms and methods for biosynthesis of adipate, hexamethylenediamine and 6-aminocaproic acid

Abstract

A non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway is provided.SOLUTION: The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, respectively. The invention further provides methods for producing 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The method includes culturing a 6-aminocaproic acid, caprolactam or hexamethylenediamine producing microbial organism, wherein the microbial organism expresses at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme in a sufficient amount, under conditions and for a sufficient period of time to produce the respective product.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 61 / 176,196, filed May 7, 2009, and U.S. Provisional Patent Application No. 61 / 176,196, filed June 22, 2009. U.S. Provisional Patent Application No. 61 / 219,365, filed Sep. 22, 2009; No. 61 / 244,844, U.S. Provisional Patent Application No. 61 / 246,973, filed September 29, 2009, and U.S. Provisional Patent Application No. 200 Each of the applications claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 247,533, filed September 30, 1999, each of which is hereby incorporated by reference. and the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The present invention relates generally to biosynthetic processes, particularly to the synthesis of adipate, hexamethylenediamine, This invention relates to an organism capable of biosynthesizing 6-aminocaproic acid and caprolactam.

[0003] Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. Nylon is a linear polyamide made by condensing diamines and adipic acid. It can be used to produce 6,6-diol, which can be used to produce various types of fibers. Other uses of adipic acid include as a plasticizer, in unsaturated polyesters, and in polyolefins. Further uses include its use in ester polyols. Food ingredients for the production of lubricating components, as well as flavorants and gelling aids Included.

[0004] Historically, adipic acid was prepared from a wide variety of fats using oxidation. Some current processes for synthesis use an excess of strong nitric acid to produce ketones or K Cyclohexanone and alcohol as components or cyclohexanol as component A It depends on the oxidation of the mixture KA oil or pure cyclohexanol. There are several variations of this main component, which differ in the route of hexanol production. For example, phenol is an alternative raw material for KA oil production, and A process for the synthesis of dipic acid has been described. Other versions of this process tend to use oxidizing agents other than nitric acid, such as hydrogen peroxide, air, or oxygen.

[0005] As described above, hexamethylenediamine (HMDA) is In addition to being used in the production of iron-6,6, it is also used in the production of polyurethanes. To make hexamethylene diisocyanate, the monomer feedstock used in This diamine also serves as a crosslinking agent in epoxy resins. HMDA is currently produced by the hydrogenation of adiponitrile.

[0006] Caprolactam is a derivative of 6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocaproic acid) It is an organic compound that is a lactam of the formula 1. It can alternatively be considered as a cyclic amide of caproic acid. One use of caprolactam is as a monomer in the production of nylon-6. Caprolactam is used as an oxidizer using hydroxylammonium sulfate. Catalytically using a shimization process followed by a Beckmann rearrangement process step It can be synthesized from cyclohexanone via rearrangement.

[0007] Hexamethylenediamine, 6-aminocaproic acid, levulinic acid, and caprolactam, etc. Methods for efficiently producing commercial quantities of compounds such as Including related benefits. Summary of the Invention

[0008] The present invention provides a 6-aminocaproic acid, caprolactam, or hexamethylenediamine pathway. The microbial organism is a non-naturally occurring microbial organism having 6-aminocaproic acid. , caprolactam, hexamethylenediamine, or levulinic acid pathways, respectively The present invention includes at least one exogenous nucleic acid encoding 6-aminocaproic acid. Further provided are methods for producing methyl methyl ketone, caprolactam, or hexamethylenediamine. The method involves the use of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. The method can include culturing a microbial organism that produces 6-phosphate. Aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzymes and at least one exogenous nucleic acid encoding each of the following in an amount sufficient to produce the respective product. 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid It is expressed under conditions and for a period of time sufficient for production. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows an exemplary pathway for adipate degradation in peroxisomes of Penicillium chrysogenum. [Figure 2]FIG. 1 shows an exemplary pathway for adipate formation via a reverse degradation pathway. Several options are provided for the final conversion of adipyl-CoA to adipate. [Figure 3] FIG. 1 shows an exemplary pathway for adipate formation via the 3-oxoadipate pathway. [Figure 4] FIG. 1 shows the similar enzymatic chemistry of the last three steps of the 3-oxoadipate pathway and the reductive TCA cycle for adipate synthesis. [Figure 5]

[0023] Figure 1 shows an exemplary pathway for the synthesis of adipic acid from glucose via cis,cis-muconic acid. Biosynthetic intermediates (abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvate (PEP), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP), 3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuic acid (PCA). Enzymes (encoding genes) or reaction conditions: (a) DAHP synthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c) 3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ), (e) protocatechuate decarboxylase (aroY), (f) catechol 1,2-dioxygenase (catA), (g) 10% Pt / C, H2, 3400 kPa, 25°C. Figure adapted from Niu et al., Biotechnol. Prog. 18:201-211 (2002). [Figure 6] FIG. 1 shows an exemplary pathway for adipate synthesis via alpha-ketoadipate using alpha-ketoglutarate as a starting point. [Figure 7] FIG. 1 shows an exemplary pathway for the synthesis of adipate using lysine as a starting point. [Figure 8] FIG. 1 shows an exemplary caprolactam synthesis pathway using adipyl-CoA as the starting point. [Figure 9] FIG. 1 shows an exemplary adipate synthesis pathway using alpha-ketoadipate as a starting point. [Figure 10]

[0023] Figure 1 shows exemplary pathways from succinyl-CoA and acetyl-CoA to hexamethylenediamine (HMDA) and caprolactam. Pathways for the production of adipate, 6-aminocaproate, caprolactam, and hexamethylenediamine from succinyl-CoA and acetyl-CoA are depicted. Abbreviations: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase, E) 3-oxoadipyl-CoA / acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G) 3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I) 3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentanoate reductase, K) adipyl-CoA / acyl -CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoA hydrolase, N) adipyl-CoA reductase (aldehyde forming), O) 6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q) 6-aminocaproyl-CoA / acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase, S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoA reductase (aldehyde forming), V) HMDA transaminase, W) HMDA dehydrogenase. [Figure 11]

[0023] Figure 1 shows exemplary pathways from 4-aminobutyryl-CoA and acetyl-CoA to hexamethylenediamine and caprolactam. Pathways for the production of 6-aminocaproate, caprolactam, and hexamethylenediamine from 4-aminobutyryl-CoA and acetyl-CoA are depicted. Abbreviations: A) 3-oxo-6-aminohexanoyl-CoA thiolase, B) 3-oxo-6-aminohexanoyl-CoA reductase, C) 3-hydroxy-6-aminohexanoyl-CoA dehydratase, D) 6-aminohex-2-enoyl-CoA reductase, E) 3-oxo-6-aminohexanoyl-CoA / acyl-CoA transferase, F) 3-oxo-6-aminohexanoyl-CoA synthase, G) 3-oxo-6-aminohexanoyl-CoA hydrolase, H) 3-oxo-6-aminohexanoyl-CoA I) 3-hydroxy-6-aminohexanoate reductase, I) 3-hydroxy-6-aminohexanoate dehydratase, J) 6-aminohex-2-enoate reductase, K) 6-aminocaproyl-CoA / acyl-CoA transferase, L) 6-aminocaproyl-CoA synthase, M) 6-aminocaproyl-CoA hydrolase, N) 6-aminocaproyl-CoA reductase (aldehyde forming), O) HMDA transaminase, P) HMDA dehydrogenase, Q) spontaneous cyclization, R) amidohydrolase. [Figure 12]1 shows a pathway to 6-aminocaproate from pyruvate and succinic semialdehyde. The enzymes are A) HODH aldolase, B) OHED hydratase, C) OHED reductase, D) 2-OHD decarboxylase, E) adipate semialdehyde aminotransferase and / or adipate semialdehyde oxidoreductase (aminating), F) OHED decarboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferase and / or 2-OHD oxidoreductase (aminating), I) 2-OHD decarboxylase, J) OH K) 2-AHE reductase, L) HODH formate-lyase and / or HODH dehydrogenase, M) 3-hydroxyadipyl-CoA dehydratase, N) 2,3-dehydroadipyl-CoA reductase, O) adipyl-CoA dehydrogenase, P) OHED formate-lyase and / or OHED dehydrogenase, Q) 2-OHD formate-lyase and / or 2-OHD dehydrogenase. Abbreviations are: HODH = 4-hydroxy-2-oxoheptane-1,7-dioate, OHED = 2-oxohept-4-ene-1,7-dioate, 2-OHD = 2-oxoheptane-1,7-dioate, 2-AHE = 2-aminohept-4-ene-1,7-dioate, 2-AHD = 2-aminoheptane-1,7-dioate, and 6-OHE = 6-oxohex-4-enoate. [Figure 13]1 shows a pathway from 6-aminocaproate to hexamethylenediamine. The enzymes are A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproic semialdehyde aminotransferase and / or 6-aminocaproic semialdehyde oxidoreductase (aminating), D) 6-aminocaproate N-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G) 6-acetamidohexanal aminotransferase and / or 6-acetamidohexanal oxidoreductase (aminating), H) 6-acetamidohexane. I) 6-acetamidohexanoate CoA transferase and / or 6-acetamidohexanoate CoA ligase, J) 6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and / or 6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoA oxidoreductase. Abbreviations: 6-AAHOP = [(6-acetamidohexanoyl)oxy]phosphonate and 6-AHOP = [(6-aminohexanoyl)oxy]phosphonate. [Figure 14] A) The acetyl-CoA cycle of arginine biosynthesis. Reactions (1) and (2) are catalyzed by ornithine acetyltransferase, which has the functionality of acetylglutamate synthase and ornithine acyltransferase. Reaction 3 is a lumped reaction catalyzed by acetylglutamate kinase, N-acetylglutamylphosphate reductase, and acetylornithine aminotransferase; B) The acetyl-CoA cycle of HMDA biosynthesis. Reactions (1) and (2) are catalyzed by HMDA acetyltransferase. Reaction (3) is a lumped reaction encompassing the entire pathway from 6-acetamidohexanoate to 6-acetamidohexanamine shown in Figure 13. [Figure 15] Figure 1 shows the growth of E. coli in media containing various concentrations of 6-ACA. E. coli was inoculated into the media and grown under aerobic (left and right bars) or anaerobic (middle bar) conditions. Cultures were grown under aerobic conditions (right bar) for 48 hours during the first test and 30 hours for the second test. [Figure 16] Figure 1 shows the resistance of E. coli when exposed to 6-ACA. Mid-logarithmic phase (OD = 0.3, below the dotted line) or early stationary (OD = 0.6, above the dotted line) cells were spun down and resuspended in fresh M9-glucose medium with various concentrations of 6-ACA. After overnight growth, cultures were monitored for growth by measuring OD. [Figure 17] (Figure 1 shows ethanol production from cultures exposed to various concentrations of 6-ACA. Mid-logarithmic or early quiescent cells were spun down and resuspended in fresh M9-glucose medium with various concentrations of 6-ACA. After overnight growth, cultures were monitored for growth by measuring OD600, and metabolic activity was assayed by ethanol production. [Figure 18] Panels A and B show growth in various concentrations of 6-ACA with and without glycine betaine. Panel A. OD600 measurements of media inoculated with mid-logarithmic phase cultures of E. coli with various concentrations of 6-ACA with (right bar) and without (left bar) 2 mM glycine betaine. Panel B. Photograph showing growth of the same culture in anaerobic bottles. [Figure 19] LC / MS analysis of in vitro thiolase reactions. Succinyl-CoA and acetyl-CoA were added to His-tagged purified thiolase at a ratio of 2:1 (succinyl-CoA:acetyl-CoA). Reactions were analyzed by LC / MS and quantified by comparison to standards for acetoacetyl-CoA or by determined peak area for 3-oxoadipyl-CoA (β-ketoadipyl-CoA). [Figure 20]1 shows an exemplary pathway from glutamate to hexamethylenediamine (HMDA) and 6-aminocaproate. Enzymes are indicated as follows: A) glutamyl-CoA transferase and / or ligase, B) beta-ketothiolase, C) 3-oxo-6-aminopimeloyl-CoA oxidoreductase, D) 3-hydroxy-6-aminopimeloyl-CoA dehydratase, E) 6-amino-7-carboxyhept-2-enoyl-CoA reductase, F) 6-aminopimeloyl-CoA reductase (aldehyde forming), G) 2-amino-7-oxoheptanoate aminotransferase and / or aminating oxidoreductase, H) homolysine decarboxylase, I) 6-aminopimeloyl-CoA hydrolase, transferase, and / or ligase, J) 2-aminopimelate decarboxylase. The enzyme commission numbers indicated for each reaction are listed below in Example XXVI. [Figure 21]1 shows an exemplary pathway from glutaryl-CoA to hexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes are shown as follows: A) glutaryl-CoA beta-ketothiolase, B) 3-oxopimeloyl-CoA hydrolase, transferase, and / or ligase, C) 3-oxopimelate reductase, D) 3-oxo-1-carboxyheptanal 7-aminotransferase and / or 7-aminating oxidoreductase, E) 3-oxo-7-aminoheptanoate 3-aminotransferase and / or 3-aminating oxidoreductase, F) 3-oxopimelate kinase. , G) 5-oxopimeloylphosphonate reductase, H) 3-oxopimelate CoA transferase and / or ligase, I) 5-oxopimeloyl-CoA reductase (aldehyde forming), J) 3-oxopimelate 3-aminotransferase and / or 3-aminating oxidoreductase, K) 3-aminopimelate CoA transferase and / or ligase, L) 5-aminopimeloyl-CoA reductase (aldehyde forming), M) 3-aminopimelate kinase, N) 5-amino Pimeloylphosphonate reductase, O) 3-aminopimelate reductase, P) 3-amino-7-oxoheptanoate 2,3-aminomutase, Q) 2-amino-7-oxoheptanoate 7-aminotransferase and / or aminating oxidoreductase, R) 3,7-diaminoheptanoate 2,3-aminomutase, S) homolysine decarboxylase, T) 3-aminopimelate 2,3-aminomutase, U) 2-aminopimelate kinase, V) 2-aminopimelate CoA transferase and / or ligase, W) 2-aminopimelate reductase, X) 6-aminopimeloylphosphonate reductase, Y) 6-aminopimeloyl-CoA reductase (aldehyde forming), Z) 3-amino-7-oxoheptanoate 7-aminotransferase and / or 7-aminating oxidoreductase, AA) 2-aminopimelate decarboxylase, and AB) 3-oxo-1-carboxyheptanal 3-aminotransferase and / or 3-aminating oxidoreductase.The enzyme commission numbers indicated for each reaction are listed below in XXVI. [Figure 22]

[0023] Figure 2 shows an exemplary pathway to hexamethylenediamine (HMDA) from pyruvate and 4-aminobutanal. The enzymes are designated as follows: A) 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, B) 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, C) 2-oxo-7-aminohept-3-enoate reductase, D) 2-oxo-7-aminoheptanoate aminotransferase and / or aminating oxidoreductase, E) homolysine decarboxylase, F) 2-oxo-7-aminoheptanoate decarboxylase, and G) 6-aminohexanal aminotransferase and / or 6-aminohexanal aminating oxidoreductase. The enzyme commission numbers designated for each reaction are described below in Example XXVI. [Figure 23]

[0023] Figure 1 shows an exemplary pathway from homolysine to 6-aminocaproate. Step A is catalyzed by homolysine 2-monooxygenase. Step B is hydrolysis catalyzed by dilute acid or base. [Figure 24]

[0033] Figure 1 shows an exemplary pathway from 6-aminocaproate to hexamethylenediamine. This diagram represents an additional pathway to that shown in Figure 13. The enzymes are shown as follows: A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproic semialdehyde aminotransferase and / or 6-aminocaproic semialdehyde oxidoreductase (aminating), D) 6-aminocaproate N-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G) 6-acetamidohexanal aminotransferase and / or 6-acetamidohexanal oxidoreductase (aminating), H) 6-acetamidohexanamine N-acetyltransferase. I) 6-acetamidohexanoate CoA transferase and / or 6-acetamidohexanoate CoA ligase, J) 6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and / or 6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoA oxidoreductase, O) 6-aminocaproate reductase, and P) 6-acetamidohexanoate reductase. Abbreviations are: 6-AAHOP = [(6-acetamidohexanoyl)oxy]phosphonate and 6-AHOP = [(6-aminohexanoyl)oxy]phosphonate. The enzyme commission numbers indicated for each reaction are listed below in Example XXVI. [Figure 25]

[0033] Figure 1 shows exemplary pathways from succinyl-CoA and acetyl-CoA to hexamethylenediamine (HMDA), caprolactam, or levulinic acid. Pathways for the production of adipate, 6-aminocaproate, caprolactam, hexamethylenediamine, and levulinic acid from succinyl-CoA and acetyl-CoA are depicted. This diagram also depicts additional pathways to those shown in Figure 10. The enzymes are designated as follows: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase, E) 3-oxoadipyl-CoA / acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G) 3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I) 3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentanoate reductase, K) adipyl-CoA / acyl-CoA transferase, L) adipyl-CoA synthase. M) adipyl-CoA hydrolase, N) adipyl-CoA reductase (aldehyde forming), O) 6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q) 6-aminocaproyl-CoA / acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase, S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoA reductase (aldehyde forming), V) HMDA transaminase, W) HMDA dehydrogenase, X) adipate reductase, Y) adipate kinase, Z) adipylphosphate reductase, and AA) 3-oxoadipate decarboxylase. [Figure 26]FIG. 1 shows exemplary pathways from 2-amino-7-oxosubarate to hexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes are shown as follows: A) 2-amino-7-oxosubarate keto acid decarboxylase, B) 2-amino-7-oxoheptanoate decarboxylase, C) 6-aminohexanal aminating oxidoreductase and / or 6-aminohexanal aminotransferase, D) 2-amino-7-oxoheptanoate oxidoreductase, E) 2-aminopimelate decarboxylase, F) 6-aminohexanal oxidoreductase, G) 2-amino-7-oxoheptanoate decarboxylase, H) homolysine decarboxylase, I) 2-amino-7-oxoheptanoate decarboxylase, J) homolysine decarboxylase, K) 2-amino-7-oxoheptanoate decarboxylase, K) homolysine decarboxylase, L) 2-amino-7-oxoheptanoate ... J) 2-oxo-7-aminoheptanoate aminating oxidoreductase and / or 2-oxo-7-aminoheptanoate aminotransferase, K) 2-amino-7-oxosubarate aminating oxidoreductase and / or 2-amino-7-oxosubarate aminotransferase, L) 2,7-diaminosubarate decarboxylase, and M) 2-amino-7-oxoheptanoate aminating oxidoreductase and / or 2-amino-7-oxoheptanoate aminotransferase. The enzyme commission numbers shown for each reaction are described below in Example XXVI. [Figure 27] FIG. 1 shows exemplary pathways from glutamate-5-semialdehyde to 2-amino-7-oxosubarate. The enzymes are shown as follows: A) 2-amino-5-hydroxy-7-oxosubarate aldolase, B) 2-amino-5-hydroxy-7-oxosubarate dehydratase, and C) 2-amino-5-ene-7-oxosubarate reductase. [Figure 28] FIG. 1 shows growth yields for 6-ACA versus ADHEr, LDH_D mutants. [Figure 29] FIG. 1 shows 6-ACA versus growth yield for ADHEr, MDH, ASPT, LDH_D mutants. [Figure 30]FIG. 1 shows 6-ACA versus growth yield for ADHEr, MDH, ASPT, LDH_D, THD2 mutants. [Figure 31] FIG. 1 shows 6-ACA versus growth yield for ADHEr, MDH, ASPT, LDH_D, GLUDy mutants. [Figure 32] FIG. 1 shows growth yields for 6-ACA versus PGI, EDA mutants or PGI, PGDHy mutants. [Figure 33] FIG. 1 shows growth yields for 6-ACA versus PGI, EDA, ADHEr mutants or PGI, PGDHy, ADHEr mutants. [Figure 34] FIG. 1 shows 6-ACA versus growth yield for ADHEr, PGI, HEX1 mutants. [Figure 35] Figure 1 shows the growth-coupled adipate production characteristics of the prioritized strain designs (gray) compared to wild-type E. coli (black), assuming a glucose uptake rate of 10 mmol / gDW / Hr. [Figure 36] Figure 1 shows the activity of CAR889 and 891 using 20 mM adipate. Activity is shown as units per mg of total protein in crude lysate. [Figure 37] Figure 1 shows the activity of CAR720, 889, 890, 891 using 50 mM 6-aminocaproate. Activity is shown as units per mg of total protein in crude lysate. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a method for producing a 6-aminocaproic acid, a caprolactam, a hexamethylenediamine, or a levulinic acid. This specification relates to the design and production of cells and organisms that have biosynthetic production capabilities for phosphate. The results described herein demonstrate that metabolic pathways can be used to treat 6-aminocarboxylic acids in E. coli, other cells, or organisms. To achieve the biosynthesis of propranolol, caprolactam, hexamethylenediamine, or levulinic acid We demonstrate that 6-aminocaproic acid can be engineered and recombinantly engineered to Biosynthetic production of caprolactam, hexamethylenediamine, or levulinic acid was engineered. These metabolic genotypes can be confirmed by constructing strains with the Genetically engineered cells or organisms may also be grown in a 6- Aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis To further increase it, it can be subjected to adaptive evolution.

[0011] As disclosed herein, 6-aminocaproic acid, caprolactam, hexamethylene Many metabolic pathways for the production of benzodiamine, or levulinic acid, have been described. The reverse adipate degradation pathway and the 3-oxoadipate pathway contribute to (i) adipate yield (glucose (92% molar yield based on the amount of adipate), (ii) insufficient oxygen demand for adipate synthesis, (iii) related and (iv) the theoretical energy profile for producing adipate as the sole fermentation product. The ability of the α-ketoadipate or lysine to pass through the Metabolic pathways for dipate production have also been described, but are low yielding and require extensive preparation to maximize production. Aeration is required to convert adipyl-CoA, the precursor in the reverse degradation pathway, into 6-acetyl-CoA. Pathways for producing either or both of aminocaproate and caprolactam are also disclosed. Disclosed in the specification.

[0012] As disclosed herein, a number of exemplary pathways for the biosynthesis of adipate are An exemplary first pathway has been described in organisms such as P. chrysogenum. This includes adipate synthesis via a route that relies on the reversibility of adipate degradation (see Examples I and II). (See also References II and II.) A second exemplary pathway involves the reaction of 3-oxoacetamide with 3-oxoacetamide to form adipate. This involves the formation of dipate, followed by its reduction, dehydration, and reduction again (see Examples III and (See IV.) Adipate yield using either of these two pathways is The oxygen uptake is 0.92 moles per mole of glucose absorbed. The energy requirements for growth and product secretion under anaerobic conditions are not necessary to achieve the desired yield. Advantageously, a method for producing adipate from glucose-derived cis,cis-muconic acid is provided. This has been previously described (Frost et al., U.S. Pat. No. 5,487,987, issued Jan. 30, 1996) (Embodiment (See Example V.) The implementation disclosed herein of this previously described method The advantages of the embodiments are discussed below. α-Ketoadipate (Example VI) or Lysine (Example VII) Precursor The metabolic pathway for adipate production throughout the body is low yielding, and to maximize production, Aeration is required for the reverse degradation pathway, which converts adipyl-CoA, a precursor, to 6-aminocaproic acid. Pathways for producing either or both acetone and caprolactam are described (see Examples). (See Examples VIII and IX.) Additional pathways for producing adipate are described in Examples X and and XI. Succinyl-CoA and acetyl-CoA to 6-aminocaproate, Caprolactam, hexamethylenediamine, and levulinic acid, either 1, 2, 3, or 4 Pathways for producing all are described in Examples XII and XXVIII. Several pathways for the production of 6-aminocaproate from aldehyde and pyruvate include: Described in Example XIX. Hexamethylenediamine from 6-aminocaproate Several pathways for the production are described in Examples XX and XXVII. 6-aminocaproate and / or hexamethylenediamine are produced from the ester. The pathway for producing glutaryl-CoA is described in Examples XXIV and XXV. Several pathways for the production of hexamethylenediamine and 6-aminobenzoate from glutaryl-CoA At least one pathway for the production of caproate is described in Examples XXIV and XXV. A pathway for producing 6-aminocaproate from homolysine is described in Example XXV. 2-amino-7-oxosubarate to hexamethylenediamine A pathway for producing 6-aminocaproate is described in Example XXIV. Some routes for achieving this are described in Example XXV. Exemplary Genes and Enzymes Needed to Construct Organisms and Cloning and Traits Methods for transformation, methods for monitoring product formation, and genetically engineered microorganisms Methods for use in producing the

[0013] As disclosed herein, an enzyme that uses glucose / sucrose as a carbon substrate Six different routes for the synthesis of dipic acid are described. For all maximum yield calculations: The missing reaction in a given pathway was previously described (Reed et al., Genome Biol. 4:R5 4 (2003)) was added to the E. coli stoichiometric network in SimPheny. Adipate becomes a charged molecule under physiological conditions and is secreted from the network. It was hypothesized that it required energy in the form of a proton-based symport system. Such transport systems are thermodynamically viable when performed at neutral or near-neutral pH. Low pH adipic acid formation opposes ATP-dependent transport mechanisms, e.g., proton symport. The pathways and methods of these pathways will require the ABC system as a The reactions are described in Examples I to XI.

[0014] As used herein, the term "microorganism" refers to a microorganism or microorganism of the invention. The term "non-naturally occurring" in this context refers to a microbial organism that is not naturally occurring, including wild-type strains of the species mentioned above. possessing at least one genetic variation not normally found in naturally occurring strains of the species Genetic variations are intended to mean, for example, variations that encode metabolic polypeptides. Modifications that introduce expressible nucleic acids, other nucleic acid additions, nucleic acid deletions, and / or genetic modifications of the microorganism. Such modifications include, for example, heterologous, homologous, or other functional disruption of the substance. or coding regions for heterologous and homologous polypeptides and functional fragments thereof. Further modifications include, for example, modifications in non-coding regulatory regions that alter the expression of a gene or operon. Exemplary metabolic polypeptides include 6-aminocaproic acid, caprolactam, hexahydroxybenzoates, and the like. Contains enzymes in the methylenediamine or levulinic acid biosynthetic pathway.

[0015] Metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Non-naturally occurring microorganisms may be designed to encode nucleic acids encoding metabolic polypeptides or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

[0016] As used herein, the term "isolated" when used in reference to a microbial organism. "Contained" means that the microbial organism described above is found in nature, but contains at least one component. The term is intended to mean an organism that is substantially free of The term also includes microbial organisms found in food, but from which some or all components have been removed. Microbial organisms are found in environments where they do not occur naturally, but some or all of the components An isolated microbial organism also includes a microbial organism from which components have been removed. Conserved species found in the world or growing in environments where they do not occur naturally Living or viable, but partially or completely separated from other substances. Isolated Specific examples of microbial organisms include partially pure microorganisms, substantially pure microorganisms, and naturally occurring microorganisms. This includes microorganisms cultured in a medium that does not contain any microbial organisms.

[0017] As used herein, the terms "microbial," "microbial organism," or "microorganism" refer to " refers to any organism that exists as a microscopic cell within the scope of archaea, bacteria, or eukaryotes. The term is therefore intended to mean any living organism, including prokaryotic organisms having microscopic sizes. or eukaryotic cells or prokaryotic or eukaryotic organisms, and is intended to include bacteria of all species This term includes eukaryotic microorganisms such as bacteria, archaea, and eubacteria, as well as yeasts and fungi. also includes cell cultures of any species that can be cultivated for the production of biochemicals. .

[0018] As used herein, the term "CoA" or "coenzyme A" refers to the active enzyme system. An organic cofactor, the presence of which is required for the activity of many enzymes (apoenzymes), to form It is intended to mean the prosthetic group (the non-protein part of an enzyme). Coenzyme A is Functions in certain condensing enzymes, in acetyl or other acyl group transfer and in fatty acid It acts in the synthesis and oxidation of pyruvate and in the acetylation of other enzymes.

[0019] As used herein, a compound having the formula -OOC-(CH2)4-COO- (see Figure 2) (IUPAC "Adipate" (IUPAC name hexanedioate) is a derivative of adipic acid (IUPAC name hexanedioic acid). In its ionized form, adipate and adipic acid, including all salt forms thereof, Used interchangeably throughout to refer to compounds in either neutral or ionized form It is understood that the particular form will depend on the pH. It will be understood by those skilled in the art.

[0020] As used herein, a compound of the formula -OOC-(CH2)5-NH2 (see Figures 8 and 12) "6-aminocaproate" is 6-aminocaproic acid (IUPAC name 6-aminohexanoic acid) ionized form of 6-aminocaproate and 6-aminocaproic acid, From the outset, to refer to a compound in either its neutral or ionized form, including salt forms. It is understood that the specific forms may be used interchangeably. It will be understood by those skilled in the art that this would be the case.

[0021] As used herein, "caprolactam" (IUPAC name azepan-2-one) refers to 6- It is the lactam of aminohexanoic acid (see Figure 8).

[0022] As used herein, 1,6-diaminohexane or 1,6-hexanediamine The so-called "hexamethylenediamine" has the chemical formula HN(CH)NH (Figures 10, 11, and 13). (See

[0023] As used herein, the term "culture" when used in reference to culture or growth conditions "Qualitatively anaerobic" means that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in the liquid medium. The term is also intended to mean a temperature maintained by an atmosphere of less than about 1% oxygen. The term "culture medium" is intended to include a sealed chamber of liquid or solid medium.

[0024] As used herein, the term "immersion" when used in reference to culture or growth conditions. "Osmoprotectants" are substances that act as osmolytes and allow the microbial organisms described herein to It is intended to mean a compound that helps to withstand osmotic stress. These include, for example, betaine, amino acids, and the sugar trehalose. Non-limiting examples include glycine betaine, praline betaine, dimethyl Tetin, dimethylslfonioproprionate, 3-dimethyl Methyl 2-sulfoniopropionate, Pipecolic acid, Dimethylsulfonioacetate, Choline , L-carnitine, and ectoine.

[0025] As used herein, the term "enhancement" when used in reference to the production of a biochemical. "Growth-linked" refers to the biosynthesis of the aforementioned biochemicals produced during the growth phase of microorganisms. In certain embodiments, growth-coupled production is unavoidable. It is understood that the biosynthesis of the above-mentioned biochemicals is an inevitable product produced during the growth phase of microorganisms. This means:

[0026] As used herein, a "metabolic modification" refers to a compound that is altered from its naturally occurring state. Metabolic modification is intended to refer to a biochemical reaction that has been modified, for example, by modifying the enzymes involved in the reaction. This can include loss of biochemical reaction activity due to functional disruption of one or more genes encoding the An exemplary set of metabolic modifications is described herein (see Example XXX). .

[0027] As used herein, the term "gene disruption" or its grammatical equivalents refers to a gene disruption. The term "genetic mutation" is intended to mean a genetic mutation that renders a gene product inactive. For example, deletion of the entire gene, deletion of regulatory sequences required for transcription or translation, truncated gene products a variety of mutations that result in deletion of parts of genes or inactivation of the coding gene product One particularly useful method of gene disruption is by It is a complete gene deletion because it is This is because it reduces or eliminates the occurrence of genetic reversion.

[0028] "Exogenous," as used herein, refers to a molecule or activity that is present in a host It is intended to mean introduced into a microbial organism. For example, the molecule may be introduced into a host By introduction of the coding nucleic acid into the host genetic material, such as by integration into a chromosome. It can be introduced as a vector or as non-chromosomal genetic material such as a plasmid. Therefore, the term, as used in reference to expression of an encoding nucleic acid, is intended to include the expression of an encoding nucleic acid into a microbial organism. Refers to the introduction of an encoding nucleic acid in an expressible form. When used in reference to a biosynthetic activity, this The term refers to an activity that is introduced into a host reference organism. The encoding nucleic acid may be homologous or heterologous, and expresses the above-mentioned activity after introduction into the organism. Therefore, the term "endogenous" refers to a molecule or activity that is present in the host. Likewise, the term when used in reference to expression of an encoding nucleic acid contained within a microbial organism. The term "heterologous" refers to the expression of a molecule derived from a source other than the species mentioned above. "Homologous" refers to a molecule or activity that is derived from the host microbial organism. Thus, exogenous expression of an encoding nucleic acid of the present invention can be achieved by either heterologous or homologous encoding nucleic acid. can use both.

[0029] When more than one exogenous nucleic acid is contained in a microbial organism, the more than one exogenous nucleic acid The nucleic acid may be associated with the encoding nucleic acid or biosynthetic activity described above, as discussed above. As disclosed herein, more than one exogenous nucleic acid may be expressed as separate nucleic acid molecules. The vector may be introduced into a host microbial organism on a nucleic acid molecule, on a polycistronic nucleic acid molecule, or a combination thereof. It is further understood that multiple nucleic acids can be introduced and still be considered to be more than one exogenous nucleic acid. It is understood that, for example, as disclosed herein, a microbial organism can be adapted to express a desired pathway enzyme. or genetically engineered to express two or more exogenous nucleic acids encoding proteins. Two exogenous nucleic acids encoding desired activities are introduced into a host microbial organism. In some cases, the two exogenous nucleic acids may be present on separate plasmids, e.g., on a single plasmid. The vectors can be introduced as a single nucleic acid into the host chromosome at a single site or multiple sites. It is understood that the nucleic acid sequence of the nucleic acid fragment can be integrated into the nucleic acid fragment of ... Similarly, more than two exogenous nucleic acids may be present on separate plasmids, e.g., on a single plasmid. The genes can be introduced into the host organism in any desired combination on a single site or It can be integrated into the host chromosome at multiple sites, and more than one exogenous nucleic acid, e.g. For example, it is understood that the exogenous nucleic acids can be considered as three exogenous nucleic acids. The number of responsible nucleic acids or biosynthetic activities is not the number of separate nucleic acids introduced into the host organism. It refers to the number of encoding nucleic acids or the number of biosynthetic activities.

[0030] The non-naturally occurring microbial organisms of the invention can contain stable genetic variations. This refers to microorganisms that can be cultured for more than five generations without loss of variation. Generally, stable genetic variations include modifications that persist for more than 10 generations, and in particular, stable The modifications will persist for approximately 25 generations or more, and especially stable genetic modifications may persist indefinitely. Including these, the generations will be over 50 years old.

[0031] In the case of gene disruption, a particularly useful stable genetic variation is a gene deletion. The use of gene deletion to introduce genetic mutations in a plant increases the possibility of reversion to the phenotype before the genetic mutation. It is particularly useful for reducing the possibility of, for example, growth-linked changes in biochemical stability. The production may involve, for example, the production of genes encoding enzymes that catalyze one or more reactions in a set of metabolic modifications. This can be achieved by the deletion of the gene. The stability of growth-linked production of biochemicals is , and can be enhanced through multiple deletions, with each disrupted activity This significantly reduces the likelihood of multiple compensatory returns occurring.

[0032] Those skilled in the art will appreciate that genetic alterations, including the metabolic modifications exemplified herein, can be used in bacteria such as E. coli. Suitable host organisms and their corresponding metabolic reactions or genes for desired metabolic pathways. It will be understood that any desired genetic material is described with respect to suitable source organisms. However, in the field of complete genome sequencing and genomics for a wide variety of organisms, Given the high level of skill in the art, those skilled in the art will be able to readily understand the teachings and methods inherently provided herein. The same principles and guidance could be readily applied to all other organisms. The metabolic variations of E. coli exemplified in are not conserved in the presence of the same or similar codes from species other than those mentioned above. By incorporating nucleic acids, it can be easily applied to other species. Variations include, for example, genetic variations of species, generally homologs, particularly orthologs, paralogs, or non-homologs. Includes orthologous gene replacement.

[0033] Orthologs are genes that are closely related by vertical descent and exist in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase perform substantially the same or identical functions. Epoxide hydrolases share orthologues for the biological function of epoxide hydrolysis. Genes can be considered to be related to one another, for example, because they are homologous or have a common ancestor. If they share a sufficient amount of sequence similarity to indicate that they are closely related by evolution, Genes are closely related by primary sequence similarity, not necessarily sequence similarity. sufficient amounts of triadic ancestry to show that they evolved from a common ancestor within which no identifiable If genes share a common dimensional structure, they can also be considered orthologs. Homologous genes are proteins with sequence similarity of approximately 25% to 100% amino acid sequence identity. can encode proteins that share less than 25% amino acid similarity. If the genes encoding them and their three-dimensional structures also show similarities, then they are related by vertical descent. Tissue plasminogen activator and elastase Members of the serine protease family of enzymes, including ATPases, are descended vertically from a common ancestor. is deemed to have occurred by

[0034] Orthologs are genes or sequences that have diverged in structure or overall activity, e.g., through evolution. For example, if the first species encodes a gene product that exhibits two functions, , if such functions are separated into distinct genes in the second species, three genes and their corresponding products are considered to be orthologs. In this manner, one skilled in the art can easily identify orthologous genes with metabolic activity to be introduced or disrupted. It will be understood that the following will be selected for the construction of non-naturally occurring microorganisms: An example of an ortholog that exhibits separable activities is one in which distinct activities are expressed between two or more species or within a single A particular example is the separation of two types of serine proteases into distinct gene products within a species. Protease activity, elastase proteolysis and plasminogen proteolysis The separation of plasminogen activator and elastase into distinct molecules. The second example is the Mycoplasma 5'-3' exonuclease and the Drosophila DNA polymerase. The separation of DNA polymerase III activity from a first species is achieved by the separation of DNA polymerase III activity from a second species. Considered orthologous to xonuclease and / or polymerase and vice versa.

[0035] In contrast, paralogs are closely related, e.g., by duplication followed by evolutionary divergence. Homologs are not identical but have similar or common functions. Paralogs are e.g. They may originate from or be derived from the same or different species. For example, microsomal epoxides Hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide Hydrolases II) are classified as enzymes that catalyze distinct reactions and have distinct functions in the same species. They correspond to two distinct enzymes that coevolved from a common ancestor and can therefore be considered paralogs. Paralogs are proteins from the same species that share significant sequence similarity with each other. This suggests that they are conspecific or closely related through coevolution from a common ancestor. The group of Larogas protein families includes HipA homologs, luciferase genes, and peptidases. thidase, and others.

[0036] Nonorthologous gene replacement can substitute for the above gene functions in different species. Substitutions are non-orthologous genes from one species that can be substituted. performs substantially the same or similar functions in the species of origin compared to the functions described above in the Generally, non-orthologous gene replacements include those that can replace genes encoding the functions described above. It can be identified as structurally related to known genes, but is not structurally related. However, functionally similar genes and their corresponding gene products are still and still fall within the meaning of the term as it is used herein. Functional similarity can be achieved, for example, by comparing the gene encoding the function to be replaced. , at least some structure in the active site or binding region of the nonorthologous gene product Therefore, non-orthologous genes are considered to be, for example, paralogs or non-close relatives. Contains edge genes.

[0037] Therefore, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or lev Constructing and Producing Non-Naturally Occurring Microbial Organisms of the Invention Capable of Phosphate Biosynthesis In determining the species of interest, one of skill in the art will be able to apply the teachings and guidance provided herein to the particular species. This allows identification of metabolic modifications to include identification and inclusion or inactivation of orthologs. It will be understood that enzymes that catalyze similar or substantially similar metabolic reactions can be used. Coding paralogs and / or non-orthologous gene displacements are present in the above-mentioned microorganisms. To the extent possible, those skilled in the art can also utilize genes that are evolutionarily closely related to these genes. Gene disruption strategies involve the disruption of any functional significance in the enzymatic activity targeted for disruption. To ensure that the complexities do not interfere with the designed metabolic modifications, Genes that are evolutionarily closely related also have similarities with the host microbial organism, paralogs, or can also be disrupted or deleted in the ortholog.

[0038] Orthologs, paralogs, and nonorthologous gene displacements are well known to those skilled in the art. For example, the nucleic acid or amino acid sequence for the two polypeptides can be determined by a method such as Examination of amino acid sequences reveals sequence identities and similarities between the compared sequences. Based on such similarities, proteins may be closely related through evolution from a common ancestor. Those skilled in the art can determine whether the similarity is high enough to indicate that Al Algorithms well known to those skilled in the art, such as ign, BLAST, Clustal W, and others, are Pure sequence similarity or identity is compared and determined, and a weight or score is assigned. The presence or significance of gaps in the sequence that can be fitted is also determined. Algorithms are also known in the art and are used to determine nucleotide sequence similarity or identity. It is equally applicable to determining whether there is sufficient similarity to determine relevance. The parameter is used to find statistical similarity or similar matches in random polypeptides. Based on well-known methods for calculating the likelihood of making a match and the significance of the match determined. A computer comparison of two or more sequences may also be performed, if desired, by one skilled in the art. Related gene products or proteins can be identified by visual optimization. Similarity, for example, 25% to 100% sequence identity, can be expected. Proteins are expected to occur by chance when a database of sufficient size is scanned. The sequence may have an identity that is essentially the same as that expected (approximately 5%). Sequences between 5% and 24% are relatively may correspond to or correspond to sufficient homology to conclude that the compared sequences are closely related The size of the dataset must be taken into account to determine the significance of such a match. Further statistical analysis can be performed to determine the relevance of these sequences. .

[0039] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm are: The meter can be, for example, as described below. Amino acid sequence alignment was performed using BLASTP version 2.0.8 (January 5, 1999) with the following parameters: ter:Matrix:0 BLOSUM62;GapOpen:11;GapExtension:1;x_dr It can be done using opoff:50;expect:10.0;wordsize:3;filter:on Nucleic acid sequence alignments were performed using BLASTN version 2.0.6 (September 16, 1998) with the following parameters: tar:match:1;mismatch:-2;gapopen:5;gapextension:2;x_drop It can be run using off:50; expect:10.0; wordsize:11; filter:off. Those skilled in the art may wish to increase or decrease the stringency of the comparison, e.g., by adjusting the sequence of two or more sequences. Any modifications can be made to the above parameters to determine relevance. You will understand.

[0040] Adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or Disclosed herein are non-naturally occurring microbial organisms capable of producing buric acid. For example, the adipate pathway can be a reverse adipate degradation pathway (see Examples I and II). (See, e.g., J. Am. Soc. Soc. 11:111-1122.) For example, non-naturally occurring microbial organisms produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to The adipate pathway can include succinyl-CoA:acetate. 3-hydroxyacyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase oxiadipyl-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, the adipate pathway involves the 3-oxoadipate pathway. (See Examples III and IV). Non-naturally occurring microorganisms The organism encodes adipate pathway enzymes that are expressed in sufficient amounts to produce adipate. and an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway. The acetyl-CoA pathway is characterized by succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl- CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydrogenase hydratase, and 2-enoate reductase.

[0041] Additionally, non-naturally occurring microbial organisms have been shown to be capable of producing 6-aminocaproic acid sufficiently. at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in an amount The 6-aminocaproic acid pathway can include a CoA-dependent The enzymes include aldehyde dehydrogenase and transaminase (see Examples VIII and IX). Alternatively, 6-aminocaproate dehydrogenase can decompose adipate semiconducting enzymes. The aldehyde can be converted to form 6-aminocaproate ( (See Figure 8.) Non-naturally occurring microbial organisms also produce caprolactam. at least one exogenous gene encoding a caprolactam pathway enzyme expressed in an amount sufficient to The caprolactam pathway can also include a CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate aldehyde These include carboxygenases, and amidohydrolases (see Examples VIII and IX).

[0042] As disclosed herein, microorganisms that produce 6-aminocaproic acid or caprolactam The organism produces 6-aminocaproic acid and / or caprolactam from the adipyl-CoA precursor. (See Figure 8 and Examples VIII and IX.) Microbial organisms that produce caproic acid or caprolactam also produce adipyl-CoA. For example, the adipyl-CoA pathway can include a pathway for Schematic diagram of the use of succinyl-CoA and acetyl-CoA as precursors through the production of dipyr-CoA. Two enzymes can be involved, namely the final step for converting adipyl-CoA to adipate. Therefore, one exemplary adipyl-CoA pathway is 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase enzyme, 3-hydroxyadipyl-CoA dehydratase, and 5-carboxy-2-pentenoyl- CoA reductase may be included.

[0043] Furthermore, as shown in Figure 1, the adipate degradation pathway is catalyzed by adipate CoA ligase. The adipate-CoA conversion step involves converting adipate to adipyl-CoA. The oA pathway includes, for example, the adipate-CoA ligase activity in the first step of FIG. Any of the enzymes in the final step of Figure 2, such as adipyl-CoA synthetase, phosphotransadipylase / adipate (also called adipate Co-A ligase) kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase and further comprising an enzyme activity that converts adipate to adipyl-CoA, including either an enzyme or a phosphoenzyme. The adipate pathway can be considered as a pathway for converting adipate to adipyl-CoA. The enzyme may be an endogenous activity or may be co-activated as disclosed herein. Therefore, any adipate pathway can be provided as an exogenous nucleic acid. is utilized along with the enzyme activity from adipate to adipyl-CoA to obtain the adipyl-CoA pathway. It is understood that such a pathway can be used to prepare 6-aminocaproic acid and / or was synthesized by the addition of 6-aminocaproate to provide the adipyl-CoA precursor for caprolactam production. The acid or caprolactam producing microbial organisms may be included.

[0044] A further exemplary adipate pathway utilizes alpha-ketoadipate as a precursor. (See Figure 6 and Example VI.) For example, non-naturally occurring microbial organisms include asiatic microbial organisms. and at least one gene encoding an adipate pathway enzyme expressed in sufficient amounts to produce adipate. The adipate pathway can include one exogenous nucleic acid, and the adipate pathway can include a homozygous adipate pathway. Citrate synthase, homoaconitase, homoisocitrate dehydrogenase, 2-ketoaconitase adipate reductase, alpha-hydroxyadipate dehydratase, and oxido A further exemplary adipate pathway utilizes the lysine degradation pathway ( (See Figure 7 and Example VII.) Other naturally occurring microbial organisms have been shown to produce adipate. at least one enzyme encoding an adipate pathway enzyme expressed in a sufficient amount to produce The adipate pathway can include an exogenous nucleic acid, and the adipate pathway can include a carbon-nitrogen linker. enzymes, oxidoreductases, transaminases, and oxidoreductases .

[0045] Another exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (Figure 1). 9 and Examples X and XI). Thus, non-naturally occurring microbial organisms and a small number of genes encoding adipate pathway enzymes that are expressed in sufficient amounts to produce adipate. and an adipate pathway comprising at least one exogenous nucleic acid, the adipate pathway comprising , alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase (phosphot alpha-ketoadipylase) / alpha-ketoadipate kinase, or alpha-ketoadipyl-Co A: Acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxy 5-carboxy-2-pentenoyl-CoA reductase; and and adipyl-CoA synthetase, phosphotransadipylase / adipate kinase, adipate Adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Additionally, non-naturally occurring microbial organisms can be expressed in sufficient amounts to produce adipate. an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme; and the adipate pathway can have 2-hydroxyadipate dehydrogenase; Hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase transhydroxyadipylase) / 2-hydroxyadipate kinase, or 2-hydroxyadipyl- CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-calcium phosphate dehydrogenase carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotran Adipylase / adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase.

[0046] As disclosed herein, the present invention provides a method for producing 6-aminocaproic acid. at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in an amount and non-naturally occurring microbial organisms, including microbial organisms having a 6-aminocaproic acid pathway comprising The 6-aminocaproic acid pathway includes 3-oxo-6-aminohexanoyl-CoA thiol. 3-Oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA reductase 6-aminohex-2-enoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproic acid caproyl-CoA / acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or The present invention provides a non-naturally occurring microbial organism that contains a 6-aminocaproyl-CoA hydrolase. (See Examples XII and XIII; Steps A / B / C / D / K / L / M in Figure 11). and 6-aminocaproic acid pathway enzymes expressed in sufficient amounts to produce 6-aminocaproic acid. A microorganism having a 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding an enzyme. a non-naturally occurring microbial organism, including a microbial organism, in which the 6-aminocaproic acid pathway is , 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA / acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3 -Oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reader 3-hydroxy-6-aminohexanoate dehydratase; and 6-aminohexa-2- Non-naturally occurring microbial organisms containing enoate reductase are provided (see Examples XII and XIII). See steps A / E / F / G / H / I / J of Figure 11).

[0047] In another embodiment, the present invention provides a method for producing caprolactam by the use of a recombinant protein comprising: Caprolactam containing at least one exogenous nucleic acid encoding a prolactam pathway enzyme.

[0023] Non-naturally occurring microbial organisms, including microbial organisms having a pathway The tam pathway is mediated by 6-aminocaproyl-CoA / acyl-CoA transferase or 6-aminocaproyl-CoA

[0023] Non-naturally occurring microbial organisms containing methyloyl-CoA synthase are provided (see Examples XII and XIII). See steps XV; steps K / L of Figure 11). Microbial organisms not present in the present invention can further contain the 6-aminocaproic acid pathway (Figure 11 (See, for example, the 6-aminocaproic acid pathway.) An exemplary 6-aminocaproic acid pathway is the CoA-dependent aldehyde dehydrogenase pathway. and transaminase or 6-aminocaproate dehydrogenase 6-aminocaproic acid pathway or 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo -6-aminohexanoyl-CoA / acyl-CoA transferase, 3-oxo-6-aminohexanoyl 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-Hydroxy-6-aminohexanoate reductase; 3-Hydroxy-6-aminohexanoate dehydrogenase 6-Aminocaproic acid-catalyzed enzymes, including 6-aminohex-2-enoate reductase and 6-aminohex-2-enoate reductase These and other examples disclosed herein include steps A / E / F / G / H / I / J of FIG. The exemplary 6-aminocaproic acid pathway may further include a caprolactam pathway, if desired. It is understood that the present invention can be carried out in a microbial organism. Encoding hexamethylenediamine pathway enzymes expressed in sufficient amounts to produce the amine. A microbial organism having a hexamethylenediamine pathway comprising at least one exogenous nucleic acid a non-naturally occurring microbial organism, including an organism, wherein the hexamethylenediamine pathway comprises a 6- Aminocaproyl-CoA / acyl-CoA transferase or 6-aminocaproyl-CoA synthase 6-aminocaproyl-CoA reductase (aldehyde formation); and hexamethylenediamine naturally occurring enzymes, including amine transaminase or hexamethylenediamine dehydrogenase Non-resident microbial organisms are also provided (see Examples XII and XVI; steps K / L / L of Figure 11). N / O / P). Such non-naturally occurring microbial organisms containing the hexamethylenediamine pathway The body can further include a 6-aminocaproic acid pathway (see Figure 11). The 6-aminocaproic acid pathway is composed of CoA-dependent aldehyde dehydrogenase; 6-Aminocaproic acid pathway involving 6-aminocaproate dehydrogenase or 6-aminocaproate dehydrogenase or 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-Co A / acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoyl 3-hydroxy-6-aminohexanoate dehydratase; and 6-amino 6-aminocaproic acid pathway containing hex-2-enoate reductase (step 11 in Figure 11) A / E / F / G / H / I / J). These and other exemplary 6-aminocaproic acids disclosed herein The pathway may further be incorporated into a microbial organism having a hexamethylenediamine pathway, if desired. It is understood that it can include.

[0048] In yet another embodiment, the present invention provides a method for producing caprolactam by the use of a recombinant yeast strain of the present invention. caprolactam comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme. 1. A non-naturally occurring microbial organism having a caprolactam pathway, the caprolactam pathway comprising: 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA thiolase ductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohexanoyl A non-naturally occurring microbial organism containing 2-enoyl-CoA reductase is provided (in practice). See Examples XII and XVII; Steps A / B / C / D of Figure 11). and at least one hexamethylenediamine pathway enzyme encoding the hexamethylenediamine pathway enzyme expressed in sufficient amounts to Non-naturally occurring microbial organisms having a hexamethylenediamine pathway containing one exogenous nucleic acid In a biological organism, the hexamethylenediamine pathway is A thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-amino Hexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; 6-amino Nocaproyl-CoA reductase (aldehyde formation); and hexamethylenediamine trans Non-naturally occurring microbial enzymes containing aminase or hexamethylenediamine dehydrogenase A biological sample is also provided (see Examples XII and XVIII; steps A / B / C / D / D of Figure 11). N / O / P).

[0049] In yet another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) from a plant. and a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in an adequate amount. A non-naturally occurring microbial organism having a 6-ACA pathway, wherein the 6-ACA pathway comprises 4-hydroxy- 2-Oxoheptane-1,7-dioate (HODH) aldolase, 2-oxohept-4-ene-1,7-di Oate (OHED) hydratase, 2-oxohept-4-ene-1,7-dioate (OHED) reductase 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, adipate semial dehyde aminotransferase, adipate semialdehyde oxidoreductase (adipate 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, 6-oxohept-4-ene-1,7-dioate decarboxylase Hex-4-enoate (6-OHE) reductase, 2-oxoheptane-1,7-dioate (2-OHD) reductase aminotransferase, 2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase Amination, 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, 2-oxo Hept-4-ene-1,7-dioate (OHED) aminotransferase, 2-oxohept-4-ene 2-aminohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), 2-aminohept-4-ene-1,7 -dioate (2-AHE) reductase, 4-hydroxy-2-oxoheptane-1,7-dioate (HOD H) Formate lyase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase , adipyl-CoA dehydrogenase, 2-oxohept-4-ene-1,7-dioate (OHED) formate ase, 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, 2-oxohept- 2-oxoheptane-1,7-dioate (2-OHD) formate lyase, 2-oxoheptane-1,7-dioate (2-OHD) dehydrogenase the non-naturally occurring enzyme, including a hydrogenase or a pyruvate formate lyase activating enzyme. A microbial organism is provided (see Examples XIX and XXI; Steps A-Q of Figure 12). In some embodiments, the 6-ACA pathway includes succinic semialdehyde dehydrogenase, alpha-keto Glutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase Includes ze.

[0050] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in ;OHED reductase;2-OHD decarboxylase;or adipate semialdehyde aminotransferase containing acetyltransferase or adipate semialdehyde oxidoreductase (aminating) Non-naturally occurring microbial organisms are provided, including those described in Examples XIX and XXI; In a further aspect, the 6-ACA pathway comprises the steps of succinic acid semialdehyde carboxylase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate In another aspect of the invention, the non-naturally occurring microorganism comprises a phospholipid (PEP) carboxykinase. The organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set including HODH Aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; and azide Adipate semialdehyde aminotransferase or adipate semialdehyde oxide Encodes reductase (amination).

[0051] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in ;OHED decarboxylase;6-OHE reductase;or adipate semialdehyde aminotransferase containing acetyltransferase or adipate semialdehyde oxidoreductase (aminating) Non-naturally occurring microbial organisms are provided, including those described in Examples XIX and XXI; In a further aspect, the 6-ACA pathway comprises the steps of succinic acid semialdehyde carboxylase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate In another aspect of the invention, the non-naturally occurring microorganism comprises a phospholipid (PEP) carboxykinase. The organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set including HODH Aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase; and azide Adipate semialdehyde aminotransferase or adipate semialdehyde oxide Encodes reductase (amination).

[0052] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE and a non-naturally occurring microbial organism containing a 2-AHD decarboxylase. (See Examples XIX and XXI; Steps A / B / J / D / I of Figure 12). In a further aspect, The 6-ACA pathway is composed of succinic semialdehyde dehydrogenase, alpha-ketoglutarate decaaldehyde, and The present invention includes carboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In certain other embodiments, the naturally occurring microbial organism does not contain exogenous genes encoding 6-ACA pathway enzymes. and a set of nucleic acids comprising: an OHDH aldolase; an OHED hydratase; an OHED amino acid; 2-AHE reductase or OHED oxidoreductase (aminating) and 2-AHD decarboxylase.

[0053] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in ;OHED reductase;2-OHD aminotransferase or 2-OHD oxidoreductase (amination); or 2-AHD decarboxylase. (See Examples XIX and XXI; Steps A / B / C / H / I of Figure 12). In a further aspect, The 6-ACA pathway is composed of succinic semialdehyde dehydrogenase, alpha-ketoglutarate decaaldehyde, and The present invention includes carboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In certain other embodiments, the naturally occurring microbial organism does not contain exogenous genes encoding 6-ACA pathway enzymes. The set includes a set of nucleic acids having a specific function, the set including: an HODH aldolase; an OHED hydratase; an OHED reductase; 2-OHD aminotransferase or 2-OHD oxidoreductase (amination); and Encodes 2-AHD decarboxylase.

[0054] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; HODH-aldolase; HODH-formate-lyase; and pyruvate formate lyase activating enzyme or HODH dehydrogenase; 3-hydroxyadipate 2,3-Dehydroadipyl-CoA dehydratase; 2,3-Dehydroadipyl-CoA reductase; Adipyl-CoA dehydrogenase enzyme; or adipate semialdehyde aminotransferase or adipate semialdehyde aminotransferase A non-naturally occurring microbial organism containing an aminating aldehyde oxidoreductase (See Examples XIX and XXI; Steps A / L / M / N / O / E of Figure 12). In , the 6-ACA pathway involves succinic semialdehyde dehydrogenase, alpha-ketoglutaric acid dehydrogenase, and Contains acid decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase In another embodiment of the invention, the non-naturally occurring microbial organism encodes a 6-ACA pathway enzyme. The set includes a set of exogenous nucleic acids comprising HODH aldolase; HODH formate lyase; and Pyruvate formate lyase activating enzyme or HODH dehydrogenase; 3-hydroxyadipyl-Co A dehydratase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase and adipate semialdehyde aminotransferase or adipate semialdehyde Encodes a dehydoreductase (aminating).

[0055] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in OHED formate-lyase and pyruvate formate-lyase activating enzyme or OHED dehydrogenase; 2, 3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; or adipate Semialdehyde aminotransferase or adipate semialdehyde oxidizer

[0023] Non-naturally occurring microbial organisms containing aminating enzymes are provided (Examples XIX and XX). 12 steps A / B / P / N / O / E). In a further aspect, the 6-ACA pathway is acid semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding the 6-ACA pathway enzymes. The set comprises an HODH aldolase; an OHED hydratase; an OHED formate lyase; and a pyruvate lyase. Phosphate formate-lyase activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA redox Adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase Encoding sucrose or adipate semialdehyde oxidoreductase (aminating) do.

[0056] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; 6-ACA pathway is a microbial organism not present in ;OHED reductase;2-OHD formate lyase and pyruvate formate lyase activating enzyme or 2-OHD Dehydrogenase; adipyl-CoA dehydrogenase; or adipate semialdehyde amino Transferase or adipate semialdehyde oxidoreductase (amination) (See Examples XIX and XXI; Figure 1). In a further aspect, the 6-ACA pathway comprises the steps A / B / C / Q / O / E of 12. dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenol In another aspect of the invention, non-naturally occurring The microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising , HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyruvate Phosphate formate-lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase and adipate semialdehyde aminotransferase or adipate semialdehyde The present invention further provides a method for the preparation of a 6-aminocarboxylic acid oxidoreductase (aminating enzyme). at least one gene encoding a 6-ACA pathway enzyme that is expressed in sufficient amounts to produce 6-ACA and a non-naturally occurring microbial organism having a 6-ACA pathway containing one exogenous nucleic acid. The 6-ACA pathway involves glutamyl-CoA transferase, glutamyl-CoA ligase, and base Taketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxybenzoate 6-amino-7-carboxyhept-2-enoyl-CoA dehydratase, 6-amino-7-carboxyhept-2-enoyl-CoA dehydratase 6-aminopimeloyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde forming), or 2-aminopimeloyl-CoA reductase A non-naturally occurring microbial organism containing minopimelate decarboxylase is provided. See Examples XXV and XXVI; steps A / B / C / D / E / I / J of Figure 20). In other words, the non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding the 6-ACA pathway enzymes. The set includes a glutamyl-CoA transferase or a glutamyl-CoA ligase ;beta-ketothiolase;3-oxo-6-aminopimeloyl-CoA oxidoreductase;3- Hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-eno 6-aminopimeloyl-CoA reductase (aldehyde formation); and 2-aminopimeloyl-CoA reductase It encodes minopimelate decarboxylase.

[0057] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; microbial organisms not present in the wild-type microbial organisms, wherein the 6-ACA pathway is mediated by glutaryl-CoA beta-ketothiol. 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase , 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3- Oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomuta Non-naturally occurring microbial organisms containing 2-aminopimelate decarboxylase or 2-aminopimelate decarboxylase (See Examples XXV and XXVI; Steps A / B / J / T / AA of Figure 21). In some embodiments, the non-naturally occurring microbial organism comprises an exogenous nucleic acid encoding the 6-ACA pathway enzymes. The set includes a set of acids, the set including glutaryl-CoA beta-ketothiolase; 3-oxopimelo 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA 3-oxopimelate aminotransferase or 3-oxopimelate 3-aminopimelate 2,3-aminomutase; and 2-aminopimelate aminating oxidoreductase. The present invention further relates to a method for the production of 6-aminocaproic acid At least one encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA a non-naturally occurring microbial organism having a 6-ACA pathway comprising an exogenous nucleic acid of The CA pathway provides a non-naturally occurring microbial organism containing homolysine 2-monooxygenase. (See Examples XXV and XXVI; Step A of Figure 23). In a further embodiment, 6-ACA The route involves the use of dilute acid or base to convert 6-aminohexanamide to 6-aminocaproate. (See Examples XXV and XXVI.) ;Step B in Figure 23).

[0058] The present invention further provides a method for producing 6-aminocaproic acid (6-ACA) by the use of a 6-aminocaproic acid-containing enzyme. - a naturally occurring 6-ACA pathway comprising at least one exogenous nucleic acid encoding an ACA pathway enzyme; In a microbial organism not present in the wild, the 6-ACA pathway is mediated by adipate reductase, adipate or adipylphosphate reductase (see Example XXVIII) (See steps X / Y / Z of Figure 25 and Example XXXI). In a further aspect, the 6-ACA pathway can be catalyzed by adipate synthesis. In another further aspect, the 6-aminocaproic acid (6-ACA) pathway comprises an azide reductase. In another embodiment, the 6- Microbial organisms having an ACA pathway include those having the adipate pathway, Caprolactam, and / or the hexamethylenediamine pathway (see Example XXVIII). (Steps A to W in Figure 25).

[0059] In one embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities to produce 6-aminocaproic acid (6-ACA). and a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme. a non-naturally occurring microbial organism, wherein the 6-ACA pathway is catalyzed by the synthesis of 2-amino-7-oxo- keto acid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase , 2-amino-7-oxoheptanoate oxidoreductase, 2-aminopimelate decal carboxylase, 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanol Noate decarboxylase, or 2-amino-7-oxosubarate amino acid decarboxylase

[0023] In one embodiment, a non-naturally occurring microbial organism is provided, comprising a microbial enzyme (see Examples XXV and XXVI). 26 steps A / B / D / E / F / G / I). In a further aspect, the microbial organism is 2-amino-7- 2-amino-7-oxosubarate expressed in sufficient amounts to produce oxosubarate 2-amino-7-oxosubarate having at least one exogenous nucleic acid encoding a tract enzyme The 2-amino-7-oxosubarate pathway is a pathway that subarate aldolase, 2-amino-5-hydroxy-7-oxosubarate dehydratase, or contains 2-amino-5-ene-7-oxosuberativ reductase (see Examples XXV and XXVI) (Steps A / B / C in Figure 27).

[0060] In another embodiment of the invention, the non-naturally occurring microbial organism encodes the 6-ACA pathway enzymes. the set of exogenous nucleic acids encoding 2-amino-7-oxosubarate keto acid decarboxylase; 2-amino-7-oxoheptanoate oxidoreductase; and 2-amino- Encoding minopimelate decarboxylase (see Example XXV; see steps in Figure 26 In another embodiment of the invention, the non-naturally occurring microbial organism is capable of converting 6-ACA pathway The set includes a set of exogenous nucleic acids encoding an enzyme, the set including 2-amino-7-oxo-substrate 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanal oxidoreductase (see Example XXV). 26 steps A / B / F). In other embodiments of the invention, the non-naturally occurring microbial organism is , a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising 2-amino-7-ol Hexosubarate amino acid decarboxylase; 2-amino-7-oxoheptanoate decarboxylase xylanase; and 6-aminohexanal oxidoreductase (see Example XXV). See steps I / G / F of Figure 26). In a further aspect of each of the above embodiments, The microbial organism may comprise a 2-amino-7-oxosubarate complex expressed in sufficient amounts to produce 2-amino-7-oxosubarate. 2- with a second set of exogenous nucleic acids encoding amino-7-oxosubarate pathway enzymes. The 2-amino-7-oxosubarate pathway is 2-amino-5-hydroxy-7-oxosubarate aldolase; 2-amino-5-hydroxy-7-oxosubarate aldolase and 2-amino-5-ene-7-oxosabreductase ( See Examples XXV and XXVI; steps A / B / C of Figure 27).

[0061] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). and HMDA, including at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in the a non-naturally occurring microbial organism, wherein the HMDA pathway comprises 6-aminocaproate kinase oxidoreductase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, 6-aminohexanoyloxy Aminocaproic semialdehyde aminotransferase, 6-aminocaproic semialdehyde Dehyde oxidoreductase (aminating), 6-aminocaproate N-acetyltransferase enzyme, 6-acetamidohexanoate kinase, [(6-acetamidohexanoyl)oxidase 6-Acetamidohexanalamino]phosphonate (6-AAHOP) oxidoreductase transferase, 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanal oxidoreductase 6-acetamidohexanamine N-acetyltransferase Acetaminohexanoate hydrolase (amide), 6-acetamidohexanoate CoA transferase, 6-acetamidohexanoate CoA transferase Acetamidohexanoate CoA ligase, 6-acetamidohexanoyl-CoA oxidoreductase ectase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase Sulfuric acid, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase 6-aminocaproate CoA transferase, and 6-aminocaproate CoA Non-naturally occurring microbial organisms containing a ligase are provided (see Examples XX and XXI). (Steps A to N in Figure 13).

[0062] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway comprises 6-aminocaproate kinase; 6-Aminocaproate kinase; HOP oxidoreductase; or 6-aminocaproic semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde aminotransferase In addition, non-naturally occurring microbial organisms are provided (see Examples XX and XXI; see the sequence in Figure 13). In other aspects of the invention, the non-naturally occurring microbial organism expresses or encodes the HMDA pathway enzymes. The set includes a set of exogenous nucleic acids encoding 6-aminocaproate kinase 6-AHOP oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase aminotransferase (amination) or 6-aminocaproic acid semialdehyde aminotransferase Do it.

[0063] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway comprises 6-aminocaproate kinase; 6-Aminocaproate kinase; HOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; or 6-aminocaproyl-CoA oxidoreductase Aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid A non-naturally occurring microbial organism containing phosphate semialdehyde aminotransferase. (See Examples XX and XXI; Steps A / L / N / C of Figure 13). Other Aspects of the Invention In this context, non-naturally occurring microbial organisms express a set of exogenous nucleic acids encoding HMDA pathway enzymes. The set includes 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde Oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase Encodes spherase.

[0064] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway does not involve the 6-aminocaproate CoA transactivation Ferrase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxide Reductase; or 6-aminocaproic semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde aminotransferase, providing a microbial organism that is not present in the sample (see Examples XX and XXI; steps M / N / C of FIG. 13 ). In another embodiment of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. The set includes a set of exogenous nucleic acids that encode 6-aminocaproate CoA transferase. 6-aminocaproyl-CoA oxidoreductase or 6-aminocaproate CoA ligase; and 6-aminocaproic acid semialdehyde oxidoreductase (aminating) or encodes 6-aminocaproic acid semialdehyde aminotransferase.

[0065] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is Transferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductor 6-acetamidohexanal aminotransferase or 6-acetamidohexanal Sanal oxidoreductase (amination); or 6-acetamidohexanamine N-acetyl containing 6-acetamidohexanamine hydrolase (amide) Non-naturally occurring microbial organisms are provided (see Examples XX and XXI; Figure 13). Steps D / E / F / G / H). In other aspects of the invention, the naturally occurring microbial organism is not HMDA-mediated. The set includes a set of exogenous nucleic acids encoding tract enzymes, the set including 6-aminocaproate N- Acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxide Reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal Hexanal oxidoreductase (amination); and 6-acetamidohexanamine N- Acetyltransferase or 6-acetamidohexanamine hydrolase (amide) Code.

[0066] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is 6-acetamidohexanoate CoA transferase or 6-acetamidohexanoate CoA transferase 6-Acetamidohexanoate-CoA ligase; 6-Acetamidohexanoyl-CoA oxidoreductase 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Xanal oxidoreductase (amination); or 6-acetamidohexanamine N-acetate methyltransferase or 6-acetamidohexanamine hydrolase (amide) Non-naturally occurring microbial organisms comprising the microbial organisms described herein are provided (see Examples XX and XXI; Figure 13 In another aspect of the invention, the non-naturally occurring microbial organism is a set of exogenous nucleic acids encoding pathway enzymes, the set comprising 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate CoA transferase or 6-acetamidohexanoate CoA ligase; 6-acetamidohexanoyl-CoA oxidase 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Amidohexanal oxidoreductase (amination); and 6-acetamidohexanal oxidoreductase amine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amine The present invention further provides a method for producing hexamethylenediamine (HMDA) by the use of a recombinant vector. HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts. a non-naturally occurring microbial organism having a HMDA pathway, wherein the HMDA pathway comprises a 6-aminocaproic acid 6-Acetamidohexanoate N-acetyltransferase; 6-Acetamidohexanoate kinase; 6-AAHOP 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase 6-Acetamidohexanal oxidoreductase (amination); or 6-acetamidohexanal amine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI). (See steps D / E / K / J / G of Figure 13). In other aspects of the invention, non-naturally occurring microbial organisms are used. The body includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set including 6-aminocarboxylic acid Proate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AA HOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6- Acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanal Hexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase The present invention further provides a method for producing hexamethylenediamine (HMDA). the HMDA pathway comprises at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in a sufficient amount to A non-naturally occurring microbial organism having an HMDA pathway, wherein the HMDA pathway comprises glutamyl-Co A transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6- Aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydrogenase hydratase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopyridinyl meloyl-CoA reductase (aldehyde forming), 2-amino-7-oxoheptanoate amino Transferase, 2-amino-7-oxoheptanoate aminating oxidoreductase or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A-H of Figure 20). In another aspect of the invention, The non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes. , the set includes glutamyl-CoA transferase or ligase; beta-ketothiolase ;3-oxo-6-aminopimeloyl-CoA oxidoreductase;3-hydroxy-6-aminopimelo 6-amino-7-carboxyhept-2-enoyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6- Aminopimeloyl-CoA reductase (aldehyde formation); 2-amino-7-oxoheptanoate Aminotransferase or aminating oxidoreductase; and homolysine decarboxime It encodes xylase.

[0067] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; a non-naturally occurring microbial organism, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiol enzyme, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1 -carboxyheptanal aminotransferase, 3-oxo-1-carboxyheptanal aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotrans ferase, 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase 5-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxopimelate 5-oxopimeloyl-CoA transferase, 3-oxopimelate ligase, 5-oxopimeloyl-CoA Reductase (aldehyde forming), 3-oxopimelate aminotransferase, 3-oxopimelate Sopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase 3-aminopimelate ligase, 5-aminopimeloyl-CoA reductase (aldehyde form synthesis), 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3- Aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase , 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate 7-aminotransferase Heptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-amino 2,3-aminomutase, homolysine decarboxylase, 3-aminopimelate 2,3-aminomutase, 2-aminopyrimelate Aminopimelate kinase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 2-aminopimelate reductase, 6-aminopimeloylphosphonate Reductase, 6-aminopimeloyl-CoA reductase (aldehyde forming), 3-amino-7-o 3-amino-7-oxoheptanoate 7-aminotransferase, or 3-amino-7-oxoheptanoate and (b) providing a non-naturally occurring microbial organism comprising a cysteine-containing aminating oxidoreductase. (See Examples XXIV and XXVI; Figure 21).

[0068] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1- Carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal ol 7-aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, 3 ,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase Non-naturally occurring microbial organisms comprising: 21 steps A / B / C / D / E / R / S). In another aspect of the invention, the non-naturally occurring microbial organism is , a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1 -Carboxyheptanal 7-aminating oxidoreductase; 3-oxo-7-aminoheptano 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine dehydrogenase It encodes a carboxylase.

[0069] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate kinase; 5-oxopimeloyl 3-oxo-1-carboxyheptanal 7-aminotransferase 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate Ethyl 3-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomethane and a non-naturally occurring microbial organism comprising a lysine decarboxylase or a homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A / B / F / G / D / E / R / S of Figure 21). In embodiments, the non-naturally occurring microbial organism contains an exogenous nucleic acid encoding a HMDA pathway enzyme. The set includes a glutaryl-CoA beta-ketothiolase; -CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl 3-Oxopimelate kinase; 5-Oxopimeloylphosphonate reductase 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1- Carboxyheptanal 7-aminating oxidoreductase; 3-oxo-7-aminoheptanoic acid 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidase lysine reductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine deca Encodes carboxylase.

[0070] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate CoA transferase, 3- Oxopimelate CoA ligase, 5-oxopimeloyl-CoA reductase (aldehyde formation) , 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminotransferase 3-Oxo-7-aminoheptanoate 3-aminoheptanol 7-aminating oxidoreductase -Aminotransferase, 3-oxo-7-aminoheptanoate 3-amination oxidreda ctase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase Non-naturally occurring microbial organisms containing cyclosporinase are provided (see Examples XXIV and XXVI). 21 steps A / B / H / I / D / E / R / S). In another aspect of the invention, non-naturally occurring The microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA Pimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; 5-oxopimelate Iodoyl-CoA reductase (aldehyde forming); 3-oxo-1-carboxyheptanal 7-amino Transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7- Aminoheptanoate 3-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3 -aminomutase; and homolysine decarboxylase.

[0071] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1- Carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-Aminating oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferase 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3 ,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase Non-naturally occurring microbial organisms comprising: 21 steps A / B / C / AB / Z / R / S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising glutaryl-C oA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-C CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate ligase 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo -1-carboxyheptanal 3-aminating oxidoreductase; 3-amino-7-oxoheptanal 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminotransferase oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine dehydrogenase It encodes a carboxylase.

[0072] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate kinase, 5-oxopimeloyl 3-oxo-1-carboxyheptanal 3-aminotransferase 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, 3-aminating 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate Ethyl 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomethane and a non-naturally occurring microbial organism comprising a lysine decarboxylase or a homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A / B / H / I / AB / Z / R / S of Figure 21). In some embodiments, the non-naturally occurring microbial organism comprises an exogenous nucleic acid encoding a HMDA pathway enzyme. The set includes a set of: glutaryl-CoA beta-ketothiolase; 3-oxopimelloi; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Ile-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1 -Carboxyheptanal 3-aminating oxidoreductase; 3-amino-7-oxoheptano 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine dehydrogenase It encodes a carboxylase.

[0073] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase enzyme, 3-oxopimeloyl-CoA ligase, 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase, 5-oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1- Carboxyheptanal 3-aminating oxidoreductase, 3-amino-7-oxoheptano 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-amination oxidase lysine dehydrogenase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine dehydrogenase A non-naturally occurring microbial organism is provided that contains a carboxylase (Examples XXIV and XXV). See Figure 21, steps A / B / F / G / AB / Z / R / S). The non-resident microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3- Oxopimelate CoA transferase or 3-oxopimelate CoA ligase; 5-oxopimelate Pimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 3-aldehyde Aminotransferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase ductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino -7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

[0074] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase enzyme, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase, 3-aminopimelate reductase 2,3-aminomutase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate amination oxidase Non-naturally occurring microorganisms containing lysine reductase or homolysine decarboxylase Providing an organism (see Examples XXIV and XXVI; steps A / B / / J / O / P / Q / S of Figure 21) In another aspect of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. The set of exogenous nucleic acids includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimeloyl-CoA ligase Sopimelate aminating oxidoreductase; 3-aminopimelate reductase; 3-amino -7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-amino 2-amino-7-oxoheptanoate 7-amination oxidoreductase or 2-amino-7-oxoheptanoate 7-amination oxidoreductase and homolysine decarboxylase.

[0075] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase enzyme, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase, 3-aminopimelate kinase , 5-aminopimeloylphosphonate reductase, 3-amino-7-oxoheptanoate 2,3 -aminomutase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2- 7-amino-7-oxoheptanoate 7-aminating oxidoreductase, or homolysine decal

[0023] Non-naturally occurring microbial organisms containing carboxylases are provided (see Examples XXIV and XXVI). See steps A / B / J / M / N / P / Q / S of Figure 21). In another aspect of the invention, naturally occurring The microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA hydrolase 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; Pimelate aminotransferase or 3-oxopimelate aminating oxidoreductase 3-aminopimelate kinase; 5-aminopimeloylphosphonate reductase; 3-aminopimelate kinase; 2-amino-7-oxoheptanoate 7- Aminotransferase or 2-amino-7-oxoheptanoate aminating oxidizer and homolysine decarboxylase.

[0076] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase esterase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (alginate) aldehyde formation), 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate amine non-naturally occurring enzymes, including lysine oxidoreductases or homolysine decarboxylases A microbial organism is provided (see Examples XXIV and XXVI; steps A / B / J / K / L / of FIG. 21 ). In another aspect of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. The set includes a set of exogenous nucleic acids encoding glutaryl-CoA beta-ketothiol. 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase , or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate CoA trans ferase or 3-aminopimelate CoA ligase; 5-aminopimeloyl-CoA reductase 2,3-aminomutase (aldehyde-forming); 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7- Oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate and homolysine decarboxylase.

[0077] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate Heptanoate 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-amino The present invention provides a non-naturally occurring microbial organism containing a lysine decarboxylase or a homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A / B / J / O / Z / R / S of Figure 21). In some embodiments, the non-naturally occurring microbial organism comprises an exogenous nucleic acid encoding a HMDA pathway enzyme. The set includes a set of: glutaryl-CoA beta-ketothiolase; 3-oxopimelloi; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Ile-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate 3-aminopimelate reductase; 3-amino-7-oxo Heptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminotransferase 3,7-diaminoheptanoate 2,3-aminomutase; and homo It encodes lysine decarboxylase.

[0078] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase esterase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (alginate) aldehyde formation), 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino -7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2 Non-naturally occurring microbial enzymes containing 3-aminomutase or homolysine decarboxylase (See Examples XXIV and XXVI; Steps A / B / J / K / L / Z / R / of Figure 21.) In another embodiment of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. The set includes a set of exogenous nucleic acids comprising glutaryl-CoA beta-ketothiolase; Oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3- Oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminotransferase Xopimelate aminating oxidoreductase; 3-aminopimelate CoA transferase 5-aminopimeloyl-CoA-reductase (aldehyde) amide formation); 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- Oxoheptanoate aminating oxidoreductase; 3,7-diaminoheptanoate 2,3- aminomutase; and homolysine decarboxylase.

[0079] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate kinase, 5- Aminopimeloylphosphonate reductase, 3-amino-7-oxoheptanoate 7-amino Notransferase, a 3-amino-7-oxoheptanoate aminating oxidoreductase 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase

[0023] Non-naturally occurring microbial organisms containing the enzymes are provided (see Examples XXIV and XXVI). 21 steps A / B / J / M / N / Z / R / S). In another aspect of the invention, a non-naturally occurring microorganism The organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set including glutamate. 3-oxopimeloyl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimelo 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate Aminotransferase or 3-oxopimelate aminating oxidoreductase; 5-aminopimeloylphosphonate reductase; 3-amino-7-o 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and It encodes molysine decarboxylase.

[0080] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-Oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomuta 2-aminopimelate reductase, 2-amino-7-oxoheptanoate 7-aminotoxidase transferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a non-naturally occurring microbial organism containing a homolysine decarboxylase. See Examples XXIV and XXVI; steps A / B / J / T / W / Q / S of Figure 21). Other Aspects of the Invention In this context, non-naturally occurring microbial organisms express a set of exogenous nucleic acids encoding HMDA pathway enzymes. The set includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-C oA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminotransferase 2-aminopimelate oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino- amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase It encodes a serotype.

[0081] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-Oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomuta 2-aminopimelate kinase, 6-aminopimeloylphosphonate reductase, 2- Amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate a lysine aminating oxidoreductase or a homolysine decarboxylase; Provide a non-naturally occurring microbial organism (see Examples XXIV and XXVI; see steps in Figure 21). In other aspects of the invention, the non-naturally occurring microbial organism is HMDA. a set of exogenous nucleic acids encoding pathway enzymes, the set including glutaryl-CoA beta -ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA tran isothiocyanate, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase spherase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-Aminomutase; 2-Aminopimelate kinase; 6-Aminopimeloylphosphonate ductase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino -7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase It encodes an enzyme.

[0082] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In a non-naturally occurring microbial organism, the HMDA pathway is mediated by glutaryl-CoA beta-ketothiola 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase ase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-Oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomuta 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 6- Aminopimeloyl-CoA reductase (aldehyde forming), 2-amino-7-oxoheptanoic acid 2-amino-7-oxoheptanoate aminating oxidoreductase Non-naturally occurring microbial organisms containing lysine decarboxylase or homolysine decarboxylase (See Examples XXIV and XXVI; Steps A / B / J / T / V / Y / Q / S of Figure 21). In certain other aspects, the non-naturally occurring microbial organism comprises an exogenous gene encoding an HMDA pathway enzyme. The set includes a set of nucleic acids comprising glutaryl-CoA beta-ketothiolase; 3-oxopyrrolidin; pimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Pimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate 3-Aminopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; 6-aminopimelate Iodoyl-CoA reductase (aldehyde formation); 2-amino-7-oxoheptanoate 7-amino transferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase.

[0083] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; In non-naturally occurring microbial organisms, the HMDA pathway is Heptanoate aldolase, 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase 2-oxo-7-aminohept-3-enoate reductase, 2-oxo-7-aminohepta Noate aminotransferase, 2-oxo-7-aminoheptanoate aminotransferase Ferrase aminating oxidoreductase, homolysine decarboxylase, 2-oxo-7 -aminoheptanoate decarboxylase, 6-aminohexanal aminotransferase naturally occurring enzymes, including 6-aminohexanal aminating oxidoreductases, providing a microbial organism free of microbial markers (see Examples XXIV and XXVI; Steps A-G of Figure 22). In another aspect of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. a set of exogenous nucleic acids, the set comprising 2-oxo-4-hydroxy-7-aminoheptanoic acid; 2-Oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-Oxo-4-hydroxy-7-aminoheptanoate dehydratase 2-Oxo-7-aminohept-3-enoate reductase; 2-Oxo-7-aminoheptanoate amine Notransferase or 2-oxo-7-aminoheptanoate aminating oxidoreductase and homolysine decarboxylase. The non-existent microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, The set consists of 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; Hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminohept-3-enoate reductase; 2-oxo-7-aminoheptanoate decarboxylase; and 6-aminoheptanoate decarboxylase Xanal aminotransferase or 6-aminohexanal aminating oxidoreductase It encodes a tase.

[0084] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; A non-naturally occurring microbial organism in which the HMDA pathway is mediated by 6-aminocaproate reductase , 6-aminocaproic semialdehyde aminotransferase, 6-aminocaproic semialdehyde aminotransferase Aldehyde oxidoreductase (aminating), 6-aminocaproate N-acetyltransferase Spherase, 6-acetamidohexanoate reductase, 6-acetamidohexanone aminotransferase, 6-acetamidohexanal oxidoreductase (amino 6-acetamidohexanamine N-acetyltransferase, or acetamidohexanamine A non-naturally occurring microbial organism containing hexaneamine hydrolase (amide) is provided. (See Example XXVII; Steps O / C or D / P / G / H of Figure 24 and Example XXXI). In other embodiments, the naturally occurring microbial organism does not contain an exogenous gene encoding a HMDA pathway enzyme. and a set of nucleic acids, the set comprising: a 6-aminocaproate reductase; and a 6-aminocaproate reductase. Caproic acid semialdehyde aminotransferase or 6-aminocaproic acid semialdehyde In another aspect of the invention, the naturally occurring The microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Midohexanal oxidoreductase (amination); and 6-acetamidohexanamine N -acetyltransferase or 6-acetamidohexanamine hydrolase (amide) The present invention further provides a method for producing hexamethylenediamine (HMDA) comprising: a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in an amount the HMDA pathway comprises a 2-amino-7-oxosamine derivative; Valine keto acid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase 6-aminohexanal aminating oxidoreductase, 6-aminohexanal aminating oxidoreductase 2-amino-7-oxoheptanoate decarboxylase, homolysinase gin decarboxylase, 2-amino-7-oxosubarate amino acid decarboxylase, 2 -oxo-7-aminoheptanoate aminating oxidoreductase, 2-oxo-7-aminoheptanoate Butanoate aminotransferase, 2-amino-7-oxosubarate amination oxy 2-amino-7-oxosubarate aminotransferase, 2,7-diaminobenzoate reductase, Nosabarate decarboxylase, 2-amino-7-oxoheptanoate aminotransferase esterase, or 2-amino-7-oxoheptanoate aminating oxidoreductase , provides non-naturally occurring microbial organisms (see Examples XXIV and XXVI; Figure 26 In a further aspect, the microbial organism comprises 2-amino-7-oxidase (steps A / B / C / G / H / I / J / K / L / M). 2-Amino-7-oxosubarate pathway enzymes expressed in sufficient amounts to produce 2-amino-7-oxosubarate 2-amino-7-oxosubarate pathway having at least one exogenous nucleic acid encoding an enzyme and the 2-amino-7-oxosubarate pathway produces 2-amino-5-hydroxy-7-oxosubarate. 2-amino-5-hydroxy-7-oxosubarate dehydratase, 2-amino-5-hydroxy-7-oxosubarate dehydratase, or 2- amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI) (See steps A / B / C in Figure 27).

[0085] In another embodiment of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. the set comprising a set of exogenous nucleic acids that encode 2-amino-7-oxosubarate amination Oxidoreductase or 2-amino-7-oxosubarate aminotransferase; 2,7- Diaminosubarate decarboxylase; and homolysine decarboxylase (See Examples XXIV and XXVI; Steps K / L / H of Figure 26). The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding HMDA pathway enzymes. The set comprises 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7-aminoheptanoate aminating oxidoreductase or 2-oxo-7-aminoheptanoate and homolysine decarboxylase (Fig. See Examples XXIV and XXVI; Steps I / J / H of Figure 26). In another embodiment of the invention, The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding the HMDA pathway enzymes. The set includes 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7 -aminoheptanoate decarboxylase; and 6-aminohexanal amination oxide Encoding a reductase or a 6-aminohexanal aminotransferase (Example X See steps XIV and XXVI of Figure 26). In another embodiment of the invention, naturally occurring The non-existent microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, The set consists of 2-amino-7-oxosubarate keto acid decarboxylase; Heptanoate decarboxylase; and 6-aminohexanal aminating oxidoreductase 6-aminohexanal aminotransferase (Examples XXIV and XXIV) See Figure 26 and XXVI; steps A / B / C of Figure 26). In another embodiment of the invention, naturally occurring The microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, 2-amino-7-oxosubarate keto acid decarboxylase; 2-amino-7-oxohepto Heptanoate aminating oxidoreductase or 2-amino-7-oxoheptanoate amino transferase; and homolysine decarboxylase (Examples XXIV and XXIII). See VI; steps A / M / H of Figure 26). In a further aspect of each of the above embodiments. The microbial organism is expressed in sufficient amounts to produce 2-amino-7-oxosubarate. and a second set of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes. The 2-amino-7-oxosubarate pathway involves the 2-amino-7-oxosubarate pathway. 2-amino-5-hydroxy-7-oxosubarate aldolase; 2-amino-5-hydroxy-7-oxo Subarate dehydratase; and 2-amino-5-ene-7-oxosubarate reductase (See Examples XXV and XXVI; Steps A / B / C of Figure 27). The present invention further provides At least one encoding an LA pathway enzyme expressed in sufficient amounts to produce LA. a non-naturally occurring microbial organism having an LA pathway comprising an exogenous nucleic acid of 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA / acyl-CoA transferase enzyme, 3-oxoadipyl-CoA synthase, 3-oxoadipyl-CoA hydrolase, or 3- A non-naturally occurring microbial organism containing an oxoadipate decarboxylase is provided. (See Example XXIX; steps A / E / F / G / AA of Figure 25). In another embodiment of the invention, natural The non-native microbial organism comprises a set of exogenous nucleic acids encoding LA pathway enzymes, The set consists of 3-oxoadipyl-CoA thiolase; 3-oxoadipyl-CoA / acyl-CoA transactivator; ferase, 3-oxoadipyl-CoA synthase, or 3-oxoadipyl-CoA hydrolase and 3-oxoadipate decarboxylase.

[0086] The non-naturally occurring microbial organisms disclosed herein include, for example, 6-aminocaproic acid esters. It can have a carboxylic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway. Non-naturally occurring microbial organisms, as disclosed herein, can convert substrates into products. The recombinant vector contains at least one exogenous nucleic acid encoding a polypeptide that converts the recombinant vector. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid encoding a polypeptide. The polypeptide may contain a carboxylic acid, and the polypeptide may be any of the carboxylic acids shown in Figures 2, 3, 8, 9, 10, 11, 12, 13, and 20-27. 6-aminocaproic acid, caprolactam, hexamethylene, such as those shown in They are enzymes or proteins that convert substrates and products of the diamine or levulinic acid pathway.

[0087] For example, a non-naturally occurring microbial organism can have an adipate pathway, and the microbial The organism converts succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-Hydroxyadipyl-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 and a polypeptide encoding a substrate selected from the group consisting of hydroxybenzoates and adipate, to a product. It contains at least one exogenous nucleic acid (see Figure 2). The biological organism can have an adipate pathway, and the microbial organism can produce succinyl-CoA and and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipyl 3-Oxoadipate to 3-hydroxyadipate; 3-Hydroxyadipate to hexa -2-enedioate (also referred to herein as 5-carboxy-2-pentanoate); hexa- Polypeptides that convert substrates selected from 2-enedioate to adipate into products and (c) containing at least one exogenous nucleic acid encoding a nucleotide sequence (see Figure 3). Non-naturally occurring microbial organisms can have a 6-aminocaproic acid pathway, and microbial The organism converts adipyl-CoA to adipate semialdehyde; and adipate semialdehyde A polypeptide that converts a substrate selected from the group consisting of 6-aminocaproate to a product is provided. It contains at least one exogenous nucleic acid that encodes the nucleic acid (see Figure 8). The absent microbial organism can have a caprolactam pathway, and the microbial organism can Adipyl-CoA to adipate semialdehyde; adipate semialdehyde to 6-aminocarbonyl and 6-aminocaproate to produce a product selected from the group consisting of caprolactam and 6-aminocaproate. The nucleic acid sequence contains at least one exogenous nucleic acid encoding a polypeptide that converts the Additionally, non-naturally occurring microbial organisms can have an adipate pathway, The substance is converted from alpha-ketoadipate to alpha-ketoadipyl-CoA; alpha-ketoadipyl 2-hydroxyadipyl-CoA to 5-carboxy-2-pentyl-CoA; 5-Carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA a small molecule encoding a polypeptide that converts a substrate selected from the group consisting of thiamin and adipate to a product; It contains at least one exogenous nucleic acid (see Figure 9). The microbial organism can have an adipate pathway, and the microbial organism can be an alpha-keto Adipate to 2-hydroxyadipate; 2-hydroxyadipate to 2-hydroxyadipate 2-Hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy 2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate. at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product; Contains (Figure 9).

[0088] Additionally, non-naturally occurring microbial organisms have been shown to possess the 6-aminocaproyl-CoA pathway. The microbial organisms can convert 4-aminobutyryl-CoA and acetyl-CoA into 3-oxo-6-aminobutyryl-CoA. 3-Oxo-6-aminohexanoyl-CoA; 3-Oxo-6-aminohexanoyl-CoA to 3-hydroxy-6-amino 3-Hydroxy-6-aminohexanoyl-CoA to 6-aminohex-2-enoyl a substrate selected from 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA at least one exogenous nucleic acid encoding a polypeptide that converts Further substrates and products of such a pathway include 6-aminocaproyl-CoA to 6- Aminocaproate; 6-aminocaproyl-CoA to caprolactam; or 6-aminocaproyl 6-aminocaproate semialdehyde from acetyl-CoA and 6-aminocaproate semialdehyde It can contain hexamethylenediamine from the hydride (Figure 11). The entity can also have a 6-aminocaproic acid pathway, and the microbial organism can Thiyl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-Oxo-6-aminohexanoate from hexanoyl-CoA; 3-Oxo-6-aminohexano 3-Hydroxy-6-aminohexanoate; 3-Hydroxy-6-aminohexanoate 6-aminohex-2-enoate to 6-aminohex-2-enoate; and 6-aminohex-2-enoate to 6-aminocaproate at least one polypeptide encoding a polypeptide that converts a substrate selected from proates to a product; Each contains one exogenous nucleic acid (Figure 11). Additional substrates and products of such pathways include: -aminocaproate to caprolactam or 6-aminocaproate to 6-aminocapro 6-aminocaproyl-CoA, 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde, and 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde. It can be derived from minocaproate semialdehyde and contains hexamethylenediamine (Figure 11). .

[0089] Additionally, non-naturally occurring microbial organisms can possess the 6-aminocaproic acid pathway. The microbial organisms convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxo-2-hydroxybenzoate. 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) 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-aminocaproic acid and at least one polypeptide encoding a polypeptide that converts a substrate selected from the group consisting of methyl, ... The non-naturally occurring microbial organism may alternatively contain a 6- The microbial organism can have an aminocaproic acid pathway, in which pyruvate and succinate are synthesized. 4-Hydroxy-2-oxoheptane-1,7-dioate from methylaldehyde; 4-Hydroxy-2-oxoheptane-1,7-dioate 2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2- Oxohept-4-ene-1,7-dioate (OHED) to 6-oxohex-4-enoate (6-OHE);6 -oxohex-4-enoate (6-OHE) to adipate semialdehyde; and adipate semialdehyde A polypeptide that converts a substrate selected from an aldehyde to a product. The non-naturally occurring peptide contains at least one exogenous nucleic acid encoding the non-naturally occurring peptide (Figure 12). The microbial organism can alternatively have a 6-aminocaproic acid pathway, and the microbial organism , pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-diol 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene 2-Oxohept-4-ene-1,7-dioate (OHED); 2-Oxohept-4-ene-1,7-dioate (OHED) to 2-amino 2-aminohept-4-ene-1,7-dioate (2-AHE); 2-aminohept-4-ene-1,7-dioate (2-AHE) 2-aminoheptane-1,7-dioate (2-AHD); and 2-aminoheptane-1,7-dioate (2-A A polypeptide that converts a substrate selected from 6-aminocaproate to a product. (Figure 12). The organism can alternatively have a 6-aminocaproic acid pathway, and the microbial organism can 4-Hydroxy-2-oxoheptane-1,7-dioate from bate and succinic semialdehyde 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 2-aminoheptane 2-aminoheptane-1,7-dioate (2-AHD); and 2-aminoheptane-1,7-dioate (2-AHD) to 6-amino A small number of polypeptides encoding polypeptides that convert a substrate selected from caproate to a product. Non-naturally occurring microbial organisms contain at least one exogenous nucleic acid (Figure 12). The microbial organism can have a 6-aminocaproic acid pathway, and the microbial organism can produce pyruvate and succinate. 4-Hydroxy-2-oxoheptane-1,7-dioate from acid semialdehyde; 4-Hydroxy-2- Oxoheptane-1,7-dioate (HODH) to 3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA 2,3-dehydroadipyl-CoA to 2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA to adipyl-CoA; Dipyr-CoA to adipate semialdehyde; and adipate semialdehyde to 6-amino A small number of polypeptides encoding polypeptides that convert a substrate selected from caproate to a product. Non-naturally occurring microbial organisms contain at least one exogenous nucleic acid (Figure 12). The microbial organism can have a 6-aminocaproic acid pathway, and the microbial organism can produce pyruvate and succinate. 4-Hydroxy-2-oxoheptane-1,7-dioate from acid semialdehyde; 4-Hydroxy-2- Oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED );2-oxohept-4-ene-1,7-(OHED) to 2,3-dehydroadipyl-CoA;2,3-dehydroadipyl-CoA Pyr-CoA to adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate A polymer that converts a substrate selected from semialdehyde to 6-aminocaproate into a product. The polypeptides contain at least one exogenous nucleic acid encoding a naturally occurring polypeptide (Figure 12). Microbial organisms that do not have a 6-aminocaproic acid pathway may alternatively have a microbial organism The body converts pyruvate and succinic semialdehyde into 4-hydroxy-2-oxoheptane-1,7- 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohepta- 4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 2-oxohept-4-ene-1,7-dioate 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to azide Adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipyl-CoA to adipate semialdehyde and 6-aminocaproate. It contains at least one exogenous nucleic acid that encodes the target gene (Figure 12).

[0090] Additionally, non-naturally occurring microbial organisms can possess the 6-aminocaproic acid pathway. Microbial organisms convert glutamic acid 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- 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimethan 6-aminopimeloyl-CoA to 2-aminopimelate; and 2-aminopimelate and 6-aminocaproate. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid that encodes the microbial protein (Figure 20). Alternatively, the microbial organism may have a 6-aminocaproic acid pathway, and the microbial organism may -CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimeloyl-CoA to 3-oxopimelate; Xopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; and and the conversion of a substrate selected from 2-aminopimelate to 6-aminocaproate to a product. The nucleic acid sequence of the present invention contains at least one exogenous nucleic acid encoding a polypeptide that performs the function of the present invention (Figure 21). The absent microbial organisms may alternatively have a 6-aminocaproic acid pathway, The product organism is homolysine to 6-aminohexanamide; and 6-aminohexanamide to 6 -aminocaproate to a product. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid that encodes a nucleotide sequence encoding the nucleotide sequence of interest (Figure 23). Alternatively, the microbial organism can have a 6-aminocaproic acid pathway, in which the microbial organism can convert adipate to from adipate semialdehyde; from adipate to adipylphosphate; and A polymer that converts a substrate selected from pyruvic acid to adipate semialdehyde into a product. The polypeptides contain at least one exogenous nucleic acid encoding the polypeptide (Figure 25).

[0091] Additionally, non-naturally occurring microbial organisms can possess the 6-aminocaproic acid pathway. The microbial organisms convert 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate. 2-Amino-7-oxoheptanoate to 6-aminohexanal; 6-Aminohexanal from 6-aminocaproate; from 2-amino-7-oxosubarate to 2-amino-7-oxohepta 2-Amino-7-oxoheptanoate to 6-aminohexanal; 2-Amino-7-oxoheptanoate to 6-aminohexanal Soheptanoate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate at least one polypeptide encoding a substrate selected from the group consisting of a ester and a methyl ester; The non-naturally occurring microbial organism further contains one exogenous nucleic acid (Figure 26). The microbial organism can have a glutamate-5-semicarbarate pathway, 2-Amino-5-hydroxy-7-oxosubarate from aldehydes; 2-Amino-5-hydroxy-7- Oxosubarate to 2-amino-5-ene-7-oxosubarate; and 2-amino-5-ene-7-oxosubarate Conversion of substrates selected from 2-amino-7-oxosubarate to products selected from 2-amino-7-oxosubarate The present invention further comprises at least one exogenous nucleic acid encoding a polypeptide that performs the steps of the method of the present invention (FIG. 27). In addition, non-naturally occurring microbial organisms have been shown to possess the hexamethylenediamine (HMDA) pathway. The microbial organisms can convert 6-aminocaproate to [(6-aminohexanoyl)oxy]hydroxybenzoate. [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) from 6-amino and 6-aminocaproic semialdehyde from hexamethyl and at least one polypeptide encoding a polypeptide that converts a substrate selected from benzodiamines to a product. Each of the non-naturally occurring microbial organisms contains one exogenous nucleic acid (Figure 13). The microbial organism can have a HMDA pathway, in which 6-aminocaproate is converted to [(6-aminocaproate)]. [(6-aminohexanoyl)oxy]phosphonate (6-AHOP); [(6-aminohexanoyl)oxy]phosphonate 6-Aminocaproyl-CoA from 6-aminocaproate (6-AHOP); 6-Aminocaproyl-CoA from 6-aminocaproate and 6-aminocaproic semialdehyde to hexamethylenediamine At least one polypeptide encoding a polypeptide that converts a substrate selected from the group consisting of The non-naturally occurring microbial organism may alternatively express exogenous nucleic acids via HMDA (Figure 13). The microbial organism can have a pathway for converting 6-aminocaproate to 6-aminocaproyl -CoA; 6-aminocaproyl-CoA to 6-aminocaproic semialdehyde; and 6-aminocaproic Conversion of selected substrates from ronic acid semialdehyde to hexamethylenediamine to products. The natural product contains at least one exogenous nucleic acid encoding a polypeptide that performs the steps of: Alternatively, a microbial organism not present in the present invention may have a HMDA pathway, and the microbial organism may , 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate from [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP); 6-Acetamidohexanal from 6-hexanoyloxyphosphonate (6-AAHOP); 6-Acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexane Polypeptides that convert substrates selected from amines to hexamethylenediamines into products. The non-naturally occurring microorganism contains at least one exogenous nucleic acid encoding a peptide (Figure 13). Alternatively, the microbial organism can have the HMDA pathway, and the microbial organism can have the 6-aminocaproic acid 6-Acetamidohexanoate from 6-acetamidohexanoate; 6-Acetamidohexanoate from 6-acetamidohexanoate 6-Acetamidohexanoyl-CoA; 6-Acetamidohexanoyl-CoA to 6-Acetamidohexanoyl-CoA nal; 6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanal Conversion of a substrate selected from hexamethylenediamine to a product. The nucleic acid sequence of the present invention contains at least one exogenous nucleic acid encoding a polypeptide that performs the function of the present invention (Figure 13). Alternatively, the microbial organism may have a HMDA pathway, and the microbial organism may 6-Aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP); 6-Acetamidohexanoyl-CoA; 6-A 6-Acetamidohexanal from cetoamidohexanoyl-CoA; 6-Acetamidohexanal 6-Acetamidohexanamine from hexane; and 6-Acetamidohexanamine from hexa Encoding a polypeptide that converts a substrate selected from methylenediamine to a product. Contains at least one exogenous nucleic acid (Figure 13).

[0092] Additionally, non-naturally occurring microbial organisms possess the hexamethylenediamine (HMDA) pathway. Glutamate can be converted to glutamyl-CoA; glutamyl-CoA can be converted to 3-amino-3-hydroxybenzoates; -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-cal 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-amino- 6-Aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; a group selected from: amino-7-oxoheptanoate to homolysine; and homolysine to HMDA at least one exogenous nucleic acid encoding a polypeptide that converts a protein to a product. (Figure 20). Non-naturally occurring microbial organisms may alternatively possess the HMDA pathway. Microbial organisms convert glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA A to 3-oxopimelate; 3-oxopimelate to 3-oxo-1-carboxyheptanal; 3-Oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoate; 3-oxo-7- Aminoheptanoate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to and homolysine to HMDA; It contains at least one exogenous nucleic acid encoding a peptide (Figure 21). Alternatively, microbial organisms may have the HMDA pathway, and the microbial organisms may have glutaryl -CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimeloyl-CoA to 3-oxopimelate; 5-Oxopimeloylphosphonate from oxopimelate; 5-Oxopimeloylphosphonate from 3-oxo-1-carboxyheptanal; from 3-oxo-1-carboxyheptanal 3-Oxo-7-aminoheptanoate; 3-Oxo-7-aminoheptanoate to 3,7-diaminoheptanoate 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA. At least one exogenous gene encoding a polypeptide that converts a selected substrate to a product. Non-naturally occurring microbial organisms alternatively possess the HMDA pathway (Figure 21). The microbial organism can convert glutaryl-CoA to 3-oxopimeloyl-CoA; meloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA; 5-Oxopimeloyl-CoA to 3-oxo-1-carboxyheptanal; 3-Oxo-7-aminoheptanoate from cyheptanal; 3-Oxo-7-aminoheptanoate 3,7-diaminoheptanoate to homolysine; 3,7-diaminoheptanoate to homolysine; and Encoding a polypeptide that converts a substrate selected from molysin to HMDA into a product Non-naturally occurring microbial organisms contain at least one exogenous nucleic acid (Figure 21). Alternatively, the microbial organism can have an HMDA pathway, which converts glutaryl-CoA to 3-oxopimethicone. 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3- Oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal to 3-amino-7- Oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA. At least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product. Non-naturally occurring microbial organisms may alternatively possess the HMDA pathway (Figure 21). Microbial organisms convert glutaryl-CoA to 3-oxopimeloyl-CoA; CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA; 5-oxo Pimeloyl-CoA to 3-oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal 3-Amino-7-oxoheptanoate from nal; 3-Amino-7-oxoheptanoate from 3,7 -diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine and at least one polypeptide encoding a polypeptide that converts a substrate selected from the group consisting of HMDA and HMDA to a product. The non-naturally occurring microbial organism may alternatively contain one or more exogenous nucleic acids (Figure 21). Pathway A, in which the microbial organism can convert glutaryl-CoA to 3-oxopimeloyl-CoA. A; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimelate Pimeloyl phosphonates; 5-oxopimeloyl phosphonates to 3-oxo-1-carboxyheptanes Tanal; 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate; 3- Amino-7-oxoheptanoate to 3,7-diaminoheptanoate; 3,7-diaminoheptano and homolysine to HMDA. The nucleic acid sequence of the present invention contains at least one exogenous nucleic acid encoding a polypeptide that performs the function of the present invention (Figure 21). Alternatively, the microbial organism may have a HMDA pathway, and the microbial organism may 3-Oxopimeloyl-CoA to 3-oxopimeloyl-CoA; 3-Oxopimeloyl-CoA to 3-oxopimele 3-Aminopimelate to 3-Aminopimelate; 3-Aminopimelate to 3-Amino-7- Oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7-oxo(axo) 2-amino-7-oxoheptanoate to homolysine; and homolysine to homolysine at least one molecule encoding a polypeptide that converts a substrate selected from the group consisting of HMDA and HMDA to a product; The non-naturally occurring microbial organism may alternatively contain one exogenous nucleic acid (Figure 21). The microbial organism can have a pathway for converting glutaryl-CoA to 3-oxopimeloyl-CoA. ;3-Oxopimeloyl-CoA to 3-oxopimelate;3-Oxopimelate to 3-aminopimelate 3-Aminopimelate to 5-Aminopimeloylphosphonate; 5-Aminopimeloylphosphonate Sulfonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to from 2-amino-7-oxo(axo)heptanoate; from 2-amino-7-oxo(axo)heptanoate A polypeptide that converts a substrate selected from homolysine; and homolysine to HMDA to a product. The non-naturally occurring peptide contains at least one exogenous nucleic acid encoding the non-naturally occurring peptide (Figure 21). The microbial organism can alternatively have an HMDA pathway, and the microbial organism can produce glutaryl-C oA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimeloyl-CoA to 3-oxopimelate Sopimelate to 5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7-oxo 3-Amino-7-oxoheptanoate to 2-amino-7-oxoheptanoate 2-amino-7-oxoheptanoate to homolysine; and homolysine to HM At least one encoding a polypeptide that converts a substrate selected from DA to a product. The non-naturally occurring microbial organism alternatively contains exogenous nucleic acids encoding the HMDA pathway (Figure 21). and the microbial organism can have glutaryl-CoA to 3-oxopimeloyl-CoA; Oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate 3-Aminopimelate to 3-amino-7-oxoheptanoate; 3-Aminopimelate to 3-amino-7-oxoheptanoate; 3,7-Diaminoheptanoate to 3,7-diaminoheptanoate; 3,7-Diaminoheptanoate to homodiaminoheptanoate and a polypeptide that converts a substrate selected from homolysine to HMDA into a product. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid encoding the The entity can alternatively have a HMDA pathway, in which the microbial organism converts glutaryl-CoA to 3- Oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimele 3-aminopimelate to 3-aminopimelate; 3-aminopimelate to 5-aminopimeloyl-CoA; 5-amino Pimeloyl-CoA to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate 3,7-diaminoheptanoate to homolysine; 3,7-diaminoheptanoate to homolysine; and Encoding a polypeptide that converts a substrate selected from molysin to HMDA into a product Non-naturally occurring microbial organisms contain at least one exogenous nucleic acid (Figure 21). Alternatively, the microbial organism can have an HMDA pathway, which converts glutaryl-CoA to 3-oxopimethicone. 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3- Aminopimelate; 3-aminopimelate to 5-aminopimeloylphosphonate; 5-aminopi Melloylphosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxohepta 3,7-diaminoheptanoate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; and a polypeptide that converts a substrate selected from homolysine and HMDA to a product. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid that encodes the microbial protein (Figure 21). Alternatively, a microbial organism may have a HMDA pathway, in which glutaryl-CoA is converted to 3-oxo-3-hydroxybenzoate. Sopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate from 3-aminopimelate; from 3-aminopimelate to 2-aminopimelate; from 2-aminopimelate from 2-amino-7-oxoheptanoate; from 2-amino-7-oxoheptanoate and a polypeptide that converts a substrate selected from homolysine to HMDA into a product. (Figure 21). The organism can alternatively have a HMDA pathway, in which the microbial organism converts glutaryl-CoA to 3-Oxopimeloyl-CoA; 3-Oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate 3-aminopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate 6-Aminopimeloylphosphonate from methylate; 6-Aminopimeloylphosphonate from 2- Amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine; and A polypeptide encoding a substrate selected from homolysine to HMDA to a product. The non-naturally occurring microbial organism contains at least one exogenous nucleic acid (Figure 21). Alternatively, the HMDA pathway can be mediated by the microbial organism converting glutaryl-CoA to 3-oxopyranoside. meloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to from 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoic acid Selected from: 2-amino-7-oxoheptanoate to homolysine; and homolysine to HMDA at least one exogenous gene encoding a polypeptide that converts the substrate to a product; Non-naturally occurring microbial organisms have alternatively been shown to possess the HMDA pathway. The microbial organisms can convert pyruvate and 4-aminobutanal to 2-oxo-4-hydroxybutanal. 2-Oxo-4-hydroxy 7-aminoheptanoate; 2-Oxo-4-hydroxy 7-aminoheptanoate to 2-oxo 2-oxo-7-aminohept-3-enoate; 2-oxo-7-aminohept-3-enoate to 2-oxo- 7-aminoheptanoate; 2-oxo-7-aminoheptanoate to homolysine; and homolysine A small number of genes encoding polypeptides that convert a substrate selected from HMDA to a product. Non-naturally occurring microbial organisms contain at least one exogenous nucleic acid (Figure 22). The HMDA pathway can be mediated by microbial organisms, which can convert pyruvate and 4-aminobutanal into 2-Oxo-4-hydroxy-7-aminoheptanoate; 2-Oxo-4-hydroxy-7-aminoheptanoate 2-Oxo-7-aminohept-3-enoate from 2-oxo-7-aminohept-3-enoate; 2-oxo-7-aminoheptanoate to 2-oxo-7-aminoheptanoate; 2-oxo-7-aminoheptanoate to 6 -aminohexanal; and 6-aminohexanal to HMDA (Figure 22) Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms The body converts 6-aminocaproate to 6-aminocaproic acid semialdehyde; and 6-aminocaproate Polypeptides that convert substrates selected from uronic acid semialdehyde to HMDA into products (Figure 24). The organism may alternatively have the HMDA pathway, and may be a microorganism Organisms convert 6-aminocaproate to 6-acetamidohexanoate; Xanoate to 6-acetamidohexanal; 6-acetamidohexanal to 6-acetamidohexanal 6-Acetamidohexanamine; 6-Acetamidohexanamine to HMDA as a substrate containing at least one exogenous nucleic acid encoding a polypeptide that converts the product (Figure 24). Non-naturally occurring microbial organisms can alternatively possess the HMDA pathway, The bioorganism converts 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate; 2- Amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HM DA; 2-amino-7-oxosubarate to 2-oxo-7-aminoheptanoate; 2-amino-7-oxo Homolysine from 2-oxoheptanoate; Homolysine from HMDA; 2-oxo-7-aminoheptanoate 2-oxo-7-aminoheptanoate to homolysine; 2-oxo-7-aminoheptanoate to 6-aminohexanal; 2-oxo-7-aminoheptanoate to 6-aminohexanal 2,7-diaminosubarate to 2,7-diaminosubarate; and 2,7-diaminosubarate to At least one polypeptide encoding a substrate selected from homolysine to a product Each of the non-naturally occurring microbial organisms contains one exogenous nucleic acid (Figure 26). The microbial organism can have an amino-7-oxosubarate pathway, and the microbial organism can have a glutamate-5- 2-Amino-5-hydroxy-7-oxosubarate from semialdehyde; 2-Amino-5-hydroxy -7-oxosubarate to 2-amino-5-ene-7-oxosubarate; and 2-amino-5-ene-7- Substrate to product conversion selected from oxosubarate to 2-amino-7-oxosubarate The recombinant vector contains at least one exogenous nucleic acid encoding a polypeptide that performs the recombinant function (Figure 27).

[0093] Additionally, non-naturally occurring microbial organisms can have a levulinic acid pathway, and The organism converts succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; pyridin-CoA to 3-oxoadipate; and 3-oxoadipate to levulinic acid. At least one exogenous nucleic acid encoding a polypeptide that converts the substrate to a product. Any of the pathways disclosed herein that produce a pathway intermediate contains an acid. can be used to produce intermediates for other pathways, if desired. For example, as disclosed herein, the alpha-ke The adipate to adipate pathway produces the intermediate adipyl-CoA, which is also shown in Figure 1. It is also an intermediate in the pathway depicted in Figure 10. Therefore, an alternative pathway is 10, which includes adipate to adipyl-CoA. It can be converted to 6-aminocaproate, caprolactam, or hexamethylenediamine. It is understood that any of the pathways disclosed herein that produce the desired intermediates can be used. may be combined with any other pathway disclosed herein as long as the desired product is produced. For example, the naturally occurring The microbial organisms that are absent express 2-AHD decarboxylase (Step I of Figure 12) and 6-acetamido dihexanoate kinase (Step E of Figure 13) or alternatively 2-oxohept-4-ene-1,7- adipate (OHED) decarboxylase (Step F in Figure 12), adipate semialdehyde methyltransferase (Step E of Figure 12), and 6-acetamidohexanoyl-CoA oxidase. 5-carboxy-2-pentenoyl-CoA reductase (Step J of Figure 13) or alternatively 5-carboxy-2-pentenoyl-CoA reductase tase (Step D of Figure 10), adipyl-CoA dehydrogenase (Step O of Figure 12), and 6-adipyl-CoA dehydrogenase (Step O of Figure 12). Aminocaproyl-CoA oxidoreductase (Step N of Figure 13) or alternatively 2-amino-7-ol Hexoheptanoate aminotransferase (Step G of Figure 20) and 3,7-diaminoheptanoate thanoate 2,3-aminomutase (Step R of Figure 21) or alternatively 6-aminocaproate inductase (Step O of Figure 24) and 6-aminohex-2-enoate reductase (Step O of Figure 11) 25) or alternatively adipate reductase (Step X of Figure 25) and 6-acetamide 6-aminocaproic acid pathway enzymes, such as xanoate reductase (Step P of Figure 24) at least one nucleic acid encoding a hexamethylenediamine pathway enzyme; It can have at least one nucleic acid.

[0094] In a further embodiment, the present invention provides a method for producing a hydroxybenzoate comprising the steps of: 6-aminocaproic acid, caprolactam, hexamethylenediamine; A non-naturally occurring microbial organism having a diamine or levulinic acid pathway, The non-naturally occurring microbial organisms are those disclosed herein or depicted in any of Figures 1-14 and 20-27. An enzyme that converts a substrate into a product selected from any of the substrates or products shown in The invention also includes at least one exogenous nucleic acid encoding a desired gene or protein. The present invention is suitable for producing a product, and adequate activity is available for the conversion of a substrate to a product. Any of the substrate-product pairs disclosed herein can be synthesized by one of skill in the art based on the teachings herein. Therefore, it will be understood that the present invention can be easily determined by the enzyme. Non-naturally occurring vectors containing at least one exogenous nucleic acid encoding a gene or protein. microbial organisms, wherein the enzyme or protein is one of those shown in Figures 1-14 and 20-27. Any of the following: 6-aminocaproic acid, caprolactam, hexamethylenediamine and a non-naturally occurring microbial organism that converts substrates and products of the levulinic acid pathway. do.

[0095] 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway Although generally described herein as a microbial organism containing 6-aminocaproic acid, the present invention also relates to microbial organisms containing 6-aminocaproic acid. Produces intermediates in the protic acid, caprolactam, hexamethylenediamine, or levulinic acid pathways 6-aminocaproic acid, caprolactam, hexamethylene a natural product containing at least one exogenous nucleic acid encoding a diamine or levulinic acid pathway enzyme; It is understood that non-naturally occurring microbial organisms are further provided. For example, As disclosed, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or The levulinic acid pathway is illustrated in Figures 1-14 and 20-27. 6-aminobenzoates that produce benzoylcholine, caprolactam, hexamethylenediamine, or levulinic acid Contains caproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathways In addition to the microbial organisms, the present invention further provides 6-aminocaproic acid, caprolactam, hexyl hydroxybenzoates, and the like. At least one exogenous nucleic acid encoding a methylendiamine or levulinic acid pathway enzyme A non-naturally occurring microbial organism comprising 6-aminocaproic acid, wherein the microbial organism , caprolactam, hexamethylenediamine, or levulinic acid pathway intermediates, e.g., Figure 1 A non-naturally occurring microbial organism that produces any of the intermediates shown in ~14 and 20~27. Offer your body.

[0096] Those described in the Examples and illustrated in the Figures, including pathways 1-14 and 20-27, Any of the routes disclosed herein, including those described above, may be used in combination with any other route, as desired. Utilizing non-naturally occurring microbial organisms to produce intermediates or products It is understood that the process can be carried out in a manner that produces intermediates, as disclosed herein. Microbial organisms interact with other microbial organisms that express downstream pathway enzymes to produce a desired product. However, 6-aminocaproic acid, caproic acid, Naturally occurring enzymes that produce lactams, hexamethylenediamine, or levulinic acid pathway intermediates Microbial organisms that do not exist can be utilized to produce intermediates as desired products. It is understood that:

[0097] The present invention relates generally to metabolic reactions, their reactants or products, or in particular to the above-mentioned alternatives. one or more nucleic acids encoding enzymes that are associated with or catalyze a catalytic reaction, reactant, or product Described herein with respect to acids or genes. Not expressly mentioned herein To the extent that the term "reaction" is used herein, those skilled in the art will understand that reference to a reaction also refers to the reactants and products of the reaction. Similarly, unless expressly stated otherwise in this specification, it will be understood that reactants or Reference to a product also refers to a reaction, and not to any of these metabolic components. Reference to also refers to genes encoding enzymes that catalyze the reactions, reactants, or products described above (e.g., Similarly, the term refers to the general knowledge of metabolic biochemistry, enzymology, and genomics. Given the known art, reference herein to a gene or encoding nucleic acid is References to the corresponding coding enzymes and the reactions they catalyze, as well as the reactants and products of the reactions, are also included. become.

[0098] The naturally occurring microbial organisms of the invention may produce one or more 6-aminocaproic acid, caprolactone, One or more enzymes involved in the biosynthetic pathway of tam, hexamethylenediamine, or levulinic acid are co-produced. For biosynthesis, the vector can be produced by introducing an expressible nucleic acid encoding the vector. Depending on the host microbial organism chosen, specific 6-aminocaproic acid, caprolactam, Nucleic acids for some or all of the hexamethylenediamine or levulinic acid biosynthetic pathways For example, the host of choice may contain one or more genes for the desired biosynthetic pathway. If an enzyme is deficient, expressible nucleic acids for the deficient enzyme(s) are subsequently exogenously added. Alternatively, the host of choice may be one that contains several pathway genes. Indicating endogenous expression of the gene, but lacking others, the coding nucleic acid is 6-aminocarboxylic acid. Achieve protic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis Therefore, the non-naturally occurring microbial organisms of the present invention require the deficient enzyme(s) for the Biosynthetic pathways can be achieved by introducing exogenous enzyme activities into the organism to obtain the desired biosynthetic pathway. The biosynthetic pathway, which can be generated or is desired, comprises, together with one or more endogenous enzymes, a 6-amino Caproic acid, caprolactam, hexamethylenediamine, or levulinic acid This can be achieved by introducing one or more exogenous enzymatic activities that produce the desired product. .

[0099] 6-aminocaproic acid, caprolactam, hexamethylene in selected host microbial organisms The non-naturally occurring microbial organisms of the invention depend on diamine or levulinic acid biosynthetic pathway components. The organism may produce at least one exogenously expressed 6-aminocaproic acid, caprolactam, Hexamethylenediamine, or levulinic acid pathway-encoding nucleic acid and one or more adipate, 6-amylidene phosphate-binding proteins. It includes any coding nucleic acid for the aminocaproic acid or caprolactam biosynthetic pathway. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or Levulinic acid biosynthesis can be mediated by deficient pathway enzymes through exogenous expression of the corresponding coding nucleic acids. It can be established in the host. 6-aminocaproic acid, caprolactam, hexamethion In hosts deficient in all enzymes of the diamine or levulinic acid pathway, It can involve exogenous expression of all enzymes, but it is important that the host contains at least one of the pathway enzymes. It is understood that all enzymes in a pathway can be expressed even if the enzyme has a specific function.

[0100] For example, exogenous expression of all enzymes in the pathway for the production of adipate is 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase Enzyme, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase / adipylase Glutamate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydride In particular, the host organism may contain a soluble ... Pathway enzymes: succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxybenzoate 3-hydroxyadipyl-CoA dehydrogenase, 5-carboxy-2 -pentenoyl-CoA reductase, and adipyl-CoA synthetase Alternatively, the host organism may express the adipate pathway enzyme succinyl-CoA:acetyl-CoA acyl Transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl -CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotransferase In addition, the host organism may contain adipyrase / adipate kinase. Adipate pathway enzyme, succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxybenzoate hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-calcium carboxy-2-pentenoyl-CoA reductase and adipyl-CoA:acetyl-CoA transferase Additionally, the host organism can contain the adipate pathway enzyme succinyltransferase. -CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA redox The enzyme may contain catabolites, coenzymes, and adipyl-CoA hydrolase.

[0101] In the case of 6-aminocaproic acid producing microbial organisms, the production of 6-aminocaproic acid Exogenous expression of all enzymes in the pathway for CoA-dependent aldehyde dehydrogenase Aldehyde dehydrogenase and transaminase or CoA-dependent aldehyde dehydrogenase and 6-amino Caproate dehydrogenase and the like can be contained in the host organism. For lactam-producing microbial organisms, the pathway for the production of caprolactam Exogenous expression of all enzymes involved in the synthesis of aldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase, transalcohol-dependent aldehyde dehydrogenase, and hydroxybenzoate Aminocaproate dehydrogenase or 6-aminocaproate dehydrogenase, and amidohydrolase In another example, the production of 6-aminocaproic acid (6-ACA) can be achieved by incorporating the 6-aminocaproic acid Exogenous expression of all enzymes in the pathway for biosynthesis is required: HODH aldolase; OHED hydratase 2-OHD decarboxylase; OHED reductase; 2-OHD decarboxylase; and adipate semialdehyde amine aminotransferase or adipate semialdehyde oxidoreductase (amino OHED decarboxylase; 6-OHED aldolase; 6-OHED hydratase; 6-OHED decarboxylase; ductase; and adipate semialdehyde aminotransferase or adipate OHE semialdehyde oxidoreductase (aminating) or alternatively HODH aldolase; D hydratase; OHED aminotransferase or OHED oxidoreductase (amination) 2-AHE reductase; and 2-AHD decarboxylase or alternatively HODH aldolase; OHE D hydratase; OHED reductase; 2-OHD aminotransferase or 2-OHD oxytransferase and 2-AHD decarboxylase or alternatively HODH aldolase HODH formate-lyase and pyruvate formate-lyase activating enzyme or HODH dehydrogenase 3-Hydroxyadipyl-CoA dehydratase; 2,3-Dehydroadipyl-CoA reductase Adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase adipate semialdehyde oxidoreductase or adipate semialdehyde oxidoreductase (aminating) or alternatively HO DH aldolase; OHED hydratase; OHED formate-lyase and pyruvate formate-lyase activation Enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase hydrogenase; and adipate semialdehyde aminotransferase or Dipate semialdehyde oxidoreductase (aminating) or alternatively HODH aldolase ;OHED hydratase;OHED reductase;2-OHD formate-lyase and pyruvate formate-lyase activities dehydrogenase or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase; and adipyl Adipate semialdehyde aminotransferase or adipate semialdehyde oxytransferase Further aspects of the present invention include the inclusion of enzymes such as thiazolinone, ... Similarly, all of the 6-ACA pathways described above produce succinic semialdehyde dehydrogenase. enzyme, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) In another example, a carboxykinase may be included to promote the production of 6-aminocaproic acid (6-ACA). Exogenous expression of all enzymes in the pathway for glutamyl-CoA transferase beta-ketothiolase; 3-oxo-6-aminopimeloyl-C oA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-aminopimeloyl-CoA 6-aminopimeloyl-CoA reductase; 7-carboxyhept-2-enoyl-CoA reductase and 2-aminopimelate decarboxylase, or glutaryl- CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl- CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate amino Transferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2-aminopimelate decarboxylase; and 2-aminopimelate decarboxylase. , can be contained in a host organism.

[0102] In another example, exogenous expression of all enzymes in the pathway for the production of hexamethylenediamine. Expression of 6-aminocaproate kinase; 6-AHOP oxidoreductase; and 6-aminocaproate 6-aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde aldehyde aminotransferase, or 6-aminocaproate kinase; 6-AHOP 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproyl-CoA 6-aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde Dehyde aminotransferase, or 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid Propanol semialdehyde aminotransferase, or 6-aminocaproate N-acetate 6-Acetamidohexanoate kinase; 6-AAHOP oxidoreductase 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Xanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetate methyltransferase or 6-acetamidohexanamine hydrolase (amide), or 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate C CoA transferase or 6-acetamidohexanoate CoA ligase; Xanoyl-CoA oxidoreductase; 6-acetamidohexanal aminotransferase 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanal oxidoreductase Amidohexanamine N-acetyltransferase or 6-acetamidohexanamine Hydrolase (amide), or 6-aminocaproate N-acetyltransferase; 6- Acetamide hexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamide hexanoate kinase Xanal aminotransferase or 6-acetamidohexanal oxidoreductase and 6-acetamidohexanamine N-acetyltransferase or 6 -Acetamidohexanamine hydrolase (amide), etc., in the host organism In another example, all of the pathways for the production of hexamethylenediamine can be Exogenous expression of enzymes such as glutamyl-CoA transferase or ligase; beta-ketothiol; 3-Oxo-6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA oxidoreductase Nopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase 6-Aminopimeloyl-CoA reductase (aldehyde forming); 2-amino-7-oxoheptase thanoate aminotransferase or aminating oxidoreductase; and homozygous Decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl 3-Oxopimelate reductase; 3-oxo-1-carboxyheptanal 7 -aminotransferase or 3-oxo-1-carboxyheptanal 7-amination oxide 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo 3,7-Diaminoheptanoate 3-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase, or glutaryl-CoA-based Taketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA tra 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloyl-CoA ligase; Hexopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 7-amino Transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7- Aminoheptanoate 3-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3 -aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-ketase thiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA trans ferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate CoA transferase 5-oxopimeloyl-CoA reductase (aldehyde) hydride formation); 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; 3-oxo-7-aminoheptanal Heptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amino 3,7-diaminoheptanoate 2,3-aminomutase; and homodimer Decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl 3-Oxopimelate reductase; 3-oxo-1-carboxyheptanal 3 -aminotransferase or 3-oxo-1-carboxyheptanal 3-amination oxide reductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino 3,7-Diaminoheptanoate 7-aminating oxidoreductase; 2,3-aminomutase; and homolysine decarboxylase, or glutaryl-CoA-based Taketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA tra 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloyl-CoA ligase; Hexopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 3-amino Transferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- Oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3 -aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-ketase thiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA trans ferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate CoA transferase 5-oxopimeloyl-CoA reductase (aldehyde) hydride formation), 5-oxopimeloyl-CoA hydrolase or 5-oxopimeloyl-CoA ligase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal 3-aminotransferase 3-Aminoheptanal 3-aminating oxidoreductase; 3-amino-7-oxoheptanoate 7- Aminotransferase or 3-amino-7-oxoheptanoate 7-amination oxidizer ductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrothiolase enzyme, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidase 3-aminopimelate reductase; 3-amino-7-oxoheptanoate 2 , 3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2 -amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decal carboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydroxylase dolorase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA Gauze; 3-oxopimelate aminotransferase or 3-oxopimelate amination Oxidoreductase; 3-aminopimelate kinase; 5-aminopimeloylphosphonate ductase; 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate Heptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminotransferase and homolysine decarboxylase, or glutaryl-CoA vector 3-Oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA thiolase transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase 3-aminopimelate aminating oxidoreductase or 3-oxopimelate aminating oxidoreductase; 5-aminopimeloyl-CoA transferase or 3-aminopimelate CoA ligase; A reductase (aldehyde formation);3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate 7-aminotransferase butanoate aminating oxidoreductase; and homolysine decarboxylase, or is glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductor 3-Aminopimelate reductase; 3-amino-7-oxoheptanoate 7-aminotransferase sferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase;3, 7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase, or or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; Pimelate aminotransferase or 3-oxopimelate aminating oxidoreductase 3-aminopimelate CoA transferase or 3-aminopimelate CoA ligase; 5- Aminopimeloyl-CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoate 7-Aminotransferase or 3-amino-7-oxoheptanoate aminating oxidore ductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrothiolase enzyme, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidase 5-Aminopimeloylphosphonate reductase; 3-aminopimelate kinase; 5-aminopimeloylphosphonate reductase 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- Oxoheptanoate aminating oxidoreductase; 3,7-diaminoheptanoate 2,3- aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-keto Thiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase 3-oxopimeloyl-CoA ligase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase 3-Aminopimelate 2,3- Aminomutase; 2-aminopimelate reductase; 2-amino-7-oxoheptanoate 7- Aminotransferase or 2-amino-7-oxoheptanoate aminating oxidizer and homolysine decarboxylase, or glutaryl-CoA beta-ketothiolase 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-amino Mutase; 2-aminopimelate kinase; 6-aminopimeloylphosphonate reductase; 2 -amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate butanoate aminating oxidoreductase; and homolysine decarboxylase, or is glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductor 2,3-aminomutase; 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; 6-aminopimeloyl-CoA ligase ductase (aldehyde formation); 2-amino-7-oxoheptanoate 7-aminotransferase 2-amino-7-oxoheptanoate aminating oxidoreductase; and homodimer Decarboxylase, or 2-oxo-4-hydroxy-7-aminoheptanoate aldolase 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminoheptanoate dehydratase 2-oxo-7-aminoheptanoate aminotransferase ferase or 2-oxo-7-aminoheptanoate aminating oxidoreductase; and Morizin decarboxylase, or 6-aminocaproate reductase; and 6-amino Caproic semialdehyde aminotransferase or 6-aminocaproic semialdehyde Hydroxidoreductase (amination), or 6-aminocaproate N-acetyltransferase 6-Acetamidohexanoate reductase; 6-Acetamidohexanal reductase aminotransferase or 6-acetamidohexanal oxidoreductase (amino and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine can be contained in the host organism, such as hexaneamine hydrolase (amide) .

[0103] 6-aminocaproic acid, caprolactam, hexamethylene in selected host microbial organisms The non-naturally occurring microbial organisms of the invention depend on diamine or levulinic acid biosynthetic pathway components. The organism may produce at least one exogenously expressed 6-aminocaproic acid, caprolactam, Hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and one or more 6-aminocaproic acid Acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathways For example, 6-aminocaproic acid, caprolactam, hexyl 2-(4-aminomethyl-2-benzo[a]pyrimidine)-4-hydroxybenzo[b]pyrimidine ... and can be established in hosts deficient in pathway enzymes or proteins. Any enzyme in the caproate, caprolactam, hexamethylenediamine, or levulinic acid pathway In a host deficient in an enzyme or protein, the addition of all enzymes or proteins in the pathway The expression of the pathway enzyme or protein may be inducible, but the host may contain at least one of the pathway enzymes or proteins. It is understood that all enzymes or proteins in a pathway can be expressed even if the vector has a specific structure. For example, as disclosed herein, 6-aminocaproic acid, caprolactam, All enzymes or enzymes in the pathway for the production of hexamethylenediamine or levulinic acid The present invention can involve exogenous expression of a protein.

[0104] Given the teachings and guidance provided herein, one of ordinary skill in the art will be able to derive an expressible form of The number of encoding nucleic acids to be introduced should be at least as large as the adipate concentration of the selected host microbial organism. , 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway It will be understood that the naturally occurring amino acid sequence of the present invention may be equivalent to a deletion. Microbial organisms that do not produce 6-aminocaproic acid, caprolactam, and hexamethylenediamine or at least 1, 2, 3, 4 or 5 encoding the above enzymes constituting the levulinic acid biosynthetic pathway , 5, 6, 7, 8, 9, 10, 11, or 12 of any of the nucleic acids. In embodiments, the non-naturally occurring microbial organism also produces 6-aminocaproic acid, caprolactone, or to promote or optimize the biosynthesis of tam, hexamethylenediamine, or levulinic acid. The host microbial organism may contain other genetic modifications that confer other useful functions to the host microbial organism. One such functionality is, for example, succinyl-CoA and / or ATP in the case of adipate synthesis. is acetyl-CoA or 6-aminocaproate, including the adipate pathway enzymes disclosed herein Adipyl-CoA or adipate or 6-aminobenzoate in the synthesis of carboxylic acid or caprolactam Pyruvate and succinic semialdehyde in the case of caproate synthesis, glutamic acid, glutamic acid Thallyl-CoA, homolysine, or 2-amino-7-oxosubarate or hexamethylenediamine 6-aminocaproate, glutamic acid, glutaryl-CoA, pyruvate for amine synthesis and 6-aminobutanal, such as 4-aminobutanal or 2-amino-7-oxosubarate. of the nocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway precursors It may involve one or more synthetic increases.

[0105] Generally, the host microbial organism is selected from those that are capable of producing the desired precursor or precursors de novo. As a naturally produced molecule that provides increased production of precursors naturally produced by the body 6-aminocaproic acid, caprolactam, hexamethosinate, or as a genetically engineered product The strains are selected to produce precursors of the diamine or levulinic acid pathway. As disclosed in, the host organism can be genetically engineered to increase production of the precursor. Furthermore, microbial organisms genetically engineered to produce desired precursors can be used to It can be used as the main product, 6-aminocaproic acid, caprolactam, hexamethylene Further genetically engineered to express enzymes or proteins of the benzodiamine or levulinic acid pathways. It can be made.

[0106] In some embodiments, the non-naturally occurring microbial organisms of the invention comprise 6-aminocaproic acid. Enzymes for synthesizing carboxylic acid, caprolactam, hexamethylenediamine, or levulinic acid In this particular embodiment, the host cell is produced from a host cell containing the ability to lectins, caprolactam, hexamethylenediamine, or levulinic acid pathway reactions to 6-amino For the production of caproic acid, caprolactam, hexamethylenediamine, or levulinic acid To drive it, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or It may be useful to increase the synthesis or accumulation of levulinic acid pathway products. The increase in the product can be achieved by, for example, adding one or more of the above 6-aminocaproic acid, caprolactam, hexamethylenediamine, This can be achieved by overexpression of nucleic acids encoding diamine or levulinic acid pathway enzymes. 6-aminocaproic acid, caprolactam, hexamethylenediamine, or lev Overexpression of phosphate pathway enzyme(s) can be achieved by, for example, overexpressing the endogenous gene(s). It can arise through the exogenous expression of a heterologous gene or genes. Organisms that produce, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 encoding levulinic acid biosynthetic pathway enzymes , 11, 12, 13, 14, i.e., through overexpression of any nucleic acid, 6-aminocaproic acid The naturally occurring bacteria of the present invention that produce hydroxybenzoates, caprolactam, hexamethylenediamine, or levulinic acid They can be easily produced to be non-existent microbial organisms. Organisms that do not produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or Mutagenesis of endogenous genes leading to increased activity of enzymes in the buric acid biosynthetic pathway Therefore, it can be generated.

[0107] In particularly useful embodiments, exogenous expression of the encoding nucleic acid is utilized. Expression and / or regulatory elements can be added to achieve desired expression levels controlled by the user. This provides the ability to tailor the structure to the host and application. While endogenous expression also allows for the removal of negative regulatory effectors or inducible promoters, by induction of the promoter of a gene when bound to a target or other regulatory element. Therefore, naturally occurring derivatives can be utilized in other embodiments, such as Endogenous genes with exogenous promoters can be up-regulated by providing the appropriate inducer. The regulatory region of an endogenous gene can be regulated by an inducible regulatory element. can be genetically engineered to incorporate, when desired, the endogenous gene Similarly, inducible promoters allow for the regulation of increased expression. The gene can be included as a regulatory element for an exogenous gene introduced into an organism. do.

[0108] The present invention relates to the use of one or more genetically modified organisms, such as gene disruptions, as disclosed in Example XXX and Tables 14-16. Further provided is a non-naturally occurring microbial organism comprising a protease inhibitor, wherein the organism is capable of producing 6-ACA, adipocytes, and the like. The gene disruption produces adipate, 6-ACA, and / or HMDA. Gene disruption reduces the activity of an enzyme, conferring increased production to a non-naturally occurring organism. When disrupted, the disruption couples the production of adipate, 6-ACA, and / or HMDA to the growth of the organism. Therefore, the present invention provides a method for the treatment of a malaria parasite by disrupting one or more genes. wherein the one or more gene disruptions are One or more gene disruptions occur in genes encoding the enzymes, resulting in the production of adipate, 6-A, and 6-C. The present invention provides a non-naturally occurring microbial organism that confers increased CA and / or HMDA production. As disclosed herein, such organisms are exemplified in Example XXX and Tables 14-16. In addition to gene disruptions such as those that disrupt the ATP-dependent ATPase pathway, It contains a pathway for

[0109] In the methods of the invention, any of the one or more exogenous nucleic acids may be expressed in a non-naturally occurring microbial It is understood that the microbial organism can be introduced into a microbial organism to produce a biologically active agent. Nucleic acids may be prepared from, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or A levulinic acid biosynthetic pathway can be introduced into the microbial organism. The encoding nucleic acid may be 6-aminocaproic acid, caprolactam, hexamethylenediamine, or Biosynthesis is required to catalyze some of the reactions required to confer urinary acid biosynthesis capability. For example, the 6-amino acid sequence may be introduced to produce an intermediate microbial organism having the ability to synthesize the 6-amino acid sequence. The biosynthetic pathways for caproic acid, caprolactam, hexamethylenediamine, or levulinic acid The non-naturally occurring microbial organism comprises at least two exogenous genes encoding desired enzymes. In the case of adipate production, at least two exogenous nucleic acids can be included. The acid is reacted with succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyacyl- CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyltransferase, and 3-hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoA and 5-carbo hydroxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA and adipyl-CoA It can encode enzymes such as combinations of synthetases, and the like. In the case of caprolactam production, at least two exogenous nucleic acids are involved in the CoA-dependent alcohol synthesis. Dehyde dehydrogenase and transaminase or CoA-dependent aldehyde dehydrogenase Combinations of enzymes and amidohydrolases or transaminases and amidohydrolases In the case of 6-aminocaproic acid production, at least At least two exogenous nucleic acids are 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) amide. hydratase and 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, or 2-oxohept-4-ene-1,7-dioate (OHED) hydratase OHED hydratase and 2-aminoheptane-1,7-dioate (OHED) hydratase 2-Adipic acid decarboxylase, 3-hydroxyadipyl-CoA dehydratase, and adipyl-CoA decarboxylase Glutamyl-CoA dehydrogenase, glutamyl-CoA transferase and 6-aminopimeloyl-C oA hydrolase, or glutaryl-CoA beta-ketothiolase and 3-aminopimelate 2,3 It is possible to encode enzymes such as the combination of hexamethylene In the case of diamine production, at least two exogenous nucleic acids are 6-aminocaproate quinone. and [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or 6-acetamidohexanoate kinase and [(6-acetamidohexanoyl)oxy] Phosphonate (6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase sferase and 6-acetamidohexanoyl-CoA oxidoreductase, 3-hydroxy 6-Aminopimeloyl-CoA dehydratase and 2-amino-7-oxoheptanoate amino transferase, or 3-oxopimeloyl-CoA ligase and homolysine decarboxylase Thus, two of the biosynthetic pathways can be encoded using enzymes such as a combination of enzymes. Any combination of one or more enzymes can be included in a non-naturally occurring microbial organism of the invention. It is understood that this can be done.

[0110] Similarly, as long as the combination of enzymes of a desired biosynthetic pathway results in the production of the corresponding desired product. , any combination of three or more enzymes of a biosynthetic pathway, e.g., in the case of adipate production, the enzymes The enzyme succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl- CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase; or succinyl-C oA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase; or succinyl-CoA:acetyl-CoA Acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-Co A dehydratase and adipyl-CoA:acetyl-CoA transferase, as well as other enzymes Combinations can be included in the non-naturally occurring microbial organisms of the invention, as desired. In the case of 6-aminocaproic acid production, it is understood that at least three exogenous nuclei The acid is 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, 2-oxoheptane-1,7-dioate 4-Butane-1,7-dioate (OHED) hydratase and 2-oxoheptane-1,7-dioate ( 2-OHD) decarboxylase, or 2-oxohept-4-ene-1,7-dioate (OHED) hydratase 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase and 2-aminohepta 2-AHD decarboxylase, or 3-hydroxyadipyl-CoA dehydratase tase, 2,3-dehydroadipyl-CoA reductase and adipyl-CoA dehydrogenase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA Hydrolase and 2-aminopimelate decarboxylase, or glutaryl-CoA beta- Ketothiolase, 3-aminating oxidoreductase and 2-aminopimelate decarboxylase enzyme, or 3-oxoadipyl-CoA thiolase, 5-carboxy-2-pentenoate reductase The present invention can encode enzymes such as a combination of taurate and adipate reductase. In the case of hexamethylenediamine production, at least three exogenous nucleic acids are 6-amino Caproate kinase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxide reductase and 6-aminocaproic acid semialdehyde aminotransferase, or 6- Aminocaproate N-acetyltransferase, 6-acetamidohexanoate quinone and [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase 6-aminocaproate N-acetyltransferase, or 6-aminocaproate N-acetyltransferase, xanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and 6-acetamido hexanoyl-CoA oxidoreductase, or 3-oxo-6-aminopimeloyl-CoA oxidoreductase thiocyanate reductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase and homolyzed Decarboxylase, or 2-oxo-4-hydroxy-7-aminoheptanoate aldolase 2-oxo-7-aminohept-3-enoate reductase and homolysine decarboxylase 6-acetamidohexanoate reductase, or 6-acetamidohexanal reductase amine transferase and 6-acetamidohexanamine N-acetyltransferase Similarly, enzymes of a desired biosynthetic pathway can be encoded. As long as the combination of elements results in the production of the corresponding desired product, the biosynthetic Any combination of four or more enzymes of the pathway can be used in the non-naturally occurring methods of the present invention, as desired. It can be contained in a microbial organism.

[0111] 6-aminocaproic acid, caprolactam, hexamethylenediamine as described herein In addition to the biosynthesis of levulinic acid, the non-naturally occurring microbial organisms and methods of the invention They also have been used in conjunction with each other and with other methods in the art to achieve product biosynthesis by other routes. It can be used in a wide variety of combinations with other microbial organisms and methods well known in the art. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or 6-aminocaproic acid, caprolactam, hexamethosinate, other than the use of levulinic acid-producing strains. One alternative to producing diamine or levulinic acid is adipate, 6-aminocarboxylic acid. Caproic acid or caprolactam pathway intermediates are converted to 6-aminocaproic acid, caprolactam, Addition of other microbial organisms capable of converting hexamethylenediamine or levulinic acid One such procedure is via, for example, the synthesis of 6-aminocaproic acid, caprolactam, of microbial organisms producing methicone, hexamethylenediamine, or levulinic acid pathway intermediates. Fermentation involves the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, Levulinic acid pathway intermediates include 6-aminocaproic acid, caprolactam, and hexamethylenediamine. Amine or levulinic acid pathway intermediates, such as 6-aminocaproic acid, caprolactam, and hexametazolinone. Use as a substrate for a second microbial organism that converts it to ethylenediamine or levulinic acid 6-aminocaproic acid, caprolactam, hexamethylenediamine, or The levulinic acid pathway intermediates can be added directly to another culture of a second organism or 6- Aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediate The original culture of the microbial producing strain can be purified by removing these microbial organisms, for example, by cell separation. and then subsequent addition of a second organism to the fermentation broth followed by an intermediate purification step. can be utilized to produce the final product without further modification.

[0112] In other embodiments, the non-naturally occurring microbial organisms and methods of the invention provide, for example, 6-aminobutyric acid. Biosynthesis of aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid To achieve this, various subroutines can be assembled. In such cases, the biosynthetic pathways for the desired products of the invention can be isolated in different microbial organisms. Different microbial organisms can be co-cultured to produce an end product. In such a biosynthetic scheme, the product of the first microbial organism is synthesized until the final product is synthesized. , which serves as a substrate for a second microbial organism. For example, 6-aminocaproic acid, caprolactam The biosynthesis of benzophenone, hexamethylenediamine, or levulinic acid is dependent on other pathway intermediates or products. By constructing a microbial organism containing a biosynthetic pathway for the conversion of a pathway intermediate. Alternatively, 6-aminocaproic acid, caprolactam, hexamethoxazole, Diamine or levulinic acid may also be used in co-cultures or co-cultures using two organisms in the same vessel. The first microbial organism can be biosynthetically produced through fermentation, , 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid intermediate The second microbial organism converts the intermediate into 6-aminocaproic acid, caprolactam, Convert to hexamethylenediamine or levulinic acid.

[0113] Given the teachings and guidance provided herein, one of skill in the art would be able to identify 6-aminocaproic acid , caprolactam, hexamethylenediamine, or levulinic acid. Co-cultures of microbial organisms, other non-naturally occurring microbial organisms with sub-pathways, and In addition to a combination of other chemical and / or biochemical procedures well known in the art. A wide variety of combinations and permutations are possible with the non-naturally occurring microbial organisms and methods of the invention. You will understand that it exists.

[0114] Similarly, the host organism produces 6-aminocaproic acid, caprolactam, and hexamethylenediamine. or for the introduction of one or more gene disruptions desired to increase production of levulinic acid. It will be understood by those skilled in the art that selection can be made based on characteristics. When a genetic modification is to be introduced into a host organism to disrupt a gene, Any homolog, ortholog, or paralog that catalyzes a similar, but not identical, metabolic reaction The enzymes can be similarly disrupted to ensure that the desired metabolic reactions are sufficiently disrupted. It is understood that certain differences exist in the metabolic networks between different organisms. Those skilled in the art will recognize that the actual gene that is disrupted in a given organism may vary between organisms. However, given the teachings and guidance provided herein, Taking into consideration, those skilled in the art can also necessary to construct organisms of the target species that would increase amine or levulinic acid biosynthesis. The method of the present invention can be applied to any suitable host organism to identify the relevant homologous metabolic perturbations. It will be understood that the present invention can be applied to any of the above-mentioned methods. In certain embodiments, the increased production is , 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid The synthesis can be coupled to the growth of an organism, and, if desired, 6-amino Organisms that produce caproic acid, caprolactam, hexamethylenediamine, or levulinic acid This can inevitably lead to growth in the

[0115] 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway The source of encoding nucleic acid for the enzyme can be, for example, a gene whose encoding gene product performs the reaction described above. The present invention includes any species capable of catalyzing the catalysis of bacteria, such as archaea and eubacteria. Eukaryotic organisms, including bacteria, yeast, plants, insects, animals, and mammals, including humans Such sources include both prokaryotes and eukaryotes, including, but not limited to, Exemplary species for sources are, for example, Escherichia coli, E. coli strain K12, E. coli, Bacterium C, Escherichia coli W, Pseudomonas species, Pseudomonas knackmu ssii), Pseudomonas species strain B13, Pseudomonas putida, Pseudomonas Pseudomonas fluorescens, Pseudomonas stuze Pseudomonas stutzeri, Pseudomonas mendocina, Rhodopseudomonas palustris, Mycobacterium tuberculosis tuberculosis, Vibrio cholera, Helicobacter pylori, Klebsiella pneumoniae Klebsiella pneumoniae, Serratia proteamaculans s), Streptomyces species 2065, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1, Lars Ralstonia eutropha, Ralstonia eutropha H16, Cross Clostridium acetobutylicum, Euglena gracilis (Euglena gracilis), Treponema denticola, Clostridium Clostridium kluyveri, Homo sapiens, rat (Rattus norvegicus), Acinetobacter species ADP1, Acinetobacter species strain M-1, Strep Streptomyces coelicolor, Eubacterium barkeri erium barkeri, Peptostreptococcus asaccharolyticus asaccharolyticus), Clostridium botulinum, Clostridium botulinum strain A3, Cross Clostridium tyrobutyricum, Clostridium pastoris Clostridium pasteurianum, Clostridium thermoaceticum Moorella thermoaceticum (Moorella thermoaceticum), Moorella Moorella thermoacetica, Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), mouse (Mus musculus), wild boar (Sus scrofa), Arthrobacter species, Arthrobacter aurescens, Penicillium species Penicillium chrysogenum, Aspergillus niger r), Aspergillus nidulans, Bacillus subtilis ), Saccharomyces cerevisiae, Zymomonas mobilis , Mannheimia succiniciproducens, Clostridium Clostridium ljungdahlii, Clostridium carboxy Clostridium carboxidivorans, Geobacillus stearothermophilus ( Geobacillus stearothermophilus, Agrobacterium tumefaciens ium tumefaciens), Achromobacter denitrificans ns), Arabidopsis thaliana, Haemophilus influenzae ae), Acidaminococcus fermentans, Crost Rhizobium species M62 / 1, Fusobacterium nucleatum, cattle (B os taurus), Zoogloea ramigera, Rhodobacter sphaeroides Rhodobacter sphaeroides, Clostridium beijerinckii inckii), Metallosphaera sedula, Thermoanaerobacter - species, Thermoanaerobacter brockii, Acinetoba Acinetobacter baylyi, Porphyromonas gingivalis onas gingivalis, Leuconostoc mesenteroides , Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sulfolobus Sulfolobus solfataricus, Sulfolobus solfataricus Cass, Sulfolobus acidocaldarius, Murine typhus Salmonella typhimurium, Salmonella enterica, Thermotoga mali Thermotoga maritima, Halobacterium salinaru m), Bacillus cereus, Clostridium difficile icile), Alkaliphilus metalliredigens, Thermo Thermoanaerobacter tengcongensis, Saccharomyces cerevisiae Saccharomyces kluyveri, Helicobacter pylori, Corynebacterium glutamicum, Clostridium spp. Clostridium saccharoperbutylacetonicum, Pseudomonas Pseudomonas chlororaphis, Streptomyces clubligera Streptomyces clavuligerus, Campylobacter jejuni ), Thermus thermophilus, Pelotomaculum thermopropionicum maculum thermopropionicum), Bacteroides capillosus, Anaerotruncus colihominis, Natlanaerobius Natranaerobius thermophilius, Archaeglobus fulgidus haeoglobus fulgidus), Archaeoglobus fulgidus DSM 4304, Haloarcula mali Haloarcula marismortui, Pyrobaculum aerophyllum erophilum), Pyrobaculum aerophilum strain IM2, tobacco (Nicotiana tabacum), paper - Mint (Menthe piperita), Loblolly pine (Pinus taeda), Barley (Hordeum vulgare), Zea mays, Rhodococcus opacus, Capriavida Cupriavidus necator, Bradyrhizobium japonicum Bradyrhizobium japonicum USDA110, Ascarius suum ), butyrate-producing bacteria L2-50, Bacillus megaterium, Methanococcus mali Methanococcus maripaludis, Methanosarcina mazei azei), Methanosarcina mazei, Methanosarcina bark Methanocarcina barkeri, Methanocaldococcus januschii jannaschii), Caenorhabditis elegans, Leishmania sylvatica major), Methylomicrobium alcaliphilum 20 Z, Chromohalobacter salexigens, Archaeogro Archaeglobus fulgidus, Chlamydomonas reinhardti i), Trichomonas vaginalis G3, Trypanosoma brucei a brucei, Mycoplana ramosa, Micrococcus luteus ococcus luteus, Acetobacter pasteurians, Kluy Kluyveromyces lactis, Mesorhizobium loti um loti), Lactococcus lactis, Lysinibacillus sphaeri Lysinibacillus sphaericus, Candida boidinii, Candida Candida albicans SC5314, Burkholderia ambifaria olderia ambifaria)AMMD, Ascaris suum, Acinetobacter baumannii nii), Acinetobacter calcoaceticus, Burkholderia phymatum (Burkhol deria phymatum), Candida albicans, Clostridium subterminare (Clostr idium subterminale), Cupriavidus taiwanensis, Flavobacterium lutescens, Lacancea kluyveri (Lachancea kluyveri), Lactobacillus species 30a, Leptospira interrogans (Leptos pira interrogans), Moorella thermoacetica, Myxococcus xanthus xanthus), Nicotiana glutinosa, Nocardia ioensis Nocardia iowensis (species NRRL 5646), Pseudomonas rei nekei) MT1, Ralstonia eutropha JMP134, Ralstonia metallidurans (Rals tonia metallidurans, Rhodococcus jostii, and fission yeast (S chizosaccharomyces pombe, Selenomonas ruminantium, Streptomyces clavuligerus, Syntrophus Syntrophus aciditrophicus, Vibrio parahaemolyticus ticus, Vibrio vulnificus, and the bacteria disclosed herein or other exemplary species available as source organisms for the corresponding genes (see Examples). However, the complete genome sequences available to date include 395 Over 550 species, including microbial genomes and various yeast, fungal, plant, and mammalian genomes (More than half of these are available in public databases such as NCBI), e.g. Near-missing genes, including homologs, orthologs, paralogs, and nonorthologous gene displacements of known genes Essential 6-aminocaproic acid, caproic acid, for one or more genes in related or distantly related species Identification of genes encoding lactam, hexamethylenediamine, or levulinic acid biosynthetic activity The identification of, and exchange of genetic modifications between, organisms is routine and well known in the art. Thus, the methods described herein with respect to specific organisms such as E. coli are Production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid The metabolic changes that allow for synthesis are similarly accessible to other microorganisms, including prokaryotes and eukaryotes. Those skilled in the art will be able to readily apply the teachings and guidance provided herein. One can see that metabolic changes exemplified in one organism can be equally applied to other organisms. You will understand that.

[0116] 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis In some cases, such as when the synthesis pathway exists in unrelated species, 6-amino Nocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis are e.g. For example, unrelated enzymes that catalyze similar, but not identical, metabolic reactions to replace those mentioned above. The host species can be conferred by exogenous expression of paralog(s) from the species. Since certain differences in metabolic networks exist between different organisms, those skilled in the art will However, it will be understood that the actual gene usage among different organisms may differ. However, given the teachings and guidance provided herein, one skilled in the art will also be able to carboxylic acid, caprolactam, hexamethylenediamine, or levulinic acid. To construct a microbial organism of interest, one may use a combination of the methods exemplified herein. Using the same metabolic variations, the teachings and methods of the present invention can be applied to all microbial organisms. You will understand that you can do this.

[0117] The host microbial organism may be, for example, a bacterium, yeast, fungus, or any of a variety of organisms applicable to fermentation processes. The non-naturally occurring microbial organism can be selected from any of a variety of microorganisms, including, but not limited to, any of the following: Exemplary bacteria include Escherichia coli, Klebsiella oxytoca, and Anaerobic Bacteria. Anaerobiospirillum succiniciproducens ), Actinobacillus succinogenes, Mannheimia succiniciproducens, In Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter Gluconobacter oxydans, Zymomonas mobilis, Lactococcus Lactobacillus lactis, Lactobacillus plantarum, Strep Tomyces coelicolor, Clostridium acetobutylicum, Pseudomonas flu Exemplary yeasts include those selected from the group consisting of Saccharomyces cerevisiae, ... Fungi include budding yeast, fission yeast, Kluyveromyces lactis, Kluyveromyces maltophilus, and Kluyveromyces marxianus, Aspergillus terreus eus), Aspergillus niger, Pichia pastoris, Rhizopus arizus (Rh Rhizopus arrhizus, Rhizopus oryzae, and other similar species For example, E. coli is a well-characterized microorganism suitable for genetic engineering. Another particularly useful host organism is Saccharomyces cerevisiae. and the like. Metabolic and / or genetic modifications can be introduced to produce the desired product. It will be appreciated that any suitable microbial host organism can be used for the production of the cells of the present invention.

[0118] Not naturally occurring, 6-aminocaproic acid, caprolactam, hexamethylenediamine, Alternatively, a method for constructing a host that produces levulinic acid and testing its expression level may include: For example, by recombination and detection methods well known in the art. Such methods are described, for example, in Sambrook et al., Molecular Cloning: Laboratory Methods. Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory Press, 1999. Spring Harbor Laboratory, New York (2001); and Ausubel et al., In Molecular Biology Current Protocols in Molecular Biology, John Wiley and Sons s, Baltimore, MD (1999).

[0119] Production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid The exogenous nucleic acid sequences involved in the pathway for viability can be expressed by conjugation, electroporation, or other methods. These include, but are not limited to, transformation, chemical transformation, transduction, transfection, and ultrasonic transformation. The host cells can be transformed using techniques well known in the art, including but not limited to: The exogenous vector can be stably or transiently introduced into E. coli or other prokaryotic cells. For sexual expression, some nucleic acid sequences in eukaryotic nucleic acid genes or cDNAs are If desired, an N-terminal mitochondrial domain can be removed prior to transformation into a prokaryotic host cell. It can encode a targeting signal, such as a targeting signal or other targeting signal. For example, removal of the mitochondrial leader sequence led to increased expression in E. coli (H Offmeister et al., J. Biol. Chem. 280:4329-4338 (2005). For endogenous expression, the gene is expressed in the cytosol without the addition of a leader sequence. can be directed to mitochondria or other organelles or The nucleic acid sequence may include a suitable signal, such as a mitochondrial targeting signal or secretion signal, that is suitable for the host cell. The target sequence can be removed to direct the vector for secretion. Appropriate modifications to nucleic acid sequences, to remove or include exogenous factors, can be made to confer desired properties. It is understood that genes can be incorporated into the nucleic acid sequence of interest. To achieve optimal protein expression, techniques well known in the art are used. This can be used for codon optimization.

[0120] The expression vector(s) are operably linked to expression control sequences functional in the host organism. , one or more of 6-aminocaproic acid, caprolactam, hexamethasone, as exemplified herein Constructed to include nucleic acids encoding ethylenediamine or levulinic acid biosynthetic pathways. Expression vectors applicable for use in the microbial host organisms of the present invention include, for example, , a vector operable for stable integration into a host chromosome and a selection sequence or marker Plasmids, phage vectors, viral vectors, episomes, and artificial dyes, including Additionally, the expression vector may contain one or more selectable marker genes and an appropriate expression vector. The gene may contain regulatory sequences that confer resistance to, for example, antibiotics or toxins. Selectable to complement auxotrophic deficiencies or to supply critical nutrients not present in the culture medium. Expression control sequences may also include marker genes that can be used to express the gene. well-known constitutive and inducible promoters, transcription enhancers, transcription terminators, Two or more exogenous encoding nucleic acids may be used in the same If both nucleic acids are to be expressed simultaneously, then both nucleic acids may be, for example, in a single expression vector. or can be inserted into separate expression vectors. For single vector expression, The code nucleic acids can be operably linked to one common expression control sequence or can be linked to one common expression control sequence. different expression control sequences, such as one exogenous promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be Such confirmation can be achieved using methods well known in the art. Methods include, for example, Northern blot or polymerase chain reaction (PCR) amplification of mRNA. Nucleic acid analysis such as immunoblotting for expression of gene products or transfected Other suitable analytical methods for testing the expression of a nucleic acid sequence or its corresponding gene product are The exogenous nucleic acid can be expressed in sufficient amounts to produce a desired product, as determined by one of skill in the art. It is understood that expression levels are well known in the art and are disclosed herein. It is further shown that the method can be optimized to obtain sufficient expression. It is understood.

[0121] Directed evolution targets specific genes to improve and / or alter the properties of an enzyme. One approach involves the introduction of mutations to produce improved and / or altered enzymes. The mutants are identified through screening assays of the present invention that allow for the identification of useful mutants. A particularly useful screening method involves screening a large number of enzyme variants (e.g., >10 4 ) These include sensitive, high-throughput assays that allow automated screening of Mutagenesis and screening of enzymes to identify enzymes with optimized properties Typically, the more mutants that are screened, the more likely it is that the ideally suited mutation will be identified. This increases the likelihood of identifying specific areas of the gene for mutagenesis. Computational algorithms that can assist in the generation and screening of This can significantly reduce the number of enzyme variants that need to be tested.

[0122] Numerous directed evolution techniques have been developed (for review, see Hibbert et al., Biomol. Eng 22:11 -19 (2005); Huisman and Lalonde, Biocatalysis in the Pharmaceutical and Biotechnology Industries In Biocatalysis in the pharmaceutical and biotechnology industries 717-742 Page (2007), Patel (ed.), CRC Press; Otten and Quax et al., Biomol. Eng 22:1-9 (2005); and See, for example, Sen et al., Appl Biochem. Biotechnol 143:212-223 (2007)), various variants These methods are useful for generating libraries across many enzyme classes. and has been successfully applied to improve a wide range of properties.

[0123] Enzyme characteristics that are improved and / or altered by directed evolution techniques include, for example, selectivity / specificity. isomerism (for conversion of unnatural substrates); temperature stability (for intense high-temperature processing); pH stability ( for bioprocessing under low or high pH conditions; substrate or product tolerance (high product titers) so that it can be achieved); bond(K m ) (expanding substrate binding to include non-natural substrates); Harm (K i ) (to remove inhibition by product, substrate, or key intermediate); activity (kcat) (desired flux increase the enzyme reaction rate to achieve the desired results; expression levels (protein yield and overall Increased pathway flow rate); oxygen stability (for operation of air-sensitive enzymes under aerobic conditions); and and anaerobic activity (due to the operation of aerobic enzymes in the absence of oxygen).

[0124] The following exemplary methods involve genetic mutations to target desired properties of specific enzymes. These have been developed for allogeneic and diversified purposes. can be used to alter / optimize the activity of

[0125] EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005)) 2+ By adding ions By varying the dNTP concentration, or other conditional variations, PCR reactions can be In the reaction, the fidelity of the DNA polymerase is reduced, resulting in random Introduce point mutations. A five-step process to restrict mutagenesis to the target gene of interest. The cloning process 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; and 5) transfection into a suitable host. Transformation of offspring mutants and screening of the library for improved performance This method can be useful for simultaneously generating multiple mutations in a single gene. Many mutants can be generated by EpPCR, and therefore High-throughput screening assays or selection methods (especially those using robotics) , are useful for identifying those with desirable characteristics.

[0126] Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:el45 (2004); and Fujii et al., Nat. Protoc. 1:2493 -2497(2006)) was prepared by using the whole circular plasmid as a template and cleaving the nucleotides on the last two nucleotides. Random 6-mers with exonuclease-resistant thiophosphate bonds at the base are used for protease amplification. It is used to amplify the plasmid, in which the tandem repeats are then inserted. It contains many of the same elements as epPCR, except that it is recircularized by PCR followed by transformation into cells. Mn 2+ By adjusting the concentration, the mutation rate can be varied somewhat. To generate complete copies of the plasmid with 3-4 mutations / kbp, use simple error Use a simple single-step method: restriction enzyme digestion or specific primers Furthermore, this method is typically available as a kit.

[0127] DNA or family shuffling (Stemmer, Proc Natl Acad Sci USA 91: 10747-107 51 (1994); and Stemmer, Nature 370:389-391 (1994)) are typically used for the ligation of chimeric genes. Annealing and extension are carried out in the presence of DNA polymerase to generate a fragment. To generate a pool of random fragments that can be reassembled by cycling, use either Dnase I or Endonucleases. The process involves digestion of two or more mutant genes with a nuclease such as oV. The fragments are then separated from each other. When one copy primes the other, recombination occurs (template switching). This method can be used with DNA sequences >1 kbp. In addition to the mutant recombinants generated, this method generates extension sequences at a rate similar to error-prone PCR. This method introduces point mutations at the step. This method may result in antigenicity, can be used to remove random neutral mutations.

[0128] Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)) , involving template priming, followed by denaturation and a very short period of annealing / extension (5 This is followed by repeated cycles of two-step PCR (approximately 100 s). The growing fragments are then amplified by PCR using different templates. The template is annealed to the target DNA and further extended, and this is repeated until the full-length sequence is generated. Switching means that most of the resulting fragments have more than one parent. A combination of low-fidelity polymerases (Taq and Mutazyme) allows for the generation of opposing mutation sequences. The spectrum reduces error-prone bias.

[0129] Random Priming Recombination (RPR) involves the random Array primers generate many short DNA fragments complementary to different segments of the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Incorporation and mispriming result in point mutations. Short DNA fragments are identified based on homology. These primers prime each other, recombine, and regenerate to full length by repeated thermocycling. Removal of the template prior to this step ensures that parental recombinants are rare. This method, like most others, requires multiple iterations to evolve distinct traits. This technique avoids sequence bias and is gene-length-independent. It is free of parental DNA and requires very little parental DNA for application.

[0130] In heteroduplex recombination, the linearized plasmid DNA is repaired by mismatch repair. (Volkov et al., Nucleic Acids Res 27: e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)). Heteroduplexes are at least somewhat mutagenic. This method is suitable for large genes and entire operons.

[0131] Random Chimeragenesis on Transient Templates tes) (RACHITT) (Coco et al., Nat. Biotechnol 19:354-359 (2001)) is a method for the Dnase I fragmentation of ssDNA and Homologous fragments are bound to complementary ssDNA scaffolds in the absence of polymerase. Any overlapping non-hybridizing fragment ends are hybridized under The gaps between the fragments are filled and then ligated. full-length, gated and hybridized to a scaffold (containing U to prevent amplification) The scaffold is then broken and the diverse strands are amplified by PCR. The method involves the use of a single strand (scaffold) derived from only one parent. The fragments that are read and primed are derived from other genes; selection is made to exclude the parent scaffold. Therefore, reannealing with the parent fragments does not occur. In other cases, this is achieved by DNA shuffling and StE It is conceptually similar to P. Therefore, no populations share the same parents, and there are few inactive individuals. There should be no unshuffled parents. This technique produces a clone in which little or no parental genes are produced. It has the advantage over standard DNA shuffling in that many more crossovers can be generated. It has.

[0132] Recombinant Extension on Truncated Templates (RETT) , in the presence of unidirectional ssDNA fragments used as a pool of templates, This involves template switching of the unidirectionally growing chain (Lee et al., J. Molec. Catalysis 26: 1 19-129(2003)). DNA endonucleases are not used. One-way ssDNA is randomly pro- duced. DNA polymerase- or exonuclease-mediated sequential deletion using primers The unidirectional ssDNA is only a template, not a primer. Priming and exonuclease correspond to the enzymatic cleavage of DNA shuffling / RACHITT. RETT uses normal PCR conditions instead of very short extensions. Therefore, it may be easier to optimize than StEP. Recombination is a component of the PCR step. This method also has the advantage that there is no pause. , and can be more random than StEP.

[0133] Degenerate Oligonucleotide Gene Shuffling In dsDNA (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 2 2:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)). Other methods can be used to control the tendency of the parent to reproduce genes. The method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a suitable method to block rearrangement of the parental sequences. No nuclease is required. By adjusting the input concentration of the segments to be created, This method can be used without restriction enzyme digestion to bias the target backbone. Allows DNA shuffling from unrelated parents, allowing the choice of random mutagenesis methods To do so.

[0134] Incremental truncation for the generation of hybrid enzymes ncation for the Creation of Hybrid Enzymes (ITCHY) is a gene or gene fragment of interest. Generate a combinatorial library with a single base pair deletion of atl Acad Sci USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol 17: 1205 -1209(1999)). The truncations are introduced into two different gene segments in opposite orientations. are ligated together and the fusion is cloned. This technique No homology between genes is required. When ITCHY is combined with DNA shuffling, This system is called SCRATCHY (see below). The main advantage of both is that parental inheritance is Homology between genes is not required; for example, functional similarity between E. coli and human genes is not required. Fusions were generated via ITCHY. Once the ITCHY library was generated, all possible The intersection type is obtained.

[0135] Thio-Increme Truncation for the Generation of Hybrid Enzymes Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is a It is similar to ITCHY (Lu) except that phosphorothioate dNTPs are used to generate the nucleotide sequence. (Zetz et al., Nucleic Acids Res 29:E16 (2001)). Compared to ITCHY, THIO-ITCHY is optimized This can be easier to do and offers more reproducibility and adaptability.

[0136] SCRATCHY combines two methods for recombining genes: ITCHY and DNA shuffling. (Lutz et al., Proc Natl Acad Sci USA 98:11248-11253 (2001)). SCRATCHY is a Itchy combines the best features of Y and DNA shuffling. First, it is DNA homology independent. used to generate a comprehensive set of fusions between fragments of genes in a systematic manner. This artificial family is then subjected to a DNA shuffling step to increase the number of crossovers. The computational predictions can be used in optimization. If it is less than 80%, SCRATCHY is more effective than DNA shuffling.

[0137] In Random Drift Mutagenesis (RNDM), mutations are via epPCR followed by screening / selection for those that retain usable activity (Bergquist et al., Biomol. Eng 22:63-72 (2005)). These are then Fusions between active mutants or between active mutants and some other desired parent Used in DOGS to generate recombinants and facilitate the isolation of neutral mutations The purpose is to determine whether this activity is higher or lower than that of the original gene. The goal is to screen for retained catalytic activity regardless of whether or not the enzyme is present. If the run can detect activity above background, RNDM is considered to be a hybridization assay. RNDM can be used in high-throughput assays. This technique was used as a step to detect activity before shuffling or other subsequent steps. Neutral drift libraries are often used to identify the activity of smaller libraries. Published results using epPCR have been shown to result in higher / more immediate improvement in However, it can be applied to other large-scale mutagenesis methods.

[0138] Sequence Saturation Mutagenesis (SeSaM) is a method for the following: 1) phosphothioesterase A pool of random length fragments is generated using random incorporation of nucleotides and cleavage. This pool is used as a template, and 2) 3) extension in the presence of "uniform" bases such as inosine; This process results in random base incorporation and consequent 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. 3 41:187-189(2005)). Using this technique, a simple method can be used to suddenly detect the disease within 2-3 days. This technique may allow the generation of large libraries of mutants. The difference in this approach is that it is nonspecific compared to the mutation bias of the enzyme. This makes the technique complementary (or alternative) to epPCR.

[0139] In synthetic shuffling, overlapping oligonucleotides are used to "shuffle all of the target The shuffled progeny are designed to encode "genetic diversity" and are highly This technique allows for a wide variety of applications (Ness et al., Nat. Biotechnol 20: 1251-1255 (2002)). The fragments to be shuffled can be designed. This helps increase the diversity that occurs when more distantly related sequences are observed in more closely related sequences. The sequence / codon bias can be designed to recombine at rates close to those expected. Furthermore, the technique does not require the physical presence of the template gene.

[0140] Nucleotide Exchange and Excision Technology (NexT) uracil DNA glycosylase and then Utilizing a combination of dUTP incorporation followed by treatment with piperidine (Muller et al., Nucl eic Acids Res 33:ell7(2005)). The gene was synthesized using a proofreading polymerase and internal PCR primers. The size for shuffling was determined by varying the dUPT::dTTP ratio. This allows for direct control using a simple method for uracil incorporation and cleavage. It is an endpoint reaction that uses other nucleotides such as 8-oxo-guanine. Analogs can be used with this method. Furthermore, the technique allows for the detection of very short fragments (86 bp). The chemical cleavage of DNA used in this technique is Produces few non-shuffle clones.

[0141] Sequence Homology-Independent Protein Recombination In SHIPREC, a linker facilitates fusion between two distantly related / unrelated genes. Nuclease treatment is used to generate a series of chimeras between two genes. These fusions result in a library of single-crossover hybrids. (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)). This is a limited type of Schaffer Mutagenesis produces a distinct mutation, and a separate process is required for mutagenesis. Therefore, this technique allows for the creation of genes with various parts of each of the two unrelated parent genes. SHIPREC is a library of enzymes targeting the N-terminal region of mammalian CP450. This was tested using a fused heme-binding domain of bacterial CP450; this is a more soluble enzyme. It resulted in mammalian activity in

[0142] Gene Site Saturation Mutagenesis™ (GSSM™) In this study, the starting material was a supercoiled dsDNA plasmid containing the insert and the site of the mutation. Two primers are degenerate at the desired site (Kretz et al., Methods Enzymol. 388:3-11 (2 004)). 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, with approximately 20 nucleotides on each side. The sequences in the primers are NNN or NNK (coding) and MNN (non-coding). (N=all four, K=G, T, M=A, C). After extension, DpnI displaces the wild-type template. This technique is used to digest dam-methylated DNA, as in the case of damma-methylated DNA. Search for possible amino acid substitutions (i.e., single codons). The technique is free of nonsense codons. Facilitates the generation of all possible substitutions at a single site, resulting in equivalent ~ This technique provides a nearly equivalent representation of the structure, mechanism, or domain of the target enzyme. No prior knowledge is required. If followed by shuffling or gene rearrangement, this technique A diverse library of recombinants containing all possible combinations of single-site up-mutations The combinatorial utility of this technique has been demonstrated in the successful evolution of over 50 different enzymes. Furthermore, more than one property has been demonstrated for a given enzyme.

[0143] Combinatorial Cassette Mutagenesis (CCM) uses short oligonucleotides to replace restricted regions with many possible amino acid sequence changes. with the use of a leutidine cassette (Reidhaar-Olson et al., Methods Enzymol. 208:564-586 (1991) and Reidhaar-Olson et al., Science 241:53-57 (1988)). Using this technique, two or three Furthermore, the method allows simultaneous substitution at a limited range of sites. This technique tests a large number of sequence variations in the lambda repressor DNA binding domain. was used to investigate the amount of information in

[0144] Combinatorial Multiple Cassette Mutagenesis Mutagenesis (CMCM) is a method for identifying C mutations, except when it is used as part of a larger program. Similar in nature to CM: 1) 2) High mutation rates for ID hotspots and hot regions 3) the use of epPCR to cover defined regions of protein sequence space; Elongation by MCM (Reetz, M.T., S. Wilensek, D. Zha, and K.E. Jaeger, 2001, "Con Directed advancement of enantioselective enzymes through binatorial multiple cassette mutagenesis Directed Evolution of an Enantioselective Enzyme through Combinatorial Multip le-Cassette Mutagenesis.)" Angew. Chem. Int. Ed Engl. 40:3589-3591.) As in CCM This method allows testing virtually all possible variations on the target site. When used in conjunction with methods for generating mutated and shuffled genes, it This approach provides an excellent means to generate highly shuffled proteins. We succeeded in increasing the enantioselectivity of

[0145] In the mutator strain technique, a conditional ts mutator plasmid is used to induce the growth of the larvae during selection. Randomly allows for a 20-4000-fold increase in the frequency of spontaneous mutations, and selection is not required. When the β-glucan is added to the β-glucan, it blocks the accumulation of deleterious mutations (Selifonova et al., Appl Environ Microbiol 67:364 5-3649(2001)). This technique involves the use of a gene encoding a mutant subunit of DNA polymerase III. This subunit is based on the plasmid-borne mutD5 gene, which encodes the endogenous DNA polymerase III and impairs the proofreading ability of polymerase III in all strains carrying the plasmid. A broad spectrum of base substitutions and frameshift mutations occurs. Once the desired phenotype is achieved, the mutator plasmid should be eliminated; This is achieved through a temperature-sensitive origin of replication, which allows the plasmid to cure at 41°C. It is worth noting that mutator strains have been investigated for quite some time. (See, e.g., Low et al., J. Mol. Biol. 260:359-3680 (1996)). In this technique, A very high spontaneous mutation rate is observed. Conditional traits are not a cause of unwanted background mutations. This technique enhances the mutagenesis rate and increases the likelihood of a desired phenotype. It can be combined with adaptive evolution to achieve rapid

[0146] Look-Through Mutagenesis (LTM) is a method for the generation of selected amino acids. "This is a multidimensional mutagenesis method to evaluate and optimize combinatorial mutations of acids." (Ajpal et al., Proc Natl Acad Sci USA 102:8466-8471 (2005)). Rather than saturating each site with a specific cation, nine sets of cations are used to determine the chemical properties of the amino acid R groups. The smaller number of changes per site allows multiple sites to be selected to cover this range. For low nanomolar to picomolar antibodies, An >800-fold increase in binding affinity of was achieved through this method. This is a rational approach to minimize the number of possible combinations that can be screened. To find improved traits by significantly reducing the number of potential clones This can, among other things, increase binding affinity and / or enhance resolution. The technique has been applied to antibody engineering to reduce isolation. They can be combined.

[0147] Gene rearrangements can be performed on multiple genes at once or on many libraries of single gene chimeras ( It is a DNA shuffling method (Verenium) that can be applied to the generation of multiple mutations. Tunable GeneReassembly™ (TGR™) Technology supplied by. Typically, this technique involves searching the sequence space represented for the desired refinement. This technology is used in combination with ultra-high throughput screening. Allows multiple genetic recombinations independent of sex. The exact number and location of crossover events is determined by the biological information. The fragments can be predetermined using fragments designed through informatics analysis. The procedure involves virtually no parental gene rearrangement and low levels of inactive genes, resulting in non- consistently leads to high levels of diversity. In combination with GSSM™, a wide range of mutations can be achieved. The method involves the "blend" of DNA shuffling. and allows for "fine tuning", for example, to optimize codon usage.

[0148] In silico Protein Design Automation (PDA) A structurally determined protein backbone with a specific fold is fixed and the fold and and sequences for amino acid substitutions that can stabilize the overall protein energetics. It is an optimization algorithm that searches the column space (Hayes et al., Proc Natl Acad Sci USA 99:15926-15931(2002)). This technology allows structural analysis of protein amino acid variations. It is used in in silico structure-based entropy prediction to search for resistance of Statistical mechanics is applied to calculate the binding interactions at each position. The resistance of the structure to exchange is an indicator of binding. Ultimately, this technique allows for the complete characterization of the structure. The method is designed to obtain the desired modification of protein properties while maintaining the integrity. Allows computational evaluation of many possible sequence variants at any given time and filtering of them (10 50 The selection of sequence variants to test is based on the most favorable thermodynamic predictions. On the surface, only stability or properties associated with stability can be efficiently detected using this technique. The method can be used for several therapeutic proteins, particularly immunoglobulins. In silico prediction has been used successfully to engineer globulins. Avoids testing potential mutants. Predictions based on existing 3D structures are based on hypothesized structures. This technique appears to be more successful than structure-based predictions. Mutations can be easily predicted, allowing for their targeted screening, which ,Due to the exponential growth in numbers, it is sometimes not possible even using purely experimental techniques.

[0149] Iterative Saturation Mutagenesis (ISM) is: 1) Enzyme Improvement 2) Use structure / function information to select promising sites; 3) Stratagene QuikChange (or 3) saturation mutagenesis at selected sites using a gene encoding a desired property; screening / selection; and 4) using improved clones to re-introduce and replicate at other sites. with continued repetition (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. m. Int. Ed Engl. 45:7745-7751(2006)). This is a proven methodology, which Ensure that all possible substitutions at the positions are generated for screening / selection. do.

[0150] Any of the above methods for mutagenesis may be used alone or in any combination. Furthermore, any one or combination of directed evolution methods can be used in adaptive evolution techniques. can be used together with

[0151] The present invention relates to a method for producing a hydroxybenzoate comprising the steps of: adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine The present invention further provides methods for producing desired intermediates or products such as levulinic acid or levulinic acid. For example, methods for producing adipate include selecting naturally occurring organisms that have an adipate pathway. The pathway can include culturing a microbial organism that does not contain adipate, and the pathway can include culturing a microbial organism that does not contain adipate. and is expressed under conditions and for a period of time sufficient to produce adipate in an amount sufficient to produce at least one exogenous nucleic acid encoding an adipate pathway enzyme, The pathway involves succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl- CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-penten Thenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipyl Adipase / adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl Additionally, the method for producing adipate includes the use of an adipate pathway. The method can include culturing a naturally occurring microbial organism having a pathway, the pathway comprising: and in an amount sufficient to produce adipate, under conditions to produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme that is expressed for a sufficient period of time The adipate pathway includes succinyl-CoA:acetyl-CoA acyltransferase, 3 -oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxybenzoate hydroxyadipate dehydratase, and 2-enoate reductase.

[0152] Additionally, methods for producing 6-aminocaproic acid include those having a 6-aminocaproic acid pathway. The pathway can include culturing a non-naturally occurring microbial organism comprising 6-aminocarboxylic acid. under conditions to produce 6-aminocaproic acid in an amount sufficient to produce 6-aminocaproic acid, and at least one 6-aminocaproic acid pathway enzyme encoding a 6-aminocaproic acid pathway enzyme that is expressed for a period of time sufficient to The 6-aminocaproic acid pathway involves two exogenous nucleic acids and is a CoA-dependent aldehyde dehydrogenase pathway. It contains genase and transaminase or 6-aminocaproate dehydrogenase. Additionally, methods for producing caprolactam include integrating naturally occurring plants with caprolactam pathways. The pathway can include culturing a microbial organism that does not produce caprolactam. under conditions and for a period of time sufficient to produce caprolactam in a quantity sufficient to at least one exogenous nucleic acid encoding an expressed caprolactam pathway enzyme; The caprolactam pathway involves CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase.

[0153] The present invention provides a method for producing 6-aminocaproic acid (6-ACA) under conditions and with sufficient time to do so. Cultivating a non-naturally occurring microbial organism having a 6-ACA pathway described herein for a period of time. In one embodiment, a method for producing 6-ACA is further provided. The pathway consists of HODH aldolase, OHED hydratase, OHED reductase, and 2-OHD decarboxylase. and adipate semialdehyde aminotransferase or adipate semialdehyde In another aspect, the 6-ACA pathway includes an HODH aldolase (aminating). OHED hydratase; OHED decarboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase and 2-AHD decarboxylase. In another aspect, the 6-ACA pathway includes HODH alginate. 2-OHD hydratase; 2-OHD reductase; 2-OHD aminotransferase or 2-OH In another embodiment, the enzymes include 2-AHD decarboxylase, 2-AHD oxidoreductase (aminating enzyme); and 2-AHD decarboxylase. The 6-ACA pathway activates HODH aldolase, HODH formate-lyase, and pyruvate formate-lyase. Enzyme or HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehyd Droadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semia aldehyde aminotransferase or adipate semialdehyde oxidoreductase In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an O HED formate-lyase and pyruvate formate-lyase activating enzyme or OHED dehydrogenase; 2,3- Dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; 2-OHD formate-lyase and pyruvate formate-lyase activating enzyme or 2-O HD dehydrogenase; adipyl-CoA dehydrogenase; and adipate semialdehyde Aminotransferase or adipate semialdehyde oxidoreductase (amination) In a further aspect, the 6-ACA pathway described above comprises succinic semialdehyde dehydrogenase. hydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpidem It may contain benzoyl peroxidase (PEP) carboxykinase.

[0154] The present invention provides a method for producing hexamethylenediamine (HMDA) under conditions and sufficient for the production of HMDA. Cultivating a non-naturally occurring microbial organism having the HMDA pathway described herein for a period of time. In one embodiment, the HMDA pathway is further provided. 6-aminocaproate kinase; 6-AHOP oxidoreductase; and 6-aminocaproate Acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde In another aspect, the HMDA pathway includes a 6-aminocaproate aminotransferase. Kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase and 6-aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid semialdehyde oxidoreductase (aminating) In another embodiment, the HMDA pathway includes a nocaproate semialdehyde aminotransferase. 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde Oxidoreductase (amination) or 6-aminocaproic semialdehyde aminotransferase In another aspect, the HMDA pathway includes 6-aminocaproate N-acetyltransferase. 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6- Acetamidohexanal aminotransferase or 6-acetamidohexanal Oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase In another embodiment, the enzyme includes 6-acetamidohexanamine hydrolase or 6-acetamidohexanamine hydrolase (amide). The HMDA pathway involves 6-aminocaproate N-acetyltransferase; 6-acetamide Hexanoate CoA transferase or 6-acetamidohexanoate CoA ligase;6 -Acetamidohexanoyl-CoA oxidoreductase; 6-acetamidohexanalamine 6-acetamidohexanal oxidoreductase or 6-acetamidohexanal oxidoreductase (aminating) and 6-acetamidohexanamine N-acetyltransferase or 6-acetamido In another aspect, the HMDA pathway includes 6-aminocaproic acid esterase (HMDA). 6-Acetamidohexanoate N-acetyltransferase; 6-Acetamidohexanoate kinase; 6-AAHO P oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Cetoamidohexanal oxidoreductase (amination); and 6-acetamidohexane Amine N-acetyltransferase or 6-acetamidohexanamine hydrolase ( amides).

[0155] Additionally, methods for producing adipate include culturing non-naturally occurring organisms that have an adipate pathway. The method can include culturing a microbial organism sufficient to produce adipate, wherein the pathway comprises culturing a microbial organism sufficient to produce adipate. Adipate is expressed under conditions and for a period of time sufficient to produce adipate in sufficient amounts. at least one exogenous nucleic acid encoding an adipate pathway enzyme, , alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase / alpha -Ketoadipate kinase, or alpha-ketoadipyl-CoA:acetyl-CoA transferase 2-Hydroxyadipyl-CoA dehydrogenase; 2-Hydroxyadipyl-CoA dehydrase 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase , phosphotransadipylase / adipate kinase, adipyl-CoA:acetyl-CoA trans Furthermore, it contains the enzymes phosphodiesterase and adipyl-CoA hydrolase to produce adipate. The method includes culturing a non-naturally occurring microbial organism having an adipate pathway. and the pathway can be used to produce adipate in sufficient quantities to produce adipate. and a minimum of 100 genes encoding adipate pathway enzymes that are expressed under conditions and for a period of time sufficient to and the adipate pathway includes at least one exogenous nucleic acid, and genase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipyl 2-hydroxyadipate kinase, or 2-hydroxyadipyl-CoA:acetyl-CoA Transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-penteno and adipyl-CoA synthetase, phosphotransadipylase / adipylase Adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA Contains hydrolases.

[0156] As disclosed herein, the present invention also provides a method for producing 6-aminocaproic acid. at least one exogenous gene encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient amounts; Culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway containing nucleic acids and a method for producing 6-aminocaproic acid, The pathway is 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl- CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohexanoyl 2-enoyl-CoA reductase; and 6-aminocaproyl-CoA / acyl-CoA transferase enzyme, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase (See Examples XII and XIII; Steps A / B / C / D / K / L of FIG. 11 ). The present invention further provides a 6-aminocaproic acid derivative expressed in sufficient amounts to produce 6-aminocaproic acid. 6-aminocaproate pathway enzymes, including at least one exogenous nucleic acid encoding the aminocaproate pathway enzyme. By culturing a naturally occurring microbial organism that possesses the uronate pathway, 6-aminocarbonyl carboxylates were obtained. 1. A method for producing 6-aminocaproic acid, the 6-aminocaproic acid pathway comprising: 3-Oxo-6-aminohexanoyl-CoA thiolase; 3-Oxo-6-aminohexanoyl-CoA / acyl-CoA tran sferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-amino Hexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy- 6-aminohexanoate dehydratase; and 6-aminohex-2-enoate dehydratase (See Examples XII and XIV; Steps A / E / F of Figure 11 ). F / G / H / I / J).

[0157] In another embodiment, the present invention provides a method for producing caprolactam by the use of a recombinant protein comprising: Caprolactam containing at least one exogenous nucleic acid encoding a prolactam pathway enzyme. by culturing a non-naturally occurring microbial organism having a pathway a caprolactam pathway comprising 6-aminocaproyl-CoA / acyl The method further comprises the step of: (See Examples XII and XV; Steps K / L of Figure 11). Lactams are produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam. (See Example XII; Step Q of Figure 11). The present invention also provides a method for producing a hexamer. Hexamethylenediamine pathway enzymes expressed in sufficient amounts to produce ethylenediamine. having a hexamethylenediamine pathway comprising at least one exogenous nucleic acid encoding A non-naturally occurring microbial organism, wherein the hexamethylenediamine pathway comprises 6-aminocaproic acid. 6-aminocaproyl-CoA / acyl-CoA transferase or 6-aminocaproyl-CoA synthase; Aminocaproyl-CoA reductase (aldehyde formation); and hexamethylenediaminetransferase Non-naturally occurring microorganisms containing benzodiazepines, ... A biological organism is also provided (see Examples XII and XVI; steps K / L / N / O / P of Figure 11).

[0158] In yet another embodiment, the present invention provides a method for producing caprolactam by the use of a recombinant yeast strain of the present invention. caprolactam comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme. The caprolactam pathway has been identified by culturing a non-naturally occurring microbial organism. A method for producing caprolactam, the caprolactam pathway comprising: 3-Hydroxy-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy- 6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoA redoxase (See Examples XII and XVII; Steps A / B of Figure 11 ). In such a process, caprolactam is converted to 6-aminocaproyl caprolactam. It can be produced by the spontaneous cyclization of acetyl-CoA (see Example XII; see Figure 11). Step Q). Hexamethylenediamine is expressed in sufficient amounts to produce hexamethylenediamine. a hexamethylenediamine pathway enzyme comprising at least one exogenous nucleic acid encoding a diamine pathway enzyme; Hexamethasone was synthesized by culturing a non-naturally occurring microbial organism that possessed an amine pathway. A method for producing hexamethylenediamine, 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase 3-Hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl 6-aminocaproyl-CoA reductase (aldehyde formation); and hexa containing methylenediamine transaminase or hexamethylenediamine dehydrogenase Methods are also provided (see Examples XII and XVIII; steps A / B / C / D of FIG. 11). D / N / O / P).

[0159] In another embodiment, the present invention provides a naturally occurring plant that has a 6-aminocaproic acid (6-ACA) pathway. A method for producing 6-ACA by culturing a microbial organism that does not contain microbial The plant organism contains at least one gene encoding a 6-ACA pathway enzyme that is expressed in sufficient amounts to produce 6-ACA. The 6-ACA pathway includes at least one exogenous nucleic acid and is enzyme, alpha-ketoglutarate decarboxylase, phosphoenolpyruvate (PEP) Carboxykinase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase , 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, 2-oxohept-4-ene- 1,7-dioate (OHED) reductase, 2-oxoheptane-1,7-dioate (2-OHD) decarboxime xylase, adipate semialdehyde aminotransferase, adipate semialdehyde Dehyde oxidoreductase (aminating), 2-oxohept-4-ene-1,7-dioate (OHED) Decarboxylase, 6-oxohex-4-enoate (6-OHE) reductase, 2-oxoheptyl Tan-1,7-dioate (2-OHD) aminotransferase, 2-oxoheptane-1,7-dioate 2-aminoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), 2-aminoheptane-1,7-dioate (2-AHD ) decarboxylase, 2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase 2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (amino 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, 4-hydroxy-2- 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) formate lyase, -dioate (HODH) dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 2,3- Dehydroadipyl-CoA reductase, adipyl-CoA dehydrogenase, 2-oxohepta-4 -ene-1,7-dioate (OHED) formate lyase, 2-oxohept-4-ene-1,7-dioate (OHE D) Dehydrogenase, 2-oxoheptane-1,7-dioate (2-OHD) formate lyase, 2-oxo Heptane-1,7-dioate (2-OHD) dehydrogenase or pyruvate formate lyase activation Methods are provided that include the enzyme (see Examples XIX and XXI; Steps A-Q of Figure 12).

[0160] In another embodiment, the present invention provides a naturally occurring plant that has a 6-aminocaproic acid (6-ACA) pathway. A method for producing 6-ACA by culturing a microbial organism that does not contain microbial The plant organism contains at least one gene encoding a 6-ACA pathway enzyme that is expressed in sufficient amounts to produce 6-ACA. In one embodiment, the 6-ACA pathway comprises at least one exogenous nucleic acid. OHED hydratase; OHED reductase; 2-OHD decarboxylase; and adipase Adipate semialdehyde aminotransferase or adipate semialdehyde oxidizer (amination) (see Examples XIX and XXI; steps A / B / C / D / E of Figure 12) In another aspect of the invention, the 6-ACA pathway comprises an HODH aldolase; an OHED hydratase; an OHED decahydroxybenzoate; carboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase Enzyme or adipate semialdehyde oxidoreductase (aminating) (Example XIX and XXI; steps A / B / F / G / E of Figure 12). In another embodiment of the invention, the 6-ACA pathway OHED aldolase; OHED hydratase; OHED aminotransferase or OHED oxytocin 2-AHE reductase (aminating); 2-AHE reductase; and 2-AHD decarboxylase (actual See Examples XIX and XXI; steps A / B / J / D / I of Figure 12). In another embodiment of the present invention, 6-A The CA pathway consists of HODH aldolase, OHED hydratase, OHED reductase, and 2-OHD aminotransferase. ferase or 2-OHD oxidoreductase (aminating); and 2-AHD decarboxylase (See Examples XIX and XXI; Steps A / B / C / H / I of Figure 12). The 6-ACA pathway involves HODH aldolase, HODH formate-lyase, and pyruvate formate-lyase activities. hydroxyadipyl-CoA dehydratase or HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydrogenase Hydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semi Aldehyde aminotransferase or adipate semialdehyde oxidoreductase (See Examples XIX and XXI; Steps A / L / M / N / O / E of Figure 12). The ACA pathway is composed of HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate. Lyase-activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; Adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase and adipate semialdehyde oxidoreductase (aminating) (Examples XIX and See also steps A / B / P / N / O / E of Figure 12. In another embodiment of the invention, the 6-ACA pathway HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyridinyltransferase Vinyl formate lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase and adipate semialdehyde aminotransferase or adipate semialdehyde Dehydoreductase (aminating) (see Examples XIX and XXI; see Figure 12 In a further aspect, the 6-ACA pathway described above comprises the steps of succinate synthesis. aldehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or It may contain phosphoenolpyruvate (PEP) carboxykinase.

[0161] In another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities. a 6-ACA pathway comprising at least one exogenous nucleic acid encoding an expressed 6-ACA pathway enzyme; A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having The method, wherein the 6-ACA pathway includes glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase , 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhepta -2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation) or 2-aminopimelate decarboxylase (Examples XXV and XX). See VI; steps A / B / C / D / E / I / J of Figure 20). In another embodiment of the invention, naturally occurring The microbial organism contains a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, The enzymes are glutamyl-CoA transferase or glutamyl-CoA ligase; beta-ketothiol 3-Oxo-6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA oxidoreductase Nopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase 6-aminopimeloyl-CoA reductase (aldehyde formation); and 2-aminopimelate Encodes a decarboxylase.

[0162] In another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities. a 6-ACA pathway comprising at least one exogenous nucleic acid encoding an expressed 6-ACA pathway enzyme; A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having The method, wherein the 6-ACA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase oxidoreductase, 3-aminopimelate 2,3-aminomutase, or 2-aminopimelate and methods for producing guanine-3-phosphate decarboxylases (see Examples XXV and XXVI); 21 steps A / B / J / T / AA). In other aspects of the invention, the non-naturally occurring microbial organism is , a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set including glutaryl-Co A beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-Co A transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate amino Transferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate Encoding 2-aminopimelate 2,3-aminomutase; and 2-aminopimelate decarboxylase .

[0163] In another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities. a 6-ACA pathway comprising at least one exogenous nucleic acid encoding an expressed 6-ACA pathway enzyme; A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having The 6-ACA pathway includes homolysine 2-monooxygenase. See Examples XXV and XXVI; Step A of Figure 23). In a further aspect, the 6-ACA pathway , with dilute acid or base to convert 6-aminohexanamide to 6-aminocaproate This involves hydrolysis of the 6-aminohexanamide product (see Example XXV; step 2 of Figure 23). Pub B).

[0164] In another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities. a 6-ACA pathway comprising at least one exogenous nucleic acid encoding an expressed 6-ACA pathway enzyme; A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having The method, wherein the 6-ACA pathway includes an adipate reductase, an adipate kinase, or an adipate kinase.

[0023] Methods are provided for the preparation of cyclohexyl phosphate reductase (see Example XXVIII; Figure 2). In a further aspect, the 6-aminocaproic acid (6-ACA) pathway comprises adipate. In another further aspect, the 6-ACA pathway includes adipate kinase and In another embodiment, the microorganisms having the 6-ACA pathway described above contain adipylphosphate reductase. The organism may comprise an adipate pathway, a caprolactam pathway, and / or Further included are hexamethylenediamine pathways (see Example XXVIII; steps in Figure 25). A~W).

[0165] In another embodiment, the present invention provides a method for producing 6-aminocaproic acid (6-ACA) in sufficient quantities. a 6-ACA pathway comprising at least one exogenous nucleic acid encoding an expressed 6-ACA pathway enzyme; 6-aminocaproic acid (6-AAC) was obtained by culturing a non-naturally occurring microbial organism having 2-amino-7-oxosubarate keto acid (6-ACA), Decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 2-amino- 7-oxoheptanoate oxidoreductase, 2-aminopimelate decarboxylase , 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoate deca carboxylase, or 2-amino-7-oxosubarate amino acid decarboxylase , a method is provided (see Examples XXV and XXVI; steps A / B / D / E / F / G / I of Figure 26). In a further aspect, the microbial organism is sufficient to produce 2-amino-7-oxosubarate. at least one exogenous gene encoding a 2-amino-7-oxosubarate pathway enzyme expressed in sufficient amounts; The 2-amino-7-oxosubarate pathway has a nucleotide-dependent The hydroxylase pathway is 2-amino-5-hydroxy-7-oxosubarate aldolase, 2-amino-5-hydroxy-7-oxosubarate aldolase, hydroxy-7-oxosubarate dehydratase, or 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps A / B / C of Figure 27).

[0166] In another embodiment of the invention, the invention provides a method for producing a 6-aminocaproic acid (6-ACA) pathway enzyme encoding A naturally occurring 6-aminocaproic acid (6-ACA) pathway protein comprising a set of exogenous nucleic acids that encodes the 6-aminocaproic acid (6-ACA) pathway. 1. A method for producing 6-ACA by culturing a microbial organism that does not The set consists of 2-amino-7-oxosubarate keto acid decarboxylase; Heptanoate oxidoreductase; and 2-aminopimelate decarboxylase (See Example XXV; Steps A / D / E of Figure 26). In other embodiments, the naturally occurring microbial organism does not contain exogenous genes encoding the 6-ACA pathway enzymes. The set includes a set of nucleic acids having a structure comprising 2-amino-7-oxosubarate keto acid decarboxylate 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanoate decarboxylase 26, steps A / B / F of Figure 26. In another embodiment of the invention, the non-naturally occurring microbial organism encodes the 6-ACA pathway enzymes. The set includes a set of exogenous nucleic acids encoding 2-amino-7-oxosubarate amino acids. 2-amino-7-oxoheptanoate decarboxylase; and 6-amino- It encodes methylaminohexanal oxidoreductase (see Example XXV; see Figure 26). In a further aspect of each of the above embodiments, the microbial organism comprises a 2-amino acid 2-amino-7-oxosubarate expressed in sufficient amounts to produce 2-amino-7-oxosubarate 2-amino-7-oxo-salvatore with a second set of exogenous nucleic acids encoding glycoprotein pathway enzymes. The 2-amino-7-oxosubarate pathway is the 2-amino-5-hydroxy-7-oxosubarate pathway. Subabarate aldolase; 2-amino-5-hydroxy-7-oxosubarate dehydratase; and 2-amino-5-ene-7-oxosuberativ reductase (see Examples XXV and XXVI) (See steps A / B / C of Figure 27).

[0167] In another embodiment, the present invention provides a naturally occurring hexamethylenediamine (HMDA) pathway. A method for producing HMDA by culturing a microbial organism that does not The host organism contains at least one gene encoding an HMDA pathway enzyme that is expressed in sufficient amounts to produce HMDA. The HMDA pathway involves the use of 6-aminocaproate kinase and [(6-aminocaproate) kinase]. (6-hexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, 6-aminocaproic acid Acid semialdehyde aminotransferase, 6-aminocaproic acid semialdehyde oxy 6-aminocaproate N-acetyltransferase, 6-aminocaproate reductase (aminating), Cetamidohexanoate kinase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, 6-acetamidohexanal aminotransferase 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanal Hexanamine N-acetyltransferase, 6-acetamidohexanamine hydrolase 6-acetamidohexanoate CoA transferase, 6-acetamidohexanoate CoA transferase xanoate-CoA ligase, 6-acetamidohexanoyl-CoA oxidoreductase, [(6 -acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, 6-aminohexanoyloxy containing aminocaproate CoA transferase and 6-aminocaproate CoA ligase, A method is provided (see Examples XX and XXI; Steps A-N of Figure 13).

[0168] In another embodiment, the present invention provides a naturally occurring hexamethylenediamine (HMDA) pathway. A method for producing HMDA by culturing a microbial organism that does not The host organism contains at least one gene encoding an HMDA pathway enzyme that is expressed in sufficient amounts to produce HMDA. In one aspect, the HMDA pathway comprises a 6-aminocarboxylic acid. Proate kinase; 6-AHOP oxidoreductase; and 6-aminocaproic semialdehyde Oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase (See Examples XX and XXI; Steps A / B / C of Figure 13). In other aspects, the HMDA pathway comprises 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semial Dehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde amination transferase (see Examples XX and XXI; steps A / L / N / C of Figure 13). In another aspect of the invention, the HMDA pathway includes 6-aminocaproate CoA transferase or 6- Aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; and 6- Aminocaproic acid semialdehyde oxidoreductase (aminating) or 6-aminocaproic acid Acid semialdehyde aminotransferase (see Examples XX and XXI; Figure 1 In another aspect of the invention, the HMDA pathway can be catalyzed by the synthesis of 6-aminocaproate N-acetate. 6-Acetamidohexanoate kinase; 6-AAHOP oxidoreductase 6-acetamidohexanal aminotransferase or 6-acetamidohexanal aminotransferase Xanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetate containing methyltransferase or 6-acetamidohexanamine hydrolase (amide) (See Examples XX and XXI; Steps D / E / F / G / H of Figure 13). In another aspect of the invention, H The MDA pathway consists of 6-aminocaproate N-acetyltransferase; 6-acetamidohexa 6-acetamidohexanoate CoA transferase or 6-acetamidohexanoate CoA ligase; 6-Acetamidohexanoyl-CoA oxidoreductase; 6-Acetamidohexanalamino transferase or 6-acetamidohexanal oxidoreductase (amination); and and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine amine hydrolase (amide) (see Examples XX and XXI; see steps in Figure 13). In another aspect of the invention, the HMDA pathway is a pathway that converts 6-aminocaproate N-acetyltransferase (6-aminocaproate N-acetyltransferase). Transferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductor 6-acetamidohexanal aminotransferase or 6-acetamidohexanal 6-Acetamidohexanamine N-acetyltransferase (amination); transferase or 6-acetamidohexanamine hydrolase (amide) (implementation See Examples XX and XXI; steps D / E / K / J / G in Figure 13).

[0169] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway includes glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhepta-2 -enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde forming), 2 -amino-7-oxoheptanoate aminotransferase, 2-amino-7-oxoheptanoate a method comprising: a lysine aminating oxidoreductase or a homolysine decarboxylase; (See Examples XXIV and XXVI; Steps A-H of Figure 20). In embodiments, the non-naturally occurring microbial organism contains an exogenous nucleic acid encoding a HMDA pathway enzyme. The set includes a glutamyl-CoA transferase or ligase; 3-Oxo-6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6- Aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA dehydratase ductase; 6-aminopimeloyl-CoA reductase (aldehyde forming); 2-amino-7-oxo heptanoate aminotransferase or aminating oxidoreductase; and It encodes lysine decarboxylase.

[0170] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal Aminotransferase, 3-oxo-1-carboxyheptanal amination oxidizer ctase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7- Aminoheptanoate 3-aminating oxidoreductase, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxopimelate CoA transferase 3-oxopimeloyl-CoA reductase, 3-oxopimelate ligase, 5-oxopimeloyl-CoA reductase (aldehyde formation), 3-oxopimelate aminotransferase, 3-oxopimelate amination Oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate Ligase, 5-aminopimeloyl-CoA reductase (aldehyde-forming), 3-aminopimelate Kinase, 5-aminopimeloylphosphonate reductase, 3-aminopimelate reductase 2,3-aminomutase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate amination oxidase lysine reductase, 3,7-diaminoheptanoate 2,3-aminomutase, homolysine decal carboxylase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate kinase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 2-amino Pimelate reductase, 6-aminopimeloylphosphonate reductase, 6-aminopime Iodoyl-CoA reductase (aldehyde forming), 3-amino-7-oxoheptanoate 7-amino Transferase or 3-amino-7-oxoheptanoate aminating oxidoreductor

[0043] Methods are provided for the preparation of ELISA kits, including the use of ELISA kits (see Examples XXIV and XXVI; Figure 21).

[0171] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 7 -aminotransferase, 3-oxo-1-carboxyheptanal 7-amination oxidore ductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo- 7-aminoheptanoate 3-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase. See Examples XXIV and XXVI; Steps A / B / C / D / E / R / S of Figure 21). The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding HMDA pathway enzymes. The set includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydroxylase; lorase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Gauze; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 7-amino Transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7- Aminoheptanoate 3-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3 -aminomutase; and homolysine decarboxylase.

[0172] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminotransferase Carboxyheptanal 7-aminating oxidoreductase, 3-oxo-7-aminoheptanoic acid 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-amination oxy lysine decareductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decareductase and methods for producing carboxylases, including carboxylases (see Examples XXIV and XXVI; steps in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism is HMDA. a set of exogenous nucleic acids encoding pathway enzymes, the set including glutaryl-CoA beta -ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA tran spherase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimelate kinase; Hexopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 7-amino Transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7- Aminoheptanoate 3-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3 -aminomutase; and homolysine decarboxylase.

[0173] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase 5-oxopimeloyl-CoA reductase (aldehyde-forming), 3-oxo-1-carboxy Heptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminotransferase Oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, 3,7-diamino 2,3-aminomutase, or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A / B / H / I / D / E / R / S of Figure 21). In certain other aspects, the non-naturally occurring microbial organism comprises an exogenous gene encoding an HMDA pathway enzyme. The set includes a set of nucleic acids comprising glutaryl-CoA beta-ketothiolase; 3-oxopyrrolidin; pimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Pimeloyl-CoA ligase; 3-oxopimelate CoA transferase or 3-oxopimelate merate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo- 1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal Tanal 7-aminating oxidoreductase; 3-oxo-7-aminoheptanoate 3-amino Transferase or 3-oxo-7-aminoheptanoate 3-aminating oxidoreductor 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase Code the following.

[0174] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 3 -aminotransferase, 3-oxo-1-carboxyheptanal 3-amination oxidore ductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino- 7-Oxoheptanoate 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase. See Examples XXIV and XXVI; Steps A / B / C / AB / Z / R / S of Figure 21). The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding HMDA pathway enzymes. The set includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydroxylase; lorase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA Gauze; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 3-amino Transferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- Oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3- aminomutase; and homolysine decarboxylase.

[0175] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-aminotransferase Carboxyheptanal 3-aminating oxidoreductase, 3-amino-7-oxoheptanoic acid 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminotransferase lysine decareductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decareductase and methods for producing carboxylases, including carboxylases (see Examples XXIV and XXVI; steps in Figure 21). In other aspects of the invention, the non-naturally occurring microbial organism is a HMD The set includes a set of exogenous nucleic acids encoding glutaryl-CoA pathway enzymes, Taketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA tra 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5- Oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 3-amino 3-oxo-1-carboxyheptanal 3-amination oxidoreductase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7 -oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2, 3-aminomutase; and homolysine decarboxylase.

[0176] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate CoA transferase or 3-oxopimelate C CoA ligase, 5-oxopimeloyl-CoA reductase (aldehyde-forming), 3-oxo-1-cal 3-Oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-aminotransferase -aminating oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferase 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3,7-diamino- aminoheptanoate 2,3-aminomutase, or homolysine decarboxylase, (See Examples XXIV and XXVI; Steps A / B / F / G / AB / Z / R / S of Figure 21) In another aspect of the invention, the non-naturally occurring microbial organism encodes a HMDA pathway enzyme. The set of exogenous nucleic acids includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3- Oxopimeloyl-CoA ligase; 3-oxopimelate CoA transferase or 3-oxopimeloyl-CoA ligase Pimelate CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxopimeloyl-CoA reductase 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal Butanal 3-aminating oxidoreductase; 3-amino-7-oxoheptanoate 7-amino Transferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductor 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase Encodes ze.

[0177] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate 3-aminopimelate reductase, 3-amino-7-oxo-3-aminopimelate reductase, 2-amino-7-oxoheptanoate 7-aminotransferase 2-amino-7-oxoheptanoate aminating oxidoreductase, The method further provides a method for the production of a lysine decarboxylase-containing lysine decarboxylase (see Examples XXIV and XXVI). 21 steps A / B / / J / O / P / Q / S). In another aspect of the invention, the naturally occurring microbial The human organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: Tharyl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA hydrolase 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminating oxidoreductase; Aminopimelate reductase;3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate 7-aminotransferase butanoate 7-aminating oxidoreductase; and homolysine decarboxylase Load.

[0178] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloyl phosphate sulfonate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate ester aminating oxidoreductase, or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A / B / J / M / N / P / Q / S of Figure 21). In certain other aspects, the non-naturally occurring microbial organism comprises an exogenous gene encoding an HMDA pathway enzyme. The set includes a set of nucleic acids comprising glutaryl-CoA beta-ketothiolase; 3-oxopyrrolidin; meloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA meloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate 3-aminopimelate kinase; 5-aminopimeloyl Phosphonate reductase; 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate and homolysine decarboxylase. do.

[0179] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate carboxylate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde-forming), 3-amino- 7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-amino Notransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase and homolysine decarboxylase or homolysine decarboxylase (see Examples XXIV and XXVI). See steps A / B / J / K / L / P / Q / S of Figure 21). In another embodiment of the invention, non-naturally occurring The microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: , glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; Pimelate aminotransferase or 3-oxopimelate aminating oxidoreductase 3-aminopimelate CoA transferase or 3-aminopimelate CoA ligase 5-Aminopimeloyl-CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoic acid 2-amino-7-oxoheptanoate 7-aminotransferase 2-amino-7-oxoheptanoate 7-aminating oxidoreductase; and homodimer It encodes a decarboxylase.

[0180] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptase Heptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-amination oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homodimer and methods for producing a soluble decarboxylase comprising the steps of: In another aspect of the invention, the non-naturally occurring microbial organism comprises: The set includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set including glutaryl-CoA vectors. 3-Oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA thiolase transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate amino transferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 3-amino-7-oxoheptanoate 7-aminotransferase or 3-Amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate noate 2,3-aminomutase; and homolysine decarboxylase.

[0181] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate carboxylate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde-forming), 3-amino- 7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or The method further provides a method for the production of a lysine decarboxylase-containing lysine decarboxylase (see Examples XXIV and XXVI). 21 steps A / B / J / K / L / Z / R / S). In another aspect of the invention, a naturally occurring microorganism is The organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 3-oxopimeloyl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; -aminopimelate CoA transferase or 3-aminopimelate CoA ligase; Pimeloyl-CoA-reductase (aldehyde formation); 3-amino-7-oxoheptanoate 7-aldehyde Aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase It encodes an enzyme.

[0182] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphoryl nate reductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3 -amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate and a method for producing a lysine decarboxylase comprising administering to a subject ... (See Examples XXIV and XXVI; Steps A / B / J / M / N / Z / R / S of Figure 21). Similarly, non-naturally occurring microbial organisms can express exogenous nucleic acids encoding HMDA pathway enzymes. The set includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-C oA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA oA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminotransferase Oxidoreductase; 3-aminopimelate kinase; 5-aminopimeloylphosphonate reductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino 3,7-Diaminoheptanoate aminating oxidoreductase; and homolysine decarboxylase.

[0183] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate reductase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2- 7-oxoheptanoic acid aminating oxidoreductase, or homolysine decarboxime (See Examples XXIV and XXVI; steps of Figure 21). In other aspects of the invention, the non-naturally occurring microbial organism expresses or produces HMDA pathway enzymes. The set includes a set of exogenous nucleic acids encoding glutaryl-CoA beta-keto Thiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase 3-aminopimelate 2,3 -aminomutase; 2-aminopimelate reductase; 2-amino-7-oxoheptanoate 7- Aminotransferase or 2-amino-7-oxoheptanoate aminating oxidizer and homolysine decarboxylase.

[0184] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate tokinase, 6-aminopimeloylphosphonate reductase, 2-amino-7-oxoheptase 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate amination oxidase and a method for producing a lysine decarboxylase comprising the steps of: (a) lysine decarboxylase; (b) a lysine decarboxylase; See XIV and XXVI; steps A / B / J / T / U / X / Q / S of Figure 21). In another aspect of the invention, The non-naturally occurring microbial organism contains a set of exogenous nucleic acids encoding the HMDA pathway enzymes. The set includes glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; enzyme, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidase Oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate kinase 6-Aminopimeloylphosphonate reductase; 2-amino-7-oxoheptanoate 7- Aminotransferase or 2-amino-7-oxoheptanoate aminating oxidizer and homolysine decarboxylase.

[0185] In another embodiment, the present invention provides a method for producing hexamethylenediamine (HMDA) in an amount sufficient to produce hexamethylenediamine (HMDA). a HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in and (c) culturing a non-naturally occurring microbial organism having a hydroxybenzoate derivative of the formula (I) for producing HMDA. The method, wherein the HMDA pathway comprises glutaryl-CoA beta-ketothiolase, 3-oxopimeloi 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase Oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 6-aminopimeloyl-CoA ligase ductase (aldehyde-forming), 2-amino-7-oxoheptanoate 7-aminotransferase enzyme, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homoribonucleotide The present invention provides methods for the preparation of ribozymes, including ribozymes containing ... and ribozymes containing ribozymes (see Examples XXIV and XXVI; Figure 1). 21 steps A / B / J / T / V / Y / Q / S). In another aspect of the invention, a non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising glutaryl-C oA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-C CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate amine 3-aminotransferase or 3-oxopimelate aminating oxidoreductase; Pimelate 2,3-aminomutase; 2-aminopimelate CoA transferase or 2-amino Pimelate CoA ligase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-aminopimeloyl-CoA reductase 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate and homolysine decarboxylase; .

[0186] The present invention further provides a method for producing hexamethylenediamine (HMDA) expressed in sufficient amounts to produce hexamethylenediamine (HMDA). a natural HMDA pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme; Hexamethylenediamine (HMDA) was synthesized by culturing non-naturally occurring microbi...

Claims

1. Containing hexamethylenediamine (HMDA) pathway enzymes expressed in sufficient amounts to produce HMDA. A non-naturally occurring microbial organism comprising a HMDA pathway comprising a set of exogenous nucleic acids that encodes the HMDA pathway. There was, The set of exogenous nucleic acids comprises 6-aminocaproate reductase; and 6-aminocaproate Caproic acid semialdehyde aminotransferase or 6-aminocaproic acid semialdehyde aminotransferase Encodes oxidoreductase (amination), The microbial organism is (a) a 6-aminocaproic acid pathway comprising: (i) adipate reductase; and (ii) adipate reductase. Adipate semialdehyde aminotransferase, adipate semialdehyde oxidizer ductase (aminating), 6-aminocaproate transaminase, or 6-aminocaproate the 6-aminocaproic acid pathway, which includes an acetone dehydrogenase; and (b) the adipate pathway, which involves succinyl-CoA:acetyl-CoA acyltransferase ase, 3-oxoadipyl-CoA / acyl-CoA transferase, 3-oxoadipyl-CoA synthase tase, 3-oxoadipyl-CoA hydrolase, 3-oxoadipate reductase, 3-hydroxybenzoate hydroxyadipate dehydratase, and 5-carboxy-2-pentanoate reductase The adipate pathway comprises an enzyme selected from the group consisting of: The non-naturally occurring microbial organism further comprises:

2. 10. The method of claim 1, wherein the set of exogenous nucleic acids comprises at least two exogenous nucleic acids. A non-naturally occurring microbial organism.

3. the 6-aminocaproic acid pathway includes the adipate reductase and the adipate semiconducting enzyme; The non-naturally occurring microorganism of claim 1 or 2, comprising an aldehyde aminotransferase. Biological organism.

4. the 6-aminocaproic acid pathway includes the adipate reductase and the adipate semiconducting enzyme; 3. The naturally occurring enzyme according to claim 1 or 2, comprising an aldehyde oxidoreductase (aminating). No microbial organisms.

5. The 6-aminocaproic acid pathway comprises the adipate reductase and the 6-aminocaproic acid 3. The non-naturally occurring microbial organism of claim 1 or 2, comprising an ate transaminase. 。

6. The 6-aminocaproic acid pathway comprises the adipate reductase and the 6-aminocaproic acid 3. The non-naturally occurring microbial organism of claim 1 or 2, comprising an ate dehydrogenase.

7. The 6-aminocaproic acid pathway includes the adipate reductase, the adipate semidiazepine, Aldehyde aminotransferase, adipate semialdehyde oxidoreductase amination, the 6-aminocaproate transaminase, or the 6-aminocaproate 3 to 5, comprising at least one exogenous nucleic acid encoding an ethoxylate dehydrogenase.

7. The non-naturally occurring microbial organism of any one of 6.

8. The 6-aminocaproic acid pathway includes the adipate reductase, the adipate semidiazepine, Aldehyde aminotransferase, adipate semialdehyde oxidoreductase amination, the 6-aminocaproate transaminase, and the 6-aminocaproate at least two encoding two enzymes selected from the group consisting of acetone dehydrogenase The non-naturally occurring microbial organism of any one of claims 3 to 6, comprising an exogenous nucleic acid of body.

9. The adipate pathway includes the succinyl-CoA:acetyl-CoA acyltransferase , the 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-oxoadipyl-CoA A synthase, the 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase at least one exogenous gene encoding an enzyme selected from the group consisting of:

9. The non-naturally occurring microbial organism of any one of claims 1 to 8, comprising a nucleic acid of the same or similar nature.

10. The adipate pathway includes the succinyl-CoA:acetyl-CoA acyltransferase , the 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-oxoadipyl-CoA A synthase, the 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase at least two encoding two enzymes selected from the group consisting of benzoate reductases, The non-naturally occurring microbial organism of any one of claims 1 to 8, comprising an exogenous nucleic acid. 。

11. The adipate pathway includes the succinyl-CoA:acetyl-CoA acyltransferase , the 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-oxoadipyl-CoA A synthase, the 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase at least three encoding three enzymes selected from the group consisting of benzoate reductases, The non-naturally occurring microbial organism of any one of claims 1 to 8, comprising an exogenous nucleic acid. 。

12. The adipate pathway includes the succinyl-CoA:acetyl-CoA acyltransferase , the 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-oxoadipyl-CoA A synthase, the 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase at least four encoding four enzymes selected from the group consisting of benzoate reductases, The non-naturally occurring microbial organism of any one of claims 1 to 8, comprising an exogenous nucleic acid. 。

13. The adipate pathway includes the succinyl-CoA:acetyl-CoA acyltransferase , the 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-oxoadipyl-CoA A synthase, the 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase at least five encoding five enzymes selected from the group consisting of benzoate reductases, The non-naturally occurring microbial organism of any one of claims 1 to 8, comprising an exogenous nucleic acid. 。

14. The five enzymes are the succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA / acyl-CoA transferase, the 3-hydroxyadipate dehydratase, and the 5-carboxy-2-pentatase 14. The non-naturally occurring microbial organism of claim 13, comprising a noate reductase.

15. The five enzymes are the succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA synthase, the 3-oxoadipate reductase, the 3-hydroxybenzoate hydroxyadipate dehydratase, and the 5-carboxy-2-pentanoate reductase 14. The non-naturally occurring microbial organism of claim 13, comprising:

16. The five enzymes are the succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA hydrolase, the 3-oxoadipate reductase, the 3-hydroxy hydroxyadipate dehydratase, and the 5-carboxy-2-pentanoate reductor 14. The non-naturally occurring microbial organism of claim 13, comprising a microbial strain selected from the group consisting of lactic acid bacteria, ...

17. 17. The method of claim 1, wherein at least one exogenous nucleic acid is a heterologous nucleic acid.

2. The non-naturally occurring microbial organism described herein.

18. 18. The method according to claim 1, wherein the microbial organism is a eubacterium, a yeast, or a fungus.

2. The non-naturally occurring microbial organism of claim 1.

19. The microbial organism is selected from the group consisting of Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succini cyproducts, Phaseolus vulgaris, Bacillus subtilis, Corynebacterium glutamicum, Glycerol Conobacter oxydans, Zymomonas mobilis, Lactococcus lactis, La Lactobacillus plantarum, Streptomyces coelicolor, Clostridium aeruginosa setobutylicum, Pseudomonas fluorescens, and Pseudomonas putida groups 20. The non-naturally occurring microbial organism of claim 18, which is a eubacterium selected from the group consisting of:

20. The microbial organism may be selected from the group consisting of budding yeast, fission yeast, Kluyveromyces lactis, Kluyveromyces lactis, Rhommyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia patens yeast or fungus selected from the group consisting of Rhizopus suris, Rhizopus aristos, and Rhizopus oryzae 16. The non-naturally occurring microbial organism of claim 15, which is a species of Bacillus subtilis.

21. A method for producing hexamethylenediamine (HMDA), comprising: A naturally occurring strain of any one of claims 1 to 17 in a culture medium under conditions and for a period sufficient therefor. The method comprises culturing a non-native microbial organism.

22. 22. The method of claim 21, wherein the conditions comprise substantially anaerobic culture conditions.

23. 23. The method of claim 21 or 22, further comprising separating the HMDA from other components in the medium. The method described.

24. The HMDA can be purified by continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, Distillation, crystallization, centrifugation, extraction filtration, ion exchange chromatography, size exclusion chromatography Other compounds in the medium can be isolated by methods such as chromatographic, adsorption chromatography, or ultrafiltration.

24. The method of claim 23, wherein the component is separated.

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