WO2012177721A1

WO2012177721A1 - Microorganisms for producing 6-aminocaproic acid

Info

Publication number: WO2012177721A1
Application number: PCT/US2012/043283
Authority: WO
Inventors: Anthony P. Burgard; Robin E. Osterhout; Jun Sun; Priti Pharkya
Original assignee: Genomatica, Inc.
Priority date: 2011-06-22
Filing date: 2012-06-20
Publication date: 2012-12-27
Also published as: AU2013203175A1; AU2013203175A9; AU2012273093A1

Abstract

The invention provides a non-naturally occurring microbial organism having an adipate, a 6-aminocaproic acid, caprolactam, or hexametheylenediamine pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine pathway and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2. The invention additionally provides a method for producing adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine. The method can include culturing an adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine producing microbial organism described herein under conditions and for a sufficient period of time to produce adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine.

Description

MICROORGANISMS FOR PRODUCING 6-AMINOCAPROIC ACID

This application claims the benefit of priority of United States Provisional application serial No. 61/500,127, filed June 22, 2011, and United States Provisional application serial No. 61/502,294, filed June 28, 2011, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, and more specifically to organisms having adipate, hexamethylenediamine, 6-aminocaproic acid and caprolactam biosynthetic capability.

Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. It can be used is to produce nylon 6,6, a linear polyamide made by condensing adipic acid with

hexamethylenediamine. This is employed for manufacturing different kinds of fibers. Other uses of adipic acid include its use in plasticizers, unsaturated polyesters, and polyester polyols. Additional uses include for production of polyurethane, lubricant components, and as a food ingredient as a flavorant and gelling aid.

Historically, adipic acid was prepared from various fats using oxidation. Some current processes for adipic acid synthesis rely on the oxidation of KA oil, a mixture of

cyclohexanone, the ketone or K component, and cyclohexanol, the alcohol or A component, or of pure cyclohexanol using an excess of strong nitric acid. There are several variations of this theme which differ in the routes for production of KA or cyclohexanol. For example, phenol is an alternative raw material in KA oil production, and the process for the synthesis of adipic acid from phenol has been described. The other versions of this process tend to use oxidizing agents other than nitric acid, such as hydrogen peroxide, air or oxygen.

In addition to hexamethylenediamine (HMD A) being used in the production of nylon-6,6 as described above, it is also utilized to make hexamethylene diisocyanate, a monomer feedstock used in the production of polyurethane. The diamine also serves as a cross-linking agent in epoxy resins. HMD A is presently produced by the hydrogenation of adiponitrile. Caprolactam is an organic compound which is a lactam of 6-aminohexanoic acid (ε- aminohexanoic acid, 6-aminocaproic acid). It can alternatively be considered cyclic amide of caproic acid. One use of caprolactam is as a monomer in the production of nylon-6.

Caprolactam can be synthesized from cyclohexanone via an oximation process using hydroxylammonium sulfate followed by catalytic rearrangement using the Beckmann rearrangement process step.

Methods for effectively producing commercial quantities of compounds such as

hexamethylenediamine, 6-aminocaproic acid, levulinic acid and carpolactamare described herein and include related advantages. SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexametheylenediamine pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of adipate, 6- aminocaproic acid, caprolactam or hexametheylenediamine by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2. The invention additionally provides a method for producing adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine. The method can include culturing an adipate, 6- aminocaproic acid, caprolactam or hexametheylenediamine producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an adipate, 6-aminocaproic acid, caprolactam, or hexametheylenediamine pathway enzyme in a sufficient amount to produce the respective product and and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2, under conditions and for a sufficient period of time to produce adiapte, 6-aminocaproic acid, caprolactam or

hexametheylenediamine. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an exemplary pathway for adipate degradation in the peroxisome of

Penicillium chrysogenum.

Figure 2 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 shows an exemplary pathway for adipate formation via the 3-oxoadipate pathway.

Figure 4 show the similar enzyme chemistries of the last three steps of the 3-oxoadipate pathway for adipate synthesis and the reductive TCA cycle.

Figure 5 shows an exemplary pathway for synthesis of adipic acid from glucose via cis,cis- muconic acid. Biosynthetic intermediates (abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvic acid (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 ( roFFBR), (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 taken from Niu et al, Biotechnol. Prog.

18:201-211 (2002)).

Figure 6 shows an exemplary pathway for adipate synthesis via alpha-ketoadipate using alpha-ketoglutarate as a starting point. Figure 7 shows an exemplary pathway for synthesis of adipate using lysine as a starting point.

Figure 8 shows an exemplary caprolactam synthesis pathway using adipyl-CoA as a starting point.

Figure 9 shows exemplary adipate synthesis pathways using alpha-ketoadipate as a starting point. Figure 10 shows exemplary pathways from succinyl-CoA and acetyl-CoA to

hexamethylenediamine (HMD A) 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- pentenoate 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 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-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 shows pathways to 6-aminocaproate from pyruvate and succinic semialdehyde. 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-AHD decarboxylase, J) OHED aminotransferase and/or OHED oxidoreductase (aminating), 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-l,7-dioate, OHED = 2-oxohept-4-ene- 1,7-dioate, 2-OHD = 2-oxoheptane-l,7-dioate, 2-AHE = 2-aminohept-4-ene-l,7-dioate, 2- AHD = 2-aminoheptane- 1,7-dioate, and 6-OHE = 6-oxohex-4-enoate.

Figure 13 shows pathways to hexamethylenediamine from 6-aminocapropate. 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-acetamidohexanamine N-acetyltransferase and/or 6- acetamidohexanamine hydrolase (amide), 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 are: 6- AAHOP = [(6-acetamidohexanoyl)oxy]phosphonate and 6-AHOP = [(6- aminohexanoyl)oxy]phosphonate.

Figure 14 shows: A) the acetyl-CoA cycle of arginine biosynthesis. Reactions (1) and (2) are catalyzed by ornithine acetyltransferase with acetylglutamate synthase and ornithine acyltransferase functionality. Reaction 3 is a lumped reaction catalyzed by acetylglutamate kinase, N-acetylglutamylphosphate reductase, and acetylornithine aminotransferase; B) the acetyl-CoA cycle of HMD A biosynthesis. Reactions (1) and (2) are catalyzed by HMD A acetyltransferase. Reaction (3) is a lumped reaction that includes all pathways to 6- acetamidohexanamine from 6-acetamidohexanoate shown in Figure 13.

Figure 15 shows the growth of E. coli in media containing various concentrations of 6-ACA. E. coli was inoculated into media and grown in either aerobic (left and right bars) or anaerobic (middle bars) conditions. The cultures were grown for 48 hrs during the first trial and 30 hrs for a second trial under aerobic conditions (right bars).

Figure 16 shows the tolerance of E. coli when exposed to 6-ACA. Midlog (OD600 = 0.3, lower dashed line) or early stationary (OD600 = 0.6, upper dashed line) cells were spun down and resuspended in fresh M9-Glucose medium with various concentrations of 6-ACA. After overnight growth, cultures were measured for growth by measuring OD600. Figure 17 shows the ethanol production from cultures exposed to various concentrations of 6- ACA. Midlog or early stationary cells were spun down and resuspended in fresh M9-Glucose medium with various concentrations of 6-ACA. After overnight growth, cultures were measured for growth by measuring OD600 and metabolic activity assayed by ethanol production.

Figure 18, panels A and B, show the growth in various concentrations of 6-ACA with and without glycine betaine. Panel A. OD600 measurements of medium inoculated with midlog cultures of E. coli with various concentrations of 6-ACA with (right bars) and without (left bars) 2 mM glycine betaine. Panel B. Photograph showing the growth of same cultures in the anaerobic bottles.

Figure 19 shows LC/MS analysis of in vitro thiolase reactions. Succinyl-CoA and acetyl-CoA were added to His-tagged, purified thiolases at a ratio of 2: 1 (succinyl-CoA:acetyl-CoA). Reactions were analyzed by LC/MS and quantified by comparison to a standard for acetoacetyl-CoA or peak area determined for 3-oxoadipyl-CoA (β-ketoadipyl-CoA).

Figure 20 shows exemplary pathways from glutamate to hexamethylenediamine (HMD A) and 6-aminocaproate. The enzymes are designated 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) homo lysine decarboxylase, I) 6-aminopimeloyl-CoA hydrolase, transferase and/or ligase, J) 2- aminopimelate decarboxylase. The enzyme commission number indicated for each reaction is described in Example XXVI below.

Figure 21 shows exemplary pathways from glutaryl-CoA to hexamethylenediamine (HMD A) and 6-aminocaproate. The enzymes are designated as follows: A) glutaryl-CoA beta- ketothiolase, B) 3-oxopimeloyl-CoA hydrolase, transferase and/or ligase, C) 3-oxopimelate reductase, D) 3-oxo-l-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 Co A 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-aminopimeloylphosphonate 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-l-carboxyheptanal 3 -aminotransferase and/or 3-aminating oxidoreductase. The enzyme commission number indicated for each reaction is described in XXVI below. Figure 22 shows an exemplary pathway from pyruvate and 4-aminobutanal to

hexamethylenediamine (HMD A). 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, G) 6-aminohexanal aminotransferase and/or 6-aminohexanal aminating oxidoreductase. The enzyme commission number indicated for each reaction is described in Example XXVI below.

Figure 23 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 shows exemplary pathways from 6-aminocaproate to hexamethylenediamine. This figure depicts additional pathways further to those presented in Figure 13. The enzymes are designated 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 and/or 6-acetamidohexanamine hydrolase (amide), 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 number indicated for each reaction is described in Example XXVI below. Figure 25 shows exemplary pathways from succinyl-CoA and acetyl-CoA to

hexamethylenediamme (HMD A), caprolactam or levulinic acid. Pathways for the production of adipate, 6-aminocaproate, caprolactam, hexamethylenediamme and levulinic acid from succinyl-CoA and acetyl-CoA are depicted. This figure depicts additional pathways further to those presented 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-pentenoate 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 shows exemplary pathways from 2-amino-7-oxosubarate to hexamethylenediamme (HMDA) and 6-aminocaproate. The enzymes are designated 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-oxosubarate amino acid decarboxylase, 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 number indicated for each reaction is described in Example XXVI below. Figure 27 shows an exemplary pathway from glutamate-5-semialdehyde to 2-amino-7- oxosubarate. The enzymes are designated as follows: A) 2-amino-5-hydroxy-7-oxosubarate aldolase, B) 2-amino-5-hydroxy-7-oxosubarate dehydratase, C) 2-amino-5-ene-7-oxosubarate reductase. Figure 28 shows the 6-ACA versus growth yields for an ADHEr, LDH D mutant.

Figure 29 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT, LDH D mutant.

Figure 30 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT, LDH D, THD2 mutant. Figure 31 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT, LDH D, GLUDy mutant.

Figure 32 shows the 6-ACA versus growth yields for a PGI, EDA mutant or a PGI, PGDHy mutant.

Figure 33 shows the 6-ACA versus growth yields for a PGI, EDA, ADHEr mutant or a PGI, PGDHy, ADHEr mutant.

Figure 34 shows the 6-ACA versus growth yields for an ADHEr, PGI, HEX1 mutant.

Figure 35 shows growth-coupled adipate production characteristics of high priority strain designs (grey) compared with that of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. Figure 36 shows the activity of CAR 889 and 891 using 20 mM Adipate. Activity is shown as units per mg of total protein in the crude lysate.

Figure 37 shows the activity of CAR 720, 889, 890, 891 using 50mM 6-aminocaproate. Activity is shown as units per mg of total protein in the crude lysate.

Figure 38, panels A and B, show an exemplary pathway for fixation of C02 to acetyl-CoA and the biosynthesis of adipate, 6-aminocapropate and hexamethylenediamine from acetyl- CoA. Panel A shows the pathways for fixation of C02 to acetyl-CoA using the reductive TCA cycle. Panel B shows exemplary pathways for the biosynthesis of adipate, 6- aminocapropate, and hexamethylenediamine from acetyl-CoA. The enzymatic transformations shown are carried out by the following enzymes: conversion of malonyl-CoA to malonyl-CoA methyl ester by A) malonyl-CoA methyltransferase, conversion of malonyl- CoA methyl ester to 3-oxo-glutaryl-[acp] methyl ester by B) 3-oxo-glutaryl-[acp] mthylester synthase, conversion of 3-oxo-glutaryl-[acp] methyl ester to 3-hydroxyglutaryl-[acp] methyl ester by C) 3-oxo-glutaryl-[acp] methyl ester reductase, conversion of 3 -hydroxy glutaryl- [acp] methyl ester to enoylglutaryl-[acp] methyl ester by D) 3-hydroxyglutaryl-[acp] methylester dehydratase, conversion of enoylglutaryl-[acp] methyl ester to glutaryl-[acp] methyl ester by E) enoylglutaryl-[acp] methyl ester reductase, conversion of glutaryl-[acp] methyl ester to 3-oxo-pimelyl-[acp] methyl ester by F) 3-oxo-pimoloyl-[acp] methyl ester synthase, conversion of 3-oxo-pimelyl-[acp] methyl ester to 3-hydroxypimelyl-[acp] methyl ester by G) 3-oxo-pimeloyl-[acp] methyl ester reductase, conversion of 3-hydroxypimelyl- [acp] methyl ester to enoylpimelyl-[acp] methyl ester by H) 3-hydroxypimeloyl-[acp] methyl ester dehydratase, conversion of enoylpimelyl-[acp] methyl ester to pimelyl-[acp] methyl ester by I) enoylpimeloyl-[acp] methyl ester reductase, conversion of pimelyl-[acp] methyl ester to pimelyl-[acp] by J) pimeloyl-[acp] methyl ester esterase, conversion of pimelyl-[acp] to pimelate by K) thioesterase, conversion of pimelate to 2-hydroxyheptanedioic acid by L) pimelate-2-monooxygenase, conversion of 2-hydroxyheptanedioic acid to a-ketopimelate by M) 2-hydroxyheptanedioic acid dehydrogenase, conversion of a-ketopimelate to 6 oxohexanoate by N) α-ketopimelate decarboxylase, conversion of 6 oxohexanoate to adipate by O) 6-oxohexanoate dehydrogenase, conversion of 6 oxohexanoate to 6-aminocaproate by P) 6-oxohexanoate dehydrogenase (aminating) or 6-oxohexanoate aminotransferase, conversion of 6-aminocaproate to 6-aminohexanal by Q) 6-aminocaproate reductase, conversion of 6-aminohexanal to hexamethylenediamine by R) 6-aminohexanal

dehydrogenase (aminating) or 6-aminohexanal aminotransferase, conversion of a- ketopimelate to 2-aminopimelate by S) α-ketopimelate dehydrogenase (aminating) or a- ketopimelate amino transferase, conversion of 2-aminopimelate to 2-amino7-oxoheptanoate by T) 2-aminopimelate, conversion of 2-aminopimelate to 6-aminocaproate by U) 2- aminopimelate decarboxylase, conversion of 2-amino7-oxoheptanoate to 2,7

diaminoheptanoate by V) 2-amino-7-oxoheptanoate dehydrogenase, conversion of 2,7 diaminoheptanoate to hexamethylenediamine by W) 2,7-diaminoheptanoate decarboxylase and conversion of acetyl-CoA to malonyl-CoA by X) acetyl-CoA carboxylase. Figure 39, panels A and B, show an exemplary pathway for fixation of C02 to succinyl-CoA and acetyl-CoA and the biosynthesis of adipate, 6-aminocapropate, caprolactam and hexamethylenediamine. Panel A shows the pathways for fixation of C02 to succinyl-CoA and acetyl-CoA using the reductive TCA cycle. Panel B shows exemplary pathways for the biosynthesis of adipate, 6-aminocapropate, caprolactam and hexamethylenediamine from succinyl-CoA and acetyl-CoA. The enzymatic transformations shown are carried out by the following enzymes: 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-pentenoate reductase, K) adipyl-CoA-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.

Figure 40, panels A and B, show an exemplary pathway for for fixation of C02 to alpha- ketoglutarate and the biosynthesis of adipate, 6-aminocapropate, caprolactam and

hexamethylenediamine. Panel A shows the pathways for fixation of C02 to alpha- ketoglutarate using the reductive TCA cycle. Panel B shows exemplary pathways for the biosynthesis of adipate, 6-aminocapropate, caprolactam and hexamethylenediamine from alpha-ketoglutarate. The enzymatic transformations shown are carried out by the following enzymes: A) Homocitrate synthase, B) Homocitrate dehydratase, C) cis-Homoaconitate hydratase, D) Threo-isohomocitrate dehydrogenase, E) Alpha-ketoadipyl-CoA synthetase, Alpha-ketoadipyl-CoA transferase or Phosphotransketoadipylase/Alpha-ketoadipate kinase, F) Alpha-ketoadipyl-CoA reductase, G) 2-Hydroxyadipate dehydrogenase, H) 2- Hydroxyadipyl-CoA synthtase, 2-Hydroxyadipyl-CoA transferase, or

phosphotranshydroxyadipylase/2-hydroxyadipate kinase, I) 2-Hydroxyadipyl-CoA

dehydratase, J) 5-Carboxy-2-pentenoyl-CoA reductase, K) adipyl-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.

Figure 41 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. thermoacetica CODH (Moth_1202/1203) or Mtr (Moth l 197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).

Figure 42 shows CO oxidation assay results. Cells (M. thermoacetica or E. coli with the CODH/ ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared. Assays were performed at 55oC at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course.

Figure 43 shows the nucleotide sequence (SEQ ID NO:) of carboxylic acid reductase from Nocardia iowensis (GNM_720), and Figure 43 shows the encoded amino acid sequence (SEQ ID NO:).

Figure 44 shows the nucleotide sequence (SEQ ID NO:) of phosphpantetheine transferase, which was codon optimized, and Figure 44B shows the encoded amino acid sequence (SEQ ID NO:).

Figure 45A shows the nucleotide sequence (SEQ ID NO:) of carboxylic acid reductase from Mycobacterium smegmatis mc(2)155 (designated 890), and Figure 45B shows the encoded amino acid sequence (SEQ ID NO:). Figure 46A shows the nucleotide sequence (SEQ ID NO:) of carboxylic acid reductase from Mycobacterium avium subspecies paratuberculosis K-10 (designated 891), and Figure 46B shows the encoded amino acid sequence (SEQ ID NO:).

Figure 47A shows the nucleotide sequence (SEQ ID NO:) of carboxylic acid reductase from Mycobacterium marinum M (designated 892), and Figure 47B shows the encoded amino acid sequence (SEQ ID NO:).

Figure 48A shows the nucleotide sequence (SEQ ID NO:) of carboxylic acid reductase designated 891GA, and Figure 48B shows the encoded amino acid sequence (SEQ ID NO:). DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cells and organisms having biosynthetic production capabilities for adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The results described herein indicate that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in Escherichia coli and other cells or organisms. Biosynthetic production of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis, including under conditions approaching theoretical maximum growth.

As disclosed herein, a number of metabolic pathways for the production of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid are described. Two routes, the reverse adipate degradation pathway and the 3-oxoadipate pathway, were found to be beneficial with respect to: (i) the adipate yields (92% molar yield on glucose), (ii) the lack of oxygen requirement for adipate synthesis, (iii) the associated energetics, and (iv) the theoretical capability to produce adipate as the sole fermentation product. Metabolic pathways for adipate production that pass through -ketoadipate or lysine are also described but are lower yielding and require aeration for maximum production. A pathway for producing either or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is also disclosed herein.

As disclosed herein, a number of exemplary pathways for biosynthesis of adipate are described. One exemplary pathway involves adipate synthesis via a route that relies on the reversibility of adipate degradation as described in organisms such as P. chrysogenum (see Examples I and II). A second exemplary pathway entails the formation of 3-oxoadipate followed by its reduction, dehydration and again reduction to form adipate (see Examples III and IV). The adipate yield using either of these two pathways is 0.92 moles per mole glucose consumed. The uptake of oxygen is not required for attaining these theoretical maximum yields, and the energetics under anaerobic conditions are favorable for growth and product secretion. A method for producing adipate from glucose-derived cis,cis-muconic acid was described previously (Frost et al., United States Patent No. 5,487,987, issued January 30, 1996)(see Example V). Advantages of the embodiments disclosed herein over this previously described method are discussed. Metabolic pathways for adipate production that pass through -ketoadipate (Example VI) or lysine (Example VII) precursors are lower yielding and require aeration for maximum production. A pathway for producing either or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is described (see Example VIII and IX). Additional pathways for producing adipate are described in Examples X and XI. Pathways for producing any one, two, three or all four of 6-aminocaproate, caprolactam, hexamethylenediamine and levulinic acid from succinyl-CoA and acetyl-CoA are described in Examples XII, XXVIII. Several pathways for the production of 6-aminocaproate from succinic semialdehyde and pyruvate are described in Example XIX. Several pathways for the production of

hexamethylenediamine from 6-aminocaproate are described in Examples XX and XXVII. A pathway for producing either or both 6-aminocaproate and hexamethylenediamine from glutamate is described in Examples XXIV and XXV. Several pathways for the production of hexamethylenediamine from glutaryl-CoA and at least one pathway for production of 6- aminocaproate from glutaryl-CoA are described in Examples XXIV and XXV. A pathway for producing 6-aminocaproate from homolysine is described in Example XXV. Pathways for producing hexamethylenediamine from 2-amino-7-oxosubarate are described in Example XXIV. Several pathways for producing 6-aminocaproate are described in Example XXV. Exemplary genes and enzymes required for constructing microbes with these capabilities are described as well as methods for cloning and transformation, monitoring product formation, and using the engineered microorganisms for production.

As disclosed herein, six different pathways for adipic acid synthesis using glucose/sucrose as a carbon substrate are described. For all maximum yield calculations, the missing reactions in a given pathway were added to the E. coli stoichiometric network in SimPheny that is similar to the one described previously (Reed et al., Genome Biol. 4:R54 (2003)). Adipate is a charged molecule under physiological conditions and was assumed to require energy in the form of a proton-based symport system to be secreted out of the network. Such a transport system is thermodynamically feasible if the fermentations are carried out at neutral or near- neutral pH. Low pH adipic acid formation would require an ATP-dependant export mechanism, for example, the ABC system as opposed to proton symport. The reactions in the pathways and methods of implementation of these pathways are described in Examples I- XI. As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes within a adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms "microbial," "microbial organism" or "microorganism" is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

As used herein, "adipate," having the chemical formula -OOC-(CH2)4-COO- (see Figure 2) (IUPAC name hexanedioate), is the ionized form of adipic acid (IUPAC name hexanedioic acid), and it is understood that adipate and adipic acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH.

As used herein, "6-aminocaproate," having the chemical formula -OOC- (CH2)5-NH2 (see Figures 8 and 12), is the ionized form of 6-aminocaproic acid (IUPAC name 6- aminohexanoic acid), and it is understood that 6-aminocaproate and 6-aminocaproic acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. As used herein, "caprolactam" (IUPAC name azepan-2-one) is a lactam of 6-aminohexanoic acid (see Figure 8).

As used herein, "hexamethylenediamine," also referred to as 1 ,6-diaminohexane or 1,6- hexanediamine, has the chemical formula H₂N(CH₂)₆NH₂ (see Figures 10, 11 and 13).

As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1 % oxygen. As used herein, the term "osmoprotectant" when used in reference to a culture or growth condition is intended to mean a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, for example, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine.

As used herein, the term "growth-coupled" when used in reference to the production of a biochemical is intended to mean that the biosynthesis of the referenced biochemical is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.

As used herein, "metabolic modification" is intended to refer to a biochemical reaction that is altered from its naturally occurring state. Metabolic modifications can include, for example, elimination of a biochemical reaction activity by functional disruptions of one or more genes encoding an enzyme participating in the reaction. Sets of exemplary metabolic modifications are described herein (see Example XXX).

As used herein, the term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention. "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are

homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others. A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. In gene disruption strategies, evolutionally related genes can also be disrupted or deleted in a host microbial organism, paralogs or orthologs, to reduce or eliminate activities to ensure that any functional redundancy in enzymatic activities targeted for disruption do not short circuit the designed metabolic modifications.

Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: - 2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

Disclosed herein are non-naturally occurring microbial organisms capable of producing adipate, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid . For example, an adipate pathway can be a reverse adipate degradation pathway (see

Examples I and II). For example, a non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. In addition, an adipate pathway can be through a 3-oxoadipate pathway (see Examples III and IV). A non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3- oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.

Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6- aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase and transaminase (see Examples VIII and IX). Alternatively, 6-aminocaproate dehydrogenase can be used to convert adipate semialdehyde to form 6-aminocaproate (see Figure 8). A non- naturally occurring microbial organism can also have a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including CoA- dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase (see Examples VIII and IX).

As disclosed herein, a 6-aminocaproic acid or caprolactam producing microbial organism can produce 6-aminocaproic acid and/or caprolactam from an adipyl-CoA precursor (see Figure 8 and Examples VIII and IX). Therefore, it is understood that a 6-aminocaproic acid or caprolactam producing microbial organism can further include a pathway to produce adipyl- CoA. For example an adipyl-CoA pathway can include the enzymes of Figure 2 that utilize succinyl-CoA and acetyl-CoA as precursors through the production of adipyl-CoA, that is, lacking an enzyme for the final step of converting adipyl-CoA to adipate. Thus, one exemplary adipyl-CoA pathway can include succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2- pentenoyl-CoA reductase.

In addition, as shown in Figure 1 , an adipate degradation pathway includes the step of converting adipate to adipyl-CoA by an adipate CoA ligase. Therefore, an adipyl-CoA pathway can be an adipate pathway that further includes an enzyme activity that converts adipate to adipyl-CoA, including, for example, adipate-CoA ligase activity as in the first step of Figure 1 or any of the enzymes in the final step of Figure 2 carried out in the reverse direction, for example, any of adipyl-CoA synthetase (also referred to as adipate Co-A ligase), phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl- CoA hydrolase. An enzyme having adipate to adipyl-CoA activity can be an endogenous activity or can be provided as an exogenous nucleic acid encoding the enzyme, as disclosed herein. Thus, it is understood that any adipate pathway can be utilized with an adipate to adipyl-CoA enzymatic activity to generate an adipyl-CoA pathway. Such a pathway can be included in a 6-aminocaproic acid or caprolactam producing microbial organism to provide an adipyl-CoA precursor for 6-aminocaproic acid and/or caprolactam production.

An additional exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see Figure 6 and Example VI). For example, a non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including homocitrate synthase, homoaconitase, homoisocitrate dehydrogenase, 2- ketoadipate reductase, alpha-hydroxyadipate dehydratase and oxidoreductase. A further exemplary adipate pathway utilizes a lysine dedgradation pathway (see Figure 7 and Example VII). Another non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including carbon nitrogen lyase, oxidoreductase, transaminase and oxidoreductase.

Yet another exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see Figure 9 and Examples X and XI). Thus, a non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase/alpha-ketoadipate kinase or alpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2- hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Additionally, a non-naturally occurring microbial organism can have an adipate pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, the adipate pathway including 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase,

phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl- CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase.

As disclosed herein, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3- hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6- aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl- CoA hydrolase (see Examples XII and XIII; steps A/B/C/D/K/L/M of Figure 11). The invention additionally provides a non-naturally occurring microbial organism, including a microbial organism having a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including 3-oxo-6- aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6- aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6- aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehydratase; and 6-aminohex-2- enoate reductase (see Examples XII and XIV; steps A/E/F/G/H/I/J of Figure 11).

In another embodiment, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 6-aminocaproyl- CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase (see Examples XII and XV; steps K/L of Figure 11). Such a non-naturally occurring microbial organism containing a caprolactam pathway can further comprise a 6-aminocaproic acid pathway (see Figure 11). Exemplary 6-aminocaproic acid pathways include the 6-aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase; and transaminase or 6-aminocaproate dehydrogenase or the 6-aminocaproic acid pathway including 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 reductase; 3- hydroxy-6-aminohexanoate dehydratase; and 6-aminohex-2-enoate reductase (steps

A/E/F/G/H/I/J of Figure 11). It is understood that these or other exemplary 6-aminocaproic acid pathways disclosed herein can additionally be included in a microbial organism having a caprolactam pathway, if desired. The invention also provides a non-naturally occurring microbial organism, including a microbial organism having a hexamethylenediamine pathway including at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in a sufficient amount to produce hexamethylenediamine, the hexamethylenediamine pathway including 6-aminocaproyl-CoA/acyl-CoA transferase or 6- aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde forming); and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase (see Example XII and XVI; steps K/L/N/O/P of Figure 11). Such a non-naturally occurring microbial organism containing a hexamethylenediamine pathway can further comprise a 6- aminocaproic acid pathway (see Figure 11). Exemplary 6-aminocaproic acid pathways include the 6-aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase; and transaminase or 6-aminocaproate dehydrogenase or the 6-aminocaproic acid pathway including 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 reductase; 3-hydroxy-6-aminohexanoate dehydratase; and 6-aminohex-2-enoate reductase (steps A/E/F/G/H/I/J of Figure 11). It is understood that these or other exemplary 6-aminocaproic acid pathways disclosed herein can additionally be included in a microbial organism having a hexamethylenediamine pathway, if desired. In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3- oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and 6- aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps A/B/C/D of Figure 11). Also provided is a non-naturally occurring microbial organism having a

hexamethylenediamine pathway including at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in a sufficient amount to produce hexamethylenediamine, the hexamethylenediamine pathway including 3-oxo-6- aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6- aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase (aldehyde forming); and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase (see Examples XII and XVIII; steps A/B/C/D/N/O/P of Figure 11).

In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase, a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase, a 2-oxohept-4-ene-l,7-dioate (OHED) reductase, a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, an adipate semialdehyde oxidoreductase (aminating), a 2- oxohept-4-ene-l,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate (6-OHE) reductase, a 2-oxoheptane-l,7-dioate (2-OHD) aminotransferase, a 2-oxoheptane-l,7-dioate (2-OHD) oxidoreductase (aminating), a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase, a 2- oxohept-4-ene-l,7-dioate (OHED) aminotransferase, a 2-oxohept-4-ene-l,7-dioate (OHED) oxidoreductase (aminating), a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase, a 4- hydroxy-2-oxoheptane-l,7-dioate (HODH) formate-lyase, a 4-hydroxy-2-oxoheptane-l,7- dioate (HODH) dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl- CoA reductase, a adipyl-CoA dehydrogenase, a 2-oxohept-4-ene-l,7-dioate (OHED) formate-lyase, a 2-oxohept-4-ene-l,7-dioate (OHED) dehydrogenase, a 2-oxoheptane-l,7- dioate (2-OHD) formate-lyase, a 2-oxoheptane-l,7-dioate (2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme (see Examples XIX and XXI; steps A-Q of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde

dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase.

The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/D/E of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set encodes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase

(aminating). The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6- OHE reductase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/F/G/E of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase

(aminating).

The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; or a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/J/D/I of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate

decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); or a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/C/H/I of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate

decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD

oxidoreductase (aminating); and a 2-AHD decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3- dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/L/M/N/O/E of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non- naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6- ACA pathway enzymes, where the set encode an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3- hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating).

The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl- CoA reductase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/E of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a

phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non- naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6- ACA pathway enzymes, where the set encode an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED

dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase

(aminating). The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/Q/O/E of Figure 12). In a further aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a 6-amino-7-carboxyhept- 2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), or a 2- aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode a glutamyl-CoA transferase or glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6- aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6- amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate

aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/J/T/AA of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; and a 2-aminopimelate decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a homolysine 2- monooxygenase (see Examples XXV and XXVI; steps A of Figure 23). In a further aspect, the 6-ACA pathway includes hydrolysis of the 6-aminohexanamide product by a dilute acid or base to convert 6-aminohexanamide to 6-aminocaproate (see Examples XXV and XXVI; step B of Figure 23). The invention additionally provides a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an adipate reductase, an adipate kinase or an adipylphosphate reductase (see Example XXVIII; steps X/Y/Z of Figure 25 and Example XXXI). In a further aspect, the 6-ACA pathway includes an adipate reductase. In another further aspect, the 6-ACA pathway includes an adipate kinase and an adipylphosphate reductase. In still another aspect, the microbial organism having the 6-aminocaproic acid (6-ACA) pathway above further comprises an adipate pathway, a caprolactam pathway and/or a hexamethylenediamine pathway described here (see Example XXVIII; steps A-W of Figure 25).

In one embodiment, the invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino- 7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate oxidoreductase, a 2- aminopimelate decarboxylase, a 6-aminohexanal oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a 2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and XXVI; steps A/B/D/E/F/G/I of Figure 26). In a further aspect, the microbioal organism has a 2-amino-7-oxosubarate pathway having at least one exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2- amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7- oxosubarate aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a 2-amino-5-ene- 7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).

In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a 2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of Figure 26). In yet another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2- amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of Figure 26). In still yet another embodiment of the invention, the the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal oxidoreductase (see Example XXV; steps I/G/F of Figure 26). In a further aspect of each of the above embodiments, the microbioal organism has a 2-amino-7-oxosubarate pathway having a second set of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the 2-amino-7- oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate aldolase; a 2-amino-5- hydroxy-7-oxosubarate dehydratase; and a 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).

In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase, an

[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), a 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase, an [(6- acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a 6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase (aminating), a 6- acetamidohexanamine N-acetyltransferase, a 6-acetamidohexanamine hydrolase (amide), a 6- acetamidohexanoate CoA transferase, a 6-acetamidohexanoate CoA ligase, a 6- acetamidohexanoyl-CoA oxidoreductase, a [(6-acetamidohexanoyl)oxy]phosphonate (6- AAHOP) acyltransferase, a [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, a 6-aminocaproate CoA transferase and a 6-aminocaproate CoA ligase (see Examples XX and XXI; steps A-N of Figure 13).

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; or a 6- aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid

semialdehyde aminotransferase (see Examples XX and XXI; steps A/B/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6- aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6- aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase (animating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/L/N/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate kinase; a 6-AHOP

acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps M/N/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); or a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/H of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal

aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); or a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide). The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); or a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/G of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce

HMDA, the HMDA pathway includinga glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6- aminopimeloyl-CoA dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6- aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A-H of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutamyl-CoA transferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3- hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine

decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-l-carboxyheptanal aminotransferase, a 3-oxo-l-carboxyheptanal aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7- aminoheptanoate 3 -aminating oxidoreductase, a 3-oxopimelate kinase, a 5- oxopimeloylphosphonate reductase, a 3-oxopimelate Co A transferase, a 3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxopimelate

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7- aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7- diaminoheptanoate 2,3-aminomutase, a homolysine decarboxylase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a 2- aminopimelate CoA ligase, a 2-aminopimelate reductase, a 6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7- oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI; Figure 21).

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-l-carboxyheptanal 7-aminotransferase, a 3-oxo-l-carboxyheptanal 7- aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl- CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l- carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating

oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-l-carboxyheptanal 7- aminotransferase, a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7- aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating

oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3 -oxo- 1 -carboxyheptanal 7-aminotransferase or a 3 -oxo- 1 -carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-l-carboxyheptanal 7-aminotransferase, 3-oxo-l-carboxyheptanal 7- aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homo lysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l-carboxyheptanal 7- aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3-oxo-l-carboxyheptanal 3 -aminotransferase, a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMD A) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-l-carboxyheptanal 3- aminotransferase, a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7- oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-l-carboxyheptanal 3 -aminotransferase, a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7- oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7- aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B//J/0/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7- oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2- amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2- amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7- aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5- aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3- amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3- aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/W/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate kinase, a 6-aminopimeloylphosphonate reductase, a 2- amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/U/X/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, a 6- aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate 7- aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2- aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMD A) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A, the HMDA pathway including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a 2-oxo-4- hydroxy-7-aminoheptanoate dehydratase, a 2-oxo-7-aminohept-3-enoate reductase, a 2-oxo- 7-aminoheptanoate aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate decarboxylase, a 6- aminohexanal aminotransferase or a 6-aminohexanal aminating oxidoreductase (see

Examples XXIV and XXVI; steps A-G of Figure 22). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-oxo-4-hydroxy-7- aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7- aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7- aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2- oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminotransferase or a 6-aminohexanal aminating oxidoreductase.

The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase (aminating), 6- aminocaproate N-acetyltransferase, a 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or D/P/G/H of Figure 24 and Example XXXI). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes 6-aminocaproate N-acetyltransferase; 6- acetamidohexanoate reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and 6-acetamidohexanamine N-acetyltransferase or 6- acetamidohexanamine hydrolase (amide). The invention additionally provides a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-amino-7- oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 6- aminohexanal aminating oxidoreductase, a 6-aminohexanal aminotransferase, a 2-amino-7- oxoheptanoate decarboxylase, a homolysine decarboxylase, a 2-amino-7-oxosubarate amino acid decarboxylase, a 2-oxo-7-aminoheptanoate aminating oxidoreductase, a 2-oxo-7- aminoheptanoate aminotransferase, a 2-amino-7-oxosubarate aminating oxidoreductase, a 2- amino-7-oxosubarate aminotransferase, a 2,7-diaminosubarate decarboxylase, a 2-amino-7- oxoheptanoate aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI; Steps A/B/C/G/H/I/J/K/L/M of Figure 26). In a further aspect, the microbioal organism has a 2-amino-7-oxosubarate pathway having at least one exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).

In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/H of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7- oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a 2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps I/J/H of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7- oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6- aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase (see

Examples XXIV and XXVI; steps I/G/C of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7- oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6- aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase (see

Examples XXIV and XXVI; steps A/B/C of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7- oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H of Figure 26). In a further aspect of each of the above embodiments, the microbioal organism has a 2-amino-7-oxosubarate pathway having a second set of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the 2-amino-7- oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate aldolase; a 2-amino-5- hydroxy-7-oxosubarate dehydratase; and a 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).The invention additionally provides a non-naturally occurring microbial organism having a levulinic acid (LA) pathway including at least one exogenous nucleic acid encoding a LA pathway enzyme expressed in a sufficient amount to produce LA, the LA pathway including a 3-oxoadipyl-CoA thiolase, a 3- oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, or a 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of Figure 25). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding LA pathway enzymes, wherein the set encodes a 3-oxoadipyl-CoA thiolase; a 3-oxoadipyl-CoA/acyl-CoA transferase, a 3- oxoadipyl-CoA synthase, or a 3-oxoadipyl-CoA hydrolase; and a 3-oxoadipate

decarboxylase.

A non-naturally occurring microbial organism disclosed herein can have, for example, a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, wherein the non-naturally occurring microbial organism includes at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product, as disclosed herein. Thus, a non-naturally occurring microbial organism can contain at least one exogenous nucleic acid encoding a polypeptide, where the polypeptide is an enzyme or protein that converts the substrates and products of an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, such as that shown in Figures 2, 3, 8, 9, 10, 11, 12, 13, 20-27 and 38-40.

For example, a non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from succinyl-CoA and acetyl- CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate (see Figure 2). Additionally, a non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from succinyl- CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; 3-oxoadipate to 3-hydroxyadipate; 3-hydroxyadipate to hexa-2-enedioate (also referred to herein as 5- carboxy-2-pentenoate); hexa-2-enedioate to adipate (see Figure 3). Also, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (see Figure 8). Furthermore, a non-naturally occurring microbial organism can have a caprolactam pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from adipyl-CoA to adipate semialdehyde; adipate

semialdehyde to 6-aminocaproate; and 6-aminocaproate to caprolactam. Additionally, a non- naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from alpha-ketoadipate to alpha-ketoadipyl-CoA; alpha- ketoadipyl-CoA to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl- CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (see Figure 9). Also, a non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from alpha-ketoadipate to 2-hydroxyadipate; 2- hydroxyadipate to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl- CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (Figure 9).

Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproyl-CoA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 4-aminobutyryl- CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to 3- hydroxy-6-aminohexanoyl-CoA; 3-hydroxy-6-aminohexanoyl-CoA to 6-aminohex-2-enoyl- CoA; 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA (Figure 11). Additional substrates and products of such a pathway can include 6-aminocaproyl-CoA to 6-aminocaproate; 6- aminocaproyl-CoA to caprolactam; or 6-aminocaproyl-CoA to 6-aminocaproate

semialdehyde and 6-aminocaproate semialdehyde to hexamethylenediamine (Figure 11). A non-naturally occurring microbial organism also can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to 3-oxo-6- aminohexanoate; 3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate; 3-hydroxy-6- aminohexanoate to 6-aminohex-2-enoate; and 6-aminohex-2-enoate to 6-aminocaproate (Figure 11). Additional substrates and products of such a pathway can include 6- aminocaproate to caprolactam or 6-aminocaproate to 6-aminocaproyl-CoA, 6-aminocaproyl- CoA to 6-aminocaproate semialdehyde, and 6-aminocaproate semialdehyde to

hexamethylenediamine (Figure 11).

Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-l,7-dioate; 4-hydroxy-2-oxoheptane-l,7- dioate (HODH) to 2-oxohept-4-ene-l,7-dioate (OHED): 2-oxohept-4-ene-l,7-dioate (OHED) to 2-oxoheptane-l,7-dioate (2-OHD); 2-oxoheptane-l,7-dioate (2-OHD) to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (Figure 12). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4- hydroxy-2-oxoheptane-l,7-dioate; 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) to 2- oxohept-4-ene-l,7-dioate (OHED); 2-oxohept-4-ene-l,7-dioate (OHED) to 6-oxohex-4- enoate (6-OHE): 6-oxohex-4-enoate (6-OHE) to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (Figure 12). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane- 1,7-dioate; 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) to 2-oxohept-4-ene-l,7-dioate (OHED); 2-oxohept-4-ene-l,7-dioate (OHED) to 2-aminohept-4-ene-l,7-dioate (2-AHE); 2- aminohept-4-ene-l,7-dioate (2-AHE) to 2-aminoheptane-l,7-dioate (2-AHD); and 2- aminoheptane-l,7-dioate (2-AHD) to 6-aminocaproate (Figure 12). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4- hydroxy-2-oxoheptane-l,7-dioate; 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) to 2- oxohept-4-ene-l,7-dioate (OHED); 2-oxohept-4-ene-l,7-dioate (OHED) to 2-oxoheptane- 1,7-dioate (2-OHD); 2-oxoheptane-l,7-dioate (2-OHD) to 2-aminoheptane-l,7-dioate (2- AHD); and 2-aminoheptane-l,7-dioate (2-AHD) to 6-aminocaproate (Figure 12). A non- naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-l,7-dioate; 4-hydroxy-2-oxoheptane-l,7- dioate (HODH) to 3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA to adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (Figure 12). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane- 1,7-dioate; 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) to 2-oxohept-4-ene-l,7-dioate (OHED); 2-oxohept-4-ene-l,7-dioate (OHED) to 2,3-dehydroadipyl-CoA; 2,3- dehydroadipyl-CoA to adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (Figure 12). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane- 1,7-dioate; 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) to 2-oxohept-4-ene-l,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 adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (Figure 12). Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate to glutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl- CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7- carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (Figure 20). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3- oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (Figure 21). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from homolysine to 6- aminohexanamide; and 6-aminohexanamide to 6-aminocaproate (Figure 23). A non-naturally occurring microbial organism alternatively can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from adipate to adipate semialdehyde; adipate to adipylphospate; and adipylphospate to adipate semialdehyde (Figure 25). Additionally, a non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 2-amino-7- oxosubarate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal; 6- aminohexanal to 6-aminocaproate; 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate; 2- amino-7-oxoheptanoate to 6-aminohexanal; 2-amino-7-oxoheptanoate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (Figure 26). A non-naturally occurring microbial organism can further have a 2-amino-7-oxosubarate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate-5-semialdehyde to 2-amino-5-hydroxy-7- oxosubarate; 2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosubarate; and 2- amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (Figure 27).Additionally, a non- naturally occurring microbial organism can have an hexamethylenediamine (HMD A) pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to [(6-aminohexanoyl)oxy]phosphonate (6-AHOP); [(6-aminohexanoyl)oxy]phosphonate (6- AHOP) to 6-aminocaproaic semialdehyde; and 6-aminocaproaic semialdehyde to

hexamethylenediamine (Figure 13). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to [(6-aminohexanoyl)oxy]phosphonate (6-AHOP); [(6- aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA to 6-aminocaproaic semialdehyde; and 6-aminocaproaic semialdehyde to

hexamethylenediamine (Figure 13). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA to 6- aminocaproic semialdehyde; and 6-aminocaproic semialdehyde to hexamethylenediamine (Figure 13). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to [(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP); [(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanal; 6- acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to

hexamethylenediamine (Figure 13). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to 6- acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal; 6- acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to

hexamethylenediamine (Figure 13). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to [(6- acetamidohexanoy)oxy]phosphonate (6-AAHOP); [(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6- acetamidohexanal; 6-acetamidohexanal to 6-acetamidohexanamine; and 6- acetamidohexanamine to hexamethylenediamine (Figure 13).

Additionally, a non-naturally occurring microbial organism can have an

hexamethylenediamine (HMD A) pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate to glutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3- oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino- pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl- CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; -amino- 7-oxoheptanoate to homolysine; and homolysine to HMDA (Figure 20). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-oxo-l-carboxy heptanal; 3-oxo-l- carboxy heptanal to 3-oxo-7-amino heptanoate; 3-oxo-7-amino heptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3- oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl phosponate; 5-oxopimeloyl phosponate to 3-oxo-l-carboxy heptanal; 3-oxo-l-carboxy heptanal to 3-oxo-7-amino heptanoate; 3-oxo-7-amino heptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine and homolysine to HMDA (Figure 21). A non- naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA; 5-oxopimeloyl- CoA to 3-oxo-l-carboxy heptanal; 3-oxo-l-carboxy heptanal to 3-oxo-7-amino heptanoate; 3-oxo-7-amino heptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine and homo lysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3- oxopimelate to 3-oxo-l-carboxy heptanal; 3-oxo-l-carboxy heptanal to 3-amino-7- oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homo lysine; and homo lysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3- oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA; 5-oxopimeloyl-CoA to 3-oxo-l-carboxy heptanal; 3-oxo-l-carboxy heptanal to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homo lysine; and homo lysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3- oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl phosponate; 5-oxopimeloyl phosponate to 3-oxo-lcarboxy heptanal; 3-oxo-l-carboxy heptanal to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA (Figure 21). A non- naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate; 2-amino- 7-axoheptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 5-aminopimeloyl phosphonate; 5-aminopimeloyl phosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate; 2-amino-7-axoheptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3- oxopimelate; 3-oxopimelate to 5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7- oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate; 2-amino-7- axoheptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl- CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 5- aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate; 3-amino-7- oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism

alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3- oxopimelate to 3-aminopimelate; 3-aminopimelate to 5-aminopimeloyl phosphonate; 5- aminopimeloyl phosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7- diamino heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3- oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to 2-amino-7-oxoheptanoate; 2- amino-7-oxoheptanoate to homolysine; and homolysine to HMDA (Figure 21). A non- naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3- oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to 6-aminopimeloylphosphonate; 6- aminopimeloylphosphonate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homo lysine; and homo lysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3- oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; 2- aminopimelate to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7- oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine; and homolysine to HMDA (Figure 21). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate; 2-oxo-4-hydroxy 7-aminoheptanoate to 2-oxo-7- amino hept-3 -enoate; 2-oxo-7-amino hept-3-enoate to 2-oxo-7-amino heptanoate; 2-oxo-7- amino heptanoate to homolysine; andhomo lysine to HMDA (Figure 22). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from pyruvate and 4-aminobutanal to 2-oxo-4- hydroxy 7-aminoheptanoate; 2-oxo-4-hydroxy 7-aminoheptanoate to 2-oxo-7-amino hept-3 - enoate; 2-oxo-7-amino hept-3 -enoate to 2-oxo-7-amino heptanoate; 2-oxo-7-aminoheptanoate to 6-aminohexanal; and 6-aminohexanal to HMDA (Figure 22). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-aminocaproic semialdehyde; and 6- aminocaproic semialdehyde to HMDA (Figure 24). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to 6-acetamidohexanal; 6-acetamidohexanal to 6-acetamidohexanamine; 6- acetamidohexanamine to HMDA (Figure 24). A non-naturally occurring microbial organism alternatively can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HMDA; 2-amino-7-oxosubarate to 2-oxo-7- aminoheptanoate; 2-amino-7-oxoheptanoate to homolysine; homolysine to HMD A; 2-oxo-7- aminoheptanoate to homolysine; 2-oxo-7-aminoheptanoate to 6-aminohexanal; 2-amino-7- oxosubarate to 2,7-diaminosubarate; and 2,7-diaminosubarate to homolysine (Figure 26). A non-naturally occurring microbial organism can further have a 2-amino-7-oxosubarate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from glutamate-5- semialdehyde to 2-amino-5-hydroxy-7-oxosubarate; 2-amino-5-hydroxy-7-oxosubarate to 2- amino-5-ene-7-oxosubarate; and 2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (Figure 27). This invention is also directed, in part to engineered biosynthetic pathways to improve carbon flux through a central metabolism intermediate en route to adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine. The present invention provides non-naturally occurring microbial organisms having one or more exogenous genes encoding enzymes that can catalyze various enzymatic transformations en route to adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine. In some embodiments, these enzymatic

transformations are part of the reductive tricarboxylic acid (RTCA) cycle and are used to improve product yields, including but not limited to, from carbohydrate-based carbon feedstock.

In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents and/or carbon to byproducts. In accordance with some embodiments, the present invention increases the yields of adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2. In addition to syngas, other sources of such gases include, but are not limted to, the atmosphere, either as found in nature or generated.

The C02-fixing reductive tricarboxylic acid (RTCA) cycle is an endergenic anabolic pathway of C02 assimilation which uses reducing equivalents and ATP (Figure 38A). One turn of the RTCA cycle assimilates two moles of C02 into one mole of acetyl-CoA, or four moles of C02 into one mole of oxaloacetate. This additional availability of acetyl-CoA improves the maximum theoretical yield of product molecules derived from carbohydrate- based carbon feedstock. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose and glycerol.

In some embodiments, the reductive TCA cycle, coupled with carbon monoxide

dehydrogenase and/or hydrogenase enzymes, can be employed to allow syngas, C02, CO, H2, and/or other gaseous carbon source utilization by microorganisms. Synthesis gas (syngas), in particular is a mixture of primarily H2 and CO, sometimes including some amounts of C02, that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter. Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500oC) to provide syngas as a 0.5: 1-3: 1 H2/CO mixture. In addition to coal, biomass of many types has been used for syngas production and represents an inexpensive and flexible feedstock for the biological production of renewable chemicals and fuels. Carbon dioxide can be provided from the atmosphere or in condensed from, for example, from a tank cylinder, or via sublimation of solid C02. Similarly, CO and hydrogen gas can be provided in reagent form and/or mixed in any desired ratio. Other gaseous carbon forms can include, for example, methanol or similar volatile organic solvents.

The components of synthesis gas and/or other carbon sources can provide sufficient C02, reducing equivalents, and ATP for the reductive TCA cycle to operate. One turn of the RTCA cycle assimilates two moles of C02 into one mole of acetyl-CoA and requires 2 ATP and 4 reducing equivalents. CO and/or H2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively. Reducing equivalents can come in the form of NADH, NADPH, FADH, reduced quinones, reduced ferredoxins, thioredoxins, and reduced flavodoxins. The reducing equivalents, particularly NADH, NADPH, and reduced ferredoxin, can serve as cofactors for the RTCA cycle enzymes, for example, malate dehydrogenase, fumarate reductase, alpha- ketoglutarate:ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase),

pyruvate: ferredoxin oxidoreductase and isocitrate dehydrogenase. The electrons from these reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions, protons, or an electrode. The ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme.

The reductive TCA cycle was first reported in the green sulfur photosynthetic bacterium Chlorobium limicola (Evans et al, Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways have been characterized in some prokaryotes (proteobacteria, green sulfur bacteria and thermophiUic Knallgas bacteria) and sulfur-dependent archaea (Hugler et al., J. Bacteriol. 187:3020-3027 (2005; Hugler et al, Environ. Microbiol. 9:81-92 (2007). In some cases, reductive and oxidative (Krebs) TCA cycles are present in the same organism (Hugler et al., supra (2007); Siebers et al, J. Bacteriol. 186:2179-2194 (2004)). Some methanogens and obligate anaerobes possess incomplete oxidative or reductive TCA cycles that may function to synthesize biosynthetic intermediates (Ekiel et al., J. Bacteriol. 162:905-908 (1985); Wood et al, FEMS Microbiol. Rev. 28:335-352 (2004)).

The key carbon-fixing enzymes of the reductive TCA cycle are alpha- ketoglutarate:ferredoxin oxidoreductase, pyruvate :ferredoxin oxidoreductase and isocitrate dehydrogenase. Additional carbon may be fixed during the conversion of

phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxylase or

phosphoenolpyruvate carboxykinase or by conversion of pyruvate to malate by malic enzyme.

Many of the enzymes in the TCA cycle are reversible and can catalyze reactions in the reductive and oxidative directions. However, some TCA cycle reactions are irreversible in vivo and thus different enzymes are used to catalyze these reactions in the directions required for the reverse TCA cycle. These reactions are: (1) conversion of citrate to oxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate, and (3) conversion of succinyl-CoA to alpha-ketoglutarate. In the TCA cycle, citrate is formed from the condensation of

oxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrate to oxaloacetate and acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, or citryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyase can be coupled to acetyl-CoA synthetase, an acetyl-CoA transferase, or phosphotransacetylase and acetate kinase to form acetyl-CoA and oxaloacetate from citrate. The conversion of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse reaction is catalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formed from the NAD(P)+ dependent decarboxylation of alpha- ketoglutarate by the alpha-ketoglutarate dehydrogenase complex. The reverse reaction is catalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase.

An organism capable of utilizing the reverse tricarboxylic acid cycle to enable production of acetyl-CoA-derived products on 1) CO, 2) C02 and H2, 3) CO and C02, 4) synthesis gas comprising CO and H2, and 5) synthesis gas or other gaseous carbon sources comprising CO, C02, and H2 can include any of the following enzyme activities: ATP-citrate lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoA synthetase,acetyl-CoA transferase, pyruvate:ferredoxin oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, hydrogenase, and ferredoxin (see Figure 39A and 40 A). Enzymes and the corresponding genes required for these activities are described herein above.

Carbon from syngas or other gaseous carbon sources can be fixed via the reverse TCA cycle and components thereof. Specifically, the combination of certain carbon gas-utilization pathway components with the pathways for formation of adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine from acetyl-CoA results in high yields of these products by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously or produced endogenously from CO, into acetyl-CoA.

In some embodiments, an adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine pathway in a non-naturally occurring microbial organism of the invention can utilize any combination of (1) CO, (2) C02, (3) H2, or mixtures thereof to enhance the yields of biosynthetic steps involving reduction, including addition to driving the reductive TCA cycle.

In some embodiments a non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; and at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C02, (3) H2, (4) C02 and H2, (5) CO and C02, (6) CO and H2, or (7) CO, C02, and H2.

In some embodiments a method includes culturing a non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine pathway also comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. Additionally, such an organism can also include at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C02, (3) H2, (4) C02 and H2, (5) CO and C02, (6) CO and H2, or (7) CO, C02, and H2 to produce a product.

In some embodiments a non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, a pyruvate: ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase and an alpha- ketoglutarate:ferredoxin oxidoreductase.

In some embodiments a non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of carbon monoxide and/or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. The at least one exogenous nucleic acid is selected from a carbon monoxide dehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, and a ferredoxin. In some embodiments, the present invention provides a method for enhancing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock, such as sugars or gaseous carbon sources, the method includes culturing this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine.

In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway includes two exogenous nucleic acids, each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine pathway includes three exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding an ATP- citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding a citrate lyase, a fumarate reductase, and an alpha- ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non-naturally occurring microbial organism includes four exogenous nucleic acids encoding a pyruvate :ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate

carboxykinase, a CO dehydrogenase; and an H₂ hydrogenase. In some embodiments, the non-naturally occurring microbial organism includes two exogenous nucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

In some embodiments, the non-naturally occurring microbial organisms having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further include an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin

oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, and combinations thereof.

In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further includes an exogenous nucleic acid encoding an enzyme selected from carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin, NAD(P)H:ferredoxin oxidoreductase and combinations thereof. In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway utilizes a carbon feedstock selected from (1) CO, (2) C02, (3) C02 and H2, (4) CO and H2, or (5) CO, C02, and H2. In some embodiments, the non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine pathway utilizes hydrogen for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine pathway utilizes CO for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine pathway utilizes combinations of CO and hydrogen for reducing equivalents.

In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further includes one or more nucleic acids encoding an enzyme selected from a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a pyruvate carboxylase, and a malic enzyme.

In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further includes one or more nucleic acids encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA synthetase, and a succinyl-CoA transferase.

In some embodiments, the non-naturally occurring microbial organism having an adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine pathway further includes at least one exogenous nucleic acid encoding a citrate lyase, an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, and a ferredoxin.

It is understood by those skilled in the art that the above-described pathways for increasing product yield can be combined with any of the pathways disclosed herein, including those pathways depicted in the figures. One skilled in the art will understand that, depending on the pathway to a desired product and the precursors and intermediates of that pathway, a particular pathway for improving product yield, as discussed herein above and in the examples, or combination of such pathways, can be used in combination with a pathway to a desired product to increase the yield of that product or a pathway intermediate.

In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 6-aminocaproic acid (6-ACA) pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; wherein said 6-ACA pathway comprises a pathway selected from: (A) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6-aminocaproyl-CoA/acyl-CoA transferase, a 6- aminocaproyl-CoA synthase, or a 6-aminocaproyl-CoA hydrolase; (B) a 3-oxo-6- aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo- 6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6- aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2- enoate reductase; (C) an 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase; a 2-oxohept- 4-ene-l,7-dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7-dioate (OHED) reductase; a 2- oxoheptane-l,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde

aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex-4-enoate (6-OHE) reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);(E) an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase;(F) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase; (G) an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (H) an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate

semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (I) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (J) a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta- ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl- CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase; (K) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; (L) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl- [acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3- hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6-oxohexanoate aminotransferase; (M) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3- oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate

dehydrogenase (aminating) or α-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase; (N) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (O) a homocitrate synthase; a homocitrate dehydratase; a cis-

Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (P) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; (Q) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; and an adipate reductase; (R) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and (S) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-

Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase.

In some embodiments, the invention provides a non-naturally occurring microbial organism described herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin

oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

In some embodiments, the invention provides a non-naturally occurring microbial organism of disclosed herein, wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 6-ACA pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism comprising: (A) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6-aminocaproyl-CoA/acyl-CoA transferase, a 6-aminocaproyl-CoA synthase, or a 6-aminocaproyl-CoA hydrolase; (B) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6- aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3- oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6- aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase; (C) five exogounous nucleic acids encoding an 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase; a 2- oxohept-4-ene-l,7-dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7-dioate (OHED) reductase; a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex-4-enoate (6-OHE) reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (E) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase; (F) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase; (G) seven exogounous nucleic acids encoding an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (H) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl- CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating); (I) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (J) seven exogounous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept- 2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2- aminopimelate; (K) two exogounous nucleic acids encoding a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; (L) sixteen exogounous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA

methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an

enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6- oxohexanoate aminotransferase; (M) sixteen exogounous nucleic acids encoding an acetyl- CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl- [acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3- hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate

dehydrogenase (aminating) or α-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase; (N) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (O) twelve exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (P) ten exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; (Q) nine exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (R) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; or (S) nine exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase.

In some aspects, a microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some aspects, a microbial organism comprising (ii) comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism , wherein said microbial organism comprising (K) as disclosed above further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from: (A) succinyl- CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase; (B) adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase and adipyl-CoA hydrolase; and (C) succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. In some aspects the non-naturally occurring microbial organism disclosed herein includes at least one exogenous nucleic acid that is a heterologous nucleic acid. In some aspects the non-naturally occurring microbial organism disclosed herein is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a

phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said caprolactam pathway comprises a pathway selected from: (A) a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6- aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and a 6- aminohex-2-enoyl-CoA reductase; (C) a CoA-dependent aldehyde dehydrogenase; a transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (D) a

homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (E) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an

adipylphosphate reductase; (F) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (G) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase; (H) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate

transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; (I) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (J) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase; (K) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-

Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (L) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (M) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; and an adipate reductase; (N) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; and (O) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three or four exogenous nucleic acids each encoding a caprolactam pathway enzyme. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) one exogenous nucleic acid enconding a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (B) four exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and a 6-aminohex-2-enoyl-CoA reductase; (C) three exogenous nucleic acids encoding a CoA-dependent aldehyde dehydrogenase; a transaminase or a 6-aminocaproate

dehydrogenase; and an amidohydrolase; (D) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (E) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (F) eleven exogenous nucleic acids encoding a

homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate

transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (G) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; and an adipate reductase; (H) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha- ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate

transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; (I) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (J) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase; (K) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (L) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (M) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-

Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase; (N) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; or (O) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (A) describe above further comprises a 6-aminocaproic acid pathway, wherein said 6-aminocaproic acid pathway is selected from: (A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6- aminocaproate dehydrogenase; and (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6- aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3- oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6- aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase.

In some embodiments, the invention provides a non-naturally occurring microbial organism of disclosed herein, wherein said microbial organism comprising (C) described above further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from: (A) succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase; (B) adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase and adipyl-CoA hydrolase; and (C) succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a non-naturally occurring microbial organism of disclosed here, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a hexamethylenediamine (HMD A) pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; wherein said HMDA pathway comprises a pathway selected from: (A) a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine

dehydrogenase; (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a

hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase; (C) a 6- aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (D) a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (E) a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (F) a 6- aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide); (G) a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6- acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide); (H) a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide); (I) a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3- oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA

dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase; (J) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo- 1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3- aminotransferase or a 3-oxo-7-aminoheptanoate 3 -aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (K) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (L) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl- CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3- oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l- carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (M) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo- l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (N) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homo lysine decarboxylase; (O) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homo lysine decarboxylase; (P) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7- aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Q) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (R) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (S) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (T) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (U) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5- aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3- amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (V) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (W) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (X) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Y) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2- oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Z) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal

aminotransferase or 6-aminohexanal aminating oxidoreductase; (AA) a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating); (AB) a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate reductase; a 6-acetamidohexanal aminotransferase or 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or 6-acetamidohexanamine hydrolase (amide); (AC) a 2-amino-7- oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7- diaminosubarate decarboxylase; and a homolysine decarboxylase; (AD) a 2-amino-7- oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a 2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase; (AE) a 2- amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase; (AF) a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate

decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase; (AG) a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7- oxoheptanoate aminating oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a homolysine decarboxylase; (AH) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an

enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate decarboxylase; a 6-oxohexanoate dehydrogenase (aminating) or 6- oxohexanoate aminotransferase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AI) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an a-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6- aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AJ) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl- [acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3- hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (animating) or an a-ketopimelate amino transferase; a 2-aminopimelate; a 2- amino-7-oxoheptanoate dehydrogenase; and a 2,7-diaminoheptanoate decarboxylase; (AK) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AL) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (AM) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase; (AN) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AO) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and (AP) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin

oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a non- naturally occurring microbial organism herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen exogenous nucleic acids each encoding a HMDA pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) three exogenous nucleic acids encoding a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase; (B) six exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; a 6- aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase; (C) three exogenous nucleic acids encoding a 6- aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (D) four exogenous nucleic acids encoding a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (E) three exogenous nucleic acids encoding a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (F) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide); (G) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide); (H) five exogenous nucleic acids encoding a 6- aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide); (I) eight exogenous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6- aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6- amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase; (J) seven exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7- aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7- aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3 -aminating

oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (K) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l- carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating

oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (L) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5- oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7- aminotransferase or 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7- aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating

oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (M) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo- l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (N) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5- oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3- oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (O) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (P) seven exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Q) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (R) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (S) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl- CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3- oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (T) eight exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5- aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7- aminotransferase or 3-amino-7-oxoheptanoate animating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (U) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a

3- amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (V) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2- aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (W) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (X) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6- aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7- aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Y) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7- aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7- aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7- aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Z) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-

4- hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2- oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminotransferase or 6- aminohexanal aminating oxidoreductase; (AA) two exogenous nucleic acids encoding a 6- aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6- aminocaproic semialdehyde oxidoreductase (animating); (AB) four exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase; a 6- acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide); (AC) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate aminating

oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase; (AD) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a 2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase; (AE) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase; (AF) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7- oxoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6- aminohexanal aminotransferase; (AG) three exogenous nucleic acids encoding a 2-amino-7- oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a homolysine decarboxylase; (AH) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an

enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate decarboxylase; a 6-oxohexanoate dehydrogenase (aminating) or 6- oxohexanoate aminotransferase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AI) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3- oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (animating) or an a-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase

(aminating) or a 6-aminohexanal aminotransferase; (AJ) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an α-ketopimelate amino transferase; a 2-aminopimelate; a 2- amino-7-oxoheptanoate dehydrogenase; and a 2,7-diaminoheptanoate decarboxylase; (AK) fourteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate

dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AL) fifteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (AM) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase; (AN) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AO) fourteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; or (AP) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-

Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase.

In some embodiments, the invention provides anon-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (A) further comprises a 6- aminocaproic acid pathway, wherein said 6-aminocaproic acid pathway is selected from: (A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; and (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl- CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6- aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6- aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (AC), (AD), (AE), (AF) or (AG) as disclosed above further comprises at least one exogenous nucleic acid encoding a 2- amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino- 7-oxosubarate, wherein said 2-amino-7-oxosubarate pathway comprises a 2-amino-5- hydroxy-7-oxosubarate aldolase; a 2-amino-5-hydroxy-7-oxosubarate dehydratase; and a 2- amino-5-ene-7-oxosubarate reductase.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a

phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said adipate pathway comprises a pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyl transferase; a 3 -hydroxy acyl- CoA dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (B) a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3-oxoadipate reductase; a 3- hydroxyadipate dehydratase; and a 2-enoate reductase; (C) an alpha-ketoadipyl-CoA synthetase, a phosphotransketoadipylase/alpha-ketoadipate kinase or an alpha-ketoadipyl- CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydrogenase; a 2-hydroxyadipyl- CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl- CoA hydrolase; (D) a 2-hydroxyadipate dehydrogenase; a 2-hydroxyadipyl-CoA synthetase, a phosphotranshydroxyadipylase/2-hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl- CoA transferase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (E) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; and (G) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen exogenous nucleic acids each encoding an adipate pathway enzyme.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-hydroxyacyl-CoA

dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (B) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3- oxoadipate reductase; a 3-hydroxyadipate dehydratase; and a 2-enoate reductase; (C) five exogenous nucleic acids encoding an alpha-ketoadipyl-CoA synthetase, a

phosphotransketoadipylase/alpha-ketoadipate kinase or an alpha-ketoadipyl-CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydrogenase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a

phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl- CoA hydrolase; (D) five exogenous nucleic acids encoding a 2-hydroxyadipate dehydrogenase; a 2-hydroxyadipyl-CoA synthetase, a phosphotranshydroxyadipylase/2- hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl-CoA transferase; a 2- hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl- CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (E) fourteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl- [acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3- hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) nine exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha- ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; or (G) eight exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase.

In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine or any adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine or adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine pathway intermediate including any adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine impurities generated in diverging away from the pathway at any point. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter the carbon- 12, carbon-13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, a target isotopic ratio of an uptake source can be obtained via synthetic chemical enrichment of the uptake source. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory. In some embodiments, a target isotopic ratio of an uptake source can be obtained by choice of origin of the uptake source in nature. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon- 14, or an environmental carbon source, such as C0₂, which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC) and/or high performance liquid chromatography (HPLC).

In some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme or an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon uptake source. In some such embodiments, the uptake source is C02. In some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme or an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source. In some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme or an adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamme intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Such combination of uptake sources is one means by which the carbon- 12, carbon- 13, and carbon- 14 ratio can be varied.

Additionally, a non-naturally occurring microbial organism can have a levulinic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; and 3-oxoadipate to levulinic acid. It is understood that any of the pathways disclosed herein which produce an intermediate of one pathway can be used to produce that intermediate for another pathway, if desired. For example, as disclosed herein, the alpha-ketoadipate to adipate pathway shown in Figure 9 produces the intermediate adipyl-CoA, which is also an intermediate in the pathway depicted in Figure 10. Thus, it is understood that an alternative pathway includes alpha- ketoadipate to adipyl-CoA, which can be converted to adipate, 6-aminocaporate, caprolactam or hexamethylenediamme, as depicted in Figure 10. It is understood that any of the pathways disclosed herein that produce a desired intermediate can be used in combination with any other pathways disclosed herein so long as a desired product is produced. For example, a non-naturally occurring microbial organism disclosed herein, can have at least one nucleic acid encoding a 6-aminocaproic acid pathway enzyme and at least one nucleic acid encoding a hexamethylenediamine pathway enzyme, such as 2-AHD decarboxylase (Step I of Figure 12) and 6-acetamidohexanoate kinase (Step E of Figure 13), or alternatively 2-oxohept-4-ene- 1,7-dioate (OHED) decarboxylase (Step F of Figure 12), adipate semialdehyde

aminotransferase (Step E of Figure 12) and 6-acetamidohexanoyl-CoA oxidoreductase (Step J of Figure 13), or alternatively 5-carboxy-2pentenoyl-CoA reductase (Step D of Figure 10), adipyl-CoA dehydrogenase (Step O of Figure 12) and 6-aminocaproyl-CoA oxidoreductase (Step N of Figure 13), or alternatively 2-amino-7-oxoheptanoate aminotransferase (Step G of Figure 20) and 3,7-diaminoheptanoate 2,3-aminomutase (Step R of Figure 21), or

alternatively 6-aminocaproate reductase (Step O of Figure 24) and 6-aminohex-2-enoate reductase (Step J of Figure 11), or alternatively adipate reductase (Step X of Figure 25) and 6-acetamidohexanoate reductase (Step P of Figure 24).

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from any of the substrates or products disclosed herein or shown in any of Figures 1-14, 20-27 and 38-40. One skilled in the art will understand that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid pathway, such as any of those shown in Figures 1- 14, 20-27 and 38-40.

While generally described herein as a microbial organism that contains an adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme expressed in a sufficient amount to produce an intermediate of a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. For example, as disclosed herein, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway are exemplified in Figures 1-14, 20-27 and 38-40. Therefore, in addition to a microbial organism containing a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway that produces adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme, where the microbial organism produces a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate, for example, any of the intermediates shown in Figures 1-14, 20-27 and 38-40. It is understood that any of the pathways disclosed herein, including those as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-14, 20-27 and 38-40, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway

intermediate can be utilized to produce the intermediate as a desired product.

The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction. The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes participating in one or more adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or proteins(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or proteins(s) to achieve adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid .

Depending on the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more adipate, 6-aminocaproic acid or caprolactam biosynthetic pathways. For example, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes of a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway, exogenous expression of all enzymes in the pathway can be included, although it is understood that all enzymes of a pathway can be expressed even if the host contains at least one of the pathway enzymes.

For example, exogenous expression of all enzymes in a pathway for production of adipate can be included in a host organism, such as succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2- pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. In particular, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5- carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase. Alternatively, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5- carboxy-2-pentenoyl-CoA reductase, and phosphotransadipylase/adipate kinase. In addition, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5- carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA:acetyl-CoA transferase. Further, a host organism can contain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5- carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA hydrolase.

In the case of a 6-aminocaproic acid producing microbial organism, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid can be included in a host organism, such as CoA-dependent aldehyde dehydrogenase and transaminase or CoA- dependent aldehyde dehydrogenase and 6-aminocaproate dehydrogenase. For a caprolactam producing microbial organism, exogenous expression of all enzymes in a pathway for production of caprolactam can be included in a host organism, such as CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and

amidohydrolase.In another example, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid (6-ACA) can be included in a host organism, such as an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase

(aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2- AHE reductase; and a 2-AHD decarboxylase, or alternatively an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase, or alternatively an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED

(aminating), or alternatively an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde

aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In a further aspect, all of the the 6-ACA pathway described above can include a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase.In another example, exogenous expression of all enzymes in a pathway for production of 6-aminocaproic acid (6-ACA) can be included in a host organism, such as a glutamyl-CoA transferase or glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6- aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6- amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelate decarboxylase.

In another example, exogenous expression of all enzymes in a pathway for production of hexamethylenediamine can be included in a host organism, such as a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, or alternatively a 6- aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (animating) or a 6-aminocaproic acid semialdehyde aminotransferase, or alternatively a 6-aminocaproate CoA transferase or a 6- aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde

aminotransferase, or alternatively a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal

aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide), or alternatively a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide), or alternatively a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In another example, exogenous expression of all enzymes in a pathway for production of hexamethylenediamine can be included in a host organism, such as a glutamyl- CoA transferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA

oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept- 2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine

decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7- aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l- carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating

oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l- carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3- aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7- oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating

oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5- oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3- oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3- oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 5- oxopimeloyl-CoA hydrolase or a 5-oxopimeloyl-CoA ligase; a 3-oxo-l-carboxyheptanal 3- aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7- oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7- oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2- amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5- aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3- aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5- aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7- aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5- aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3- amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate kinase; a 6- aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2- amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6- aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7- aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase, or alternatively a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4- hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo- 7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating

oxidoreductase; and a homolysine decarboxylase, or alternatively a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating), or alternatively a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate reductase; a 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

Depending on the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathways. For example, adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid can be included, as disclosed herein. Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the adipate, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, up to all nucleic acids encoding the above enzymes constituting a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthetic pathway. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway precursors such as succinyl-CoA and/or acetyl-CoA in the case of adipate synthesis, or adipyl-CoA or adipate in the case of 6-aminocaproic acid or caprolactam synthesis, including the adipate pathway enzymes disclosed herein, or pyruvate and succinic semialdehyde, glutamate, glutaryl-CoA, homolysine or 2-amino-7-oxosubarate in the case of 6-aminocaprioate synthesis, or 6-aminocaproate, glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2- amino-7-oxosubarate in the case of hexamethylenediamine synthesis.

Generally, a host microbial organism is selected such that it produces the precursor of a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, acetyl-CoA is produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid pathway.

In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid . In this specific embodiment it can be useful to increase the synthesis or accumulation of a adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway product to, for example, drive adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway reactions toward adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzymes or proteins. Overexpression of the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme or enzymes and/or protein or proteins of the adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, through overexpression of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, that is, up to all nucleic acids encoding adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway enzymes. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

The invention additionally provides a non-naturally occurring microbial organism that includes one or more gene disruptions, such as the gene disruptions disclosed in Example XXX and Tables 14-16, where the organism produces a 6-ACA, adipate and/or HMDA. The disruptions occur in genes encoding an enzyme that couples production of adipate, 6-ACA and/or HMDA to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer increased production of adipate, 6-ACA and/or HMDA onto the non-naturally occurring organism. Thus, the invention provides a non- naturally occurring microbial organism, comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding proteins or enzymes wherein the one or more gene disruptions confer increased production of adipate, 6-ACA and/or HMDA in the organism. As disclosed herein, such an organism contains a pathway for production of adipate, 6-ACA and/or HMDA, in addition to the gene disruptions, such as those exemplified in Example XXX and Tables 14-16.

It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic capability. For example, a non-naturally occurring microbial organism having a adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins. In the case of adipate production, at least two exogenous nucleic acids can encode the enzymes such as the combination of succinyl-CoA:acetyl-CoA acyl transferase and 3- hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyl transferase and 3- hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoA and 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA and adipyl-CoA synthetase, and the like. In the case of caprolactam production, at least two exogenous nucleic acids can encode the enzymes such as the combination of CoA-dependent aldehyde dehydrogenase and transaminase, or CoA- dependent aldehyde dehydrogenase and amidohydrolase, or transaminase and

amidohydrolase. In the case of 6-aminocaproic acid production, at least two exogenous nucleic acids can encode the enzymes such as the combination of an 4-hydroxy-2- oxoheptane-l,7-dioate (HODH) aldolase and a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase, or a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase and a 2-aminoheptane-l,7- dioate (2-AHD) decarboxylase, a 3-hydroxyadipyl-CoA dehydratase and a adipyl-CoA dehydrogenase, a glutamyl-CoA transferase and a 6-aminopimeloyl-CoA hydrolase, or a glutaryl-CoA beta-ketothiolase and a 3-aminopimelate 2,3-aminomutase. In the case of hexamethylenediamine production, at least two exogenous nucleic acids can encode the enzymes such as the combination of 6-aminocaproate kinase and

[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or a 6-acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, 6- aminocaproate N-acetyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, a 3- hydroxy-6-aminopimeloyl-CoA dehydratase and a 2-amino-7-oxoheptanoate

aminotransferase, or a 3-oxopimeloyl-CoA ligase and a homolysine decarboxylase. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention.

Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, in the case of adipate production, the combination of enzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, and 3- hydroxyadipyl-CoA dehydratase; or succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase; or succinyl- CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and adipyl-CoA:acetyl-CoA transferase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. In the case of 6-aminocaproic acid production, the at least three exogenous nucleic acids can encode the enzymes such as the combination of an 4- hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase, a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase and a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase, or a 2-oxohept-4-ene-l,7- dioate (OHED) hydratase, a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase and a 2- aminoheptane-l,7-dioate (2-AHD) decarboxylase, or a 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase and a adipyl-CoA dehydrogenase, or a 6-amino-7- carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA hydrolase and a 2- aminopimelate decarboxylase, or a glutaryl-CoA beta-ketothiolase, a 3-aminating

oxidoreductase and a 2-aminopimelate decarboxylase, or a 3-oxoadipyl-CoA thiolase, a 5- carboxy-2-pentenoate reductase and an adipate reductase. In the case of

hexamethylenediamine production, at least three exogenous nucleic acids can encode the enzymes such as the combination of 6-aminocaproate kinase,

[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase and 6-aminocaproic semialdehyde aminotransferase, or a 6-aminocaproate N-acetyltransferase, a 6- acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, or 6-aminocaproate N-acetyltransferase, a [(6- acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and 6-acetamidohexanoyl- CoA oxidoreductase, or a 3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6- aminopimeloyl-CoA dehydratase and a homo lysine decarboxylase, or a 2-oxo-4-hydroxy-7- aminoheptanoate aldolase, a 2-oxo-7-aminohept-3-enoate reductase and a homo lysine decarboxylase, or a 6-acetamidohexanoate reductase, a 6-acetamidohexanal aminotransferase and a 6-acetamidohexanamine N-acetyltransferase. Similarly, any combination of four or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

In addition to the biosynthesis of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various

combinations with each other and/or with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid other than use of the adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid producers is through addition of another microbial organism capable of converting an adipate, 6-aminocaproic acid or caprolactam pathway intermediate to adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. One such procedure includes, for example, the fermentation of a microbial organism that produces a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway intermediate. The adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway intermediate can then be used as a substrate for a second microbial organism that converts the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway intermediate to adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid . The adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway intermediate can be added directly to another culture of the second organism or the original culture of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid pathway

intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid . In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamme or levulinic acid intermediate and the second microbial organism converts the intermediate to adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamme or levulinic acid. Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis. In a particular embodiment, the increased production couples biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism, and can obligatorily couple production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism if desired and as disclosed herein.

Sources of encoding nucleic acids for a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction.

Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Escherichia coli str. K12, Escherichia coli C, Escherichia coli W, Pseudomonas sp, Pseudomonas knackmussii, Pseudomonas sp. Strain B13, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas mendocina,

Rhodopseudomonas palustris, Mycobacterium tuberculosis, Vibrio cholera, Heliobacter pylori, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces sp. 2065, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAOl, Ralstonia eutropha, Ralstonia eutropha HI 6, Clostridium acetobutylicum, Euglena gracilis, Treponema denticola,

Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp. ADP1,

Acinetobacter sp. Strain M-l, Streptomyces coelicolor, Eubacterium barkeri,

Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium botulinum A3 str, Clostridium tyrobutyricum, Clostridium pasteurianum, Clostridium thermoaceticum

(Moorella thermoaceticum), Moorella thermoacetica Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae,

Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens,

Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae,

Acidaminococcus fermentans, Clostridium sp. M62/1, Fusobacterium nucleatum, Bos taurus, Zoogloea ramigera, Rhodobacter sphaeroides, Clostridium beijerinckii, Metallosphaera sedula, Thermoanaerobacter species, Thermoanaerobacter brockii, Acinetobacter baylyi, Porphyromonas gingivalis, Leuconostoc mesenteroides, Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sulfolobus solfataricus, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Salmonella typhimurium, Salmonella enterica, Thermotoga maritima, Halobacterium salinarum, Bacillus cereus, Clostridium difficile, Alkaliphilus metalliredigenes,

Thermoanaerobacter tengcongensis, Saccharomyces kluyveri, Helicobacter pylori,

Corynebacterium glutamicum, Clostridium saccharoperbutylacetonicum, Pseudomonas chlororaphis, Streptomyces clavuligerus, Campylobacter jejuni, Thermus thermophilus, Pelotomaculum thermopropionicum, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilius, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Haloarcula marismortui, Pyrobaculum aerophilum, Pyrobaculum aerophilum str. IM2, Nicotiana tabacum, Menthe piperita, Pinus taeda, Hordeum vulgare, Zea mays, Rhodococcus opacus, Cupriavidus necator, Bradyrhizobium japonicum, Bradyrhizobium japonicum USDA110,Ascarius suum, butyrate-producing bacterium L2-50, Bacillus megaterium, Methanococcus maripaludis, Methanosarcma mazei, Methanosarcma mazei, Methanocarcina barkeri, Methanocaldococcus jannaschii, Caenorhabditis elegans, Leishmania major, Methylomicrobium alcaliphilum 20Z, Chromohalobacter salexigens, Archaeglubus fulgidus, Chlamydomonas reinhardtii, trichomonas vaginalis G3, Trypanosoma brucei, Mycoplana ramose, Micrococcus luteas, Acetobacter pasteurians, Kluyveromyces lactis, Mesorhizobium loti, Lactococcus lactis, Lysinibacillus sphaericus, Candida boidinii, Candida albicans SC5314, Burkholderia ambifaria AMMD, Ascaris suun, Acinetobacter baumanii,

Acinetobacter calcoaceticus, Burkholderia phymatum, Candida albicans, Clostridium subterminale, Cupriavidus taiwanensis, Flavobacterium lutescens, Lachancea kluyveri, Lactobacillus sp. 30a, Leptospira interrogans, Moorella thermoacetica, Myxococcus xanthus, Nicotiana glutinosa, Nocardia iowensis (sp. NRRL 5646), Pseudomonas reinekei MT1, Ralstonia eutropha JMP134, Ralstonia metal lidurans, Rhodococcus jostii,

Schizosaccharomyces pombe, Selenomonas ruminantium, Streptomyces clavuligenus, Syntrophus aciditrophicus, Vibrio parahaemolyticus, Vibrio vulnificus, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes (see Examples). However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms. In some instances, such as when a adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid biosynthetic pathway exists in an unrelated species, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. For example, E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metoblic and/or genetic modifications to produce a desired product. Methods for constructing and testing the expression levels of a non-naturally occurring adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid - producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Exogenous nucleic acid sequences involved in a pathway for production of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli

(Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate

modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties.

Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one or more adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co- expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

Directed evolution is one approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through implementation screening assays that allow for the identification of useful variants. Particularly useful screening methods include sensitive high-throughput assays that allow the automated screening of many enzyme variants (e.g., >10⁴). Iterative rounds of mutagenesis and screening typically are performed to identify an enzyme with optimized properties. The greater the number of variants screened, the higher the probability of identifying an ideally suitable variant. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened.

Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol.Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the

pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol.Eng 22: 1-9 (2005).; and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example, selectivity/specificity - for conversion of non-natural substrates; temperature stability - for robust high temperature processing; pH stability - for bioprocessing under lower or higher pH conditions; substrate or product tolerance - so that high product titers can be achieved; binding (Km) - broadens substrate binding to include non-natural substrates; inhibition (Ki) - to remove inhibition by products, substrates, or key intermediates; activity (kcat) - increases enzymatic reaction rates to achieve desired flux; expression levels - increases protein yields and overall pathway flux; oxygen stability - for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity - for operation of an aerobic enzyme in the absence of oxygen. The following exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Any of these can be used to alter/optimize activity of a decarboxylase enzyme.

EpPCR (Pritchard et al, J Theor.Biol 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn2+ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error- prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance.

This method can generate multiple mutations in a single gene simultaneously, which can be useful. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics. Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:el45 (2004); and Fujii et al., Nat.Protoc. 1 :2493-2497 (2006)) has many of the same elements as epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats. Adjusting the Mn2+ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3 - 4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci U.S.A. 91 : 10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >lkbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious, random and neutral mutations that might confer antigenicity.

Staggered Extension (StEP) (Zhao et al., Nat.Biotechnol 16:258-261 (1998)) entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents. Combinations of low- fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra. In Random Priming Recombination (RPR) random sequence primers are used to generate many short DNA fragments complementary to different segments of the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.

In Heteroduplex Recombination linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair. (Volkov et al, Nucleic Acids Res 27:el8 (1999); and

Volkov et al., Methods Enzymol. 328:456-463 (2000)) The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat.Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of ssDNA.

Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in, and then ligated to give a pool of full- length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates. (Lee et al., J.Molec. Catalysis 26: 119-129 (2003)) No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps—no direct shuffling. This method can also be more random than StEP due to the absence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol 352: 191- 204 (2007); Bergquist et al, Biomol.Eng 22:63-72 (2005); Gibbs et al, Gene 271 : 13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest. (Ostermeier et al., Proc Natl Acad Sci U.S.A. 96:3562-3567 (1999); and Ostermeier et al, Nat.Biotechnol 17: 1205-1209 (1999)) Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured. Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations. (Lutz et al., Nucleic Acids Res 29:E16 (2001)) Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide more reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling. (Lutz et al, Proc Natl Acad Sci U.S.A. 98: 11248-11253 (2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed by

screening/selection for those retaining usable activity. (Bergquist et al., Biomol.Eng 22:63- 72 (2005)) Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of

"universal" bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis. (Wong et al., BiotechnolJ 3:74- 82 (2008); Wong et al., Nucleic Acids Res 32:e26 (2004); and Wong et al., Anal.Biochem. 341 : 187-189 (2005)) Using this technique it can be possible to generate a large library of mutants within 2 -3 days using simple methods. This technique is non-directed in comparison to the mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or an alternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny. (Ness et al., Nat.Biotechnol 20: 1251-1255 (2002)) In this technique, one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny. One can design sequence/codon biases to make more distantly related sequences recombine at rates approaching those observed with more closely related sequences. Additionally, the technique does not require physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation. (Muller et al., Nucleic Acids Res 33:el 17 (2005)) The gene is reassembled using internal PCR primer extension with proofreading polymerase. The sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage. Other nucleotide analogs, such as 8-oxo-guanine, can be used with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. The chemical cleavage of DNA used in this technique results in very few unshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC) a linker is used to facilitate fusion between two distantly/unrelated genes. Nuclease treatment is used to generate a range of chimeras between the two genes. These fusions result in libraries of single-crossover hybrids. (Sieber et al., Nat.Biotechnol 19:456-460 (2001)) This produces a limited type of shuffling and a separate process is required for mutagenesis. In addition, since no homology is needed this technique can create a library of chimeras with varying fractions of each of the two unrelated parent genes. SHIPREC was tested with a heme- binding domain of a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme. In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials are a supercoiled dsDNA plasmid containing an insert and two primers which are degenerate at the desired site of mutations. (Kretz et al., Methods Enzymol. 388:3-11 (2004)) Primers carrying the mutation of interest, anneal to the same sequence on opposite strands of DNA. The mutation is typically in the middle of the primer and flanked on each side by ~20 nucleotides of correct sequence. The sequence in the primer is NNN or NNK (coding) and MNN

(noncoding) (N = all 4, K = G, T, M = A, C). After extension, Dpnl is used to digest dam- methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (i.e., one codon). The technique facilitates the generation of all possible replacements at a single-site with no nonsense codons and results in equal to near-equal representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations. (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar- Olson et al. Science 241 :53-57 (1988)) Simultaneous substitutions at two or three sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. This technique has been used to explore the information content of the lambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM)is essentially similar to CCM except it is employed as part of a larger program: 1) Use of epPCR at high mutation rate to 2) ID hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space. (Reetz, M. T., S. Wilensek, D. Zha, and K. E. Jaeger, 2001, Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette

Mutagenesis. Angew.Chem.Int.Ed Engl. 40:3589-3591.) As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51 -fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique conditional ts mutator plasmids allow increases of 20- to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required. (Selifonova et al., Appl Environ Microbiol 67:3645-3649 (2001)) This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur. In order for effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows for plasmid curing at 41oC. It should be noted that mutator strains have been explored for quite some time (e.g., see Low et al., J. Mol. Biol. 260:359-3680 (1996)). In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.

"Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids." (Rajpal et al., Proc Natl Acad Sci U.S.A. 102:8466-8471 (2005)) Rather than saturating each site with all possible amino acid changes, a set of nine is chosen to cover the range of amino acid R-group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800-fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This method is a rational approach to minimize the number of random combinations and can increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation). Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombinations independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM™, a large range of mutations can be tested for improved activity. The method allows "blending" and "fine tuning" of DNA shuffling, e.g. codon usage can be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics. (Hayes et al, Proc Natl Acad Sci U.S.A. 99: 15926-15931 (2002)) This technology uses in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position. Structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (1050). The choice of sequence variants to test is related to predictions based on the most favorable thermodynamics. Ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely

experimental technologies due to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) use knowledge of structure/function to choose a likely site for enzyme improvement; 2) saturation mutagenesis at chosen site using Stratagene QuikChange (or other suitable means); 3) screen/select for desired properties; and 4) with improved clone(s), start over at another site and continue repeating. (Reetz et al., Nat.Protoc. 2:891-903 (2007); and Reetz et al, Angew.Chem.Int.Ed Engl. 45:7745-7751 (2006)) This is a proven methodology, which assures all possible replacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone or in any

combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques.

The invention additionally provides methods for producing a desired intermediate or product such as adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. For example, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl- CoA transferase or adipyl-CoA hydrolase. Additionally, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including succinyl- CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3- hydroxyadipate dehydratase, and 2-enoate reductase.

Further, a method for producing 6-aminocaproic acid can involve culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway, the pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, under conditions and for a sufficient period of time to produce 6-aminocaproic acid, the 6-aminocaproic acid pathway including CoA-dependent aldehyde dehydrogenase and transaminase or 6-aminocaproate dehydrogenase. Additionally, a method for producing caprolactam can involve culturing a non-naturally occurring microbial organism having a caprolactam pathway, the pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, under conditions and for a sufficient period of time to produce caprolactam, the caprolactam pathway including CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase.

The invention additionally provides methods for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway described herein under conditions and for a sufficient period of time to produce 6-ACA. In one aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2- AHE reductase; and a 2-AHD decarboxylase. In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD

aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase. In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an HODH formate- lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3- hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating). In still yet another aspect, the 6-ACA pathway includes an

HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl- CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating). In still yet another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate- lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl- CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating). In a further aspect, the 6-ACA pathways described above can include a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase.

The invention additionally provides methods for producing hexamethylenediamine (HMD A) by culturing a non-naturally occurring microbial organism having a HMDA pathway described herein under conditions and for a sufficient period of time to produce HMDA. In one aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP

oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase. In another aspect, the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase. In yet another aspect, the HMDA pathway includes a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6- aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal

aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In still yet another aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal

aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide).

Also, a method for producing adipate can involve culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including alpha-ketoadipyl-CoA synthetase,

phosphotransketoadipylase/alpha-ketoadipate kinase or alpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5- carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,

phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Furthermore, a method for producing adipate can involve culturing a non- naturally occurring microbial organism having an adipate pathway, the pathway including at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, the adipate pathway including 2-hydroxyadipate dehydrogenase; 2- hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5- carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,

phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. As disclosed herein, the invention also provides method for producing 6-aminocaproic acid by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6-aminocaproic acid, the 6- aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6- aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex- 2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl- CoA synthase, or 6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; steps

A/B/C/D/K/L/M of Figure 11). The invention additionally provides a method for producing 6-aminocaproic acid by culturing a non-naturally occurring microbial organism having a 6- aminocaproic acid pathway including at least one exogenous nucleic acid encoding a 6- aminocaproic acid pathway enzyme expressed in a sufficient amount to produce 6- aminocaproic acid, the 6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reductase; 3- hydroxy-6-aminohexanoate dehydratase; and 6-aminohex-2-enoate reductase (see Examples XII and XIV; steps A/E/F/G/H/I/J of Figure 11).

In another embodiment, the invention provides a method for producing caprolactam by culturing a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase (see Examples XII and XV; steps K/L of Figure 11). In such a method, the caprolactam can be produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of Figure 11). The invention also provides a non-naturally occurring microbial organism having a hexamethylenediamine pathway including at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in a sufficient amount to produce hexamethylenediamine, the hexamethylenediamine pathway including 6-aminocaproyl-

CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde forming); and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase (see Examples XII and XVI; steps K/L/N/O/P of Figure 11).

In yet another embodiment, the invention provides a method for producing caprolactam by culturing a non-naturally occurring microbial organism having a caprolactam pathway including at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam, the caprolactam pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6- aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps A/B/C/D of Figure 11). In such a method, the caprolactam can be produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of Figure 11). Also provided is a method for producing hexamethylenediamine by culturing a non-naturally occurring microbial organism having a hexamethylenediamine pathway including at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in a sufficient amount to produce hexamethylenediamine, the hexamethylenediamine pathway including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6- aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex- 2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase (aldehyde forming); and

hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase (see

Examples XII and XVIII; steps A/B/C/D/N/O/P of Figure 11).

In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway, the microbial organism including at least one exogenous nucleic acid encoding a 6- ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase, a phosphoenolpyruvate (PEP) carboxykinase, a 4-hydroxy-2-oxoheptane-l,7- dioate (HODH) aldolase, a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase, a 2-oxohept-4- ene-l,7-dioate (OHED) reductase, a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, an adipate semialdehyde oxidoreductase (aminating), a 2-oxohept-4-ene-l,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate (6-OHE) reductase, a 2-oxoheptane-l,7-dioate (2-OHD) aminotransferase, a 2-oxoheptane-l,7-dioate (2-OHD) oxidoreductase (aminating), a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase, a 2-oxohept-4-ene-l,7-dioate (OHED) aminotransferase, a 2-oxohept-4-ene-l,7-dioate (OHED) oxidoreductase (aminating), a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase, a 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) formate-lyase, a 4-hydroxy-2-oxoheptane-l,7- dioate (HODH) dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl- CoA reductase, a adipyl-CoA dehydrogenase, a 2-oxohept-4-ene-l,7-dioate (OHED) formate-lyase, a 2-oxohept-4-ene-l,7-dioate (OHED) dehydrogenase, a 2-oxoheptane-l,7- dioate (2-OHD) formate-lyase, a 2-oxoheptane-l,7-dioate (2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme (see Examples XIX and XXI; steps A-Q of Figure 12). In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway, the microbial organism including at least one exogenous nucleic acid encoding a 6- ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA. In one aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2- OHD decarboxylase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/D/E of Figure 12). In another aspect of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/F/G/E of Figure 12). In another aspect of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED

aminotransferase or an OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/J/D/I of Figure 12). In another aspect of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase (see Examples XIX and XXI; steps A/B/C/H/I of Figure 12). In another aspect of the invention, the 6-ACA pathway includes an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/L/M/N/O/E of Figure 12). the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED formate- lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3- dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/E of Figure 12). In another aspect of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/C/Q/O/E of Figure 12). In a further aspect, the 6-ACA pathways described above can include a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a

phosphoenolpyruvate (PEP) carboxykinase.

In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6- aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a 6- amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode a glutamyl-CoA transferase or glutamyl-CoA ligase; a beta- ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl- CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase. In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a glutaryl- CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/J/T/AA of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encode a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelate decarboxylase.

In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a homolysine 2-monooxygenase (see Examples XXV and XXVI; steps A of Figure 23). In a further aspect, the 6-ACA pathway includes hydrolysis of the 6-aminohexanamide product by a dilute acid or base to convert 6-aminohexanamide to 6-aminocaproate (see Example XXV; steps B of Figure 23).

In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-ACA pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including an adipate reductase, an adipate kinase or an adipylphosphate reductase (see Example XXVIII; steps X/Y/Z of Figure 25). In a further aspect, the 6-ACA pathway includes an adipate reductase. In another further aspect, the 6-ACA pathway includes an adipate kinase and an

adipylphosphate reductase. In still another aspect, the microbial organism having the 6- aminocaproic acid (6-ACA) pathway above further comprises an adipate pathway, a caprolactam pathway and/or a hexamethylenediamine pathway described here (see Example XXVIII; steps A-W of Figure 25). In yet another embodiment, the invention provides a method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6- aminocaproic acid (6-ACA) pathway including at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7- oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate oxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanal oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a 2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and XXVI; steps A/B/D/E/F/G/I of Figure 26). In a further aspect, the microbioal organism has a 2-amino-7- oxosubarate pathway having at least one exogenous nucleic acid encoding a 2-amino-7- oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino-7- oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7- oxosubarate aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a 2-amino-5-ene- 7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).

In another embodiment of the invention, the invention provides a method for producing 6- aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway including a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto- acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a 2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of Figure 26). In yet another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7- oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6- aminohexanal oxidoreductase (see Example XXV; steps A/B/F of Figure 26). In still yet another embodiment of the invention, the the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal oxidoreductase (see Example XXV; steps I/G/F of Figure 26). In a further aspect of each of the above embodiments, the microbioal organism has a 2-amino-7-oxosubarate pathway having a second set of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5- hydroxy-7-oxosubarate aldolase; a 2-amino-5-hydroxy-7-oxosubarate dehydratase; and a 2- amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27). In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMD A) by culturing a non-naturally occurring microbial organism having a HMDA pathway, the microbial organism including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate kinase, an

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway, the microbial organism including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce

HMDA. In one aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/B/C of Figure 13). In another aspect of the invention, the HMDA pathway includes a 6- aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/L/N/C of Figure 13). In another aspect of the invention, the HMDA pathway includes a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps M/N/C of Figure 13). In another aspect of the invention, the HMDA pathway includes a 6-aminocaproate N- acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/H of Figure 13). In another aspect of the invention, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6- acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6- acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of Figure 13). In another aspect of the invention, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/G of Figure 13). In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway includinga glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an 3-oxo-6- aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a 6- amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A-H of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutamyl-CoA transferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA

dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3- oxo-l-carboxyheptanal aminotransferase, a 3-oxo-l-carboxyheptanal aminating

oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a 3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a 5- aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate kinase, a 5- aminopimeloylphosphonate reductase, a 3-aminopimelate reductase, a 3-amino-7- oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino- 7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, a homolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a

2- aminopimelate CoA transferase, a 2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a 6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI; Figure 21). In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3- oxo- 1-carboxyheptanal 7-aminotransferase, a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7-aminoheptanoate

3- aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l- carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMD A) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5- oxopimeloylphosphonate reductase, a 3-oxo-l-carboxyheptanal 7-aminotransferase, a 3-oxo- l-carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3- aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7- diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3 -oxo- 1 -carboxyheptanal 7-aminotransferase or a 3 -oxo- 1 -carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA

transferase, 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3 -oxo- 1 -carboxyheptanal 7-aminotransferase, 3 -oxo- 1 -carboxyheptanal 7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3 -aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate Co A transferase or 3- oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l- carboxyheptanal 7-aminotransferase or 3-oxo-l-carboxyheptanal 7-aminating

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a 3- oxo- 1 -carboxyheptanal 3 -aminotransferase, a 3 -oxo- 1 -carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l- carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating

oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a 5- oxopimeloylphosphonate reductase, a 3-oxo- 1-carboxyheptanal 3 -aminotransferase, a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7- aminotransferase, a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7- diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo- 1-carboxyheptanal 3 -aminotransferase or a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo- 1-carboxyheptanal 3 -aminotransferase, a 3-oxo- 1-carboxyheptanal 3-aminating

oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7- aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3- oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo- 1- carboxyheptanal 3 -aminotransferase or a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMD A) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7- aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B//J/0/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7- oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2- amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2- amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate 7- aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homo lysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/K/L/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase. In yet another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a HMDA pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A, the HMDA pathway including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a 3-oxopimelate

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a 5- aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3- amino-7-oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3- aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5 -aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

aminotransferase, 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate reductase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/W/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. In yet another embodiment, the invention provides a method for producing

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate kinase, a 6-aminopimeloylphosphonate reductase, a 2- amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homo lysine decarboxylase (see Examples XXIV 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 includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producing

aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3- aminomutase, a 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, a 6- aminopimeloyl-CoA reductase (aldehyde forming), a 2-amino-7-oxoheptanoate 7- aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate animating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2- aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. The invention additionally provides a method for producing hexamethylenediamine (HMD A) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMD A) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a 2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoate aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate decarboxylase, a 6-aminohexanal aminotransferase or 6-aminohexanal aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of Figure 22). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase. In another aspect of the invention, the non- naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal

aminotransferase or a 6-aminohexanal aminating oxidoreductase. The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde aminotransferase, a 6- aminocaproic semialdehyde oxidoreductase (aminating), 6-aminocaproate N- acetyltransferase, a 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or D/P/G/H of Figure 24). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes 6- aminocaproate N-acetyltransferase; 6-acetamidohexanoate reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and 6- acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanal aminating oxidoreductase, a 6-aminohexanal aminotransferase, a 2-amino- 7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, a 2-oxo-7-aminoheptanoate decarboxylase, a homolysine decarboxylase, a 2-amino-7- oxosubarate amino acid decarboxylase, a 2-oxo-7-aminoheptanoate aminating

oxidoreductase, a 2-oxo-7-aminoheptanoate aminotransferase, a 2-amino-7-oxosubarate aminating oxidoreductase, a 2-amino-7-oxosubarate aminotransferase or a 2,7- diaminosubarate decarboxylase (see Examples XXIV and XXVI; Steps

A/B/C/G/H/I/J/K/L/M of Figure 26). In a further aspect, the microbioal organism has a 2- amino-7-oxosubarate pathway having at least one exogenous nucleic acid encoding a 2- amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino- 7-oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7- oxosubarate aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a 2-amino-5-ene- 7-oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27).

In another embodiment, the invention provides a method for producing

hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a hexamethylenediamine (HMDA) pathway including a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate animating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/H of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a 2- oxo-7-aminoheptanoate aminating oxidoreductase or a 2-oxo-7-aminoheptanoate

aminotransferase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps I/J/H of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a 2- oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a

6- aminohexanal aminotransferase (see Examples XXIV and XXVI; steps I/G/C of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase (see Examples XXIV and XXVI; steps A/B/C of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a 2- amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H of Figure 26). In a further aspect of each of the above embodiments, the microbioal organism has a 2-amino-7- oxosubarate pathway having a second set of exogenous nucleic acids encoding 2-amino-7- oxosubarate pathway enzymes expressed in a sufficient amount to produce 2-amino-7- oxosubarate, the 2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7- oxosubarate aldolase; a 2-amino-5-hydroxy-7-oxosubarate dehydratase; and a 2-amino-5-ene-

7- oxosubarate reductase (see Examples XXV and XXVI; steps A/B/C of Figure 27). .

The invention additionally provides a method for producing hexamethylenediamine (HMDA) by culturing a non-naturally occurring microbial organism having a levulinic acid (LA) pathway including at least one exogenous nucleic acid encoding a LA pathway enzyme expressed in a sufficient amount to produce LA, the LA pathway including a 3-oxoadipyl- CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, a 3- oxoadipyl-CoA hydrolase, or a 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of Figure 25). In another aspect of the invention, the non-naturally occurring microbial organism includes a set of exogenous nucleic acids encoding LA pathway enzymes, wherein the set encodes a 3-oxoadipyl-CoA thiolase; a 3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, or a 3-oxoadipyl-CoA hydrolase; and a 3- oxoadipate decarboxylase.

The invention further provides methods of producing non-naturally microbial organisms having increased production of adipate, 6- AC A and/or HMD A by disruption of one or more genes to confer increased production of adiate, 6- AC A and/or HMD A. Such gene disruptions include those exemplified herein in Example XXX and Tables 14-16.

The invention additionally provides a method for producing adipate, 6-ACA and/or HMD A that includes culturing a non-naturally occurring microbial organism that includes one or more gene disruptions that confer increased production of adiapte, 6-ACA and/or HMDA. The disruptions can occur in genes encoding an enzyme obligatory to coupling adipate, 6- AC A and/or HMDA production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, such that the disruptions confer stable growth-coupled production of adipate, 6-ACA and/or HMDA onto the non-naturally microbial organism.

In some embodiments, the gene disruption can include a complete gene deletion. Methods for gene disruption are well known to those skilled in the art and are described herein (see Example XXX). In some embodiments other methods to disrupt a gene include, for example, frameshifting by omission, addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it can confer to the non-naturally occurring organism from reverting to a phenotype expressing the previously disrupted gene. In particular, the gene disruptions are selected from the gene sets that described in Tables 14-16.

In some embodiments, the invention provides a method for producing 6-aminocaproic acid, comprising culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce 6-ACA. For example, in some embodiments, the invention provides a method for producing 6-aminocaproic acid using a non-naturally occurring microbial organism, comprising a microbial organism having a 6- aminocaproic acid (6-ACA) pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6-ACA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha- ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO

dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said 6-ACA pathway comprises a pathway selected from: (A) a 3-oxo-6- aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6- aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6- aminocaproyl-CoA/acyl-CoA transferase, a 6-aminocaproyl-CoA synthase, or a 6- aminocaproyl-CoA hydrolase; (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6- aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3- oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6- aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase; (C) an 4-hydroxy-2- oxoheptane-l,7-dioate (HODH) aldolase; a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7-dioate (OHED) reductase; a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex-4-enoate (6-OHE) reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating) ;(E) an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7- dioate (2-AHD) decarboxylase;(F) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase; (G) an HODH aldolase; an HODH formate-lyase and a pyruvate formate- lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (H) an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate- lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (I) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (J) a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2- enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2- aminopimelate decarboxylase; (K) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; (L) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6-oxohexanoate aminotransferase; (M) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate dehydrogenase (aminating) or a-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase; (N) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (O) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (P) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; (Q) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (R) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-

Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and (S) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase. In some embodiments, the invention provides a method for producing 6-aminocaproic acid using a non-naturally occurring microbial organism described herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

In some embodiments, the invention provides a method for producing 6-aminocaproic acid using a non-naturally occurring microbial organism of disclosed herein, wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 6- AC A pathway enzyme.

In some embodiments, the invention provides a method for producing 6-aminocaproic acid using a non-naturally occurring microbial organism comprising: (A) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl- CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6-aminocaproyl-CoA/acyl-CoA transferase, a 6-aminocaproyl-CoA synthase, or a 6-aminocaproyl-CoA hydrolase; (B) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3- oxo-6-aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6- aminohex-2-enoate reductase; (C) five exogounous nucleic acids encoding an 4-hydroxy-2- oxoheptane-l,7-dioate (HODH) aldolase; a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7-dioate (OHED) reductase; a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex-4-enoate (6-OHE) reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (E) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2- aminohept-4-ene-l,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase; (F) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase; (G) seven exogounous nucleic acids encoding an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (H) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (I) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate- lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl- CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating); (J) seven exogounous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6- aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6- amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate; (K) two exogounous nucleic acids encoding a CoA- dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate

dehydrogenase; (L) sixteen exogounous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6-oxohexanoate aminotransferase; (M) sixteen exogounous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate dehydrogenase (aminating) or α-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase; (N) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; (Q) nine exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (R) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

In some aspects, the invention provide a method for producing 6-aminocaproic acid using a microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some aspects, a microbial organism comprising (ii) comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a method for producing 6-aminocaproic acid using a non-naturally occurring microbial organism , wherein said microbial organism comprising (K) as disclosed above further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from: (A) succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2- pentenoyl-CoA reductase; (B) adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase and adipyl-CoA hydrolase; and (C) succinyl-CoA:acetyl- CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3- hydroxyadipate dehydratase, and 2-enoate reductase.

In some aspects, the invention provides a method for producing 6-ACA, wherein the non- naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides amethod for producing caprolactam, comprising culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce caprolactam. For example, in some embodiments, the invention provides a method for producing caprolactam using a non- naturally occurring microbial organism, comprising a microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha- ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO

dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said caprolactam pathway comprises a pathway selected from: (A) a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (B) a 3-oxo-6- aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6- aminohexanoyl-CoA dehydratase; and a 6-aminohex-2-enoyl-CoA reductase; (C) a CoA- dependent aldehyde dehydrogenase; a transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (D) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; and an adipate reductase; (E) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (F) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl- CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase; (K) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (L) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (M) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

In some embodiments, the invention provides a method for producing caprolactam usinga non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate

dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three or four exogenous nucleic acids each encoding a caprolactam pathway enzyme.

In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) one exogenous nucleic acid enconding a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (B) four exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and a 6-aminohex-2-enoyl-CoA reductase; (C) three exogenous nucleic acids encoding a CoA-dependent aldehyde dehydrogenase; a transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (D) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase; (E) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase; (F) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (G) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase; (H) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; (I) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (J) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2- Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase; (K) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

adipylphosphate reductase; (L) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase; (M) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; and an adipate reductase; (N) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl- CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2- Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; or (O) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase.

In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (A) describe above further comprises a 6-aminocaproic acid pathway, wherein said 6-aminocaproic acid pathway is selected from: (A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; and (B) a 3-oxo-6- aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo- 6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6- aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2- enoate reductase. In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism of disclosed herein, wherein said microbial organism comprising (C) described above further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from: (A) succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2- pentenoyl-CoA reductase; (B) adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase and adipyl-CoA hydrolase; and (C) succinyl-CoA:acetyl- CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3- hydroxyadipate dehydratase, and 2-enoate reductase.

In some embodiments, the invention provides a method for producing caprolactam using a non-naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a non-naturally occurring microbial organism of disclosed here, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some aspects, the invention provides a method for producing caprolactam, wherein the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing HMD A, comprising culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce HMDA. For example, in some embodiments, the invention provides a method for producing HMDA using a non-naturally occurring microbial organism, comprising a microbial organism having a hexamethylenediamine (HMDA) pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMDA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha- ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO

dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said HMDA pathway comprises a pathway selected from: (A) a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a

hexamethylenediamine dehydrogenase; (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3- oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6- aminohex-2-enoyl-CoA reductase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase; (C) a 6- aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (D) a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (E) a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (F) a 6- aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase; (J) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo- 1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3- aminotransferase or a 3-oxo-7-aminoheptanoate 3 -aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (K) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (L) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl- CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3- oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l- carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (M) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (N) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3 -oxo- 1 -carboxyheptanal 3 -aminotransferase or a 3 -oxo- 1 -carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (O) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (P) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7- aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Q) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (R) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (S) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (T) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate Co A transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (U) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5- ammopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3- amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (V) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (W) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (X) a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Y) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2- oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2- oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Z) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal

enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate decarboxylase; a 6-oxohexanoate dehydrogenase (aminating) or 6- oxohexanoate aminotransferase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AI) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a

3- oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an a-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6- aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AJ) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl- [acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3- hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an α-ketopimelate amino transferase; a 2-aminopimelate; a 2- amino-7-oxoheptanoate dehydrogenase; and a 2,7-diaminoheptanoate decarboxylase; (AK) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AL) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; (AM) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha- ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase; (AN) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase; (AO) a homocitrate synthase; a homocitrate dehydratase; a cis- Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2- hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and (AP) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase.

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an

NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a method for producing HMDA using a non-naturally occurring microbial organism herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen exogenous nucleic acids each encoding a HMDA pathway enzyme.

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) three exogenous nucleic acids encoding a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine

dehydrogenase; (B) six exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase; (C) three exogenous nucleic acids encoding a 6-aminocaproate kinase; a 6- AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (D) four exogenous nucleic acids encoding a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase; (E) three exogenous nucleic acids encoding a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6- aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase; (F) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide); (G) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA

oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal

oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide); (I) eight exogenous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6- aminopimeloyl-CoA oxidoreductase; a 3 -hydroxy-6-aminopimeloyl-Co A dehydratase; a 6- amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase; (J) seven exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7- aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7- aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3 -aminating

oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (L) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a 5- oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7- aminotransferase or 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7- aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3 -animating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (M) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (N) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5- oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3- oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (O) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (P) seven exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Q) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (R) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3- oxopimelate animating oxidoreductase; a 3-aminopimelate Co A transferase or a 3- aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3- amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or

2- amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (S) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl- CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3- oxopimelate aminotransferase or 3 -oxopimelate aminating oxidoreductase; a 3- aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (T) eight exogenous nucleic acids encoding a glutaryl-CoA beta- ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3- oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5- aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7- aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (U) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a

3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase; (V) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3- oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2- aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (W) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (X) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6- aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7- aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Y) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7- aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7- aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7- aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Z) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo- 4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2- oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminotransferase or 6- aminohexanal aminating oxidoreductase; (AA) two exogenous nucleic acids encoding a 6- aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6- aminocaproic semialdehyde oxidoreductase (aminating); (AB) four exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase; a 6- acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide); (AC) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate aminating

enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate decarboxylase; a 6-oxohexanoate dehydrogenase (aminating) or 6- oxohexanoate aminotransferase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AI) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3- oxo-glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an α-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a method for producing HMDA using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (A) further comprises a 6-aminocaproic acid pathway, wherein said 6- aminocaproic acid pathway is selected from: (A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; and (B) a 3-oxo-6-aminohexanoyl- CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6- aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6- aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2- enoate reductase.

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (AC), (AD), (AE), (AF) or (AG) as disclosed above further comprises at least one exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino-7-oxosubarate, wherein said 2-amino-7-oxosubarate pathway comprises a 2-amino-5-hydroxy-7-oxosubarate aldolase; a 2-amino-5-hydroxy-7- oxosubarate dehydratase; and a 2-amino-5-ene-7-oxosubarate reductase.

In some embodiments, the invention provides a method for producing HMDA using a non- naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a method for producing HMDA using a non-naturally occurring microbial organism disclosed herein, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a method for producing HMDA, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In some embodiments, the invention provides a method for producing adipate, comprising culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce adipate. For example, in some embodiments, the invention provides a method for producing adipate using a non-naturally occurring microbial organism, comprising a microbial organism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a

phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said adipate pathway comprises a pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyl transferase; a 3 -hydroxy acyl- CoA dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (B) a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3-oxoadipate reductase; a 3- hydroxyadipate dehydratase; and a 2-enoate reductase; (C) an alpha-ketoadipyl-CoA synthetase, a phosphotransketoadipylase/alpha-ketoadipate kinase or an alpha-ketoadipyl- CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydrogenase; a 2-hydroxyadipyl- CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl- CoA hydrolase; (D) a 2-hydroxyadipate dehydrogenase; a 2-hydroxyadipyl-CoA synthetase, a phosphotranshydroxyadipylase/2-hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl- CoA transferase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (E) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo- isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl- CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha- ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl- CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; and (G) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase.

In some embodiments, the invention provides a method for producing adipate using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some embodiments, the invention provides a method for producing adipate using a non-naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

In some embodiments, the invention provides a method for producing adipate using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen exogenous nucleic acids each encoding an adipate pathway enzyme.

In some embodiments, the invention provides a method for producing adipate using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprises: (A) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5- carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a

phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl- CoA hydrolase; (B) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydratase; and a 2-enoate reductase; (C) five exogenous nucleic acids encoding an alpha- ketoadipyl-CoA synthetase, a phosphotransketoadipylase/alpha-ketoadipate kinase or an alpha-ketoadipyl-CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydrogenase; a 2- hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl- CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (D) five exogenous nucleic acids encoding a 2- hydroxyadipate dehydrogenase; a 2-hydroxyadipyl-CoA synthetase, a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl- CoA transferase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (E) fourteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo- glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) nine exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha- ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; or (G) eight exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

In some embodiments, the invention provides a method for producing adipate using a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the invention provides a non- naturally occurring microbial organism disclosed herein, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

In some embodiments, the invention provides a method for producing adipate using a non- naturally occurring microbial organism disclosed herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the invention provides a method for producing adipate using a non-naturally occurring microbial organism disclosed herein, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing adipate, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing caprolactam as disclosed here, wherein said microbial organism comprises a caprolactam pathway and wherein said caprolactam is produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam. For example, the invention provides a method for producing caprolactam, wherein said caprolactam pathway is selected from the group consisting of (G), (H), (M), (N) and (O) as disclosed above, which includes spontaneous cylcization of 6-aminocaproyl-CoA to caprolactam.

Suitable purification and/or assays to test for the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid

Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art.

The adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion

chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers can be cultured for the biosynthetic production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.

For the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication

2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In addition to renewable feedstocks such as those exemplified above, the adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H₂ and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H₂ and CO, syngas can also include C0₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0₂ and C0₂/H₂ mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H₂-dependent conversion of C0₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of C0₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2 C0₂ + 4 H₂ + n ADP + n Pi→ CH₃COOH + 2 H₂0 + n ATP

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0₂ and H₂ mixtures as well for the production of acetyl-CoA and other desired products. The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate

cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase, and these enzymes can also be referred to as methyltetrahydrofolatexorrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains a Wood-Ljungdahl pathway can confer syngas utilization ability.

Additionally, given the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, C0₂ and/or H₂ to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha- ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H₂ by carbon monoxide dehydrogenase and hydrogenase are utilized to fix C0₂ via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate

kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the p-toluate, terepathalate, or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors, glyceraldehyde-3 -phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate :ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a p- toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability. Given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the

biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid and any of the intermediate metabolites in the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. All that is required is to engineer in one or more of the required enzyme activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid when grown on a carbohydrate and produces and/or secretes any of the intermediate metabolites shown in the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway when grown on a carbohydrate or other carbon source. For example, an adipate producing microbial organisms can initiate synthesis from an intermediate, for example, 3-oxoadipyl- CoA, 3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see Figure 2), as desired. In addition, an adipate producing microbial organism can initiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA, 3-oxoadipate, 3-hydroxyadipate, or hexa-2- enedioate (see Figure 3). The 6-aminocaproic acid producing microbial organism of the invention can initiate synthesis from an intermediate, for example, adipate semialdehyde (see Figure 8). The caprolactam producing microbial organism of the invention can initiate synthesis from an intermediate, for example, adipate semialdehyde or 6-aminocaproic acid (see Figure 8), as desired. The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme in sufficient amounts to produce adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.

Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid is between about 3- 150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non- naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers can synthesize adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing microbial organisms can produce adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intracellularly and/or secrete the product into the culture medium. The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions. As described herein, one exemplary growth condition for achieving biosynthesis of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of

commercial quantities of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid will include culturing a non-naturally occurring adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose. Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers of the invention for continuous production of substantial quantities of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired. As described herein, an intermediate in the adipate pathway utilizing 3-oxoadipate, hexa-2-enedioate, can be converted to adipate, for example, by chemical hydrogenation over a platinum catalyst (see Example III).

As described herein, exemplary growth conditions for achieving biosynthesis of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid includes the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non- naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an

osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. For example, as described in Example XXII, Escherichia coli in the presence of varying amounts of 6-aminocaproic acid is suitably grown in the presence of 2 mM glycine betaine. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM. In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid or any adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway intermediate, or for side products generated in reactions diverging away from an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter the carbon- 12, carbon-13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios. In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel- derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0₂, which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

The unstable carbon isotope carbon- 14 or radiocarbon makes up for roughly 1 in 10¹² carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect".

Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by

Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio of carbon- 14 (¹⁴C) to carbon- 12 (¹²C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S- B)/(M-B), where B, S and M represent the ¹⁴C/¹²C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴C/¹²C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to 8¹³C_VPDB=-19 per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proa, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 8¹³C_VPDB=- 19 per mil. This is equivalent to an absolute (AD 1950) ¹⁴C/¹²C ratio of 1.176 ± 0.010 x 10^"12 (Karlen et al., Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one istope with respect to another, for example, the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴, and these corrections are reflected as a Fm corrected for δ¹³. An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post- 1950 injection of carbon- 14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources. As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon- 14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old. ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50% water would be considered to have a Biobased Content = 100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50%) organic carbon and is a petroleum-based product would be considered to have a Biobased Content = 0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543- 2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30%> (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable

terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011). Accordingly, in some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or an adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is C0₂. In some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or an adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon- 12, carbon-13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically produced adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate as disclosed herein, and to the products derived therefrom, wherein the adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or a adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate has a carbon- 12, carbon-13, and carbon- 14 isotope ratio of about the same value as the C0₂ that occurs in the environment. For example, in some aspects the invention provides bioderived adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or a bioderived adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate having a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or a bioderived adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, or an

intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides nylons, fibers, plastics, unsaturated polyesters, polyester polyols, polyurethanes, lubricant components, flavorants, gelling aids, pharmaceuticals, epoxy resins, nylon-like polymers, synthetic rubbers, plastics,

methyltetrahydrofuran, valerolactone or ethyl levulinate having a carbon- 12 versus carbon-13 versus carbon- 14 isotope ratio of about the same value as the C0₂ that occurs in the environment, wherein the nylons, fibers, plastics, unsaturated polyesters, polyester polyols, polyurethanes, lubricant components, flavorants, gelling aids, pharmaceuticals, epoxy resins, nylon-like polymers, synthetic rubbers, plastics, methyltetrahydrofuran, valerolactone or ethyl levulinate are generated directly from or in combination with bioderived adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid or a bioderived adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid intermediate as disclosed herein.

Adipate is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of nylons, fibers, plastics, unsaturated polyesters, polyester polyols, polyurethanes, lubricant components, flavorants or gelling aids.

Accordingly, in some embodiments, the invention provides a biobased nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid comprising one or more bioderived adipate or bioderived adipate intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

6-Aminocaproic acid is a chemical used in commercial and industrial applications. Non- limiting examples of such applications include production of nylons, fibers, plastics or pharmaceuticals. Accordingly, in some embodiments, the invention provides a biobased nylon, fiber, plastic or pharmaceutical comprising one or more bioderived 6-aminocaproic acid or bioderived 6-aminocaproic acid intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. Caprolactam is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of nylons, fibers or plastics. Accordingly, in some embodiments, the invention provides a biobased nylon, fiber or plastic comprising one or more bioderived caprolactam or bioderived caprolactam intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

Hexamethylenediamine is a chemical used in commercial and industrial applications. Non- limiting examples of such applications include production of nylons, fibers, polyurethanes or epoxy resins. Accordingly, in some embodiments, the invention provides a biobased nylon, fiber, polyurethane or epoxy resin comprising one or more bioderived hexamethylenediamine or bioderived hexamethylenediamine intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

Levulinic acid is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of nylon-like polymers, synthetic rubbers, plastics, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceuticals.

Accordingly, in some embodiments, the invention provides a biobased nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical comprising one or more bioderived levulinic acid or bioderived levulinic acid intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. As used herein, the term "bioderived" means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "biobased" means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid comprising bioderived adipate or bioderived adipate intermediate, wherein the bioderived adipate or bioderived adipate intermediate includes all or part of the adipate or adipate intermediate used in the production of nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid. Thus, in some aspects, the invention provides a biobased nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%), at least 98%> or 100%> bioderived adipate or bioderived adipate intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid wherein the adipate or adipate intermediate used in its production is a combination of bioderived and petroleum derived adipate or adipate intermediate. For example, a biobased nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid can be produced using 50%> bioderived adipate and 50%> petroleum derived adipate or other desired ratios such as 60%>/40%>, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of

bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing nylon, fiber, plastic, unsaturated polyester, polyester polyol, polyurethane, lubricant component, flavorants or gelling aid using the bioderived adipate or bioderived adipate intermediate of the invention are well known in the art. In some embodiments, the invention provides nylon, fiber, plastic or pharmaceutical comprising bioderived 6-aminocaproic acid or bioderived 6-aminocaproic acid intermediate, wherein the bioderived 6-aminocaproic acid or bioderived 6-aminocaproic acid intermediate includes all or part of the 6-aminocaproic acid or 6-aminocaproic acid intermediate used in the production of nylon, fiber, plastic or pharmaceutical. Thus, in some aspects, the invention provides a biobased nylon, fiber, plastic or pharmaceutical comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%> or 100%) bioderived 6-aminocaproic acid or bioderived 6- aminocaproic acid intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased nylon, fiber, plastic or pharmaceutical wherein the 6- aminocaproic acid or 6-aminocaproic acid intermediate used in its production is a

combination of bioderived and petroleum derived 6-aminocaproic acid or 6-aminocaproic acid intermediate. For example, a biobased nylon, fiber, plastic or pharmaceutical can be produced using 50%> bioderived 6-aminocaproic acid and 50%> petroleum derived 6- aminocaproic acid or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing nylon, fiber, plastic or pharmaceutical using the bioderived 6-aminocaproic acid or bioderived 6-aminocaproic acid intermediate of the invention are well known in the art.

In some embodiments, the invention provides nylon, fiber or plastic comprising bioderived caprolactam or bioderived caprolactam intermediate, wherein the bioderived caprolactam or bioderived caprolactam intermediate includes all or part of the caprolactam or caprolactam intermediate used in the production of nylon, fiber or plastic. Thus, in some aspects, the invention provides a biobased nylon, fiber or plastic comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived caprolactam or bioderived caprolactam intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased nylon, fiber or plastic wherein the caprolactam or caprolactam intermediate used in its production is a combination of bioderived and petroleum derived caprolactam or caprolactam intermediate. For example, a biobased nylon, fiber or plastic can be produced using 50% bioderived caprolactam and 50% petroleum derived caprolactam or other desired ratios such as

60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing nylon, fiber or plastic using the bioderived caprolactam or bioderived caprolactam intermediate of the invention are well known in the art.

In some embodiments, the invention provides nylon, fiber, polyurethane or epoxy resin comprising bioderived hexamethylenediamine or bioderived hexamethylenediamine intermediate, wherein the bioderived hexamethylenediamine or bioderived

hexamethylenediamine intermediate includes all or part of the hexamethylenediamine or hexamethylenediamine intermediate used in the production of nylon, fiber, polyurethane or epoxy resin. Thus, in some aspects, the invention provides a biobased nylon, fiber, polyurethane or epoxy resin comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived hexamethylenediamine or bioderived hexamethylenediamine intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased nylon, fiber, polyurethane or epoxy resin wherein the hexamethylenediamine or

hexamethylenediamine intermediate used in its production is a combination of bioderived and petroleum derived hexamethylenediamine or hexamethylenediamine intermediate. For example, a biobased nylon, fiber, polyurethane or epoxy resin can be produced using 50% bioderived hexamethylenediamine and 50% petroleum derived hexamethylenediamine or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing nylon, fiber, polyurethane or epoxy resin using the bioderived hexamethylenediamine or bioderived hexamethylenediamine intermediate of the invention are well known in the art. In some embodiments, the invention provides nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical comprising bioderived levulinic acid or bioderived levulinic acid intermediate, wherein the bioderived levulinic acid or bioderived levulinic acid intermediate includes all or part of the levulinic acid or levulinic acid intermediate used in the production of nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical. Thus, in some aspects, the invention provides a biobased nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%>, at least 95%, at least 98%> or 100%) bioderived levulinic acid or bioderived levulinic acid intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased nylon-like polymer, synthetic rubber, plastic,

methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical wherein the levulinic acid or levulinic acid intermediate used in its production is a combination of bioderived and petroleum derived levulinic acid or levulinic acid intermediate. For example, a biobased nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran,

valerolactone, ethyl levulinate or pharmaceutical can be produced using 50%> bioderived levulinic acid and 50%> petroleum derived levulinic acid or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing nylon-like polymer, synthetic rubber, plastic, methyltetrahydrofuran, valerolactone, ethyl levulinate or pharmaceutical using the bioderived levulinic acid or bioderived levulinic acid intermediate of the invention are well known in the art.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by

OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

The concept of growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting

substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates. The knockouts suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point should lie within its calculated solution boundary. Plots such as these allow one to visualize how close strains are to their performance limits or, in other words, how much room is available for improvement. The OptKnock framework has already been able to identify promising gene deletion strategies for biochemical overproduction, (Burgard et al. , Biotechnol Bioeng, 84(6):647-657 (2003); Pharkya et al., Biotechnol Bioeng, 84(7):887-899 (2003)) and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.

Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models.

These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the

performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.

Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components. Analysis methods such as convex analysis, linear programming and the calculation of extreme pathways as described, for example, in Schilling et al., J. Theor. Biol. 203:229-248 (2000); Schilling et al., Biotech. Bioeng. 71 :286-306 (2000) and Schilling et al, Biotech. Prog. 15:288-295 (1999), can be used to determine such phenotypic capabilities.

As described above, one constraints-based method used in the computational programs applicable to the invention is flux balance analysis. Flux balance analysis is based on flux balancing in a steady state condition and can be performed as described in, for example,

Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approaches have been applied to reaction networks to simulate or predict systemic properties of, for example, adipocyte metabolism as described in Fell and Small, J Biochem. 138:781-786 (1986), acetate secretion from E. coli under ATP maximization conditions as described in Majewski and Domach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeast as described in VanroUeghem et al., Biotech. Prog. 12:434-448 (1996). Additionally, this approach can be used to predict or simulate the growth of S. cerevisiae on a variety of single-carbon sources as well as the metabolism of H. influenzae as described in Edwards and Palsson, Proc. Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J Bio. Chem. 274: 17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130 (2001).

Once the solution space has been defined, it can be analyzed to determine possible solutions under various conditions. This computational approach is consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

The ability of a cell or organism to obligatory couple growth to the production of a biochemical product can be illustrated in the context of the biochemical production limits of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. tThe production of a desired biochemical generally is in direct competition with biomass formation for intracellular resources. Under these circumstances, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates. The knockouts suggested by the above metabolic modeling and simulation programs such as OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point will lie within its calculated solution boundary. Plots such as these allow accurate predictions of how close the designed strains are to their performance limits which also indicates how much room is available for improvement.

The OptKnock mathematical framework is exemplified herein for pinpointing gene deletions leading to growth-coupled biochemical production (see Example XXX). The procedure builds upon constraint-based metabolic modeling which narrows the range of possible phenotypes that a cellular system can display through the successive imposition of governing physico-chemical constraints, Price et al., Nat Rev Microbiol, 2: 886-97 (2004). As described above, constraint-based models and simulations are well known in the art and generally invoke the optimization of a particular cellular objective, subject to network stoichiometry, to suggest a likely flux distribution.

Briefly, the maximization of a cellular objective quantified as an aggregate reaction flux for a steady state metabolic network comprising a set N = { 1 , ... , N} of metabolites and a set M = { 1 , .. · , M) of metabolic reactions is expressed mathematically as follows: maximize v_{cellular objective}

M

subject to ∑^i/ ^V7 ⁼ 0' V i e N

7=1

Vsubstrate = V substrate _ uptake mmol/gDW-hr

V i e {limiting substrate(s)} Vatp≥ Vatp main mmol/gDW-hr

Vj > 0, V ;^' e {irrev. reactions} where Sy is the stoichiometric coefficient of metabolite i in reaction j, Vj is the flux of reaction j, v substrate _uptake represents the assumed or measured uptake rate(s) of the limiting substrate(s), and v_{atp main} is the non-growth associated ATP maintenance requirement. The vector v includes both internal and external fluxes. In this study, the cellular objective is often assumed to be a drain of biosynthetic precursors in the ratios required for biomass formation, Neidhardt, F.C. et al, 2nd ed. 1996, Washington, D.C.: ASM Press. 2 v. (xx, 2822, lxxvi ). The fluxes are generally reported per 1 gDW-hr (gram of dry weight times hour) such that biomass formation is expressed as g biomass produced/ gDW -hr or 1/hr.

The modeling of gene deletions, and thus reaction elimination, first employs the

incorporation of binary variables into the constraint-based approach framework, Burgard et al, Biotechnol Bioeng, 74: 364-375 (2001), Burgard et al, Biotechnol Prog, 17: 791-797 (2001). These binary variables, l, if reaction flux v ,. is active

y, = ' ., ₊. _fl ¹ . _{+ +}. ,V 7^' e M

¹ 0, if reaction flux v . is not active assume a value of 1 if reaction j is active and a value of 0 if it is inactive. The following constraint, min / max w · IV Λ

· .!· < »· < »' - J V ./ C M ensures that reaction flux v, is set to zero only if variable yj is equal to zero. Alternatively, when yj is equal to one, Vj is free to assume any value between a lower v™'" and an upper Vj^max bound. Here, vf"ⁿ and vf^ax are identified by minimizing and maximizing, respectively, every reaction flux subject to the network constraints described above, Mahadevan et al., Metab Eng, 5: 264-76 (2003).

Optimal gene/reaction knockouts are identified by solving a bilevel optimization problem that chooses the set of active reactions (y, = 1) such that an optimal growth solution for the resulting network overproduces the chemical of interest. Schematically, this bilevel optimization problem is illustrated in Figure 2. Mathematically, this bilevel optimization problem is expressed as the following bilevel mixed-integer optimization problem: maximize ^chemical (OptKnock)

yj subject to maximize Vbk subject to _JS_ijv_j = 0, V i e N

7=1 v_substrate = v _substrate __uptake V / G {limiting substrate(s)}

Vatp— ^atpjnain v, . > _v ^tarS^et

^Vbwmass ^ ^vbiomass

mm ^ ^ max

V - · V < V < V - V - ,

J " J J J j > V 7 e M

yj≡ {0,1 }, V y e M where v chemical is the production of the desired target product, for example adipate, 6-ACA and/or HMD A, or other biochemical product, and K is the number of allowable knockouts. Note that setting K equal to zero returns the maximum biomass solution of the complete network, while setting K equal to one identifies the single gene/reaction knockout (y, = 0) such that the resulting network involves the maximum overproduction given its maximum biomass yield. The final constraint ensures that the resulting network meets a minimum biomass yield. Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), provide a more detailed description of the model formulation and solution procedure. Problems containing hundreds of binary variables can be solved in the order of minutes to hours using CPLEX 8.0, GAMS: The Solver Manuals. 2003: GAMS Development Corporation, accessed via the GAMS, Brooke et al., GAMS Development Corporation (1998), modeling environment on an IBM RS6000-270 workstation. The OptKnock framework has already been able to identify promising gene deletion strategies for biochemical overproduction, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), Pharkya et al, Biotechnol Bioeng, 84: 887-899 (2003), and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.

The methods described above will provide one set of metabolic reactions to disrupt.

Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes. Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur. To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791- 797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®. The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum- growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US

2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

Given the teachings and guidance provided herein, those skilled in the art will understand that to disrupt an enzymatic reaction the catalytic activity of the one or more enzymes involved in the reaction is to be disrupted. Disruption can occur by a variety of means including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity disruption can occur by a genetic alteration that reduces or destroys the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits in order to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is reduced or eliminated.

Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the growth- coupled product production. Examplary disruptions to confer increased production of adipate, 6-ACA and/or HMDA are described in Example XXX and Tables 14-16.

Employing the methods exemplified above, the methods of the invention allow the construction of cells and organisms that increase production of a desired product, for example, by coupling the production of a desired product to growth of the cell or organism engineered to harbor the identified genetic alterations. As disclosed herein, metabolic alterations have been identified that couple the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of the organism. Microbial organism strains constructed with the identified metabolic alterations produce elevated levels, relative to the absence of the metabolic alterations, of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid during the exponential growth phase. These strains can be beneficially used for the commercial production of adipate, 6- aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in continuous fermentation process without being subjected to the negative selective pressures described previously. Although exemplified herein as metabolic alterations, in particular one or more gene disruptions, that confer growth coupled production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, it is understood that any gene disruption that increases the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be introduced into a host microbial organism, as desired.

Therefore, the methods of the invention provide a set of metabolic modifications that are identified by an in silico method such as OptKnock. The set of metabolic modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. For adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid production, metabolic modifications can be selected from the set of metabolic modifications listed in Tables 14-16 (see Example XXX).

Also provided is a method of producing a non-naturally occurring microbial organisms having stable growth-coupled production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The method can include identifying in silico a set of metabolic modifications that increase production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, for example, increase production during exponential growth; genetically modifying an organism to contain the set of metabolic modifications that increase production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, and culturing the genetically modified organism. If desired, culturing can include adaptively evolving the genetically modified organism under conditions requiring production of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid. The methods of the invention are applicable to bacterium, yeast and fungus as well as a variety of other cells and microorganism, as disclosed herein.

Thus, the invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions that confer increased production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. In one embodiment, the one or more gene disruptions confer growth-coupled production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, and can, for example, confer stable growth-coupled production of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid. In another embodiment, the one or more gene disruptions can confer obligatory coupling of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production to growth of the microbial organism. Such one or more gene disruptions reduce the activity of the respective one or more encoded enzymes. The non-naturally occurring microbial organism can have one or more gene disruptions included in a metabolic modification listed in Tables 14-16. As disclosed herein, the one or more gene disruptions can be a deletion. Such non-naturally occurring microbial organisms of the invention include bacteria, yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes, as disclosed herein.

Thus, the invention provides a non-naturally occurring microbial organism, comprising one or more gene disruptions, where the one or more gene disruptions occur in genes encoding proteins or enzymes where the one or more gene disruptions confer increased production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in the organism. The production of adipate, 6-aminocaproic acid, caprolactam,

hexamethylenediamine or levulinic acid can be growth-coupled or not growth-coupled. In a particular embodiment, the production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be obligatorily coupled to growth of the organism, as disclosed herein.

The invention provides non naturally occurring microbial organisms having genetic alterations such as gene disruptions that increase production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, for example, growth-coupled production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Product production can be, for example, obligatorily linked to the exponential growth phase of the microorganism by genetically altering the metabolic pathways of the cell, as disclosed herein. The genetic alterations can increase the production of the desired product or even make the desired product an obligatory product during the growth phase. Sets of metabolic alterations or transformations that result in increased production and elevated levels of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis are exemplified in Tables 14-16 (see Example XXX). Each alteration within a set corresponds to the requisite metabolic reaction that should be functionally disrupted. Functional disruption of all reactions within each set can result in the increased production of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid by the engineered strain during the growth phase. The corresponding reactions to the referenced alterations can be found in Tables 14-16 (see Example XXX), and the gene or genes that encode enzymes or proteins that carry out the reactions are set forth in Tables 14-16. For example, for each strain exemplified in Tables 14-16, the metabolic alterations that can be generated for adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production are shown in each row. These alterations include the functional disruption of the reactions shown in Tables 14-16. Each of these non-naturally occurring alterations result in increased production and an enhanced level of adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid production, for example, during the exponential growth phase of the microbial organism, compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions. Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Reverse Adipate Degradation Pathway

This example describes an exemplary adipate synthesis pathway via a reverse adipate degradation pathway.

Organisms such as Penicillium chrysogenum have the ability to naturally degrade adipate (Thykaer et al., Metab. Eng. 4: 151-158. (2002)). The mechanism is similar to the oxidation of fatty acids (see Figure 1). The first step in adipate degradation is an ATP-dependent reaction that activates adipate with CoA. The second reaction is catalyzed by a

dehydrogenase that forms 5-carboxy-2-pentenoyl-CoA from adipyl-CoA. During

peroxisomal adipate degradation, the dehydrogenase enzyme contains FAD, which accepts the electrons and then transfers them directly to oxygen. A catalase enzyme dissipates the H₂O₂ formed by the reduction of oxygen. In mitochondrial fatty acid oxidation, the FAD from the dehydrogenase transfers electrons directly to the electron transport chain. A multifunctional fatty acid oxidation protein in eukaryotes such as S. cerevisiae and P.

chrysogenum carries out the following hydratase and dehydrogenase steps. The final step is an acyl transferase that splits 3-oxoadipyl CoA into acetyl-CoA and succinyl-CoA. A highly efficient pathway for the production of adipate is achieved through genetically altering a microorganism such that similar enzymatic reactions are employed for adipate synthesis from succinyl-CoA and acetyl-CoA (see Figure 2). Successful implementation of this entails expressing the appropriate genes, tailoring their expression, and altering culture conditions so that high acetyl-CoA, succinyl-CoA, and/or redox (for example,

NADH/NAD+) ratios will drive the metabolic flux through this pathway in the direction of adipate synthesis rather than degradation. Strong parallels to butyrate formation in Clostridia (Kanehisa and Goto, Nucl. Acids Res. 28:27-30 (2000)) support that each step in the adipate synthesis pathway is thermodynamically feasible with reaction directionality governed by the concentrations of the participating metabolites. The final step, which forms adipate from adipyl-CoA, can take place either via a synthetase, phosphotransadipylase/kinase, transferase, or hydrolase mechanism.

The maximum theoretical yields of adipate using this pathway were calculated both in the presence and absence of an external electron acceptor such as oxygen. These calculations show that the pathway can efficiently transform glucose into adipate and C0₂ under anaerobic conditions with a 92% molar yield (Table 1). The production of adipate using this pathway does not require the uptake of oxygen as NAD+ can be regenerated in the two hydrogenase steps that form 3-hydroxyadipyl-CoA and adipyl-CoA (see Figure 2). Further, the pathway is favorable energetically as up to 1.55 moles of ATP are formed per mole of glucose consumed at the maximum theoretical yield of adipate assuming either a synthetase, phosphotransadipylase/kinase, or transferase mechanism for the final conversion step. The ATP yield can be further improved to 2.47 moles of ATP produced per mole of glucose if phosphoenolpyruvate carboxykinase (PPCK) is assumed to function in the ATP-generating direction towards oxaloacetate formation. Maximum ATP yield calculations were then performed assuming that the adipyl-CoA to adipate transformation is a hydrolysis step. This reduces the maximum ATP yields at maximum adipate production to 0.85 and 1.77 mole ATP per mole glucose consumed if PPCK is assumed irreversible and reversible,

respectively. Nevertheless, these ATP yields are sufficient for cell growth, maintenance, and production. Table 1 : The maximum theoretical yields of adipate and the associated ATP yields per mole of glucose using the reverse degradation pathway assuming the final step in the pathway is a synthetase, phosphotransadipylase/kinase, or transferase. Aerobic Anaerobic

Adipate Yield 0.92 0.92

Max ATP yield @ max adipate yield 1.55 1.55

Max ATP yield @ max adipate yield 2.47 2.47

PPCK assumed

Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of adipate, one or more exogenous DNA sequence(s) are expressed in a suitable host microorganism. In addition, the microorganisms can have endogenous gene(s) functionally deleted. These modifications allow the production of adipate using renewable feedstock. Below is described a number of biochemically characterized candidate genes that encode enzymes that catalyze each step of the reverse adipate degradation pathway in a production host. Although described using E. coli as a host organism to engineer the pathway, essentially any suitable host organism can be used. Specifically listed are genes that are native to E. coli as well as genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed.

Referring to Figure 2, step 1 involves succinyl CoA:acetyl CoA acyl transferase (β- ketothiolase). The first step in the pathway combines acetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. The gene products encoded by pcaF in Pseudomonas strain B13

(Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al, Proc. Natl. Acad. Sci. USA 95 :6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188: 117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiol. 153:357-365 (2007)) catalyze the conversion of 3-oxoadipyl- CoA into succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate or styrene. Since β-ketothiolase enzymes catalyze reversible

transformations, these enzymes can be employed for the first step in adipate synthesis shown in Figure 2. For example, the ketothiolase phaA from R. eutropha combines two molecules of acetyl-CoA to form acetoacetyl-CoA (Sato et al., J Biosci. Bioengineer. 103:38-44 (2007)). Similarly, a β-keto thiolase (bktB) has been reported to catalyze the condensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA (Slater et al., J Bacteriol. 180: 1979-1987 (1998)) R. eutropha. Additional candidates are found in Burkholderia ambifaria AMMD. The protein sequences for the above-mentioned gene products are well known in the art and can be accessed in the public databases such as GenBank using the following GI numbers and/or GenBank identifiers:.

These exemplary sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (for example, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to generate production hosts.

For example, orthologs oipaaJ from Escherichia coli K12 can be found using the following GI numbers and/or GenBank identifiers:

Example orthologs of pcaF from Pseudomonas knackmussii can be found using the following GI numbers and/or GenBank identifiers:

GI# GenBank Accession # Organism

4530443 AAD22035.1 Streptomyces sp. 2065

24982839 AAN67000.1 Pseudomonas putida

115589162 ABJ15177.1 Pseudomonas aeruginosa Additional native candidate genes for the ketothiolase step include atoB, which can catalyze the reversible condensation of 2 acetyl-CoA molecules (Sato et al., J Biosci. Bioengineer. 103:38-44 (2007)), and its homolog yqeF. Non-native gene candidates include phaA (Sato et al, supra, 2007) and bktB (Slater et al, J Bacteriol. 180: 1979-1987 (1998)) from ?.

eutropha, and the two ketothiolases, thiA and thiB, from Clostridium acetobutylicum (Winzer et al., J Mol. Microbiol. Biotechnol. 2:531-541 (2000)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or

GenBank identifiers:

It is less desirable to use the thiolase-encoding genes fadA and fadB, genes in fatty acid degradation pathway in E. coli, in this exemplary pathway. These genes form a complex that encodes for multiple activities, most of which are not desired in this pathway.

Referring to Figure 2, step 2 involves 3-hydroxyacyl-CoA dehydrogenase. The second step in the pathway involves the reduction of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. The gene products encoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188: 117-125 (2007)) catalyze the reverse reaction, that is, the oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene. The reactions catalyzed by such dehydrogenases are reversible and accordingly these genes represent candidates to carry out the second step of adipate synthesis as shown in Figure 2. A similar transformation is also carried out by the gene product oihbd in

Clostridium acetobutylicum (Atsumi et al., Metab. Eng. (epub Sep. 14, 2007); Boynton et al., J Bacteriol. 178:3015-3024 (1996)). This enzyme converts acetoacetyl-CoA to 3- hydroxybutyryl-CoA. Lastly, given the proximity in E. coli of paaH to other genes in the phenylacetate degradation operon (Nogales et al.,. Microbiol. 153:357-365 (2007)) and the fact that paaH mutants cannot grow on phenylacetate (Ismail et al., Eur. J. Biochem.

270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodes a 3 -hydroxy acyl- CoA dehydrogenase. The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 2, step 3 involves 3-hydroxyadipyl-CoA dehydratase. The gene product of crt from C. acetobutylicum catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (see Figure 2) (Atsumi et al., supra, 2007; Boynton et al., J Bacteriol.

178:3015-3024 (1996)). Homologs of this gene are strong candidates for carrying out the third step in the adipate synthesis pathway exemplified in Figure 2. In addition, genes known to catalyze the hydroxylation of double bonds in enoyl-CoA compounds represent additional candidates given the reversibility of such enzymatic transformations. For example, the enoyl- CoA hydratases, phaA and phaB, of P. putida are believed to carry out the hydroxylation of double bonds during phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and thus represent additional candidates for incorporation into E. coli. The deletion of these genes precludes phenylacetate degradation in P. putida. The paaA and paaB from P. fluorescens catalyze analogous transformations (Olivera et al., supra, 1998). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park and Lee, J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park and Lee, Biotechnol.

Bioeng. 86:681-686 (2004); Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346 (2004)), and paaG (Ismail et al., supra, 2003; Park and Lee, supra, 2004; Park and Lee, supra, 2004). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism

maoC 16129348 NP_415905.1 Escherichia coli paaF 16129354 NP_415911.1 Escherichia coli

paaG 16129355 NP_415912.1 Escherichia coli

cr 15895969 NP_349318.1 Clostridium acetobutylicum paaA 26990002 NP_745427.1 Pseudomonas putida

paaB 26990001 NP_745426.1 Pseudomonas putida

phaA 106636093 ABF82233.1 Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonas fluorescens

Alternatively, beta-oxidation genes are candidates for the first three steps in adipate synthesis. Candidate genes for the proposed adipate synthesis pathway also include the native fatty acid oxidation genes of E. coli and their homologs in other organisms. The E. coli genes fadA and fadB encode a multienzyme complex that exhibits ketoacyl-CoA thiolase, 3 -hydroxy acyl- CoA dehydrogenase, and enoyl-CoA hydratase activities (Yang et al., Biochem. 30:6788- 6795 (1991); Yang et al, J Biol. Chem. 265: 10424-10429 (1990); Yang et al, J Biol. Chem. 266: 16255 (1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937 (1990)). These activities are mechanistically similar to the first three transformations shown in Figure 2. The fadl and fadJ genes encode similar functions and are naturally expressed only anaerobically (Campbell et al., Mol. Microbiol. 47:793-805 (2003)). These gene products naturally operate to degrade short, medium, and long chain fatty-acyl-CoA compounds to acetyl-CoA, rather than to convert succinyl-CoA and acetyl-CoA into 5-carboxy-2-pentenoyl-CoA as proposed in Figure 2. However, it is well known that the ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase enzymes catalyze reversible transformations.

Furthermore, directed evolution and related approaches can be applied to tailor the substrate specificities of the native beta-oxidation machinery of E. coli. Thus these enzymes or homologues thereof can be applied for adipate production. If the native genes operate to degrade adipate or its precursors in vivo, the appropriate genetic modifications are made to attenuate or eliminate these functions. However, it may not be necessary since a method for producing poly[(R)-3-hydroxybutyrate] in E. coli that involves activating7¾<¾ by knocking out a negative regulator, fadR, and co-expressing a non-native ketothiolase, phaA from Ralstonia eutropha, has been described (Sato et al., J Biosci. Bioeng. 103:38-44 (2007)). This work clearly demonstrated that a beta-oxidation enzyme, in particular the gene product οΐ/adB which encodes both 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities, can function as part of a pathway to produce longer chain molecules from acetyl- CoA precursors. The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 2, step 4 involves 5-carboxy-2-pentenoyl-CoA reductase. Whereas the ketothiolase, dehydrogenase, and enoyl-CoA hydratase steps are generally reversible, the enoyl-CoA reductase step is almost always oxidative and irreversible under physiological conditions (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). FadE catalyzes this likely irreversible transformation in E. coli (Campbell and Cronan, J Bacteriol. 184:3759- 3764 (2002)). The pathway requires an enzyme that can reduce a 2-enoyl-CoA intermediate, not one such as FadE that will only oxidize an acyl-CoA to a 2-enoyl-CoA compound.

Furthermore, although it has been suggested that E. coli naturally possesses enzymes for enoyl-CoA reduction (Mizugaki et al., J Biochem. 92: 1649-1654 (1982); Nishimaki et al., J Biochem. 95: 1315-1321 (1984)), no E. coli gene possessing this function has been biochemically characterized.

One candidate gene for the enoyl-Co A reductase step is the gene product of bed from C. acetobutylicum (Atsumi et al., supra, 2007; Boynton et al., J Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA, a reaction similar in mechanism to the desired reduction of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA in the adipate synthesis pathway. Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli, resulting in an active enzyme (Hoffmeister et al., supra, 2005). This approach is well known to those skilled in the art of expressing eukarytotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci and Martin, FEBS Lett. 581 : 1561-1566 (2007)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 2, step 5 involves adipyl-CoA synthetase (also referred to as adipate-CoA ligase), phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl- CoA hydrolase. From an energetic standpoint, it is desirable for the final step in the adipate synthesis pathway to be catalyzed by an enzyme or enzyme pair that can conserve the ATP equivalent stored in the thioester bond of adipyl-CoA. The product of the sucC and sucD genes of E. coli, or homo logs thereof, can potentially catalyze the final transformation shown in Figure 2 should they exhibit activity on adipyl-CoA. The sucCD genes naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given the structural similarity between succinate and adipate, that is, both are straight chain dicarboxylic acids, it is reasonable to expect some activity of the sucCD enzyme on adipyl-CoA. An enzyme exhibiting adipyl- CoA ligase activity can equivalently carry out the ATP-generating production of adipate from adipyl-CoA, here using AMP and PPi as cofactors, when operating in the opposite physiological direction as depicted in Figure 1. Exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem. J. 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from /⁵, chrysogenum (Lamas-Maceiras et al., Biochem. J. 395, 147-155 (2005); Wang et al., Biochem. Biophy. Res. Commun. 360:453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al., J Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Bower et al., J Bacteriol. 178:4122- 4130 (1996)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Another option, using phosphotransadipylase/adipate kinase, is catalyzed by the gene products of b kl, buk2, and ptb from C. acetobutylicum (Walter et al., Gene 134: 107-111 (1993); Huang et al, J Mol. Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptb gene encodes an enzyme that can convert butyryl-CoA into butyryl-phosphate, which is then converted to butyrate via either of the buk gene products with the concomitant generation of ATP. The analogous set of transformations, that is, conversion of adipyl-CoA to adipyl-phosphate followed by conversion of adipyl-phosphate to adipate, can be carried out by the bukl, buk2, and ptb gene products. The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Alternatively, an acetyltransferase capable of transferring the CoA group from adipyl-CoA to acetate can be applied. Similar transformations are catalyzed by the gene products oicatl, cat2, and catS of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4- hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Sohling and Gottschalk, J Bacteriol. 178:871-880 (1996); Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism catl 729048 P38946.1 Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3 146349050 EDK35586.1 Clostridium kluyveri

Finally, though not as desirable from an energetic standpoint, the conversion of adipyl-CoA to adipate can also be carried out by an acyl-CoA hydrolase or equivalently a thioesterase. The top E. coli gene candidate is tesB (Naggert et al., J. Biol. Chem. 266: 11044-11050 (1991)), which shows high similarity to the human acot8, which is a dicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has also been characterized in the rat liver (Deana, Biochem. Int.

26:767-773 (1992)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Other native candidate genes include tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al, F EMS Microbiol. Rev. Ι^.Ιβ ^ (2005); Zhuang et al, FEBSLett. 516: 161-163 (2002)), paal (Song et al., J Biol. Chem. 281 : 11028-11038 (2006)), andybdB (Leduc et al., J Bacteriol. 189:71 12-7126 (2007)). The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism

tesA 16128478 NP_415027 Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paal 16129357 NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli The above description provides an exemplary adipate synthesis pathway by way of a reverse adipate degradation pathway.

EXAMPLE II

Preparation of an Adipate Producing Microbial Organism Having A Reverse

Degradation Pathway

This example describes the generation of a microbial organism capable of producing adipate using the reverse degradation pathway.

Escherichia coli is used as a target organism to engineer a reverse adipate degradation pathway as shown in Figure 2. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing adipate. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in the reverse degradation pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the paaJ (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the 6ct/ (NP_349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the reverse degradation pathway.

The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of reverse degradation pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like.

Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of adipate. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates or the adipate product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production.

For large-scale production of adipate, the above reverse degradation pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed- batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of an adipate producing microbial organism using a reverse degradation pathway.

EXAMPLE III

Adipate Synthesis Through 3-Oxoadipate

This example describes an exemplary adipate synthesis pathway through 3-oxoadipate.

An additional pathway from that described in Examples I and II that uses acetyl-CoA and succinyl-CoA as precursors for adipate formation and passes through the metabolic intermediate, 3-oxoadipate, is shown in Figure 3. The initial two transformations in this pathway are the two terminal steps of the degradation pathway for aromatic and

choloroaromatic compounds operating in the reverse direction (Kaschabek et al., J Bacteriol. 184:207-215 (2002); Nogales et al, Microbiol. 153:357-365 (2007); Ismail et al, Eur. J. Biochem. 270:3047-3054 (2003)). Specifically, the first step forms 3-oxoadipyl CoA by the condensation of succinyl- and acetyl-CoA. The second step forms 3-oxoadipate and is reported to be reversible in Pseudomonas sp. Strain B13 (Kaschabek et al., J Bacteriol.

184:207-215 (2002)).

The subsequent steps involve reduction of 3-oxoadipate to 3 -hydroxy adipate (conversion of a keto group to hydroxyl group), dehydration of 3-hydroxyadipate to yield hexa-2-enedioate, and reduction of hexa-2-enedioate to form adipate. These steps of the pathway are analogous to the conversion of oxaloacetate into succinate via the reductive TCA cycle (see Figure 4). This supports the steps in the pathway being thermodynamically favorable subject to the presence of appropriate metabolite concentrations. The final reduction step can be carried out either biochemically or by employing a chemical catalyst to convert hexa-2-enedioate into adipate. Chemical hydrogenation can be performed using Pt catalyst on activated carbon as has been described in (Niu et al., Biotechnol. Prog. 18:201-211 (2002)). The maximum theoretical yield of adipate using this pathway is 0.92 mole per mole glucose consumed, and oxygen is not required for attaining these yields (see Table 2). The associated energetics are identical to those of the reverse adipate pathway. Theoretically, ATP formation of up to 1.55 moles is observed per mole of glucose utilized through this pathway. The ATP yield improves to approximately 2.47 moles if phosphoenolpyruvate kinase (PPCK) is assumed to operate in the direction of ATP generation. Interestingly, the product yield can be increased further to 1 mole adipate per mole of glucose consumed if chemical

hydrogenation is used for the last step and a 100% efficiency of catalysis is assumed. In this scenario, up to 1.95 moles of ATP are formed theoretically without assuming the reverse functionality of PPCK.

Table 2: The maximum theoretical yields of adipate and the associated ATP yields per mole of glucose using the 3-oxoadipate pathway.

Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of adipate, one or more exogenous DNA sequence(s) can be expressed in a host microorganism. In addition, the host microorganism can have endogenous gene(s) functionally deleted. These modifications allow the production of adipate using renewable feedstock. Described below are a number of biochemically characterized candidate genes capable of encoding enzymes that catalyze each step of the 3-oxoadipate pathway for adipate synthesis. Although this method is described for E. coli, one skilled in the art can apply these teachings to any other suitable host organism. Specifically, listed below are genes that are native to E. coli as well as genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed.

Referring to Figure 3, step 1 involves succinyl Co A: acetyl Co A acyl transferase (β- ketothiolase). Gene candidates for this enzyme are listed above (Figure 2, step 1). Referring to Figure 3, step 2 involves 3-oxoadipyl-CoA transferase. In this step, 3- oxoadipate is formed by the transfer of the CoA group from 3-oxoadipyl-CoA to succinate. This activity is reported in a two-unit enzyme encoded by pcal and pcaJ in Pseudomonas (Kaschabek et al., J Bacteriol. 184:207-215 (2002)). This enzyme catalyzes a reversible transformation. The protein sequences of exemplary gene products for subunit A of this complex can be found using the following GI numbers and/or GenBank identifiers:

The protein sequences of exemplary gene products for subunit B of this complex can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 3, step 3 involves 3-oxoadipate reductase. E. coli has several candidate alcohol dehydrogenases; two that have analogous functions are malate dehydrogenase (mdh) and lactate dehydrogenase (IdhA). While it has not been shown that these two enzymes have broad substrate specificities in E. coli, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on substrates of various chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J. Biochem. 130:329-334 (1983)). An additional non-native enzyme candidate for this step is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., J Biol. Chem. 267: 15459-15463 (1992)). This enzyme is particularly interesting in that it is a dehydrogenase that operates on a 3- hydroxyacid. Given that dehydrogenases are typically reversible, it is expected that this gene product, or a homlog thereof, will be capable of reducing a 3-oxoacid, for example, 3- oxoadipate, to the corresponding 3 -hydroxyacid, for example, 3-hydroxyadipate. The protein sequences for each of these exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 3, step 4 involves 3-hydroxyadipate dehydratase. In this reaction, 3- hydroxyadipate is dehydrated to hexa-2-enedioate. Although no direct evidence for this enzymatic transformation has been identified, most dehydratases catalyze the a, β-elimination of water. This involves activation of the a-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the β-position (Martins et al, Proc. Natl. Acad .Sci. USA 101 : 15645-15649 (2004); Buckel and Golding,. FEMS Microbiol. Rev. 22:523-541 (1998)). The protein sequences for exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Other good candidates for carrying out this function are the serine dehydratases. These enzymes catalyze a very similar transformation in the removal of ammonia from serine as required in this dehydration step. The protein sequence for exemplary gene product can be found using the following GI number and/or GenBank identifier:

Non-native gene candidates for this transformation have been identified as well. For example, the multi-subunit L-serine dehydratase from Peptostreptococcus asaccharolyt was shown to complement an E. coli strain deficient in L-serine dehydratase activity

(Hofmeister et al, J Bacteriol. 179:4937-4941 (1997)). Further, a putative 2- (hydroxymethyl)glutarate dehydratase, encoded by the gene hmd in Eubacterium barkeri shows similarity to both a- and β-subunits of [4Fe-4S] -containing bacterial serine

dehydratases (Alhapel et al, Proc. Natl. Acad. Sci. USA 103: 12341-12346 (2006)). The protein sequence for exemplary gene product can be found using the following GI number and/or GenBank identifier:

Referring to Figure 3, step 5 involves 2-enoate reductase. The final step in the 3-oxoadipate pathway is reduction of the double bond in hexa-3-enedioate to form adipate. Biochemically, this transformation can be catalyzed by 2-enoate reductase (EC 1.3.1.31) known to catalyze the NADH-dependent reduction of a wide variety of a, β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J Biol. Chem. 276:5779-5787 (2001)). This enzyme is encoded by enr in several species of Clostridia (Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)) including C. tyrobutyricum and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich, et al., J Biol. Chem. 276:5779-5787 (2001)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases have been reported, out of which one has been characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and C. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., J Biol. Chem. 276:5779-5787 (2001)). Several gene candidates thus exist for catalyzing this last step in the 3-oxoadipate pathway and have been listed below. The C. thermoaceticum enr gene has also been expressed in an enzymatically active form in E. coli (Rohdich et al., supra, 2001). The protein sequences for exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism fadH 16130976 NP_417552.1 Escherichia coli

enr 169405742 AC A54153.1 Clostridium botulinum A3 str enr 2765041 CAA71086.1 Clostridium tyrobutyricum enr 3402834 CAA76083.1 Clostridium kluyveri

The above description provides an exemplary adipate synthesis pathway by way of an 3- oxoadipate pathway.

EXAMPLE IV

Preparation of an Adipate Producing Microbial Organism Having A

3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable of producing adipate using the 3-oxoadipate pathway.

Escherichia coli is used as a target organism to engineer the 3-oxoadipate pathway as shown in Figure 3. E. coli provides a good host for generating a non-naturally occurring

microorganism capable of producing adipate. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in the 3-oxoadipate pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the /? a/(NP_415915.1), /?c /J (AAN69545.1 and NP_746082.1), and bdh (AAA58352.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3- oxoadipyl-CoA transferase, and 3-oxoadipate reductase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the acnA (P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipate dehydratase and 2-enoate reductase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the 3-oxoadipate pathway.

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 3-oxoadipate pathway genes for adipate synthesis is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

For large-scale production of adipate, the 3-oxoadipate pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an

Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). This example describes the preparation of an adipate-producing microbial organism containing a 3-oxidoadipate pathway.

EXAMPLE V

Adipate Synthesis via cis,cis-Muconic Acid

This example describes an adipate synthesis pathway previously described (see Niu et al., Biotechnol. Prog. 18(2): p. 201-11. 2002; Frost et al, United States Patent No. 5,487,987, issued January 30, 1996).

Adipate synthesis via a combined biological and chemical conversion process has been previously described. (Niu et al., Biotechnol. Prog. 18:201-211 (2002)) and is shown in Figure 5. This method is further described in United States Patent No. 5,487,987. Adipate synthesis through this route entails introduction of three heterologous genes into E. coli that can convert dehydroshikimate into cis,cis-muconic acid (Niu et al., supra, 2002). A final chemical hydrogenation step leads to the formation of adipic acid. In this step, the pretreated fermentation broth that contained 150 mM cis,cis-muconate was mixed with 10% platinum (Pt) on activated carbon. The hydrogenation reaction was carried out at 3400 KPa of hydrogen pressure for two and a half hour at 250°C with stirring. The calculated adipate yields are shown in Table 3 assuming either an enzymatic or chemical catalysis step is utilized to convert cis,cis-muconate into adipate. Under aerobic conditions, an 85% molar yield of adipate can be obtained if a chemical reaction is employed for hydrogenation and a 75% molar yield is obtained if an NADH-based hydrogenase is used.

Table 3: The maximum theoretical yields of adipate per mole of glucose using the using the cis,cis-muconic acid pathway. Final step enzymatic Final step chemical

hydrogenation

Aerobic Anaerobic Aerobic Anaerobic

Adipate Yield 0.75 0.00 0.85 0.00

Although this is an exemplary method, there are disadvantages of this method compared to others, such as those described in Examples I-IV. For example, the first limitation of this method is the lower theoretical yields compared to the reverse adipate degradation and 3- oxoadipate pathways. The second limitation is that the ATP yields of this pathway are negligible. A third limitation of this pathway is that it involves a dioxygenase, necessitating a supply of oxygen to the bioreactor and precluding the option of anaerobic fermentation.

The above description provides an exemplary adipate synthesis pathway by way of a cis,cis- muconic acid pathway EXAMPLE VI

Adipate Synthesis via Alpha-Ketoadipate

This example describes an exemplary adipate synthesis pathway via an alpha-ketoadipate pathway.

Alpha-keto adipate is a known intermediate in lysine biosynthesis in S. cerevisiae, and this information was used to identify an additional pathway for adipic acid biosynthesis (see Figure 6). Conversion of alpha-ketoglutarate to alpha-ketoadipate is catalyzed by

homocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase as indicated by dashed arrows in Figure 6. Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977). Subsequent steps involve a dehydratase for the conversion of alpha-hydroxyadipate into hexa-2-enedioate followed by its reduction to adipic acid. This last step can be catalyzed either by an enzyme or can take place through a chemical reaction as described in Example II. Genes encoding the enzymes for the alpha- ketoadipate pathway are identified as described in Examples I-IV.

The adipate yields associated with this pathway are shown in Table 4. Because of the loss of two C0₂ molecules during the conversion of acetyl-CoA to adipate, only 67% of the glucose can be converted into adipate. This is reflected in the molar yields for this pathway under aerobic conditions. The yields are further reduced in the absence of oxygen uptake. Also since the maximum ATP yields under anaerobic conditions are negligible, the engineered organism will have to utilize additional substrate to form energy for cell growth and maintenance under such conditions.

Table 4: The maximum theoretical yields of adipate and the associated ATP yields per mole of glucose using the using the alpha-ketoadipate pathway.

The above description provides an exemplary adipate synthesis pathway by way of an alpha- ketoadipate pathway.

EXAMPLE VII

Adipate Synthesis via Lysine Degradation

This example describes an exemplary adipate synthesis pathway via a lysine degradation pathway.

Two additional pathways for adipate synthesis rely on lysine degradation to form adipate. One pathway starts from alpha-ketoglutarate to form lysine (pathway non-native to E. coli and found in S. cerevisiae), and the other uses aspartate as a starting point for lysine biosynthesis (pathway native to E. coli). Figure 7 shows adipate formation from lysine. The maximum theoretical yields for adipate, both in the presence and absence of oxygen, using the E. coli stoichiometric model are shown in Tables 5 and 6, with alpha-ketoglutarate and aspartate as the respective starting points for lysine. The maximum ATP yields

accompanying these theoretical yields were also calculated and are shown in the same tables. These yields are lower in comparison to the other pathways described in Examples I-TV. Genes encoding the enzymes for the alpha-ketoadipate pathway are identified as described in Examples I-TV.

Table 5: The maximum theoretical yield of adipate and the accompanying ATP yield per mole of glucose assuming the lysine biosynthesis pathway with alpha-ketoglutarate as a starting point. Aerobic Anaerobic

Adipate Yield 0.40 0.20

Max ATP yield @ max adipate yield 5.60 0.00

Table 6: The maximum theoretical yield of adipate and the accompanying ATP yield per mole of glucose assuming the lysine biosynthesis pathway with aspartate as a starting point.

The above description provides an exemplary adipate synthesis pathway by way of a lysine degradation pathway.

EXAMPLE VIII

Production of Caprolactam and 6-Aminocaproic Acid via Adipyl-CoA

This example describes an exemplary caprolactam and/or 6-aminocaproic acid synthesis pathway via an adipyl-CoA pathway.

An exemplary pathway for forming caprolactam and/or 6-aminocaproic acid using adipyl- CoA as the precursor is shown in Figure 8. The pathway involves a CoA-dependant aldehyde dehydrogenase that can reduce adipyl-CoA to adipate semialdehyde and a transaminase or 6- aminocaproate dehydrogenase that can transform this molecule into 6-aminocaproic acid. The terminal step that converts 6-aminocaproate into caprolactam can be accomplished either via an amidohydrolase or via chemical conversion (Guit and Buijs, U.S. Patent No.

6,353,100, issued March 7, 2002; Wolters et al., U.S. Patent No. 5,700,934, issued December 23, 1997; Agterberg et al., U.S. Patent No. 6,660,857, issued December 9, 2003). The maximum theoretical yield of caprolactam was calculated to be 0.8 mole per mole glucose consumed (see Table 7) assuming that the reverse adipate degradation pathway was complemented with the reaction scheme shown in Figure 8. The pathway is favorable energetically as up to 0.78 moles of ATP are formed per mole of glucose consumed at the maximum theoretical yield of caprolactam. The ATP yield can be further improved to 1.63 moles of ATP produced per mole of glucose if phosphoenolpyruvate carboxykinase (PPCK) is assumed to function in the ATP-generating direction towards oxaloacetate formation. The final amidohydrolase step is energetically and redox neutral, and thus the product and ATP molar yields associated with 6-aminocaproic acid production are equivalent to those associated with caprolactam production. Thus one can alternatively envision a

microorganism and associated fermentation process that forms 6-aminocaproic acid instead of caprolactam followed by an additional unit operation to dehydrate/cyclize 6-aminocaproic acid to caprolactam.

Table 7: The maximum theoretical yield of caprolactam and the accompanying ATP yield per mole of glucose assuming that the reverse fatty acid degradation pathway is complemented with the reaction scheme from Figure 8.

Successfully engineering this pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This entails identifying an appropriate set of enzymes, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of 6-aminocaproic acid or caprolactam, one or more exogenous DNA sequence(s) can be expressed in a host microorganism. In addition, the microorganism can have endogenous gene(s) functionally deleted. These modifications will allow the production of 6-aminocaproate or caprolactam using renewable feedstock.

Below is described a number of biochemically characterized candidate genes capable of encoding enzymes that catalyze each step of the caprolactam formation pathway described in Figure 8. Although described for E. coli, one skilled in the art can apply these teachings to any other suitable host organism. Specifically, the genes listed are native to E. coli or are genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed. Referring to Figure 8, step 1 involves CoA-dependant aldehyde dehydrogenase. Exemplary genes that encode enzymes for catalyzing the reduction of an acyl-coA to its corresponding aldehyde include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase (Reiser and Somerville,. J Bacteriol. 179:2969-2975 (1997)), t e Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol.. 68: 1192-1195 (2002)) and the sucD gene from Clostridium kluyveri (Sohling and Gottschalk, J Bacteriol. 178:871-880 (1996)), which can convert succinyl-CoA to succinate semialdehyde.

Referring to Figure 8, step 2 involves transaminase. The second step in the pathway is conversion of the 6-aldehyde to an amine. This transformation can likely be accomplished by gamma-aminobutyrate transaminase (GABA transaminase), a native enzyme encoded by gabT that transfers an amino group from glutamate to the terminal aldehyde of succinyl semialdehyde (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)). The gene product oipuuE catalyzes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J.Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scott and Jakoby, J Biol. Chem. 234:932-936 (1959)). The protein sequences for exemplary gene products can be found using the following GI numbers and/or GenBank identifiers:

Referring to Figure 8, step 2 can alternatively involve 6-aminocaproate dehydrogenase which comprises the reductive amination of adipate semialdehyde to form 6-aminocaproate. This transformation can be accomplished by lysine-6-dehydrogenase, which naturally converts L- lysine to 2-aminoadipate-6-semialdehyde. Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et a\., Appl. Environ. Microbiol. 70(2):937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J. Biochem. (Tokyo), 106(l):76-80 (1989); Misono et al., J Biochem. (Tokyo), 105(6): 1002-1008 (1989)), and Achromobacter denitrificans (Ruldeekulthamrong et al, BMB Reports 790-795 (2008)).

Referring to Figure 8, step 3 involves amidohydrolase. The final step of caprolactam synthesis is cyclization of 6-aminocaproic acid. This transformation has not been

characterized enzymatically but it is very similar to the cyclization of lysine by D-lysine lactamase (EC 3.5.2.11) from Cryptococcus laurentii (Fukumura et al., FEBS Lett. 89:298- 300 (1978)). However, the protein and nucleotide sequences of this enzyme are not currently known and, so far, lysine lactamase activity has not been demonstrated in other organisms.

Plasmids contained in several strains of Pseudomonas sp. isolated from soil have been shown to confer ability to grow on caprolactam as a sole carbon source (Boronin et al., FEMS

Microbiol. Lett. 22: 16 '-170 (1984)); however, associated gene or protein sequences have not been associated with this function to date.

The most closely related candidate enzyme with available sequence information is 6- aminohexanoate-cyclic dimer hydrolase, which has been characterized in Pseudomonas sp. and Flavobacterium sp. The nylB gene product from Pseudomonas sp NK87 was cloned and expressed in E. coli (Kanagawa et al., J Gen. Microbiol. 139:787-795 (1993)). The substrate specificity of the enzyme was tested in Flavobacterium sp K172 and was shown to react with higher-order oligomers of 6-aminohexanoate but not caprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). The reversibility and ability of 6-aminohexanoate dimer hydrolases in other organisms to react with the desired substrate in the direction of interest can be further tested. The protein sequences for exemplary gene products can be found using the following GI numbers and/or GenBank identifiers: Gene name GI# GenBank Accession # Organism

nylB 148711 AAA24929.1 Pseudomonas sp NK87 nylB 129000 P13397 Flavobacterium sp K172 nylB 119961013 YP 949627.1 Arthrobacter aurescens TCI

The above description provides an exemplary pathway to produce caprolactam and/or 6- aminocaproic acid by way of an adipyl-CoA pathway.

EXAMPLE IX

Preparation of a 6-Aminocaproate or Caprolactam Producing Microbial Organism

Having a 3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable of producing adipate using the reverse degradation pathway and converting the intracellular adipate to 6- aminocaproate and/or caprolactam. Escherichia coli is used as a target organism to engineer the necessary genes for adipate, 6- aminocaproate, and/or caprolactam synthesis (see Figure 2 and Figure 8). E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing adipate, 6-aminocaproate, and/or caprolactam. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproate and/or caprolactam, nucleic acids encoding the enzymes utilized in the reverse adipate degradation pathway and 6-aminocaproate or caprolactam synthesis pathways are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the /? <¾/ (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase, 3- hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the 6ct/ (NP_349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the acrl (YP_047869.1), g 67 (NP_417148.1), and nylB (AAA24929.1) genes encoding CoA- dependent aldehyde dehydrogenase, transaminase, and amidohydrolase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproate and/or caprolactam synthesis.

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproate and caprolactam synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6- aminocaproate and/or caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional pathway for the synthesis of 6- aminocaproate and/or caprolactam are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproate and/or caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6-aminocaproate and/or caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the products. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproate and/or caprolactam producer to further increase production.

For large-scale production of 6-aminocaproate and/or caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an

Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE X

Adipate Synthesis via 2-Hydroxyadipyl-CoA

This example describes two exemplary adipate synthesis pathways proceeding from alpha- ketoadipate and passing through a 2-hydroxyadipyl-CoA intermediate.

As described in example VI, alpha-ketoadipate is a known intermediate in lysine biosynthesis that can be formed from alpha-ketoglutarate via homocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase. Alpha-ketoadipate can be converted to 2-hydroxyadipyl-CoA by the two routes depicted in Figure 9. 2-hydroxyadipyl-CoA can be subsequently dehydrated and reduced to adipyl-CoA which can then be converted to adipate as shown in Figure 9. The maximum yield of adipate from glucose via these pathways is 0.67 mol/mol. Conversion of alpha-ketoadipate into 2-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun.

77:586-591 (1977). Alternatively, enzymes capable of reducing alpha-ketoglutarate to 2- hydroxyglutarate may also show activity on alpha-ketoadipate, which is only one carbon atom longer. One such enzyme possessing alpha-ketoglutarate reductase activity is serA of Escherichia coli (Zhao and Winkler, J Bacteriol. 178(l):232-9 (1996)). Additional exemplary enzymes can be found in Arabidopsis thaliana (Ho, et al., J Biol. Chem.

274(l):397-402 (1999)) and Haemophilus influenzae.

Referring to Figure 9, 2-hydroxyadipate can likely be converted to 2-hydroxyadipyl-CoA by the synthetases, transferases, phosphotransadipylases and kinases described in example I. Alternatively, enzymes with 2-hydroxyglutarate CoA-transferase or glutaconate CoA- transferase activity are likely suitable to transfer a CoA moiety to 2-hydroxyadipate. One example of such an enzyme is encoded by the gctA and gctB genes of Acidaminococcus fermentans (Buckel, et al., Eur. J. Biochem. 118(2):315-321 (1981); Mack, et al., Eur. J. Biochem. 226(1):41-51 (1994)). Similarly, synthetase, transferase, or phosphotransadipylase and kinase activities would be required to convert alpha-ketoadipate into alpha-ketoadipyl- CoA, as depicted in Figure 9. Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be carried out by an alpha-hydroxyacyl-CoA dehydrogenase enzyme. A similar activity was reported in propionate-adapted E. coli cells whose extracts catalyzed the oxidation of lactyl-CoA to form pyruvyl-CoA (Megraw et al, J Bacteriol. 90(4): 984-988 (1965)).

Additional hydroxyacyl-CoA dehydrogenases were described in example I.

The dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoA can be carried out by a 2-hydroxyacyl-CoA dehydratase. A 2-hydroxyglutaryl-CoA dehydratase system has been characterized in Acidaminococcus fermentans and requires both the hgdA and hgdB subunits and the activator protein, hgdC, for optimal activity (Dutscho et al., Eur. J. Biochem 181(3):741-746 (1989); Locher et al. J. Mol. Biol. 307(l):297-308; Muller and Buckel, Eur. J. Biochem. 230(2):698-704 (2001); Schweiger et al. Eur. J. Biochem. 169(2):441-448 (1987)). This enzyme system is similar in mechanism to the lactoyl-CoA dehydratase from Clostridium propionicum (Hofmeister and Buckel, Eur. J. Biochem. 206(2):547-552 (1992); Kuchta and Abeles, J. Biol. Chem. 260(24): 13181-13189 (1985)). Homologs to hgdA, hgdB, and hgdC exist in several organisms.

Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out by the enzymes described in Example I.

The above description provides an exemplary adipate synthesis pathway by way of a 2- hydroxyadipyl-CoA pathway.

EXAMPLE XI

Preparation of an Adipate Producing Microbial Organism Having a 2-Hydroxyadipyl-

CoA Pathway

This example describes the generation of a microbial organism capable of producing adipate using a 2-hydroxyadipyl-CoA pathway.

Escherichia coli is used as a target organism to engineer the necessary genes for adipate synthesis (see Figure 9). E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing adipate. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleic acids encoding the enzymes utilized in a 2-hydroxyadipyl-CoA to adipate pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001;

Ausubel, supra, 1999). In particular, the serA (NP_417388.1), gctA (Q59\ \ \), md gctB (Q591 \2)ge s encoding the 2-hydroxyadipate dehydrogenase and 2-hydroxyadipyl- CoA:acetyl-CoA transferase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the hgdA (PI 1569), hgdB (PI 1570J, and hgdC (PI 1568) genes encoding 2-hydroxyadipyl-CoA dehydratase activity, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Further, the 6ct/ (NP_349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2- pentenoyl-CoA reductase and adipyl-CoA synthetase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis.

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 2-hydroxyadipyl-CoA pathway genes for adipate synthesis is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS). Microbial strains engineered to have a functional adipate synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of adipate. Adaptive evolution also can be used to generate better producers of, for example, the alpha-ketoadipate intermediate or the adipate product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production.

For large-scale production of adipate, the 2-hydroxyadipyl-CoA pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed- batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at around a pH of 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng. 775-779 (2005)). This example describes the preparation of an adipate-producing microbial organism containing a 2-hydroxyadipyl-CoA pathway.

EXAMPLE XII

Pathways for Production of Hexamethylenediamine, Caprolactam and 6-Aminocaproic

Acid

This example describes exemplary pathways for production of hexamethylenediamine, caprolactam and 6-aminocaproic acid.

Described below are various pathways leading to the production of caprolactam,

hexamethylenediamine (HMDA), or 6-aminocaproate from common central metabolites. The first described pathway entails the activation of 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthase enzyme (Figure 10, Step Q or R) followed by the spontaneous cyclization of 6-aminocaproyl-CoA to form caprolactam (Figure 10, Step T). The second described pathway entails the activation of 6-aminocaproate to 6-aminocaproyl-CoA (Figure 10, Step Q or R), followed by a reduction (Figure 10, Step U) and amination (Figure 10, Step V or W) to form HMDA. 6-Aminocaproic acid can alternatively be activated to 6- aminocaproyl-phosphate instead of 6-aminocaproyl-CoA. 6-Aminocaproyl-phosphate can spontaneously cyclize to form caprolactam. Alternatively, 6-aminocaproyl-phosphate can be reduced to 6-aminocaproate semialdehye, which can be then converted to HMDA as depicted in Figures 10 and 11. In either this case, the amination reaction must occur relatively quickly to minimize the spontaneous formation of the cyclic imine of 6-aminocaproate semialdehyde. Linking or scaffolding the participating enzymes represents a potentially powerful option for ensuring that the 6-aminocaproate semialdehyde intermediate is efficiently channeled from the reductase enzyme to the amination enzyme.

Another option for minimizing or even eliminating the formation of the cyclic imine or caprolactam during the conversion of 6-aminocaproic acid to HMDA entails adding a functional group (for example, acetyl, succinyl) to the amine group of 6-aminocaproic acid to protect it from cyclization. This is analogous to ornithine formation from L-glutamate in Escherichia coli. Specifically, glutamate is first converted to N-acetyl-L-glutamate by N- acetylglutamate synthase. N-Acetyl-L-glutamate is then activated to N-acetylglutamyl- phosphate, which is reduced and transaminated to form N-acetyl-L-ornithine. The acetyl group is then removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase forming L-ornithine. Such a route is necessary because formation of glutamate-5 -phosphate from glutamate followed by reduction to glutamate-5-semialdehyde leads to the formation of (S)-l-pyrroline-5-carboxylate, a cyclic imine formed spontaneously from glutamate-5- semialdehyde. In the case of forming HMDA from 6-aminocaproic acid, the steps can involve acetylating 6-aminocaproic acid to acetyl-6-aminocaproic acid, activating the carboxylic acid group with a CoA or phosphate group, reducing, aminating, and

deacetylating.

Note that 6-aminocaproate can be formed from various starting molecules. For example, the carbon backbone of 6-aminocaproate can be derived from succinyl-CoA and acetyl-CoA as depicted in Figure 10 and also described in Figures 2, 3 and 8. Alternatively, 6- aminocaproate can be derived from alpha-ketoadipate, where alpha-ketoadipate is converted to adipyl-CoA (see Figure 9), and adipyl-CoA is converted to 6-aminocaproate as shown in Figure 10.

Figure 11 provides two additional metabolic pathways to 6-aminocaproate or 6- aminocapropyl-CoA starting from 4-aminobutyryl-CoA and acetyl-CoA. The first route entails the condensation of 4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6- aminohexanoyl-CoA (Step A) followed by a reduction (Step B), dehydration (Step C), and reduction (Step D) to form 6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted to 6-aminocaproate by a transferase (Step K), synthase (Step L), or hydrolase (Step M) enzyme. Alternatively, 6-aminocaproyl-CoA can be converted to caprolactam by spontaneous cyclization (Step Q) or to HMDA following its reduction (Step N) and amination (Step O or P). The second pathway described in Figure 11 entails the condensation of 4-aminobutyryl- CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (Step A) which is then converted to 3-oxo-6-aminohexanoate by a transferase (Step E), synthase (Step F), or hydrolase (Step G). 3-Oxo-6-aminohexanoate is then reduced (Step H), dehydrated (Step I), and reduced (Step J) to form 6-aminocaproate.

The starting molecule, 4-aminobutyryl-CoA, can be formed from various common central metabolites. For example, glutamate can be decarboxylated to 4-aminobutyrate, which is then activated by a CoA-transferase or synthase to 4-aminobutyryl-CoA. Alternatively, succinate semialdehyde, formed from either the reduction of succinyl-CoA or the

decarboxylation of alpha-ketoglutarate, can be transaminated to 4-aminobutyrate prior to activation by a CoA-transferase or synthase to form 4-aminobutyryl-CoA. It is noted that 4- aminobutyryl-CoA and several of the intermediates of the 4-aminobutyryl-CoA to 6- aminocaproyl-CoA pathway may spontaneously cyclize to their corresponding lactams. Thus, adding a protective functional group to the terminal amine group of 4-aminobutyryl- CoA and/or several of the amino-CoA intermediates can be used to minimize the formation of unwanted cyclic byproducts. In this case, the same general set of transformations depicted in Figure 11 would apply, although two additional steps, for example, an acetylase and deacetylase, can be added to the pathway.

All transformations depicted in Figures 10-11 fall into the 12 general categories of transformations shown in Table 8. Below is described a number of biochemically characterized candidate genes in each category. Specifically listed are genes that can be applied to catalyze the appropriate transformations in Figures 10-11 when cloned and expressed.

Table 8. Enzyme types for conversion of succinyl-CoA, acetyl-CoA, and/or 4-aminobutyryl- CoA to 6-aminocaproate, caprolactam, and/or hexamethylenediamine. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.

1.1. l .a Oxidoreductases. Four transformations depicted in Figures 10 and 11 require oxidoreductases that convert a ketone functionality to a hydroxyl group. Step B in both Figures 10 and 11 involves converting a 3-oxoacyl-CoA to a 3-hydroxyacyl-CoA. Step H both Figures 1 and 2 involves converting a 3-oxoacid to a 3-hydroxyacid. Exemplary enzymes that can convert 3-oxoacyl-CoA molecules such as 3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA into 3-hydroxyacyl-CoA molecules such as 3- hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA, respectively, include enzymes whose natural physiological roles are in fatty acid beta-oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 :403-411 (1981)). Furthermore, the gene products encoded by phaC in Pseudomonas putida C/(01ivera et al, Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188: 117-125 (2007)) catalyze the reverse reaction of step B in Figure 10, that is, the oxidation of 3 -hydroxy adipyl- CoA to form 3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene. Note that the reactions catalyzed by such enzymes are reversible. In addition, given the proximity in E. coli of paaH to other genes in the phenylacetate degradation operon (Nogales et al.,

Microbiology 153:357-365 (2007)) and the fact that paaH mutants cannot grow on

phenylacetate (Ismail et al., Eur.J Biochem. 270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA

dehydrogenases. The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J Bacteriol. 171 :6800-6807 (1989)). Additional gene candidates include Hbdl (C -terminal domain) and Hbd2 (N- terminal domain) in Clostridium kluyveri (Hillmer et al., FEBS Lett. 21 :351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al, J Biol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem. 174: 177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61 :297-309 (2006)). The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol 3:349-357 (1989)) and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al., supra).

A number of similar enzymes have been found in other species of Clostridia and in

Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)).

Various alcohol dehydrogenases represent good candidates for converting 3-oxoadipate to 3- hydroxyadipate (step H, Figure 10) or 3-oxo-6-aminohexanoate to 3-hydroxy-6- aminohexanoate (step H, Figure 11). Two such enzymes capable of converting an oxoacid to a hydroxyacid are encoded by the malate dehydrogenase (mdh) and lactate dehydrogenase (IdhA) genes in E. coli. In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on substrates of various chain lengths such as lactate, 2- oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem.

130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate for these steps is the mitochondrial 3 -hydroxy butyrate dehydrogenase {bdh) from the human heart which has been cloned and characterized (Marks et al, J Biol. Chem. 267:15459-15463 (1992)). This enzyme is a dehydrogenase that operates on a 3-hydroxyacid. Another exemplary alcohol dehydrogenase converts acetone to isopropanol as was shown in C. beijerinckii (Ismaiel et al, J Bacteriol. 175:5097-5105 (1993) and T. brockii (Lamed et al, Biochem. J. 195: 183-190 (1981); Peretz et al, Biochemistry 28:6549-6555 (1989)).

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). The transformations of adipyl-CoA to adipate semialdehyde (Step N, Figure 10) and 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde (Step U, Figure 10; Step N, Figure 1 1) require acyl-CoA dehydrogenases capable of reducing an acyl-CoA to its corresponding aldehyde. Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl- CoA reductase (Reiser et al., J Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al, Appl.Environ.Microbiol. 68: 1192-1195 (2002)), and a CoA- and NADP- dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling et al., J Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J

Bacteriol. 182:4704-4710 (2000)). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA

(Kazahaya et al., J .Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett.

27:505-510 (2005)).

An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl- CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., supra; Thauer R.K., Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been

characterized in Metallosphaera and Sulfolobus spp (Alber et al., J.Bacteriol. 188:8551-8559 (2006); Hugler et al, J.Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., supra; Berg et al., supra). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., supra). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208).

Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate- semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent

dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from

Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra).

1.3.1.a Oxidoreductase operating on CH-CH donors. Referring to Figure 10, step D refers to the conversion of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Referring to Figure 11, step D refers to the conversion of 6-aminohex-2-enoyl- CoA to 6-aminocaproyl-CoA. Enoyl-CoA reductase enzymes are suitable enzymes for either transformation. One exemplary enoyl-CoA reductase is the gene product of bed from C. acetobutylicum (Boynton et al., J Bacteriol. 178:3015-3024 (1996); Atsumi et al., Metab. Eng. 2008 10(6):305-311 (2008)(Epub Sep. 14, 2007), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister et al., supra). This approach is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci et al., FEBS Letters 581 : 1561-1566 (2007)). Gene name GI# GenBank Accession # Organism

bed 15895968 NP_349317.1 Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridium acetobutylicum etffl 15895967 NP_349316.1 Clostridium acetobutylicum

TER 62287512 Q5EU90.1 Euglena gracilis

TDE0597 42526113 NP_971211.1 Treponema denticola

Step J of both Figures 10 and 11 requires a 2-enoate reductase enzyme. 2-Enoate reductases (EC 1.3.1.31) are known to catalyze the NAD(P)H-dependent reduction of a wide variety of a, β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J.Biol. Chem. 276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr in several species of Clostridia (Giesel et al., Arch Microbiol 135:51-57 (1983)) including C. tyrobutyricum, and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich et al., supra). In the published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases have been reported, out of which one has been characterized (Sccdorf Qt εά., Proc.Natl.Acad.Sci. USA, 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and C. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel et al., supra). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enr gene has also been expressed in an enzymatically active form in E. coli (Rohdich et al., supra).

1.4.1. a Oxidoreductase operating on amino acids. Figure 10 depicts two reductive aminations. Specifically, step P of Figure 10 involves the conversion of adipate

semialdehyde to 6-aminocaproate and step W of Figure 10 entails the conversion of 6- aminocaproate semialdehyde to hexamethylenediamine. The latter transformation is also required in Figure 11, Step P.

Most oxidoreductases operating on amino acids catalyze the oxidative deamination of alpha- amino acids with NAD+ or NADP+ as acceptor, though the reactions are typically reversible. Exemplary oxidoreductases operating on amino acids include glutamate dehydrogenase

(deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by Idh, and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (McPherson et al., Nucleic.Acids Res. 11 :5257-5266 (1983); Korber et al., J.Mol.Biol. 234: 1270-1273 (1993)), gdh from Thermotoga maritima (Kort et al.,

Extremophiles 1 :52-60 (1997); Lebbink et al, J.Mol.Biol. 280:287-296 (1998); Lebbink et ah, J.Mol.Biol. 289:357-369 (1999)), and gdhAl from Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2- oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The Idh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Stoyan et al., J.Biotechnol 54:77-80 (1997); Ansorge et al, Biotechnol Bioeng. 68:557-562 (2000)). The nadX gene from Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al, J.Biol.Chem. 278:8804-8808 (2003)).

The lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes, catalyze the oxidative deamination of the ε-amino group of L-lysine to form 2-aminoadipate-6-

(Misono et al., J.Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); Misono et al., supra), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB.Rep. 41 :790-795 (2008)). Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate given the structural similarity between adipate semialdehyde and 2- aminoadipate-6-semialdehyde.

2.3.1.b Acyl transferase. Referring to Figure 10, step A involves 3-oxoadipyl-CoA thiolase, or equivalently, succinyl Co A: acetyl Co A acyl transferase (β-keto thiolase). The gene products encoded by pcaF in Pseudomonas strain B 13 (Kaschabek et al., J.Bacteriol.

184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., supra), paaE in Pseudomonas fluorescens ST Di Gennaro et al., supra), and paaJ from E. coli (Nogales et al., supra) catalyze the conversion of 3-oxoadipyl-CoA into succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate or styrene. Since β- ketothiolase enzymes catalyze reversible transformations, these enzymes can be employed for the synthesis of 3-oxoadipyl-CoA. For example, the ketothiolase phaA from ?. eutropha combines two molecules of acetyl-CoA to form acetoacetyl-CoA (Sato et al., J Biosci Bioeng 103:38-44 (2007)). Similarly, a β-keto thiolase (bktB) has been reported to catalyze the condensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA (Slater et al., J.Bacteriol. 180: 1979-1987 (1998)) in R. eutropha. In addition to the likelihood of possessing 3-oxoadipyl-CoA thiolase activity, all such enzymes represent good candidates for condensing 4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (step A, Figure 11) either in their native forms or once they have been appropriately engineered.

Gene name GI# GenBank Accession # Organism

paaJ 16129358 NP_415915.1 Escherichia coli

pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13) phaD 3253200 AAC24332.1 Pseudomonas putida

paaE 106636097 ABF82237.1 Pseudomonas fluorescens 2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymes present additional candidates for performing step A in Figures 10 and 11. AKPT is a pyridoxal phosphate-dependent enzyme participating in ornithine degradation in Clostridium sticklandii (Jeng et al, Biochemistry 13:2898-2903 (1974); Kenklies et al, Microbiology 145:819-826 (1999)). A gene cluster encoding the alpha and beta subunits of AKPT (or-2 (ortA) and or-3 (ortB)) was recently identified and the biochemical properties of the enzyme were

characterized (Fonknechten et al., J.Bacteriol. In Press (2009)). The enzyme is capable of operating in both directions and naturally reacts with the D-isomer of alanine. AKPT from Clostridium sticklandii has been characterized but its protein sequence has not yet been published. Enzymes with high sequence homology are found in Clostridium difficile,

Alkaliphilus metalliredigenes QYF, Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4 (Fonknechten et al., supra).

2.6.1. a Aminotransferase. Step O of Figures 10 and 11 and Step V of Figure 10 require transamination of a 6-aldehyde to an amine. These transformations can be catalyzed by gamma-aminobutyrate transaminase (GAB A transaminase). One E. coli GAB A transaminase is encoded by gabT and transfers an amino group from glutamate to the terminal aldehyde of succinyl semialdehyde (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)). The gene product oipuuE catalyzes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J.Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scott et al, J Biol. Chem. 234:932-936 (1959)).

Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to hexamethylenediamine. The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1 ,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K.H., J Biol Chem 239:783-786 (1964)). A putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol 184:3765-3773 (2002)).

Yet additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferases which produce malonate semialdehyde from beta-alanine (WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri was also shown to preferentially use beta-alanine as the amino group donor (Andersen et al, FEBS.J. 274: 1804-1817 (2007)). SkUGAl encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur.J.Biochem., 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both β-alanine and GABA transamination (Andersen et al., supra). 3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2- methylpropionate. This enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Tamaki et al, Methods Enzymol, 324:376-389 (2000)).

2.8.3.a Coenzyme-A transferase. CoA transferases catalyze reversible reactions that involve the transfer of a CoA moiety from one molecule to another. For example, step E of Figure 10 is catalyzed by a 3-oxoadipyl-CoA transferase. In this step, 3-oxoadipate is formed by the transfer of the CoA group from 3-oxoadipyl-CoA to succinate, acetate, or another CoA acceptor. Step E of Figure 11 entails the transfer of a CoA moiety from another 3-oxoacyl- CoA, 3-oxo-6-aminohexanoyl-CoA. One candidate enzyme for these steps is the two-unit enzyme encoded by pcal and pcaJ in Pseudomonas, which has been shown to have 3- oxoadipyl-CoA/succinate transferase activity (Kaschabek et al., supra). Similar enzymes based on homology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J.Biol. Chem.

272:25659-25667 (1997)) and Bacillus subtilis (Stols et al, Protein.Expr.Purif. 53:396-403 (2007)).

Gene name GI# GenBank Accession # Organism

pcal 24985644 AAN69545.1 Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcal 50084858 YP 046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1 pcal 21224997 NP_630776.1 Streptomyces coelicolor pcaJ 21224996 NP_630775.1 Streptomyces coelicolor

HPAG1 0676 108563101 YP_627417 Helicobacter pylori

HPAG1 0677 108563102 YP_627418 Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis

ScoB 16080949 NP_391777 Bacillus subtilis

A 3-oxoacyl-CoA transferase that can utilize acetate as the Co A acceptor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys.Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr.D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme has also been shown to transfer the Co A moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56: 1576-1583 (1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)).

The above enzymes may also exhibit the desired activities on adipyl-CoA and adipate (Figure 10, step K) or 6-aminocaproate and 6-aminocaproyl-CoA (Figure 10, step Q; Figure 2, step K). Nevertheless, additional exemplary transferase candidates are catalyzed by the gene products of catl, cat2, and catS of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., swpra;Sohling et al., EurJ Biochem. 212: 121-127 (1993);Sohling et al., J Bacteriol. 178:871-880 (1996)).

The glutaconate-Co A- transferase (EC 2.8.3.12) enzyme from anaerobic bacterium

Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al.,

Eur.J.Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al, Eur.J.Biochem. 226:41-51 (1994)).

3.1.2.a Thiolester hydrolase (CoA specific). Several eukaryotic acetyl-CoA hydrolases have broad substrate specificity and thus represent suitable candidate enzymes for hydro lyzing 3- oxoadipyl-CoA, adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA, or 6-aminocaproyl-CoA (Steps G and M of Figures 10 and 11). For example, the enzyme from Rattus norvegicus brain (Robinson et al., Biochem.Biophys.Res.Commun. 71 :959-965 (1976)) can react with butyryl- CoA, hexanoyl-CoA and malonyl-CoA.

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3- hydroxyisobutyrate during valine degradation (Shimomura et al., J Biol Chem. 269: 14248- 14253 (1994)). Genes encoding this enzyme include hibch οΐ Rattus norvegicus (Shimomura et al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Candidate genes by sequence homology include hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus.

Yet another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and

dodecanedioyl-CoA (Westin et al, J.Biol.Chem. 280:38125-38132 (2005)) and the closest E. coli homo log, tesB, which can also hydrolyze a broad range of CoA thiolesters (Naggert et al., J Biol Chem 266: 11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)).

Other potential E. coli thiolester hydrolases include the gene products of tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC (Kuznetsova et al, FEMS Microbiol Rev 29:263- 279 (2005); Zhuang et al, FEBS Lett 516: 161-163 (2002)), paal (Song et al, J Biol Chem 281 : 11028-11038 (2006)), andybdB (Leduc et al, J Bacteriol 189:7112-7126 (2007)).

Gene name GI# GenBank Accession # Organism

tesA 16128478 NP_415027 Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paal 16129357 NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli 6.3.1.a/6.3.2.a amide synthases/peptide synthases. The direct conversion of 6-aminocaproate to caprolactam (Step S, Figure 10; Step R, Figure 11) requires the formation of an

intramolecular peptide bond. Ribosomes, which assemble amino acids into proteins during translation, are nature's most abundant peptide bond-forming catalysts. Nonribosomal peptide synthetases are peptide bond forming catalysts that do not involve messenger mRNA (Schwarzer et al., Nat Prod.Rep. 20:275-287 (2003)). Additional enzymes capable of forming peptide bonds include acyl-CoA synthetase from Pseudomonas chlororaphis (Abe et al., J Biol Chem 283: 11312-11321 (2008)), gamma-Glutamylputrescine synthetase from E. coli (Kurihara et al., J Biol Chem 283: 19981-19990 (2008)), and beta-lactam synthetase from Streptomyces clavuligerus (Bachmann et al., Proc Natl Acad Sci USA 95:9082-9086

(1998);Bachmann et al., Biochemistry 39: 11187-11193 (2000);Miller et al, Nat Struct.Biol 8:684-689 (2001);Miller et al, Proc Natl Acad Sci USA 99: 14752-14757 (2002);Tahlan et al, Antimicrob. Agents. Chemother. 48:930-939 (2004)).

4.2.1.a Hydrolyase. Most dehydratases catalyze the a, β-elimination of water. This involves activation of the a-hydrogen by an electron-withdrawing carbonyl, carboxylate, or Co A- thiol ester group and removal of the hydroxyl group from the β-position. Enzymes exhibiting activity on substrates with an electron-withdrawing carboxylate group are excellent candidates for dehydrating 3-hydroxyadipate (Figure 10, Step I) or 3-hydroxy-6- aminohexanoate (Figure 11, Step I).

For example, fumarase enzymes naturally catalyze the reversible dehydration of malate to fumarate. E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001);Woods et al., Biochim Biophys Acta 954: 14-26 (1988);Guest et al, J Gen Microbiol 131 :2971-2984 (1985)). Additional enzyme candidates are found in Campylobacter jejuni (Smith et al., Int.J Biochem. Cell Biol 31 :961- 975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem.Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem. 89: 1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidops is thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum

thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

Two additional dehydratase candidates are 2-(hydroxymethyl)glutarate dehydratase and dimethylmaleate hydratase, enzymes studied for their role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci U S A 103: 12341-6 (2006)). 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate. This enzyme is encoded by hmd in Eubacterium barkeri (Alhapel et al., supra). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing bacterial serine dehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA).

Gene name GI# GenBank Accession # Organism

hmd 86278275 ABC88407.1 Eubacterium barkeri

BACCAP 02294 154498305 ZP 02036683.1 Bacteroides capillosus ANACOL_02527 167771169 ZP_02443222.1 Anaerotruncus colihominis

DSM 17241

NtherDRAFT_2368 169192667 ZP_02852366.1 Natranaerobius thermophilus

JW/NM-WN-LF

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe -dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3- dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., sw ?ra;Kollmann-Koch et al., Hoppe Seylers. Z.Physiol Chem. 365:847-857 (1984)).

An additional enzyme candidate is 2-methylmalate dehydratase, also called citramalate hydro lyase, a reversible hydro lyase that catalyzes the alpha, beta elimination of water from citramalate to form mesaconate. This enzyme has been purified and characterized in

Clostridium tetanomorphum (Wang et al., J Biol. Chem. 244:2516-2526 (1969)). The activity of this enzyme has also been detected in several bacteria in the genera Citrobacter and Morganella in the context of the glutamate degradation VI pathway (Kato et al.,

Arch. Microbiol 168:457-463 (1997)). Genes encoding this enzyme have not been identified in any organism to date. Enzymes exhibiting activity on substrates with an electron- withdrawing CoA-thiol ester group adjacent to the a-hydrogen are excellent candidates for dehydrating 3 -hydroxy adipyl- CoA (Figure 10, Step C) or 3-hydroxy-6-aminohexanoyl-CoA (Figure 11, Step C). The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carry out the

hydroxylation of double bonds during phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). The paaA and paaB from P. fluorescens catalyze analogous transformations (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., swpra;Park et al., Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., swpra;Park et al., Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681- 686 (2004)). Crotonase enzymes are additional candidates for dehydrating the required 3- hydroxyacyl-CoA molecules depicted in Figures 10 and 11. These enzymes are required for n-butanol formation in some organisms, particularly Clostridial species, and also comprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genes encoding crotonase enzymes can be found in C. acetobutylicum (Boynton et al., supra), C. kluyveri (Hillmer et al., FEBS Lett. 21 :351-354 (1972)), and Metallosphaera sedula (Berg et al., supra) though the sequence of the latter gene is not known. Enoyl-CoA hydratases, which are involved in fatty acid beta-oxidation and/or the metabolism of various amino acids, can also catalyze the hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Roberts et al., Arch.Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg.Med.Chem. 1 1 :9-20 (2003); Conrad et al, J Bacteriol. 118:103-111 (1974)).

6.2.1.a Acid- thiol ligase. Steps F, L, and R of Figure 10 and Steps F and L of Figure 11 require acid-thiol ligase or synthetase functionality (the terms ligase, synthetase, and synth; are used herein interchangeably and refer to the same enzyme class). Exemplary genes encoding enzymes likely to carry out these transformations include the sucCD genes of E. coli which naturally form a succinyl-CoA synthetase complex. This enzyme complex naturally catalyzes the formation of succinyl-CoA from succinate with the concaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given the structural similarity between succinate and adipate, that is, both are straight chain dicarboxylic acids, it is reasonable to expect some activity of the sucCD enzyme on adipyl-CoA.

Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., BiochemicalJournal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from /⁵, chrysogenum (Lamas- Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez- Blanco et al., J Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Boweret al, J Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from ws musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-Co A esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl- CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., s«pra;Brasen et al., supra).

Yet another option is to employ a set of enzymes with net ligase or synthetase activity. For example, phosphotransadipylase and adipate kinase enzymes are catalyzed by the gene products of bukl, buk2, and ptb from C. acetobutylicum (Walter et al., Gene 134: 107-111 (1993); Huang et al., J Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb gene encodes an enzyme that can convert butyryl-CoA into butyryl-phosphate, which is then converted to butyrate via either of the buk gene products with the concomitant generation of ATP.

No enzyme required - Spontaneous cyclization. 6-Aminocaproyl-CoA will cyclize spontaneously to caprolactam, thus eliminating the need for a dedicated enzyme for this step. A similar spontaneous cyclization is observed with 4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi et al, J Biol Chem 256:7642-7651 (1981)).

EXAMPLE XIII

Preparation of a 6-Aminocaproic acid Producing Microbial Organism Having a Pathway for Converting Acetyl-CoA and 4-aminobutyryl-CoA to 6-Aminocaproic acid

This example describes the generation of a microbial organism capable of producing 6- aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the 6-aminocaproic acid pathway shown in Figure 11 that starts from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing 6- aminocaproic acid. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproic acid, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the /? «J (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3- hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the 6ct/ (NP_349317.1), etfAB (NP_349315.1 and NP_349316.1), and acot8 (CAA15502) genes encoding 6-aminohex-2-enoyl-CoA reductase and 6-aminocaproyl-CoA hydrolase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the sucD (NP_904963.1), g 67 (NP_417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA

transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter, to increase the availability of 4- aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis. The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproic acid synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS). Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6- aminocaproic acid. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production. For large-scale production of 6-aminocaproic acid, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp- cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂S0₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE XIV

This example describes the generation of a microbial organism capable of producing 6- aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA. Escherichia coli is used as a target organism to engineer the 6-aminocaproic acid pathway shown in Figure 11 that starts from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing 6- aminocaproic acid. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproic acid, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the /? a/ (NP_415915.1), /?c /J (AAN69545.1 and NP_746082.1), and bdh (AAA58352.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-

CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoate reductase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the enr (CAA76083.1) and hmd (ABC88407.1) genes encoding 6- aminohex-2-enoate reductase and 3-hydroxy-6-aminohexanoate dehydratase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the sucD (NP_904963.1), gabT (NP_417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4- aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector

(Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis.

The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the 6-aminocaproic acid synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of 6- aminocaproic acid. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is performed to improve both growth and production

characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production.

For large-scale production of 6-aminocaproic acid, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp- cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE XV

Preparation of a Caprolactam Producing Microbial Organism Having a Pathway for

Converting Acetyl-CoA and Succinyl-CoA to 6-Aminocaproic acid

This example describes the generation of a microbial organism capable of producing caprolactam from acetyl-CoA and succinyl-CoA. Escherichia coli is used as a target organism to engineer the caprolactam pathway shown in Figure 10 that starts from acetyl-CoA and succinyl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing caprolactam. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the 3- oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the 6ct/ (NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the acrl (YP_047869.1), gabT (NP_417148.1), and bioW (NP_390902.2) genes encoding adipyl-CoA reductase (aldehyde forming), 6-aminocaproic acid transaminase, and 6-aminocaproyl-CoA synthase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.

The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the caprolactam synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like.

Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS). Microbial strains engineered to have a functional caprolactam synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056- 1058 (2004); Alper et al, Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the caprolactam producer to further increase production.

For large-scale production of caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE XVI

Preparation of a Hexamethylenediamine Producing Microbial Organism Having a Pathway for Converting Acetyl-CoA and Succinyl-CoA to 6-Aminocaproic acid

This example describes the generation of a microbial organism capable of producing hexamethylenediamine from acetyl-CoA and succinyl-CoA.

Escherichia coli is used as a target organism to engineer the hexamethylenediamine pathway shown in Figure 10 that starts from acetyl-CoA and succinyl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing hexamethylenediamine. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce hexamethylenediamine, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the /? «J (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the bed (NP 349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the acrl (YP_047869.1), g 67 (NP_417148.1), bioW

(NP_390902.2), and ygjG (NP_417544) genes encoding adipyl-CoA reductase (aldehyde forming), 6-aminocaproyl-CoA reductase (aldehyde forming), 6-aminocaproic acid transaminase, 6-aminocaproyl-CoA synthase, and hexamethylenediamine transaminase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylenediamine synthesis.

The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the hexamethylenediamine synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce

hexamethylenediamine is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional hexamethylenediamine synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of hexamethylenediamine. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of

hexamethylenediamine. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the hexamethylenediamine product. Adaptive evolution is performed to improve both growth and production

characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the hexamethylenediamine producer to further increase production.

For large-scale production of hexamethylenediamine, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp- cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂S0₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE XVII

Preparation of a Caprolactam Producing Microbial Organism Having a Pathway for Converting Acetyl-CoA and 4-aminobutyryl-CoA to 6-Aminocaproyl-CoA

This example describes the generation of a microbial organism capable of producing caprolactam from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the caprolactam pathway shown in Figure 11 that starts from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing caprolactam. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP_415915.1), /?a H (NP_415913.1), and maoC (NP_415905.1) genes encoding the 3-oxo- 6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6- aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the bed (NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 6-aminohex-2- enoyl-CoA reductase activity are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the sucD (NP 904963.1), gabT

(NP_417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.

Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional caprolactam synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of caprolactam. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056- 1058 (2004); Alper et al, Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the caprolactam producer to further increase production. For large-scale production of caprolactam, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an

Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE XVIII

Preparation of a Hexamethylenediamine Producing Microbial Organism Having a

Pathway for Converting Acetyl-CoA and 4-aminobutyryl-CoA to 6-Aminocaproyl-CoA

This example describes the generation of a microbial organism capable of producing hexamethylenediamine from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the hexamethylenediamine pathway shown in Figure XVII that starts from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing hexamethylenediamine. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions. To generate an E. coli strain engineered to produce hexamethylenediamine, nucleic acids encoding the requisite enzymes are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the /? a/ (NP_415915.1), /? H (NP_415913.1), and maoC (NP_415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, 3- hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. In addition, the 6ct/ (NP_349317.1), etfAB (NP_349315.1 and NP_349316.1), acrl (YP_047869.1), and ygjG (NP_417544) genes encoding 6-aminohex-2-enoyl-CoA reductase, 6-aminocaproyl- CoA reductase (aldehyde forming), and hexamethylenediamine transaminase activities are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Lastly, the sucD (NP_904963.1), gabT (NP_417148.1), and cat2 (P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GAB A transaminase, and 4- aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a third compatible plasmid, pZS23, under the PAl/lacO promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector

(Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylenediamine synthesis.

Microbial strains engineered to have a functional hexamethylenediamine synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers. To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US

For large-scale production of hexamethylenediamine, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp- cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H₂SO₄. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE XIX

Pathways for Production of 6-Aminocaproic acid from Succinic Semialdehyde and

Pyruvate

This example describes exemplary pathways for production of 6-aminocaproic acid. Novel pathways for producing 6-aminocaproic acid (6-ACA) and related products are described herein. These pathways synthesize 6-ACA from succinic semialdehyde and pyruvate, utilizing aldolase and hydratase enzymes from the 4-hydroxyphenylacetic acid degradation pathway. The candidate enzymes, and associated risks of implementation are discussed in Example XXI below. This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 6-ACA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

6-aminocaproic acid and derivatives are produced from succinic semialdehyde and pyruvate in a minimum of five enzymatic steps. In the first step of all pathways, pyruvate and succinic semialdehyde are joined by 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase. The product of this reaction, HODH, is then dehydrated by 2-oxohept-4-ene-l,7-dioate (OHED) hydratase to form OHED. In subsequent steps, OHED is transaminated, decarboxylated or reduced as shown in Figure 12.

In one route, the alkene of OHED is reduced by OHED reductase, forming 2-oxoheptane-l ,7- dioate (2-OHD) (Figure 12, Step C), a 2-ketoacid. 2-OHD is then converted to adipate semialdehyde by a ketoacid decarboxylase (Figure 12, Step D). In the final step, the aldehyde of adipate semialdehyde is converted to an amine by an aminotransferase or an aminating oxidoreductase (Figure 12, Step E).

In a similar route, the 2-keto group of 2-OHD is transaminated by an aminotransferase or an aminating oxidoreductase (Figure 12, Step H) to form 2-aminoheptane-l,7-dioate (2-AHD). This product is then decarboxylated by 2-AHD decarboxylase to form 6-aminocapropate (Figure 12, Step I). In an alternate route, OHED is first decarboxylated by OHED decarboxylase (Figure 12, Step F), resulting in the formation of 6-oxohex-4-enoate (6-OHE). The alkenal group of 6-OHE is reduced by an oxidoreductase to adipate semialdehyde (Figure 12, Step G). Adipate semialdehyde is then converted to 6-aminocaproate by an aminotransferase or aminating oxidoreductase (Figure 12, Step E).

Yet another route calls for an aminotransferase or aminating oxidoreductase to convert OHED to 2-aminohept-4-ene-l,7-dioate (2-AHE) (Figure 12, Step J). The alkene of 2-AHE is subsequently reduced by an alkene oxidoreductase (Figure 12, Step K). The product of this reaction, 2-AHD, is then decarboxylated by an amino acid decarboxylase (Figure 12, Step I) to form 6-aminocaproate.

In yet another route, HODH is converted to 3-hydroxyadipyl-CoA by either an HODH dehydrogenase or and HODH formate-lyase (Figure 12, Step L). 3-Hydroxyadipyl-CoA is subsequently dehydrated and reduced to form adipyl-CoA (Figure 12, Steps M, N). Adipyl- CoA is reduced and de-acylated to form adipate semialdehyde (Figure 12, Step O), which is then converted to 6-aminocaproate by an aminotransferase or an aminating oxidoreductase (Figure 12, Step E).

In a similar route, HODH is first converted to OHED (Figure 12, Step B), as described above. OHED is then converted to 2,3-dehydroadipyl-CoA by a dehydrogenase or an OHED formate-lyase (Figure 12, Step P). 2,3-Dihydroadipyl-CoA is then reduced to adipyl-CoA (Figure 12, Step N), which is converted to 6-aminocaproate via adipate semialdehyde (Figure 12, Steps O, E), as described previously.

In the final route, HODH is converted to 2-OHD via steps B and C, as described previously. A 2-OHD formate-lyase or dehydrogenase converts 2-OHD to adipyl-CoA (Figure 12, Step Q), which is then reduced by a CoA-dependent aldehyde dehydrogenase (Figure 12, Step O). The product, adipate semialdehyde, is converted to 6-aminocaproate by an aminotransferase or aminating oxidoreductase (Figure 12, Step E).

The routes detailed in Figure 12 are able to achieve the maximum theoretical 6-ACA yield of 0.8 moles 6-ACA per mole glucose utilized. The energetic yield is also favorable, with a maximum of 1.6 moles ATP per mole glucose utilized at the maximum product yield. The following assumptions were used to calculate yield: 1) phosphoenolpyruvate (PEP) carboxykinase is able to operate in the ATP-generating direction, 2) NH4 and 6-ACA are transported into the cell by proton antiport, and 3) succinic semialdehyde is formed from alpha-ketoglutarate and/or succinyl-CoA. Succinic semialdehyde dehydrogenase is a NAD(P)H and CoA-dependent aldehyde dehydrogenase that converts succinyl-CoA to succinic semialdehyde. Succinic semialdehyde is formed from alpha-ketoglutarate by two enzymes: alpha-ketoglutarate decarboxylase and 4-aminobutyrate transaminase.

EXAMPLE XX

Pathways for Production of Hexamethylenediamine from 6-Aminocaproate

This example describes exemplary pathways for production of hexamethylenediamine.

Novel pathways for producing hexamethylenediamine (HMDA) and related products are described herein. This pathway synthesizes HMDA from 6-Aminocaproate (6-ACA). These pathways involve activation of the acid group by phosphorylation and/or acylation.

Acetylation of the terminal amino group provides protection from spontaneous cyclization of pathway intermediates. The candidate enzymes, and associated risks of implementation are discussed in Example XXI below. This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze HMDA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

Several pathways for producing HMDA from 6-aminocaproate are detailed in Figure 13. All routes entail activation of the carboxylic acid group, followed by reduction and

transamination. In three routes, 6-aminocaproate is activated directly while in other routes, the terminal amine group is protected by N-acetylation to prevent spontaneous cyclization. In one route, 6-aminocaproate is phosphorylated to 6-AHOP by 6-aminocaproate kinase (Figure 13, Step A). 6-AHOP is then reduced to 6-aminocaproic semialdehyde (Figure 13, Step B) and subsequently transaminated (Figure 13, Step C) by an aminotransferase or an aminating oxidoreductase.

Alternately, 6-AHOP is converted to 6-aminocaproyl-CoA by an acyltransferase (Figure 13, Step L). 6-Aminocaproyl-CoA is then reduced to 6-aminocaproic semialdehyde by a Co A- dependent aldehyde dehydrogenase (Figure 13, Step N). HMDA is then formed by transamination of 6-aminocaproic semialdehyde by an aminotransferase or aminating oxidoreductase (Figure 13, Step C).

In yet another route, 6-aminocaproate is first activated to a CoA derivative by a CoA transferase or CoA ligase (Figure 13, Step M). The product, 6-aminocaproyl-CoA, may spontaneously cyclize, or be converted to 6-aminocaproic semialdehyde by an aldehyde- forming CoA-dependent aldehyde dehydrogenase (Figure 13, Step N). 6-Aminocaproic semialdehyde is converted to HMDA by an aminotransferase or an aminating oxidoreductase (Figure 13, Step C). Additional routes proceed from 6-acetamidohexanoate, the acetylated product of 6- aminocaproate N-acetyltransferase. 6-Acetamidohexanoate is converted to 6- acetamidohexanal by different routes (described below). In the final two steps of these routes, 6-acetamidohexanal is first converted to 6-acetamidohexanamine by an aminotransferase or an aminating oxidoreductase (Figure 13, Step G). 6-Acetamidohexanamine is subsequently converted to HMDA by an amide hydrolase or an N-acetyltransferase (Figure 13, Step H).

In one route, 6-acetamidohexanoate is phosphorylated by 6-acetamidohexanoate kinase (Figure 13, Step E). The product, 6-AAHOP, is reduced to form 6-acetamidohexanal (Figure 13, Step F), which is then converted to HMDA as described above.

In another route, 6-acetamidohexanoate is activated to 6-acetamidohexanoyl-CoA by a CoA transferase or CoA ligase (Figure 13, Step I). The CoA derivative is then reduced to 6- acetamidohexanal by an aldehyde-forming CoA-dependent oxidoreductase (Figure 13, Step J). 6-acetamidohexanal is then converted to HMDA as described above.

Alternately, 6-acetamidohexanoate is phosphorylated to 6-AAHOP (Figure 13, Step E) and subsequently converted to 6-acetamidohexanoyl-CoA by an acyltransferase (Figure 13, Step K). 6-Acetamidohexanoyl-CoA is then reduced to HMDA as described previously. EXAMPLE XXI

Enzyme Classification System for Production of 6-Aminocaproic acid and

Hexamethylenediamine

This example describes the enzyme classification system for the exemplary pathways described in Examples XIX and XX for production of 6-aminocaproate or

hexamethylenediamine.

All transformations depicted in Figures 12 and 13 fall into the general categories of transformations shown in Table 9. Below is described a number of biochemically characterized genes in each category. Specifically listed are genes that can be applied to catalyze the appropriate transformations in Figures 12-13 when properly cloned and expressed.

Table 9 shows the enzyme types useful to convert common central metabolic intermediates into 6-aminocaproate and hexamethylenediamine. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.

Table 9

LABEL FUNCTION

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde)

1.2.1.C Oxidoreductase (2-ketoacid to acyl-CoA)

1.2.1.d Oxidoreductase (phosphonic acid to aldehyde)

1.3. l .a Oxidoreductase (alkene to alkane)

1.4.1. a Oxidoreductase (ketone or aldehyde to amino)

2.3. l .a Acyltransferase (transferring CoA to phospho)

2.3. l .c Acyltransferase (N-acetyltransferase)

2.3. l .d Acyltransferase (formate C-acyltransferase)

2.6.1.a Aminotransferase

2.7.2.a Phosphotransferase (carboxy acceptor)

2.8.3.a Coenzyme-A transferase

3.5. l .a Hydrolase (acting on linear amides)

4.1. l .a Carboxy-lyase

4.1.2.a Aldehyde-lyase LABEL FUNCTION

4.2.1.a Hydro-lyase

6.2.1.a Acid-thiol ligase

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). The transformations of 6- acetamidohexanoyl-CoA to 6-acetamidohexanal (Figure 13, Step J) and 6-aminocaproyl-CoA to 6-aminocaproic semialdehyde (Figure 13, Step N) are catalyzed by CoA-dependent oxidoreductase enzyme in the EC class 1.2.1. Adipyl-CoA is converted to adipate semialdehyde by adipyl-CoA oxidoreductase, an enzyme with similar functionality (Figure 12, Step O). Succinic semialdehyde dehydrogenase, an enzyme that forms Figure 12 precursor succinic semialdehyde from succinyl-CoA, is also a CoA-dependent

oxidoreductase. Oxidoreductases in the EC class 1.2.1.- are capable of reducing an acyl-CoA to its corresponding aldehyde. Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase (Reiser and

Somerville, Journal of Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-l fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP- dependent succinic semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another succinic semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The acylating acetaldehyde dehydrogenase in

Pseudomonas sp, encoded by bphG, is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA

(Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)).

Gene name GI# GenBank Accession # Organism

acrl 50086359 YP 047869.1 Acinetobacter calcoaceticus acrl 1684886 AAC45217 Acinetobacter baylyi

acrl 18857901 BAB85476.1 Acinetobacter sp. Strain M-l sucD 172046062 P38947.1 Clostridium kluyveri

sucD 34540484 NP 904963.1 Porphyromonas gingivalis bphG 425213 BAA03892.1 Pseudomonas sp

adhE 55818563 AAV66076.1 Leuconostoc mesenteroides

An additional enzyme that converts an acyl-CoA to its corresponding aldehyde is malonyl- CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318: 1782-1786 (2007); and Thauer, R. K., Science. 318:1732-1733 (2007)). The enzyme utilizes NAD PH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp (Alber et al., J Bacteriol. 188:8551-8559 (2006); and Hugler et al, J Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., J Bacteriol. 188:8551-8559 (2006); and Berg et al, Science. 318: 1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WIPO Patent Application WO/2007/141208 Kind Code: A2). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate

semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)).

Gene name GI# GenBank Accession # Organism

Msed_0709 146303492 YP 001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobus tokodaii

asd-2 15898958 NP_343563.1 Sulfolobus solfataricus

Saci_2370 70608071 YP 256941.1 Sulfolobus acidocaldarius Aid 49473535 AAT66436 Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimurium eutE 2498347 P77445 Escherichia coli

1.2.1.C Oxidoreductase (2-ketoacid to acyl-CoA). Several transformations in Figure 12 require conversion of a 2-ketoacid to an acyl-CoA (Steps L, P and Q) by an enzyme in the EC class 1.2.1. Such reactions are catalyzed by multi-enzyme complexes that catalyze a series of partial reactions which result in acylating oxidative decarboxylation of 2-keto-acids.

Exemplary enzymes include 1) branched-chain 2-keto-acid dehydrogenase, 2) alpha- ketoglutarate dehydrogenase, and 3) the pyruvate dehydrogenase multienzyme complex (PDHC). Each of the 2-keto-acid dehydrogenase complexes occupies key positions in intermediary metabolism, and enzyme activity is typically tightly regulated (Fries et al., Biochemistry 42:6996-7002 (2003)). The enzymes share a complex but common structure composed of multiple copies of three catalytic components: alpha-ketoacid decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). The E3 component is shared among all 2-keto-acid dehydrogenase complexes in an organism, while the El and E2 components are encoded by different genes. The enzyme components are present in numerous copies in the complex and utilize multiple cofactors to catalyze a directed sequence of reactions via substrate channeling. The overall size of these

dehydrogenase complexes is very large, with molecular masses between 4 and 10 million Da (i.e. larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normally low or limited under anaerobic conditions in E. coli. Increased production of NADH (or NADPH) could lead to a redox-imbalance, and NADH itself serves as an inhibitor to enzyme function.

Engineering efforts have increased the anaerobic activity of the E. coli pyruvate

dehydrogenase complex (Kim et a\., Appl. Environ. Microbiol. 73: 1766-1771 (2007); Kim et al, J Bacteriol. 190:3851-3858 (2008); and Zhou et al, Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effect of NADH can be overcome by engineering an H322Y mutation in the E3 component (Kim et al, J Bacteriol. 190:3851-3858 (2008)). Structural studies of individual components and how they work together in complex provide insight into the catalytic mechanisms and architecture of enzymes in this family (Aevarsson et al, Nat. Struct. Biol. 6:785-792 (1999); and Zhou et al, Proc. Natl. Acad. Sci. U. S. A 98: 14802-14807 (2001)). The substrate specificity of the dehydrogenase complexes varies in different organisms, but generally branched-chain keto-acid dehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA and is the primary site of control of metabolic flux through the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278 (1980)). Encoded by genes sucA, sucB and Ipd in E. coli, AKGD gene expression is downregulated under anaerobic conditions and during growth on glucose (Park et al, Mol. Microbiol. 15:473-482 (1995)). Although the substrate range of AKGD is narrow, structural studies of the catalytic core of the E2 component pinpoint specific residues responsible for substrate specificity (Knapp et al., J Mol. Biol. 280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (El and E2) and pdhD (E3, shared domain), is regulated at the transcriptional level and is dependent on the carbon source and growth phase of the organism (Resnekov et al, Mol. Gen. Genet. 234:285-296 (1992)). In yeast, the LPDl gene encoding the E3 component is regulated at the transcriptional level by glucose (Roy and Dawes, J. Gen. Microbiol. 133:925-933 (1987)). The El component, encoded by KGD1, is also regulated by glucose and activated by the products of HAP2 and HAP3 (Repetto and Tzagoloff, Mol. Cell Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited by products NADH and succinyl-CoA, is well-studied in mammalian systems, as impaired function of has been linked to several neurological diseases (Tretter and dam- Vizi, Philos. Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2005)).

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as 2- oxoisovalerate dehydrogenase, participates in branched-chain amino acid degradation pathways, converting 2-keto acids derivatives of valine, leucine and isoleucine to their acyl- Co A derivatives and C0₂. The complex has been studied in many organisms including Bacillus subtilis (Wang et al., Eur. J. Biochem. 213: 1091-1099 (1993)), Rattus norvegicus (Namba et al., J Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas putida (Sokatch et al., J Bacteriol. 148:647-652 (1981)). In Bacillus subtilis the enzyme is encoded by genes pdhD (E3 component), bfmBB (E2 component), bfmBAA and bfmBAB (El component) (Wang et al., Eur. J. Biochem. 213: 1091-1099 (1993)). In mammals, the complex is regulated by phosphorylation by specific phosphatases and protein kinases. The complex has been studied in rat hepatocites (Chicco et al, J Biol. Chem. 269: 19427-19434 (1994)) and is encoded by genes Bckdha (E\ alpha , Bckdhb (E\ beta , Dbt (El), and Did (E3). The El and E3 components of the Pseudomonas putida BCKAD complex have been crystallized

(Aevarsson et al, Nat. Struct. Biol. 6:785-792 (1999); and Mattevi et al, Science. 255: 1544- 1550 (1992)) and the enzyme complex has been studied (Sokatch et al., J Bacteriol. 148:647- 652 (1981)). Transcription of the P. putida BCKAD genes is activated by the gene product oibkdR (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). In some organisms including Rattus norvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int. 31 :91 1-922 (1993)), this complex has been shown to have a broad substrate range that includes linear oxo-acids such as 2-oxobutanoate and alpha-ketoglutarate, in addition to the branched-chain amino acid precursors. The active site of the bovine BCKAD was engineered to favor alternate substrate acetyl-CoA (Meng and Chuang, Biochemistry. 33: 12879-12885 (1994)).

Gene name GI# GenBank Accession # Organism

bfmBB 16079459 NP_390283.1 Bacillus subtilis

bfmBAA 16079461 NP_390285.1 Bacillus subtilis

bfmBAB 16079460 NP_390284.1 Bacillus subtilis

pdhD 118672 P21880.1 Bacillus subtilis

IpdV 118677 P09063.1 Pseudomonas putida bkdB 129044 P09062.1 Pseudomonas putida bkdAl 26991090 NP_746515.1 Pseudomonas putida bkdA2 26991091 NP_746516.1 Pseudomonas putida

Bckdha 77736548 NP_036914.1 Rattus norvegicus

Bckdhb 158749538 NP_062140.1 Rattus norvegicus

Dbt 158749632 NP_445764.1 Rattus norvegicus

Did 40786469 NP_955417.1 Rattus norvegicus The pyruvate dehydrogenase complex, catalyzing the conversion of pyruvate to acetyl-CoA, has also been extensively studied. In the E. coli enzyme, specific residues in the El component are responsible for substrate specificity (Bisswanger, J Biol Chem. 256:815-822 (1981); Bremer, Eur. J Biochem. 8:535-540 (1969); and Gong et al., J Biol Chem. 275: 13645- 13653 (2000)). As mentioned previously, enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., Appl. Environ. Microbiol. 73: 1766-1771 (2007); Kim et al, J Bacteriol. 190:3851-3858 (2008)); and Zhou et al, Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., J Biotechnol. 56: 135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U. S. A 98: 14802-14807 (2001)) and the E2 catalytic domain from

Azotobacter vinelandii are available (Mattevi et al., Science. 255: 1544-1550 (1992)). Some mammalian PDH enzymes complexes can react on alternate substrates such as 2- oxobutanoate, although comparative kinetics oiRattus norvegicus PDH and BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton et al., Biochem. J. 234:295-303 (1986)).

Gene name GI# GenBank Accession # Organism

aceE 16128107 NP_414656.1 Escherichia coli

aceF 16128108 NP_414657.1 Escherichia coli

Ipd 16128109 NP_414658.1 Escherichia coli

pdhA 3123238 P21881.1 Bacillus subtilis

pdhB 129068 P21882.1 Bacillus subtilis

pdhC 129054 P21883.2 Bacillus subtilis

pdhD 118672 P21880.1 Bacillus subtilis

aceE 152968699 YP 001333808.1 Klebsiella pneumonia aceF 152968700 YP 001333809.1 Klebsiella pneumonia

IpdA 152968701 YP 001333810.1 Klebsiella pneumonia

Pdhal 124430510 NP_001004072.2 Rattus norvegicus

Pdhal 16758900 NP_446446.1 Rattus norvegicus

Dlat 78365255 NP_112287.1 Rattus norvegicus

Did 40786469 NP_955417.1 Rattus norvegicus As an alternative to the large multienzyme 2-keto-acid dehydrogenase complexes described above, some anaerobic organisms utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze acylating oxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenase complexes, these enzymes contain iron-sulfur clusters, utilize different cofactors, and use ferredoxin or flavodoxin as electron acceptors in lieu of NAD(P)H. While most enzymes in this family are specific to pyruvate as a substrate (POR) some 2-keto- acid:ferredoxin oxidoreductases have been shown to accept a broad range of 2-ketoacids as substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al, J Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from the thermoacidophilic archaeon Sulfolobus tokodaii 7, which contains an alpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al, J Biochem. 120:587-599 (1996)). A plasmid-based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74- 80 (2002)). Two OFORs from Aeropyrum pernix str. Kl have also been recently cloned into E. coli, characterized, and found to react with a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)). The gene sequences of these OFOR candidates are available, although they do not have GenBank identifiers assigned to date. There is bioinformatic evidence that similar enzymes are present in all archaea, some anaerobic bacteria and amitochondrial eukarya (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74- 80 (2002)). This class of enzyme is also interesting from an energetic standpoint, as reduced ferredoxin could be used to generate NADH by ferredoxin-NAD reductase (Petitdemange et al., Biochim. Biophys. Acta 421 :334-337 (1976)). Also, since most of the enzymes are designed to operate under anaerobic conditions, less enzyme engineering may be required relative to enzymes in the 2-keto-acid dehydrogenase complex family for activity in an anaerobic environment.

1.2.1.d Oxidoreductase (phosphonic acid to aldehyde). The reduction of a phosphonic acid to its corresponding aldehyde is catalyzed by an oxidoreductase in the EC class 1.2.1. Steps B and F in Figure 13 require such an enzyme for the reduction of 6-AHOP and 6-AAHOP to their corresponding aldehydes. These reactions are not catalyzed by known enzymes, but a similar reaction is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11): the NADPH-dependent reduction of 4-aspartyl phosphate to aspartate-4-semialdehyde. ASD participates in amino acid biosynthesis and recently has been studied as an antimicrobial target (Hadfield et al, Biochemistry 40: 14475-14483 (2001)). The E. coli ASD structure has been solved (Hadfield et al, JMol. Biol. 289:991-1002 (1999)) and the enzyme has been shown to accept the alternate substrate beta-3-methylaspartyl phosphate (Shames et al., J Biol. Chem. 259: 15331-15339 (1984)). The Haemophilus influenzae enzyme has been the subject of enzyme engineering studies to alter substrate binding affinities at the active site (Blanco et al, Acta Crystallogr. D. Biol. Crystallogr. 60: 1388-1395 (2004); and Blanco et al., Acta Crystallogr. D. Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are found in Mycobacterium tuberculosis (Shafiani et al., JAppl Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnle et al., J Mol. Biol. 353:1055-1068 (2005)), and the infectious microorganisms Vibrio cholera and Heliobacter pylori (Moore et al., Protein Expr. Purif. 25: 189-194 (2002)). A related enzyme candidate is acetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme that naturally reduces acetylglutamylphosphate to acetylglutamate- 5 -semialdehyde, found in S. cerevisiae (Pauwels et al., Eur. J Biochem. 270: 1014-1024 (2003)), B. subtilis (O'Reilly and Devine, Microbiology 140 (Pt 5): 1023-1025 (1994)) and other organisms.

1.3.1. a Oxidoreductase (alkene to alkane). Several transformations fall into the category of oxidoreductases that reduce an alkene to an alkane (EC 1.3.1.-). For example, Steps C, G, K and N in Figure 12, catalyzed by OHED reductase, 6-OHE reductase, 2-AHE reductase and 2,3-dehydroadipyl-CoA reductase, respectively, fall into this category. Enone reductase, alkenal reductase, and enoate reductase enzymes are suitable enzyme candidates for catalyzing the transformations of Steps C, G and K. Enoyl-CoA reductase enzymes catalyze the conversion of 2,3-dehydroadipyl-CoA to adipyl-CoA (Step N).

Enzymes with enone reductase activity have been identified in prokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur. J Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolic fraction of Saccharomyces cerevisiae were purified and characterized, and found to accept a variety of alkenals (similar to 6-OHE) and enoyl ketones (similar to OHED) as substrates (Wanner and Tressl, Eur. J Biochem. 255:271-278 (1998)). Genes encoding these enzymes have not been identified to date. Cell extracts of cyanobacterium Synechococcus sp. PCC7942 reduced a variety enone substrates to their corresponding alkyl ketones (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004)). Genes have not been associated with this activity in this organism. Enone reductases in other organisms can also catalyze this transformation.

A recombinant NADPH-dependent enone reductase from Nicotiana tabacum, encoded by NtRedl, was functionally expressed and characterized in E. coli (Matsushima et al.,

Bioorganic Chemistry 36:23-28 (2008)). This reductase was functional on the exocyclic enoyl ketone pulegone (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). An enzyme candidate in S. cerevisiae at the locus YML131 W, bears 30% identity to

NtRedl (evalxie = le-26). The amino acid sequence of NtRedl shares significant homology with 2-alkenal reductase from Arabidopsis thaliana, zeta-crystallin homolog from A.

thaliana, pulegone reductase from Menthe piperita and phenylpropenal alkene reductase irom Pinus taeda. These enzymes are known to catalyze the reduction of alkenes of α,β- unsaturated ketones and aldehydes.

Gene name GI# GenBank Accession # Organism

NtRedl 6692816 BAA89423 Nicotiana tabacum

YML131 W 45269874 AAS56318.1 Saccharomyces cerevisiae

AtDBRl 15237888 NP- 197199 Arabidopsis thaliana

P2 886430 CAA89262 Arabidopsis thaliana

PulR 34559418 AAQ75423 Menthe piperita

PtPPDBR 110816011 ABG91753 Pinus taeda 2-Alkenal reductase catalyzes the reduction of a, β-unsaturated double bonds of aldehydes and ketones. A barley alkenal hydrogenase ALH1 was identified with activity for a range of α,β-unsaturated ketones and aldehydes including trans-2-nonenal, 2-hexenal, traumatin and 1- octene-3-one (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)). The Hordeum vulgare ALH1 cDNA was cloned expressed in E. coli (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)).

2-Enoate reductase enzymes are known to catalyze the NAD(P)H-dependent reduction of a wide variety of a, β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J Biol.

Chem. 276:5779-5787 (2001)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been

characterized (Seedorf et al, Proc. Natl. Acad. Sci U. S. A 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel and Simon, Arch. Microbiol 135:51-57 (1983)). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadFf) (Rohdich et al., J Biol. Chem.

276:5779-5787 (2001)). The C. thermoaceticum enr gene has also been expressed in a catalytically active form in E. coli (Rohdich et al., J Biol. Chem. 276:5779-5787 (2001)).

Another candidate enoate reductase is 3-oxoadipate oxidoreductase (maleylacetate reductase), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. The enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke, J Bacteriol. 177:320-325 (1995); and Kaschabek. and Reineke, J Bacteriol. 175:6075-6081 (1993)), and the coding gene was cloned and sequenced (Kasberg et al., J Bacteriol. 179:3801-3803 (1997)). Candidate genes for 3- oxoadipate oxidoreductase include clcE gene from Pseudomonas sp. strain B13 (Kasberg et ah, J. Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., J Bacteriol. 180:3503-3508 (1998)), and macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)).

Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the reduction of 2,3- dehydroadipyl-CoA to adipyl-CoA (Figure 12, Step N). One exemplary enoyl-CoA reductase is the gene product oibcd from C. acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boynton et al., J Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). This approach is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci and Martin, Febs Letters 581 :1561-1566 (2007)).

Gene name GI# GenBank Accession # Organism

bed 15895968 NP_349317.1 Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridium acetobutylicum

15895967 NP_349316.1 Clostridium acetobutylicum TER 62287512 Q5EU90.1 Euglena gracilis

TDE0597 42526113 NP_971211.1 Treponema denticola

Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl- CoA. Adjacent genes in the pirn operon, pimC and pimD, bear sequence homology to C. acetobutylicum bed and are predicted to encode a flavin-containing pimeloyl-CoA

dehydrogenase (Harrison and Harwood, Microbiology 151 :727-736 (2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pirn operon composed of genes with high sequence similarity to pimC and pimD ofR. palustris (Harrison and Harwood, Microbiology 151 :727-736 (2005)).

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascarius suum and is capable of reducing a variety of linear and branched chain substrates including 2- methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et ah, J Biol. Chem. 268:22391-22396 (1993)). Two isoforms of the enzyme, encoded by genes acadl and acad, have been characterized.

1.4.1.a Oxidoreductase (ketone or aldehyde to amino). Oxidoreductases in the EC class 1.4.1 that convert an aldehyde or ketone to its corresponding amine group catalyze several biosynthetic steps in the disclosed pathways. In Figure 12, the conversions of OHED to 2- AHE (Step J), 2-OHD to 2-AHD (Step H) and adipate semialdehyde to 6-aminocaproate (Step E) are catalyzed by OHED aminating oxidoreductase, 2-OHD aminating oxidoreductase and adipate semialdehyde aminating oxidoreductase. In Figure 13, conversion of 6-aminocaproate semialdehyde to HMDA (Step H) and 6-acetamidohexanal to 6- acetamidohexanamine (Step G), are also catalyzed by aminating oxidoreductases.

Most aminating oxidoreductases catalyze the reversible oxidative deamination of alpha- amino acids with NAD+ or NADP+ as acceptor, and the reactions are typically reversible. Exemplary enzymes include glutamate dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by Idh, and aspartate dehydrogenase

(deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (Korber et al., J Mol. Biol. 234: 1270-1273 (1993); and McPherson et al., Nucleic Acids Res. 11 :5257- 5266 (1983)), gdh from Thermotoga maritime (Kort et al., Extremophiles. 1 :52-60 (1997); Lebbink et al, J Mol. Biol. 280:287-296 (1998); and Lebbink et al, J Mol. Biol. 289:357-369

(1999) ), and gdhAl from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The Idh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula, Biotechnol Bioeng 68:557-562

(2000) ; and Stoyan et al., J Biotechnol. 54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al, J Biol. Chem. 278:8804-8808 (2003)).

Lysine 6-dehydrogenase (deaminating), encoded by lysDH, catalyzes the oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde,

(Misono and Nagasaki, J Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in

Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem. 106:76-80 (1989); and Misono and Nagasaki, J Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41 :790-795 (2008)). Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate given the structural similarity between adipate semialdehyde and 2-aminoadipate-6-semialdehyde.

2.3.1.a Acyltransferase (transferring Co A to phospho). Acyltransferases that exchange a CoA moiety for a phosphate are in the EC class 2.3.1. Transformations in this category include the conversions of 6-AAHOP to 6-acetamidohexanoyl-CoA (Figure 13, Step K) and 6-AHOP to 6-aminocaproyl-CoA (Figure 13, Step L). Exemplary phosphate-transferring acyltransferases include phospho transacetylase (EC 2.3.1.8), encoded by pta, and

phosphotransbutyrylase (EC 2.3.1.19), encoded by ptb. The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191 :559-569 (1969)). This enzyme can also utilize propionyl-CoA as a substrate, forming propionate in the process (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Similarly, the ptb gene from C. acetobutylicum encodes phosphate transbutyrylase, an enzyme that reversibly converts butyryl-CoA into butyryl-phosphate (Walter et al., Gene 134: 107-111 (1993); and Wiesenborn et al, Appl Environ. Microbiol 55:317-322 (1989)). Additional ptb genes are found in butyrate-producing bacterium L2-50 (Louis et al., J Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr. Microbiol 42:345-349 (2001)).

2.3.l .c Acyltransferase (N-acetyltransferase). N-Acetyltransferases transfer an acetyl group to an amine, forming an N-acetyl group. N-Acetylation serves diverse functions in biological systems including transcriptional regulation, nuclear import, chromosome assembly and nucleosome remodeling (Kouzarides, EMBO J 19: 1176-1179 (2000)). N-Acetylation of metabolic intermediates of arginine biosynthetic pathways serves both to protect reactive intermediates from spontaneous cyclization and also to sequester pathway intermediates from competing pathways (Caldovic and Tuchman, Biochem. J 372:279-290 (2003)). Acetylation of 6- AC A (Figure 13, step D) serves a similar role in the proposed HMD A biosynthesis route of Figure 13, protecting reactive intermediates from spontaneous cyclization.

One candidate enzyme for acetylating 6-ACA is lysine N-acetyltransferase (EC 2.3.1.32), an enzyme which selectively transfers the acetyl moiety from acetyl phosphate to the terminal amino group of L-lysine, beta-L-lysine or L-ornithine. Although this enzyme is not known to acetylate 6-ACA, this substrate is structurally similar to the natural substrate. Lysine N- acetyltransferase has been characterized in Bos taurus (Paik. and Kim, Arch. Biochem.

Biophys. 108:221-229, 1964) and Methanosarcina mazei (Pfluger et al., Appl Environ.

Microbiol 69:6047-6055 (2003)). Methanogenic archaea maripaludis, M. acetivorans, M. barkeri and M. jannaschii are also predicted to encode enzymes with this functionality (Pfluger et al, Appl Environ. Microbiol 69:6047-6055 (2003)).

Alternately, 6-ACA acetylation can be catalyzed by an enzyme in the GNAT family of N- acetyltransferases. Such enzymes transfer an acetyl group from acetyl-CoA to a primary amine. The enzyme spermidine N-acetyltransferase (SSAT), also known as diamine N- acetyltransferase (EC 2.3.1.57), is capable of acetylating a variety of small molecule substrates. Purified enzymes from Ascaris suum and Onchocerca volvulus exhibit a broad substrate range that includes HMDA (Davids et al., Mol. Biochem. Parasitol. 64:341-344 (1994); and Wittich and Walter, Mol. Biochem. Parasitol. 38:13-17 (1990)), but the associated genes have not been identified to date. Other enzymes with this functionality are found in Bacillus subtilis (Forouhar et al., J Biol. Chem. 280:40328-40336 (2005)) and Homo sapiens (Casero and Pegg, FASEB J 7:653-661 (1993)). A closely related enzyme is thialysine N-acetyltransferase in C. elegans, an enzyme that accepts a range of substrates including lysine, ornithine, thialysine and others (bo-Dalo et al., Biochem. J 384: 129-137 (2004)). Amino acid residues involved in substrate binding were identified in the thialysine N-acetyltransferase from Leishmania major (Luersen, K., FEBS Lett. 579:5347-5352 (2005)). An additional candidate is the diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzyme participating in ectoine biosynthesis in Methylomicrobium alcaliphilum (Reshetnikov et al., Arch. Microbiol 184:286-297 (2006)) C. salexigens (formerly Halomonas elongata)

(Canovas et al, Syst. Appl Microbiol 21 :487-497 (1998)).

An additional enzyme candidate for acetylating 6- AC A (Figure 13, Step D) and de- acetylating 6-acetamidehexanamine (Figure 13, Step H) is ornithine acetyltransferase (OAT, EC 2.3.1.35 and EC 2.3.1.1), a bifunctional enzyme which catalyzes two steps of arginine biosynthesis (Figure 14A). The first step of arginine biosynthesis (Figure 14A, step 1) is the N-acetylation of glutamate, catalyzed by OAT with acetyl-CoA as an acetyl donor (O'Reilly and Devine, Microbiology 140 (Pt 5): 1023-1025 (1994)). OAT also catalyzes the fifth step of arginine biosynthesis (Figure 14A, step 2), in which an N-acetyl group is transferred from N- acetyl-L-ornithine to L-glutamate, the first metabolite in the arginine biosynthesis pathway. This transformation serves to recycle the acetyl group and regenerate N-acetylglutamate, conserving energy and thereby making the linear pathway a cyclic route. A similar strategy can be employed in HMDA biosynthesis from 6-aminocaproate, with a single enzyme acetylating 6-aminocaproate and de-acetylating 6-acetamidohexanamine to form HMDA (Figure 14B). Exemplary OAT enzymes are encoded by argj in Bacillus subtilis (O'Reilly and Devine, Microbiology 140 (Pt 5): 1023-1025 (1994); and Sakanyan et al., Journal of General Microbiology 138: 125-130 (1992)) and ECM40 in S. cerevisiae (Abadjieva et al., J Biol. Chem. 275: 11361-11367 (2000); and Liu et al, Eur. J Biochem. 228:291-296 (1995)). Crystal structures of the enzymes from yeast (Maes et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 62: 1294-1297 (2006)) and Mycobacterium tuberculosis (Sankaranarayanan et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 65: 173-176 (2009)) are available. Although encoded by a single open reading frame, OAT enzymes have distinct alpha and beta subunit peptides (Liu et al., Eur. J Biochem. 228:291-296 (1995)).

2.3.l .d Acyltransferase (formate C-acyltransferase). The acylation of ketoacids HODH,

OHED and 2-OHD to their corresponding CoA derivatives (Figure 12, Steps L, P and Q) and concurrent release of formate, is catalyzed by formate C-acyltransferase enzymes in the EC class 2.3.1. Enzymes in this class include pyruvate formate-lyase and ketoacid formate-lyase. Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflB in E. coli, converts pyruvate into acetyl-CoA and formate. The active site of PFL contains a catalytically essential glycyl radical that is posttranslationally activated under anaerobic conditions by PFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded by pflA (Knappe et al, Proc. Natl. Acad. Sci U. S. A 81 : 1332-1335 (1984); and Wong et al, Biochemistry 32: 14102-14110 (1993)). A pyruvate formate-lyase from Archaeglubus fulgidus encoded by pflD has been cloned, expressed in E. coli and characterized (Lehtio, L. and A. Goldman, Protein Eng Des Sel 17:545-552 (2004)). The crystal structures of the A.fulgidus and E. coli enzymes have been resolved (Lehtio et al., JMol. Biol. 357:221-235 (2006)). Additional PFL and PFL-AE candidates are found in Clostridium pasteurianum (Weidner and Sawers, J Bacteriol. 178:2440-2444 (1996)) and the eukaryotic alga Chlamydomonas reinhardtii (Cary et al., Appl. Environ. Microbiol 56: 1576- 1583 (1990)). Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate- lyase (KFL) and pyruvate formate-lyase 4, is the gene product oitdcE in E. coli. This enzyme catalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation, and can also substitute for pyruvate formate-lyase in anaerobic catabolism (Simanshu et al., J Biosci. 32: 1195-1206 (2007)). The enzyme is oxygen-sensitive and, like PflB, requires post-translational modification by PFL-AE to activate a glycyl radical in the active site (Hesslinger et al, Mol. Microbiol 27:477-492 (1998)).

Gene name GI# GenBank Accession # Organism

pflB 16128870 NP_415423.1 Escherichia coli pflA 16128869 NP_415422.1 Escherichia coli

tdcE 48994926 AAT48170.1 Escherichia coli

pflD 11499044 NP_070278.1 Archaeglubus fulgidus

Pfl 2500058 Q46266.1 Clostridium pasteurianum act 1072362 CAA63749.1 Clostridium pasteurianum

Pfll 159462978 XP OO 1689719.1 Chlamydomonas reinhardtii pflAl 159485246 XP OO 1700657.1 Chlamydomonas reinhardtii

2.6.1.a Aminotransferase. Steps E, H and J of Figure 12 and Steps C and G of Figure 13 require conversion of an aldehyde or ketone to an amino group. This transformation can be accomplished by an aminotransferase (EC 2.6.1.-). The conversion of an aldehyde to a terminal amine (Figure 12, Step E; Figure 13, Steps C and G) can be catalyzed by gamma- aminobutyrate transaminase (GABA transaminase). One E. coli GABA transaminase is encoded by gabT and transfers an amino group from glutamate to the terminal aldehyde of succinic semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)). This enzyme exhibits a broad substrate range (Liu et al., Biochemistry 43: 10896-10905 (2004)). The gene product oipuuE encodes the other 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); and Scott and Jakoby, J Biol. Chem. 234:932-936 (1959)).

Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to HMDA. The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC. Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1 ,7-diamino heptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova et al., BMC. Microbiol 3:2 (2003)). A putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol. 184:3765-3773 (2002)).

Additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferases which produce malonic semialdehyde from beta-alanine (WO08027742). The gene product oiSkPYD4 in Saccharomyces kluyveri was shown to preferentially use beta-alanine as the amino group donor (Andersen and Hansen, Gene 124: 105-109 (1993)). SkUGAl encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGAl (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both β- alanine and GABA transamination (Andersen and Hansen, Gene 124: 105-109 (1993)). 3- Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2-methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat 1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al, Methods Enzymol. 324:376-389 (2000)).

Steps J and H of Figure 12 are catalyzed by aminotransferases that transform amino acids into oxo-acids. In Step J, OHED is transaminated to form 2-AHE by OHED

aminotransferase. The transamination of 2-OHD to 2-AHD by 2-OHD aminotransferase

(Step H) is a similar reaction. An exemplary enzyme candidate for catalyzing these reactions is aspartate aminotransferase, an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate is similar in structure to OHED and 2-AHD. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84, (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (de la Torre et al, Plant J 46:414-425 (2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); and Wilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Ratt s norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589 (1980)).

Aminotransferases that work on other amino-acid substrates can catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen and Berg, C. J Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the transamination of a- ketobutyrate to generate a-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen and Berg, J Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase and

phosphohydroxythreonine aminotransferase (Lam and Winkler, J Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et al., FEBS. Lett. 390: 179-182 (1996)).

2.7.2.a Phosphotransferase (carboxy acceptor). Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. Steps A and E in Figure 13 require a phosphotransferase to activate the carboxyl groups of 6- ACA (Step A) and 6-acetamidohexanoate (Step E) to their corresponding phosphonic acids. Butyrate kinase carries out the reversible conversion of butyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56: 1576-1583

(1990)). This enzyme is encoded by either of the two buk gene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). Related enzyme isobutyrate kinase from Thermotoga maritima has also been expressed in E. coli and crystallized (Diao et al., Acta Crystallogr. D. Biol. Crystallogr. 59: 1100-1102 (2003); and Diao and Hasson, J Bacteriol. 191 :2521-2529 (2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and participates in the synthesis of several amino acids. The aspartokinase III enzyme in E. coli, encoded by lysC, has a broad substrate range and the catalytic residues involved in substrate specificity have been elucidated (Keng and Viola, Arch. Biochem. Biophys. 335:73-81 (1996)). Two additional kinases in E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA (Skarstedt and

Silverstein, J Biol. Chem. 251 :6775-6783 (1976)), phosphorylates propionate in addition to acetate (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase, encoded by proB (Smith et al., J Bacteriol. 157:545-551 (1984)), phosphorylates the gamma carbonic acid group of glutamate.

Acetylglutamate kinase phosphorylates acetylated glutamate during arginine biosynthesis and is a good candidate for phosphorylating 6-acetamidohexanoate (Figure 13, Step E). This enzyme is not known to accept alternate substrates; however, several residues of the E. coli enzyme involved in substrate binding and phosphorylation have been elucidated by site- directed mutagenesis (Marco-Martin et al., J Mol. Biol. 334:459-476 (2003); and Ramon- Maiques et al., Structure. 10:329-342 (2002)). The enzyme is encoded by argB in Bacillus subtilis and E. coli (Parsot et al., Gene 68:275-283 (1988)), and ARG5,6 in S. cerevisiae

(Pauwels et al., Eur. J Biochem. 270: 1014-1024 (2003)). The ARG5,6 gene of S. cerevisiae encodes a polyprotein precursor that is matured in the mitochondrial matrix to become acetylglutamate kinase and acetylglutamylphosphate reductase, an enzyme candidate for the reduction of 6-AAHOP (Figure 13, Step F).

Gene name GI# GenBank Accession # Organism

argB 145698337 NP_418394.3 Escherichia coli argB 16078186 NP_389003.1 Bacillus subtilis

ARG5,6 6320913 NP O 10992.1 Saccharomyces cerevisiae

2.8.3.a Coenzyme-A transferase. Coenzyme-A (CoA) transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. In Step M of Figure 13, 3- aminocaproyl-CoA is formed by the transfer of a CoA group from acetyl-CoA, succinyl- CoA, or another CoA donor. A similar transformation is catalyzed by 6-acetamidohexanoate CoA-transferase, shown in Step I of Figure 13. Exemplary CoA transferase candidates are catalyzed by the gene products of catl, cat2, and catS of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al, Proc. Natl. Acad. Sci U. S. A 105:2128-2133 (2008); and Sohling and Gottschalk, J Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J Biol. Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al, J Biol. Chem. 279:45337-45346 (2004)).

A CoA transferase that can utilize acetyl-CoA as the CoA donor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et ah, Acta Crystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); and

Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek and Frerman, Arch. Biochem. Biophys. 171 : 14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies and Schink, Appl Environ.

Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli and Overath, Eur. J Biochem. 29:553-562 (1972)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol 56: 1576-1583 (1990); and Wiesenborn et al, Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71 :58-68 (2007)).

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium

Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J Biochem. 1 18:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al, Eur. J. Biochem. 226:41-51 (1994)).

Yet another CoA transferase is the two-unit succinyl-CoA:3:oxoacid-CoA transferase encoded by peal and pcaJ in Pseudomonas putida (Kaschabek et al., J Bacteriol. 184:207- 215 (2002)). Similar enzymes based on homology exist in Acinetobacter sp. ADP1

(Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid- CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al, Protein Expr. Purif. 53:396-403 (2007)). Gene name GI# GenBank Accession # Organism peal 24985644 AAN69545.1 Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida peal 50084858 YP 046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1 peal 21224997 NP_630776.1 Streptomyces coelicolor pcaJ 21224996 NP_630775.1 Streptomyces coelicolor

HP AG 1 0676 108563101 YP_627417 Helicobacter pylori

HPAG1 0677 108563102 YP_627418 Helicobacter pylori

ScoA 16080950 NP_391778 Bacillus subtilis

ScoB 16080949 NP_391777 Bacillus subtilis

3.5.1.a Hydrolase (acting on linear amides). Deacetylation of linear acetamides is catalyzed by an amidohydrolase in the 3.5.1 family of enzymes. Such an enzyme is required for the deacetylation of 6-acetamidohexanamine to HMDA (Figure 13, Step H). An enzyme catalyzing a similar transformation is 4-acetamidobutyrate deacetylase (EC 3.5.1.63), which naturally deacetylates 4-acetamidobutyrate. The enzyme, studied for its role in putrescine degradation in Candida boidinii (Gillyon et al., Journal of General Microbiology 133:2477- 2485 (1987)), has been shown to deacetylate a variety of substrates including 6- acetamidohexanoate (Haywood and Large, Journal of General Microbiology 132:7-14 (1986)). Although 6-Acetamidohexanoate is similar in structure to the desired substrate, deacetylation of this compound (Figure 13, step D, reverse reaction) may hinder efficient production of HMDA. Protein engineering or directed evolution may be required to improve specificity for 6-acetamidohexanamine. The gene associated with this activity has not been identified to date. Acetylpolyamine amidohydrolase (EC 3.5.1.62), is another candidate enzyme that forms the diamines putrescine and cadaverine from their acetylated precursors. The acetylpolyamine deacetylase (AphA) from Mycoplana ramosa has been cloned in E. coli and characterized (Sakurada et al., J Bacteriol. 178:5781-5786 (1996)) and a crystal structure is available (Fujishiro et al., Biochem. Biophys. Res. Commun. 157: 1169-1174 (1988)). This enzyme has also been studied in Micrococcus luteus, but the associated gene has not been identified to date (Suzuki et al., Biochim. Biophys. Acta 882: 140-142 (1986)). A protein the histone deacetylase superfamily with high sequence similarity to AphA was identified in the M. luteus genome (evalue = le-18, 37% identity). The N-acetyl-L-ornithine deacetylase from E. coli is another candidate amidohydrolase (EC 3.5.1.16). The E. coli enzyme, encoded by the argE gene (McGregor et al., J Am. Chem. Soc. 127: 14100-14107 (2005); and Meinnel et al, J Bacteriol. 174:2323-2331 (1992)), removes N-acetyl groups from a variety of substrates including ornithine, lysine, glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry 39: 1285-1293 (2000)).

4.1.1.a Carboxy-lyase. Steps D and F in Figure 12 are catalyzed by 2-ketoacid decarboxylase enzymes that generate 6-OHE and adipate semialdehyde from OHED (Step F) and 2-OHD (Step D). In addition, alpha-ketoglutarate is decarboxylated to form pathway precursor succinic semialdehyde by alpha-ketoglutarate decarboxylase, a keto-acid decarboxylase. The decarboxylation of keto-acids is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to

acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2- phenylpyruvate (22). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem.

268: 1698-1704 (2001); Li, H. and F. Jordan, Biochemistry. 38: 10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64: 1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al, Eur. J. Biochem. 268: 1698-1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443- 451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263 (2002)).

Gene name GI# GenBank Accession # Organism

pdc 118391 P06672.1 Zymomonas mobilus pdcl 30923172 P06169 Saccharomyces cerevisiae pdc 20385191 Q8L388 Acetobacter pasteurians pdcl 52788279 Q12629 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al, Biochemistry 42: 1820-1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from

Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US. A 102: 10670-10675 (2005)) has been cloned and functionally expressed. Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO:) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene can be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

A fourth candidate enzyme for catalyzing this step is branched chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988); and Smit et al, Appl Environ Microbiol. 71 :303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2- oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol. 71 :303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318: 1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering.

Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis;

however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74).

Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the El subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J Biol. Chem. 267: 16601-16606 (1992); Wynn et al., J Biol. Chem. 267: 1881-1887 (1992); and Wynn et al, J Biol. Chem. 267: 12400-12403 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al, J Biol. Chem. 267: 12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.

The decarboxylation of 2-AHD to 6-aminocaproate (Figure 12, Step I) is catalyzed by an amino acid decarboxylase such as aspartate decarboxylase. Aspartate decarboxylase participates in pantothenate biosynthesis and is encoded by gene panD in Escherichia coli (Dusch et al, Appl. Environ. Microbiol 65: 1530-1539 (1999); Merke and Nichols, FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al, Biochem. J 323 (Pt 3):661-669 (1997); and Schmitzberger et al, EMBO J 22:6193-6204 (2003)). Similar enzymes from

Mycobacterium tuberculosis (Chopra et al., Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum (Dusch et al., Appl. Environ. Microbiol 65: 1530-1539 (1999)) have been expressed and characterized in E. coli.

4.1 ,2.a Aldehyde-lyase. HOHD aldolase, also known as HHED aldolase, catalyzes the conversion of 4-hydroxy-2-oxo-heptane-l,7-dioate (HOHD) into pyruvate and succinic semialdehyde (Figure 12, Step A). The enzyme is a divalent metal ion dependent class II aldolase, catalyzing the final step of 4-hydroxyphenylacetic acid degradation in E. coli C, E. coli W, and other organisms. In the native context, the enzyme functions in the degradative direction. The reverse (condensation) reaction is thermodynamically unfavorable; however the equilibrium can be shifted through coupling HOHD aldolase with downstream pathway enzymes that work efficiently on reaction products. Such strategies have been effective for shifting the equilibrium of other aldolases in the condensation direction (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995); and Pollard et ah, Appl Environ. Microbiol

64:4093-4094 (1998)). The E. coli C enzyme, encoded by hpcH, has been extensively studied and has recently been crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007); and

Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme is encoded by hpal (Prieto et al., J Bacteriol. 178: 111-120 (1996)).

4.2.1.a Hydro-lyase. The enzyme OHED hydratase participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-l,7-dioate (OHED) to 2-oxo-4- hydroxy-hepta-l,7-dioate (HODH) using magnesium as a cofactor (Burks et al., J Am. Chem. Soc. 120 (1998)) (Figure 12, Step B). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007); and Roper et al, Gene 156:47-51 (1995)) and E. coli ^(Prieto et al., J Bacteriol. 178: 111-120 (1996)). Sequence comparison reveals homologs in a range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (91% identity, evalue = 2e-138) and Salmonella enterica (91% identity, evalue = 4e-138), among others.

Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (Figure 12, Step M) is catalyzed by an enzyme with enoyl-CoA hydratase activity. 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase, dehydrates 3-hydroxyisobutyryl-CoA to form crotonoyl-CoA (Figure 14, step 2). Crotonase enzymes are required for n-butanol formation in some organisms, particularly Clostridial species, and also comprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genes encoding crotonase enzymes can be found in C. acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and

Boynton et al., J Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer and Gottschalk, FEBS Lett. 21 :351-354 (1972)), and Metallosphaera sedula (Berg et al., Science. 318: 1782- 1786 (2007)) though the sequence of the latter gene is not known.

Enoyl-CoA hydratases (EC 4.2.1.17) also catalyze the dehydration of 3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J Bacteriol. 188:8551-8559(2003); Conrad et al., J Bacteriol. 118: 103-111 (1974); and Roberts et al, Arch. Microbiol 117:99-108 (1978)). The enoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes the conversion of 3- hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al, Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P. fluorescens (Olivera et al, Proc. Natl. Acad. Sci U. S. A 95:6419-6424 (1998)). The gene product oipimF in Rhodopseudomonas palustris is predicted to encode an enoyl- CoA hydratase that participates in pimeloyl-CoA degradation (Harrison and Harwood, Microbiology 151 :727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park and Lee, J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al, J Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., J Biochem. 270:3047- 3054(2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)).

Gene name GI# GenBank Accession # Organism

ech 26990073 NP_745498.1 Pseudomonas putida

paaA 26990002 NP_745427.1 Pseudomonas putida

paaB 26990001 NP_745426.1 Pseudomonas putida

phaA 106636093 ABF82233.1 Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonas fluorescens pimF 39650635 CAE29158 Rhodopseudomonas palustris maoC 16129348 NP_415905.1 Escherichia coli

paaF 16129354 NP_415911.1 Escherichia coli paaG 16129355 NP_415912.1 Escherichia coli

Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Nakahigashi and Inokuchi, Nucleic Acids Res. 18:4937 (1990); Yang, J Bacteriol. 173:7405-7406 (1991); and Yang et al., Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38- 44 (2007)). The fadl and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

6.2.1.a Acid-thiol ligase (also called CoA synthetase). Steps I and M of Figure 13 require acid-thiol ligase or CoA synthetase functionality to transform 6-ACA and 6- acetamidohexanoate into their corresponding CoA derivatives (the terms ligase, synthetase, and synthase are used herein interchangeably and refer to the same enzyme class). Enzymes catalyzing these exact transformations have not been characterized to date; however, several enzymes with broad substrate specificities have been described in the literature. ADP- forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). A second reversible ACD in

Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on cyclic compounds phenylacetate and indoleacetate (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004); and Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl- CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

Another candidate enzyme for this step is 6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)). Other candidates are found in Bacillus subtilis (Bower et al., J Bacteriol. 178:4122-4130 (1996)) and

Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690 (1992)).

Gene name GI# GenBank Accession # Organism

pauA 15596214 NP_249708.1 Pseudomonas mendocina bioW 50812281 NP_390902.2 Bacillus subtilis

bioW 115012 P22822.1 Lysinibacillus sphaericus Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem. J230:683-693 (1985)), either of the two characterized henylacetate-Co A ligases from /⁵, chrysogenum (Lamas-Maceiras et al., Biochem. J 395: 147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun. 360:453- 458 (2007)) and the phenylacetate-Co A ligase from Pseudomonas putida (Martinez -Blanco et al., J Biol. Chem. 265:7084-7090 (1990)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.

EXAMPLE XXII

Demonstration of Escherichia coli Tolerance to 6-Aminocaproate

E. coli was assayed for tolerance, metabolic activity and growth during exposure to various concentrations of 6-aminocaproate (6-ACA). Aerobically, cultures were able to grow media with up to 10% 6-ACA, while anaerobic cultures could grow in media with approximately 6% 6-ACA (Figure 15). Because the pathway for producing 6-ACA could require anaerobic conditions, all other further testing was performed under anaerobic conditions. To assay tolerance, cultures were grown anaerobically to mid-log (0.3 OD) and early stationary phase (0.6 OD), the cells were spun down and resuspended in medium containing various concentrations of 6-ACA. The cultures were grown in capped micro fuge tubes, grown overnight and the ODs of the cultures were assayed (Figure 16). Under these conditions, cultures were able to grow (double at least 1 time) in up to 10% 6-ACA. The additional tolerance could have been from the additional glucose from resuspending the cultures in fresh M9-glucose medium or from limited oxygen that was present in the capped micro fuge tube. To determine if the cells were metabolically active in the presence of 6-ACA, samples were taken and assayed for ethanol production (Figure 17). Ethanol production (and thus metabolic activity) closely tracked with OD suggesting that if cells are present, they are likely to be metabolically active. This is helpful to understand because it suggests that even though cells may be growth inhibited by the accumulation of a product, they can still continue to produce product.

At high concentrations (>65g/L) the osmolarity of 6-ACA is -0.5 M which may cause osmotic stress. To determine osmotic stress as the basis for 6-ACA growth inhibition, cultures were grown in various concentrations of 6-ACA with and without the

osmoprotectant glycine betaine. As seen in Figure 18, anaerobic growth in medium with up to 10-12% 6-ACA can be achieved if glycine betaine is present but only 4-6% without glycine betaine. Therefore much of the toxicity of 6-ACA is likely due to the osmotic stress.

However, it should be noted that 6-ACA is similar to the amino acid lysine and could have a greater toxic effect in the cell cytoplasm vs. outside the cell.

EXAMPLE XXIII

Demonstration of Enzyme Activity for Condensing Succinyl-CoA and Acetyl-CoA to form β-ketoadipyl-CoA

Several β-ketothiolase enzymes have been shown to break β-ketoadipyl-CoA into acetyl-CoA and succinyl-CoA. For example, the gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J Bacteriol, 184(1): 207-15 (2002)), phaD in Pseudomonas putida XJ (Olivera et al, Proc Natl Acad Sci USA, 95(11), 6419-24 (1998)), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., Arch Microbiol, 188(2), 117-25 (2007)), and paaJ from E. coli (Nogales et al., Microbiology, 153(Pt 2), 357-65 (2007)) catalyze the conversion of 3- oxoadipyl-CoA into succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate or styrene. To confirm that β-ketothiolase enzymes exhibit condensation activity, several thiolases (Table 10; SEQ ID NOS: , respectively) were cloned into a derivative of pZE13(Lutz et al., Nucleic Acids Res, 29(18), 3873-81 (2001)), which results in the clones having a carboxy-terminal 6xHis tag.

Table 10: Cloned Thiolases

The genes were expressed in E. coli and the proteins purified using Ni-NTA spin columns and quantified. To assay enzyme activity in vitro, a 5X CoA:DTNB (Ellman's reagent or 5, 5'-dithiobis-(2-nitrobenzoic acid)) mixture was prepared. The mixture consisted of 10 mM succinyl-CoA, 5 mM acetyl-CoA, 30 mM DTNB in 100 mM Tris buffer, pH 7.4. Five μΐ, of the CoA:DTNB mixture was added to 0.5 μΜ purified thiolase enzyme in 100 mM Tris buffer, pH 7.8 in a final volume of 50 μΐ_^. The reaction was incubated at 30°C for 30 minutes, then quenched with 2.5 μΐ_^ 10% formic acid and samples frozen at -20°C until ready for analysis by LC/MS. Because many thiolases can condense two acetyl-CoA molecules into acetoaceytl-CoA, production of acetoacetyl-Co A was examined. Figure 19 shows that 3 thiolases demonstrated thiolase activity which resulted in acetoacetyl-CoA formation. These were fadAx from Pseudomonas putida, thiA from Clostridium acetobutylicum and thiB also from Clostridium acetobutylicum. When enzyme assays were examined for condensation of succinyl-CoA and acetyl-CoA into β-ketoadipyl-CoA, several candidates demonstrated the desired activity; paaJ from Escherichia coli (Nogales et al., Microbiol. 153:357-365 (2007)), phaD from Pseudomonas putida (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), bkt from Burkholderia ambifaria AMMD,pcaF from Pseudomonas putida KT2440 (Harwood et al., J Bacteriol. 176:6479-6488 (1994)), and pcaF from Pseudomonas aeruginosa PAOl . There was excellent specificity between the thiolases. Those that generated significant amounts of β-ketoadipyl-CoA did not produce significant amounts of acetoacetyl-CoA and likewise those that made acetoacetyl-CoA did not make detectable amounts of β-ketoadipyl-CoA.

EXAMPLE XXIV

Pathways for Production of Hexamethylenediamine from Glutamate, Glutaryl-CoA or

Pyruvate and 4-Aminobutanal

This example describes exemplary pathways for production of hexamethylenediamine (HMD A) from glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2-amino-7- oxosubarate through homolysine, the seven-carbon analog of lysine. Homolysine is an attractive precursor to HMDA. Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Homolysine can be formed

biocatalytically from the central metabolic precursors glutamate, glutaryl-CoA or pyruvate and 4-aminobutanal. Subsequent decarboxylation of homolysine by an enzyme analogous to lysine decarboxylase yields HMDA. This example describes additional pathways that proceed from 2-amino-7-oxosubarate, or pyruvate and 4-aminobutanal through the intermediate 6-aminohexanal. 6-Aminohexanal can readily be converted to HMDA by an aminotransferase or an aminating oxidoreductase.

The maximum theoretical yield of HMDA is 0.71 moles per mole glucose utilized (0.46 g/g). The pathways disclosed in Figures 20-22 and 26 achieve a maximum HMDA yield of 0.67 mol/mol (0.43 g/g).

C₆Hi₂0₆ + 1.41 NH₄ 0.71 C₆Hi₈N₂ + 1.76 C0₂ + 2.47 H₂0

Novel pathways for producing hexamethylenediamine (HMDA) and related products are described herein. The candidate enzymes, and associated risks of implementation are discussed in Example XXVI below.

This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze HMDA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

HMDA can be produced from glutamate via glutaryl-CoA in eight enzymatic steps, shown in Figure 20. In this route, glutamate is acylated to glutamyl-CoA by a Co A transferase or ligase (Step A of Figure 20). Glutamyl-CoA and acetyl-CoA are joined by a beta-ketothiolase to form the C7 compound 3-oxo-6-aminopimeloyl-CoA (Step B of Figure 20). The 3-oxo group of this product is then reduced and dehydrated, resulting in 6-amino-7-carboxyhept-2-enoyl- CoA (Steps C and D of Figure 20). An enoyl-CoA reductase reduces the double bond, forming 6-aminopimeloyl-CoA (Step E of Figure 20). 6-Aminopimeloyl-CoA is then converted to 2-amino-7-oxoheptanoate by a CoA-dependent aldehyde dehydrogenase (Step F). Transamination of the aldehyde to an amine yields homolysine (Step G of Figure 20). Finally, HMDA is formed as the decarboxylation product of homolysine (Step H of Figure 20). The maximum theoretical HMDA yield for this pathway is 0.67 moles of HMDA per mole of glucose utilized. Yield calculations assume aerobic conditions and the utilization of a CoA transferase in Step A. HMD A can also be produced from glutaryl-CoA by several routes. Exemplary routes for HMDA production are shown in Figure 21. Glutaryl-CoA is a common metabolic

intermediate in organisms that metabolize aromatic compounds. In the disclosed pathways to HMDA, glutaryl-CoA is first condensed with acetyl-CoA by a beta-ketothiolase to form 3- oxopimeloyl-CoA (Step A of Figure 21). The Co A moiety of 3-oxopimeloyl-CoA is removed by a CoA hydrolase, transferase and ligase (Step B of Figure 21). Several alternate routes for converting 3-oxopimelate to HMDA are outlined in Figure 21 and described herein. The final step of all routes to HMDA entails decarboxylation of homolysine (Step S of Figure 21).

One route entails conversion of 3-oxopimelate to 3-oxo-l-carboxyheptanal. This conversion can be catalyzed by an ATP- and NAD(P)H dependent enzyme with 3-oxopimelate reductase activity (Step C of Figure 21), or alternately can proceed through activated intermediates 5- oxopimeloyl-CoA (Steps H, I of Figure 21) or 5-oxopimeloyl-phosphonate (Steps F, G of Figure 21). Once formed, 3-oxo-l-carboxyheptanal is transaminated at the 3 -position (Step AB of Figure 21) or 7-position (Step D of Figure 21). Subsequent transamination of 3-oxo-7- aminoheptanoate (Step E of Figure 21) or 3-amino-7-oxoheptanoate (Step Z of Figure 21) yields 3,7-diaminoheptanoate. An enzyme with 3,7-diaminoheptanoate 2,3-aminomutase activity then forms homolysine (Step R of Figure 21), which is decarboxylated to HMDA (Step S of Figure 21).

In an alternate route, 3-oxopimelate is transaminated to 3-aminopimelate (Step J of Figure 21). 3-Aminopimelate is then converted to 3-amino-7-oxoheptanoate directly (Step O of

Figure 21) or via a CoA (Steps K, L of Figure 21) or phosphonic acid (Steps M, N of Figure 21) intermediate. 3-Amino-7-oxoheptanoate is subsequently converted to 2-amino-7- oxoheptanoate by a 2,3-aminomutase (Step P of Figure 21). 2-Amino-7-oxoheptanoate is converted to homolysine by an aminotransferase or aminating oxidoreductase. Alternately, 3- amino-7-oxoheptanoate is first transaminated (Step Z of Figure 21) and then converted to homolysine by an aminomutase (Step R of Figure 21).

3-Aminopimelate can be converted to 2-aminopimelate by a 2,3-aminomutase enzyme (Step T of Figure 21). An HMDA pathway involving this intermediate requires reduction of the 7- carboxylic acid to an aldehyde. This reduction is catalyzed by a bifunctional reductase (Step W of Figure 21) or by two enzymes that proceed through a CoA (Steps V, Y of Figure 21) or phosphonic acid (Steps U, X of Figure 21) intermediate. The product, 2-amino-7- oxoheptanoate is converted to HMDA as described above. Two routes for producing HMDA from pyruvate and 4-aminobutanal are shown in Figure 22. The routes achieve a maximum yield of 0.67 moles of HMDA per mole glucose utilized (0.43 g/g) under anaerobic and aerobic conditions. 4-Aminobutanal is naturally derived from ornithine by decarboxylation to putrescine and subsequent transamination. 4-Aminobutanal can also originate from 4-aminobutanoate. In one pathway, 4-aminobutanal and pyruvate are joined by aldol condensation to form 2-oxo-4-hydroxy-7-aminoheptanoate (Step A of Figure 22). The condensation product is subsequently dehydrated (Step B of Figure 22) and reduced (Step C of Figure 22). Transamination of 2-oxo-7-aminoheptanoate yields homolysine (Step D of Figure 22). HMDA is the decarboxylation product of homolysine decarboxylase (Step E of Figure 22). Alternately, pathway intermediate 2-oxo-7-aminoheptanoate is decarboxylated to form 6-aminohexanal (Step F of Figure 22). 6-Aminohexanal is subsequently converted to HMDA by an aminotransferase or aminating oxidoreductase (Step G of Figure 22).

Several routes for producing HMDA from 2-amino-7-oxosubarate are shown in Figure 26. 2- Amino-7-oxosubarate is not known to be a naturally occurring metabolite. An exemplary route for synthesizing 2-amino-7-oxosubarate is shown in Figure 27. The pathway originates with glutamate-5-semialdehyde, a metabolite naturally formed during ornithine biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed with pyruvate by an aldolase (Figure 27, Step A). The product, 2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and the resulting alkene is reduced to form 2-amino-7-oxosubarate (Figure 27, Steps B/C). In one proposed pathway to HMDA from 2-amino-7-oxosubarate, the 2-oxo acid is first decarboxylated to form 2-amino-7-oxoheptanoate (Step A of Figure 26). This product is again decarboxylated, forming 6-aminohexanal (Step B of Figure 26). Finally, 6-aminohexanal is converted to HMDA by an aminotransferase or aminating oxidoreductase (Step C of Figure 26). Alternately, the intermediate 2-amino-7-oxoheptanoate is first converted to homolysine by an aminotransferase or aminating oxidoreductase (Step M of Figure 26). Homolysine is decarboxylated to HMDA as described previously (Step H of Figure 26).

In yet another route, the 2-amino acid group of 2-amino-7-oxosubarate is decarboxylated, yielding 2-oxo-7-aminoheptanoate (Step I of Figure 26). This product can then be further decarboxylated to 6-aminohexanal (Step G of Figure 26) or transaminated to homolysine (Step J of Figure 26). Homolysine or 6-aminohexanal is then converted to HMDA as described previously. In yet another route, the 2-oxo group of 2-amino-7-oxosubarate is converted to an amino group, forming 2,7-diaminosubarate (Step K of Figure 26). Two subsequent decarboxylations yield HMDA (Steps L, H of Figure 26).

Described herein is the generation of a microbial organism that has been engineered to produce HMDA from pyruvate and 4-aminobutanal, as shown in Figure 22 (Steps A-E). This example also teaches a method for engineering a strain that overproduces HMDA.

Escherichia coli is used as a target organism to engineer a HMDA-producing pathway as shown in Figure 22. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing HMDA. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic, microaerobic or aerobic conditions.

An E. coli strain is engineered to produce HMDA from 4-aminobutanal via the route outlined in Figure 22. For the first stage of pathway construction, genes encoding enzymes to transform 4-aminobutanal and pyruvate to homo lysine (Figure 3, Steps A-D) are assembled onto vectors. In particular, the genes hpcH {CAA&ll 59), hpcG (CAA57202), enr

(YP_430895) and lysN Q genes encoding 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, 2- oxo-4-hydroxy-7-aminoheptanoate dehydratase, 2-oxo-7-aminohept-3-enoate reductase and 2-oxo-7-aminoheptanoate aminotransferase, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany), under the control of the PAl/lacO promoter. The plasmid is transformed into E. coli strain MG1655 to express the proteins and enzymes required for HMDA synthesis from 4-aminobutanal. E. coli naturally encodes two lysine decarboxylase enzymes which convert homolysine to HMDA.

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of HMDA pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA and immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce HMDA through this pathway is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional HMDA synthesis pathway from 4- aminobutanal are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

After successful demonstration of enhanced HMDA production via the activities of the exogenous enzymes, the genes encoding these enzymes are inserted into the chromosome of a wild type E. coli host using methods known in the art. Such methods include, for example, sequential single crossover (Gay et al., J Bacteriol. 3: 153 (1983)). and Red/ET methods from GeneBridges (Zhang et al., European Patent Application No. 01117 (2001))) . Chromosomal insertion provides several advantages over a plasmid-based system, including greater stability and the ability to co-localize expression of pathway genes.

2004/0009466, and in U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of HMDA. One modeling method is the bilevel optimization approach,

OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of HMDA. Adaptive evolution also can be used to generate better producers of, for example, the 2-oxo-4-hydroxy- 7-aminoheptanoate intermediate or the HMDA product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056- 1058 (2004); Alper et al, Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the HMDA producer to further increase production. For large-scale production of HMD A, the above HMDA pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp- cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2S04. The growth rate is determined by measuring optical density using a

spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al, Biotechnol. Bioeng., 775-779 (2005).

Example XXV

Pathways for Production of 6-Aminocaproate from Glutamate, Glutaryl-

CoA,Homolysine, or 2-Amino-7-oxosubarate

Novel pathways for producing 6-aminocaproate (6-ACA) and related products are described herein. The candidate enzymes, and associated risks of implementation are discussed in Example XXVI below.

This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 6-ACA production. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

6-ACA can be produced from glutamate as a starting molecule. Glutamate is transformed to 6-aminopimeloyl-CoA as described previously (Figure 20, Steps A-E). Removal of the CoA moiety of 6-Aminopimeloyl-CoA by a CoA hydrolase, transferase or ligase yields 2- aminopimelate (Step I of Figure 20). Decarboxylation of this product yields 6-ACA (Step J of Figure 20).

6-ACA can also be produced from glutaryl-CoA as a starting molecule. In the disclosed pathway to 6-ACA, similar to the HMDA pathway described above, glutaryl-CoA is first condensed with acetyl-CoA by a beta-ketothiolase to form 3-oxopimeloyl-CoA (Step A of Figure 21). The Co A moiety of 3-oxopimeloyl-CoA is removed by a Co A hydrolase, transferase and ligase (Step B of Figure 21). Then 3-oxopimelate is transaminated to 3- aminopimelate (Step J of Figure 21). 3 -Aminopimelate can be converted to 2-aminopimelate by a 2,3-aminomutase enzyme (Step T of Figure 21). Aminopimelate can then be

decarboxylated to form 6-aminocaproic acid (Step AA of Figure 21).

Homolysine is also an attractive precursor to 6-aminocaproate (6-ACA) production. Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Under aerobic conditions, oxidation of homolysine by a lysine 2- monooxygenase yields 6-aminohexanamide, which is readily hydro lyzed to 6-ACA in dilute acid or basic solution (Figure 23).

6-ACA can also be produced from 2-amino-7-oxosubarate as a starting molecule (Figure 26). 2-Amino-7-oxosubarate is not known to be a naturally occurring metabolite. An exemplary route for synthesizing 2-amino-7-oxosubarate is shown in Figure 27. The pathway originates with glutamate-5-semialdehyde, a metabolite naturally formed during ornithine biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed with pyruvate by an aldolase (Figure 27, Step A). The product, 2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and the resulting alkene is reduced to form 2-amino-7-oxosubarate (Figure 27, Steps B/C). In one proposed route, 2-amino-7-oxosubarate is decarboxylated to form 2-amino-7-oxoheptanoate (Step A of Figure 26). The aldehyde of 2-amino-7-oxoheptanoate is oxidized by an oxidoreductase to form 2-aminopimelate (Step D of Figure 26). 6-ACA is the decarboxylation product of 2- aminopimelate (Step E of Figure 26). Alternately, the 2-amino-7-oxoheptanoate intermediate is decarboxylated to form 6-aminohexanal (Step B of Figure 26), which is transaminated to 6- ACA (Step F of Figure 26). In a third proposed route, the 2-amino acid group of 2-amino-7- oxosubarate is decarboxylated, yielding 2-oxo-7-aminoheptanoate (Step I of Figure 26). This product can then be further decarboxylated to 6-aminohexanal (Step G of Figure 26). Finally, 6-aminohexanal is transaminated to 6-ACA (Step F of Figure 26). Example XXVI

Enzyme Classification System for Production of Hexamethylenediamine and 6-

Aminocaproic acid

This example describes the enzyme classification system for the exemplary pathways described in Examples XXIV and XXV for production of hexamethylenediamine or 6- aminocaproate.

All transformations depicted in Figures 20-23 and 26 fall into the general categories of transformations shown in Table 11. Below is described a number of biochemically characterized genes in each category. Specifically listed are genes that can be applied to catalyze the appropriate transformations in Figures 20-23 and 26 when properly cloned and expressed.

Table 11 shows the enzyme types useful to convert common central metabolic intermediates into 6-aminocaproate and hexamethylenediamine. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity.

Table 11

LABEL FUNCTION

1.1. La Oxidoreductase (oxo to alcohol)

1.13.12.a Monooxygenase (0₂ incorporating)

1.2. La Oxidoreductase (aldehyde to acid)

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde)

1.2.1.d Oxidoreductase (phosphonate reductase)

1.2.1.e Acid reductase

1.3. La Oxidoreductase (alkene to alkane)

1.4. La Oxidoreductase (aminating)

2.3. Lb Acyltransferase (beta-ketothiolase)

2.6.1.a Aminotransferase

2.7.2.a Phosphotransferase (carboxy acceptor)

2.8.3.a Coenzyme-A transferase

3.1.2.a CoA hydrolase

4.1. La Carboxy-lyase

4.1.2.a Aldehyde-lyase

4.2.1.a Hydro-lyase

5.4.3.a Aminomutase

6.2.1.a Acid-thiol ligase 1.1.1. a Oxidoreductase (oxo to alcohol) - The reduction of 3-oxo-6-aminopimeloyl-CoA to 3-hydroxy-6-aminopimeloyl-CoA is catalyzed by a 3-oxoacyl-CoA dehydrogenase (Figure 20, Step C). Such enzymes convert 3-oxoacyl-CoA molecules into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta-oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Furthermore, the gene products encoded by phaC in Pseudomonas putida [/(Olivera et al, Proc. Natl. Acad. Sci U. S. A 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Arch et al., Microbiol 188:117-125 (2007)) catalyze the reverse reaction of step B in Figure 10, that is, the oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene. Note that the reactions catalyzed by such enzymes are reversible. In addition, given the proximity in E. coli οΐρααΗ to other genes in the phenylacetate degradation operon (Nogales et al., Microbiology

153:357-365 (2007)) and the fact that paaH mutants cannot grow on phenylacetate (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J Bacteriol. 171 :6800-6807 (1989)). Additional gene candidates include Hbdl (C -terminal domain) and Hbd2 (N- terminal domain) in Clostridium kluyveri (Hillmer et al., FEBS Lett. 21 :351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al, J Biol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61 :297-309 (2006)).) The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol 3:349-357 (1989)) and the gene has been expressed in E. coli.

Substrate specificity studies on the gene led to the conclusion that it could accept 3- oxopropionyl-CoA as an alternate substrate (Peoples et al., Mol. Microbiol 3:349-357 (1989)).

Metallosphaera sedula (Berg et al., Science. 318: 1782-1786 (2007).

1.13.12.a Monooxygenase (0₂ incorporating) - An 0₂-incorporating monooxygenase is required to convert homolysine to 6-aminohexanamide (Step A of Figure 23). The lysine 2- monooxygenase (EC 1.13.12.2) from Pseudomonas fluorescens reacts with homolysine as a substrate (Nakazawa et al, J Biol. Chem. 247:3439-3444 (1972)). The enzyme from /⁵. putida has been characterized biochemically and the gene has been identified (Karyakin et al., Prikladnaya Biokhimiya i Mikrobiologiya 27:825-832 (1991)). Genes encoding lysine 2- monooxygenase enzymes in P. fluorescens (eval = 0.0, 90% identity), Streptomyces coelicolor (eval = 0.0, 58% identity), Rhodococcus jostii (eval = 0.0, 56% identity) and others were identified by protein sequence homology to the P. putida enzyme.

1.2.1.a Oxidoreductase (aldehyde to acid) Two transformations in Figure 26 require conversion of an aldehyde to an acid: conversion of 2-amino-7-oxoheptanoate to 2- aminopimelate (Step D) and 6-aminohexanal to 6-aminocaproate (Step F). Such reactions are catalyzed by NAD(P)+-dependent oxidoreductases that convert aldehydes to acids in the EC class 1.2.1. A candidate enzyme is the NAD+-dependent aldehyde dehydrogenase (EC 1.2.1.3). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes (Klyosov et al., Biochemistry 35:4457-4467 (1996)). Active ALDH-2 has been efficiently expressed in E. coli using the GroEL proteins as chaperonins (Lee et al., Biochem. Biophys. Res. Commun. 298:216-224 (2002)). The rat mitochondrial aldehyde dehydrogenase also has a broad substrate range that includes the enoyl-aldehyde crotonaldehyde (Siew et al., Arch. Biochem. Biophys. 176:638-649 (1976)). The E. coli gene astD also encodes an NAD+-dependent aldehyde dehydrogenase that converts succinic semialdehyde to succinate (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005)).

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) - Reductive de-acylation of 3-oxopimeloyl- CoA (Figure 21, Step I), 5-aminopimeloyl-CoA (Figure 21, Step L) and 6-aminopimeloyl- CoA (Figure 21, Step Y) to their corresponding aldehydes is catalyzed by enzymes in the EC class 1.2.1. Exemplary acyl-CoA dehydrogenases that reduce an acyl-CoA to its corresponding aldehyde include the fatty acid acyl-CoA reductase enzymes of Acinetobacter calcoaceticus (Reiser et al., Journal of Bacteriology 179:2969-2975 (1997)) and

Acinetobacter sp. M-l (Ishige et al., Appl. Environ. Microbiol. 68: 1192-1195 (2002)), and a CoA- and NADP- dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling et al., J Bacteriol. 178:871-880 (1996); and Sohling et al., J Bacteriol 178:871-80 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J Bacteriol. 182:4704-4710 (2000)). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Koo et al, Biotechnol Lett. 27:505-510 (2005)).

An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl- CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg et al., Science. 318: 1782-1786 (2007); and Thauer et al., Science. 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J Bacteriol. 188:8551-8559 (2006); and Hugler et al, J Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., J Bacteriol. 188:8551- 8559 (2006); and Berg et al, Science. 318:1782-1786 (2007)). A gene encoding a malonyl- CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius . Yet another candidate for CoA- acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth et al., Appl Environ. Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al, Appl Environ. Microbiol 65:4973-4980 (1999)).

1.2.1. d Oxidoreductase (phosphonate reductase) - The reduction of a phosphonic acid to its corresponding aldehyde is catalyzed by an oxidoreductase in the EC class 1.2.1. Steps G, N and X in Figure 21 require such an enzyme for the reduction of 5-oxopimeloyl-phosphonate, 5-aminopimeloylphosphonate and 6-aminopimeloylphosphonate to their corresponding aldehydes. These reactions are not catalyzed by known enzymes. A similar reaction is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11): the NADPH- dependent reduction of 4-aspartyl phosphate to aspartate-4-semialdehyde. ASD participates in amino acid biosynthesis and recently has been studied as an antimicrobial target (Hadfield et al, Biochemistry 40: 14475-14483 (2001)). The E. coli ASD structure has been solved (Hadfield et al, J Mol. Biol. 289:991-1002 (1999)) and the enzyme has been shown to accept the alternate substrate beta-3-methylaspartyl phosphate (Shames, et al., J Biol. Chem.

259: 15331-15339 (1984)). The Haemophilus influenzae enzyme has been the subject of enzyme engineering studies to alter substrate binding affinities at the active site (Blanco et al., Crystallogr. 60: 1388-1395 (2004)). Other ASD candidates are found in Mycobacterium tuberculosis (Shafiani et al., JAppl Microbiol 98:832-838 (2005)), Methanococcus jannaschii Faehnle et al. J Mol. 353: 1055-1068 (2005)) and the infectious microorganisms Vibrio cholera and Heliobacter pylori (Moore et al., Protein Expr. Purif. 25:189-194 (2002)). Acetylglutamylphosphate reductase (EC 1.2.1.38) is a related enzyme that naturally reduces acetylglutamylphosphate to acetylglutamate-5-semialdehyde. Genes encoding this enzyme are found in S. cerevisiae (Pauwels et al., Eur. J Biochem. 270: 1014-1024 (2003)), B. subtilis (O'Reilly et al, Microbiology 140 (Pt 5): 1023-1025 (1994)) and other organisms.

Other exemplary phosphonate reductase enzymes include glyceraldehyde 3 -phosphate dehydrogenase which converts glyceraldehyde-3 -phosphate into D-glycerate 1,3- bisphosphate (e.g., E. coli gapA (Branlant et al., Eur. J. Biochem. 150:61-66 (1985)).23)), N- acetyl-gamma-glutamyl-phosphate reductase which converts N-acetyl-L-glutamate-5- semialdehyde into N-acetyl-L-glutamyl-5 -phosphate (e.g., E. coli argC (Parsot et al., Gene. 68:275-283 (1988)), and glutamate-5 -semialdehyde dehydrogenase which converts L- glutamate-5 -semialdehyde into L-glutamyl-5-phospate (e.g., E. coliproA (Smith et al., J. Bacteriol. 157:545-551 (1984))). Genes encoding glutamate-5 -semialdehyde dehydrogenase enzymes from Salmonella typhimurium (Mahan et al., J Bacteriol. 156: 1249-1262 (1983)) and Campylobacter jejuni (Louie et al., Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E. coli.

Gene name GI# GenBank Accession # Organism

gapA 71159358 P0A9B2.2 Escherichia coli

argC 16131796 NP_418393.1 Escherichia coli proA 16128229 NP_414778.1 Escherichia coli proA 16763704 NP_459319.1 Salmonella typhimurium proA 9087222 P53000.2 Campylobacter j ejuni

1.2.1. e Acid reductase - Several transformations in Figure 21 require the conversion of an acid to an aldehyde (Figure 21, Steps C, O, W). Such a transformation is thermodynamically unfavorable and typically requires energy-rich cofactors and multiple enzymatic steps. For example, in butanol biosynthesis conversion of butyrate to butyraldehyde is catalyzed by activation of butyrate to its corresponding acyl-CoA by a Co A transferase or ligase, followed by reduction to butyraldehyde by a CoA-dependent aldehyde dehydrogenase. Alternately, an acid can be activated to an acyl-phosphate and subsequently reduced by a phosphate reductase. Direct conversion of the acid to aldehyde by a single enzyme is catalyzed by an enzyme in the 1.2.1 family. Exemplary enzymes that catalyze these transformations include carboxylic acid reductase, alpha-aminoadipate reductase and retinoic acid reductase.

Carboxylic acid reductase, found in Nocardia iowensis, catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes

(Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). This enzyme, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific

phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al.

"Biocatalytic Reduction of Carboxylic Acids: Mechanism and Applications" Chapter 15 in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, CRC Press LLC, Boca Raton, FL. (2006)).

Gene name GI# GenBank Accession # Organism

car 40796035 AAR91681.1 Nocardia iowensis (sp. NRRL 5646) npt 114848891 ABI83656.1 Nocardia iowensis (sp. NRRL 5646) An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21 : 1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J Biol. Chem 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified by sequence comparison homology searching. Directed evolution or other enzyme engineering methods may be required to enhance reactivity with the substrates in Figure 21.

1.3.1. a Oxidoreductase (alkene to alkane) - Three transformations fall into the category of oxidoreductases that reduce an alkene to an alkane (EC 1.3.1.-). The conversion of 6-amino- 7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA (Figure 20, Step E), 2-oxo-7- aminohept-3-onoate to 2-oxo-7-aminoheptanoate (Figure 22, Step C) and 2-amino-5-ene-7- oxosubarate to 2-amino-7-oxosubarate (Figure 27, Step C) are catalyzed by a 2-enoate reductase. 2-Enoate reductase enzymes are known to catalyze the NAD(P)H-dependent reduction of a wide variety of a, β-unsaturated carboxylic acids and aldehydes (Rohdich, et al., J Biol. Chem. 276:5779-5787 (2001)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been characterized (Seedorf et al, Proc. Natl. Acad. Sci U. S. A 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel et al., Arch. Microbiol 135:51- 57 (1983)). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich, et al., J Biol. Chem. 276:5779-5787 (2001)). The Moorella thermoacetica (formerly C. thermoaceticum) enr gene has also been expressed in a catalytically active form in E. coli (Ohdich, et al., J Biol. Chem. 276:5779- 5787 (2001)).

Another candidate 2-enoate reductase is maleylacetate reductase (MAR), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. MAR enzymes naturally participate in aromatic degradation pathways (Camara et al., J Bacteriol. (2009); Huang et al, Appl Environ. Microbiol 72:7238-7245 (2006)); Kaschabek et al, J Bacteriol. 177:320-325 (1995) and Kaschabek et al, J Bacteriol. 175:6075-6081 (1993)). The enzyme activity was identified and characterized in Pseudomonas sp. strain B 13

(Kaschabek et al., J Bacteriol 177:320-325 (1995); and Kaschabek et al., J Bacteriol 175:6075-6081 (1993)), and the coding gene was cloned and sequenced (Kasberg et al., J Bacteriol. 179:3801-3803 (1997)). Additional MAR gene candidates include clcE gene from Pseudomonas sp. strain B13 (Kasberg et al., J Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., J Bacteriol 180:3503-3508 (1998)), the macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha (Seibert et al, J Bacteriol. 175:6745- 6754 (1993)) and NCgllll2 in Corynebacterium glutamicum (Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)). A MAR in Pseudomonas reinekei MTl, encoded by ccaD, was recently identified and the nucleotide sequence is available under the DBJ/EMBL GenBank accession number EF159980 (Camara et al., J Bacteriol. (2009)).

Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the reduction of 6-amino- 7-carboxyhept-2-enoyl-CoA to 6-aminopimeloyl-CoA (Figure 20, Step E). One exemplary enoyl-CoA reductase is the gene product oi bcd from C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311 (2008)); and Boynton et al., JBacteriol. 178:3015-3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis

(Hoffmeister,et al, J Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister,et al., J Biol. Chem. 280:4329-4338 (2005)). This approach is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597 from the prokaryote Treponema denticola, represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci et al., Febs Letters 581 : 1561-1566 (2007)).

Gene name GI# GenBank Accession # Organism

bed 15895968 NP_349317.1 Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridium acetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum

TER 62287512 Q5EU90.1 Euglena gracilis

TDE0597 42526113 NP_971211.1 Treponema denticola Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl- CoA. Adjacent genes in the pirn operon, pimC and pimD, bear sequence homology to C. acetobutylicum bed and are predicted to encode a flavin-containing pimeloyl-CoA

dehydrogenase (Harrison et al., Microbiology 151 :727-736 (2005)). The genome of nitrogen- fixing soybean symbiont Bradyrhizobium japonicum also contains a pirn operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison et al., Microbiology 151 :727-736 (2005)).

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascarius suum and is capable of reducing a variety of linear and branched chain substrates including 2- methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993))). Two isoforms of the enzyme, encoded by genes acadl and acad, have been characterized.

1.4.1. a Oxidoreductase (aminating) - Several reactions in Figures 20-23 require the conversion of ketones or aldehydes to amine groups. Such a transformation can be accomplished by aminating oxidoreductases in the EC class 1.4.1. Enzymes in this EC class catalyze the oxidative deamination of amino groups with NAD+ or NADP+ as acceptor, and the reactions are typically reversible.

In Step D of Figure 22 the 2-oxoacid 2-oxo-7-aminoheptanoate is converted to homo lysine, a molecule resembling an amino acid (Figure 22, Step D; Figure 26, Step J). The conversion of 2-amino-7-oxosubarate to 2,7-diaminosubarate (Step K of Figure 26) is a similar

transformation. Exemplary enzymes for catalyzing these reactions include glutamate dehydrogenase (EC 1.4.1.2), leucine dehydrogenase (EC 1.4.1.9), and aspartate

dehydrogenase (EC 1.4.1.21). The gdhA gene product from Escherichia coli (Korber, .et al., J Mol. Biol. 234: 1270-1273. (1993)), gdh from Thermotoga maritime (Kort et al.,

Extremophiles 1 :52-60. 1997); Lebbink et al, J Mol. Biol. 280:287-296 (1998) and Lebbink et al., J Mol. Biol. 289:357-369 (1999))), and gdhAl from Halobacterium salinarum

(Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversible conversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The Idh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)); and Stoyan et al, J Biotechnol 54:77-80 (1997)). The nadX gene from Thermotoga maritima encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al, J Biol. Chem. 278:8804-8808 (2003)).

Two reactions entail conversion of 3-oxoacids to 3-amino acids: 3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (Figure 21, Step E), 3-oxopimelate to 3-aminopimelate (Figure 21, Step J) and 3-oxo-l-carboxyheptanal to 3-amino-7-oxoheptanoate (Figure 21, Step AB). An enzyme that reacts with 3-oxoacids is 3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11), an enzyme found in organisms that ferment lysine. The gene encoding this enzyme, kdd, was recently identified in Fusobacterium nucleatum (Kreimeyer et ah, J Biol. Chem. 282:7191- 7197 (2007)). The enzyme has been purified and characterized in other organisms (Baker et al., Chem. 247:7724-7734 (1972)); and Baker et al, Biochemistr. 13:292-299 (1974)) but the genes associated with these enzymes are not known. Candidates in Myxococcus xanthus, Porphyromonas gingivalis W83 and other sequenced organisms can be inferred by sequence homology.

The conversions of 2-amino-7-oxoheptanoate to homo lysine (Figure 20, Step G; Figure 21, Step Q; Figure 26, Step M), 3-oxo-l-carboxyheptanal to 3-oxo-7-aminoheptanoate (Figure 21, Step D) 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate (Figure 21, Step Z) and 6- aminohexanal to HMDA (Figure 26, Step C; Figure 22, Step G) are catalyzed by aminating oxidoreductases that transform aldehydes to their corresponding primary amines. An enzyme that catalyzes a similar reaction is lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH genes. This enzyme catalyzes the reversible oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde (Misono et al., J Bacteriol.

150:398-401 (1982)). Exemplary enzyme candidates are found in Geobacillus

stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)),

Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); and Misono et A., J Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al, BMP Rep. 41 :790-795 (2008)).

2.3. l.b Acyltransferase (beta-ketothiolase) - In Step A of Figure 21, Glutaryl-CoA and acetyl-CoA are condensed to form 3-oxopimeloyl-CoA by oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a beta-ketothiolase (EC 2.3.1.16). An enzyme catalyzing this transformation is found in Ralstonia eutropha (formerly known as Alcaligenes eutrophus), encoded by genes bktB and bktC (Haywood et al, FEMS Microbiology Letters 52:91-96 (1988); and Slater et al, J Bacteriol. 180: 1979-1987 (1998)). The sequence of the BktB protein is known;

however, the sequence of the BktC protein has not been reported. The pirn operon of

Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA degradation

(Harrison et al., Microbiology 151 :727-736 (2005)). A beta-ketothiolase enzyme candidate in S. aciditrophicus was identified by sequence homology to bktB (43% identity, evalue = le- 93).

Beta-ketothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl-CoA and propionyl-CoA may also be able to catalyze the formation of 3-oxopimeloyl-CoA.

Zoogloea ramigera possesses two ketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a β -oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., U.S. Patent No. 5,958,745 (1999)). The sequences of these genes or their translated proteins have not been reported, but several candidates in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from ?. eutropha. These include:

Gene name GI# GenBank Accession # Organism

phaA 113867452 YP_725941.1 Ralstonia eutropha

hl6_A1713 113867716 YP_726205.1 Ralstonia eutropha

pcaF 116694155 YP_728366.1 Ralstonia eutropha

M6 B1369 116695312 YP_840888.1 Ralstonia eutropha

hl6_A0170 113866201 YP 724690.1 Ralstonia eutropha

hl6_A0462 113866491 YP_724980.1 Ralstonia eutropha

hl6_A1528 113867539 YP_726028.1 Ralstonia eutropha

hl6_B0381 116694334 YP_728545.1 Ralstonia eutropha

hl6_B0662 116694613 YP_728824.1 Ralstonia eutropha

M6 B0759 116694710 YP_728921.1 Ralstonia eutropha

M6 B0668 116694619 YP_728830.1 Ralstonia eutropha hl6_A1720 1 13867723 YP_726212.1 Ralstonia eutropha hl6_A1887 1 13867867 YP_726356.1 Ralstonia eutropha

phbA 135759 P07097.4 Zoogloea ramigera

bktB 194289475 YP 002005382.1 Cupriavidus taiwanensis

Rmet_1362 94310304 YP_583514.1 Ralstonia metallidurans

Bphy_0975 186475740 YP OO 1857210.1 Burkholderia phymatum

Additional candidates include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products οΐαίοΒ from E. coli (Martin et al., Nat. Biotechnol 21 :796-802 (2003)), thlA and MB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol

73:7814-7818 (2007)); and Winzer et al, J Mol. Microbiol Biotechnol 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser,et al, J Biol. Chem. 269:31383-31389 (1994)).

Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta- ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J Bacteriol 176:6479- 6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B 13 (Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida £7 (Olivera et al., Proc. Natl. Acad. Sci U. S. A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di Arch et al., Microbiol 188: 1 17-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PAOl, bkt from

Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida. These enzymes can also be employed for the synthesis of 3-oxopimeloyl-CoA, a compound structurally similar to 3-oxoadipyl-CoA. Gene name GI# GenBank Accession # Organism

paaJ 16129358 NP_415915.1 Escherichia coli

pcaF 506695 AAA85138.1 Pseudomonas putida

pcaF 141777 AAC37148.1 Acinetobacter calcoaceticus paaE 106636097 ABF82237.1 Pseudomonas fluorescens bkt 115360515 YP_777652.1 Burkholderia ambifaria AMMD bkt 9949744 AAG06977.1 Pseudomonas aeruginosa P AO 1 pcaF 9946065 AAG03617.1 Pseudomonas aeruginosa P AO 1

A beta-ketothiolase is also required to condense glutamyl-CoA and acetyl-CoA (Figure 20, Step B). This transformation is not known to occur naturally. The beta-ketothiolase candidates described above are also exemplary candidates for catalyzing this transformation. 2.6.1. a Aminotransferase - Several reactions in Figures 20-26 are catalyzed by

aminotransferases in the EC class 2.6.1. Such enzymes reversibly transfer amino groups from aminated donors to acceptors such as pyruvate and alpha-ketoglutarate.

Aminotransferases selective for aldehydes are required for transaminating 2-amino-7- oxoheptanoate (Figure 20, Step G; Figure 21, Step Q; Figure 26, Step M), 3-oxo-l- carboxyheptanal (Figure 21, Step D) 3-amino-7-oxoheptanoate (Figure 21, Step Z) and 6- aminohexanal (Figure 26, Step C; Figure 22, Step G). An exemplary enzyme for converting aldehydes to primary amines is lysine-6-aminotransferase (EC 2.6.1.36). This enzyme function, converting lysine to alpha-aminoadipate semialdehyde, has been demonstrated in yeast and bacteria. Candidates from Candida utilis (Hammer et al., J Basic Microbiol 32:21- 27 (1992)), Flavobacterium lutescens (Fujii et al., J Biochem. 128:391-397 (2000)) and Streptomyces clavuligenus (Romero et al., Microbiol Biotechnol 18:241-246 (1997)) have been characterized. A recombinant lysine-6-aminotransferase from S. clavuligenus was functionally expressed in E. coli (Tobin et al., J Bacteriol. 173:6223-6229 (1991)). The F. lutescens enzyme is specific to alpha-ketoglutarate as the amino acceptor (Soda et al., Biochemistry 7:4110-4119 (1968)). Other enzymes which convert aldehydes to terminal amines include the dat gene product in Acinetobacter baumanii encoding 2,4- diaminobutanoate:2-ketoglutarate 4-transaminase (Ikai et ah, J Bacteriol. 179:5118-5125 (1997)). In addition to its natural substrate, 2,4-diaminobutyrate, DAT transaminates the terminal amines of lysine, 4-aminobutyrate and ornithine.

Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. The E. coli putrescine aminotransferase is encoded by the ygjG gene and the purified enzyme was also able to transaminate cadaverine and spermidine (Samsonova et al., Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim et al., J Biol. Chem. 239:783-786 (1964); and Samsonova et al., Microbiol 3:2 (2003)). The spuC gene of Pseudomonas aeruginosa encodes a putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate (Lu et al., J Bacteriol. 184:3765-3773 (2002)).

The conversion of an aldehyde to a terminal amine can also be catalyzed by gamma- aminobutyrate transaminase (GABA transaminase). This enzyme naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Liu et al., Biochemistry 43: 10896-10905 (2004); and Schulz et al., Appl Environ Microbiol 56: 1-6 (1990)). The two GABA transaminases in E. coli are encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al, J Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with a range of alternate substrates including 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985)); and Scott et al., J Biol. Chem. 234:932-936 (1959)). Gene name GI# GenBank Accession # Organism

gabT 16130576 NP_417148.1 Escherichia coli

puuE 16129263 NP_415818.1 Escherichia coli

abat 37202121 NP_766549.2 Mus musculus

gabT 70733692 YP_257332.1 Pseudomonas fluorescens abat 47523600 NP_999428.1 Sus scrofa

Enzymes that transaminate 3-oxoacids are required to convert 3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (Figure 21, Step E), 3-oxopimelate to 3-aminopimelate (Figure 21, Step J) and 3-oxo-l-carboxyheptanal to 3-amino-7-oxoheptanoate (Figure 21, Step AB). Enzymes catalyzing these exact transformations have not been identified to date. Beta- alanine/alpha-ketoglutarate aminotransferase (WO08027742) reacts with beta-alanine to form malonic semialdehyde, a 3-oxoacid. The gene product ofSkPYD4 in Saccharomyces kluyveri was shown to preferentially use beta-alanine as the amino group donor (Andersen et al., Gene. 124: 105-109 (1993)). SkUGAl encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al, Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both beta-alanine and GABA

transamination (Andersen et al., Gene. 124: 105-109 (1993)). 3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2- methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al, Methods Enzymol. 324:376-389 (2000)).

Several aminotransferases transaminate the amino groups of 2-oxo acids to form amino acids. Such an enzyme is required for the transamination of 2-oxo-7-aminoheptanoate to

homolysine (Figure 22, Step D; Figure 26, Step M) and 2-amino-7-oxosubarate to 2,7- diaminosubarate (Figure 26, Step K). A promising enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39), an enzyme that participates in lysine biosynthesis and degradation in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadipate, using alpha-ketoglutarate as the amino acceptor. Gene candidates are found in Homo sapiens (Okuno et al., Enzyme Protein 47: 136-148 (1993)) and Thermus thermophilus (Miyazaki et al., Microbiology 150:2327-2334 (2004)). The Thermus thermophilus enzyme, encoded by lysN, is active with several alternate substrates including oxaloacetate, 2-oxoisocaproate, 2- oxoisovalerate, and 2-oxo-3-methylvalerate.

Another candidate is aspartate aminotransferase, an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate aminotransferase activity is catalyzed by, for example, the gene products oiaspC from Escherichia coli (Yagi et al, FEBS Lett. 100:81-84 (1979); and Yagi et al, Methods

Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (de la et al, Plant J46:414-425 (2006); Kwok et al, J Exp. Bot. 55:595-604 (2004) and Wilkie et al, Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that work on other amino-acid substrates may also be able to catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen et al., J Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the transamination of a-ketobutyrate to generate a-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen et al., J Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam, J. et al., Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et al, FEBS. Lett. 390: 179-182 (1996)).

Gene name GI# GenBank Accession # Organism aspC 16128895 NP_415448.1 Escherichia coli

AAT2 1703040 P23542.3 Saccharomyces cerevisiae

ASP5 20532373 P46248.2 Arabidopsis thaliana

Got2 112987 P00507 Rattus norvegicus

avtA 49176374 YP 026231.1 Escherichia coli

serC 16128874 NP_415427.1 Escherichia coli

2.7.2.a Phosphotransferase (carboxy acceptor) - Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. Steps F, M and U in Figure 21 require a phosphotransferase to activate the carboxyl groups of 3-oxopimelate (Step F), 3-aminopimelate (Step M) and 2-aminopimelate (Step U) to their corresponding phosphonic acids. Butyrate kinase (EC 2.7.2.7) carries out the reversible conversion of butyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56: 1576-1583 (1990)). This enzyme is encoded by either of the two buk gene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found in C. butyricum and C. tetanomorphum (Twarog et al., J Bacteriol. 86: 112-117 (1963)). Related enzyme isobutyrate kinase from Thermotoga maritima has also been expressed in E. coli and crystallized (Diao et al., E. Biol. Crystallogr. 59: 1100-1102 (2003); and Diao et al, J Bacteriol. 191 :2521-2529 (2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and participates in the synthesis of several amino acids. The aspartokinase III enzyme in E. coli, encoded by lysC, has a broad substrate range and the catalytic residues involved in substrate specificity have been elucidated (Keng et al., Arch. Biochem. Biophys. 335:73-81 (1996)). Two additional kinases in E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA (Skarstedt et al., J Biol. Chem. 251 :6775-6783 (1976)), phosphorylates propionate in addition to acetate (Hesslinger et al., Mol. Microbiol 27:477- 492 (1998)). The E. coli gamma-glutamyl kinase, encoded by proB (Smith et al., J

Bacteriol. 157:545-551 (1984)), phosphorylates the gamma carbonic acid group of glutamate.

Gene name GI# GenBank Accession # Organism

bukl 15896326 NP_349675 Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum buk2 6685256 Q9X278.1 Thermotoga maritima lysC 16131850 NP_418448.1 Escherichia coli

ackA 16130231 NP_416799.1 Escherichia coli

proB 16128228 NP_414777.1 Escherichia coli

2.8.3. a Coenzyme-A transferase - CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Several transformations in Figures 20 and 21 require a CoA transferase to activate carboxylic acids to their corresponding acyl-CoA derivatives (Figure 20, Steps A and I; Figure 21, Steps H, J, V). Candidate enzymes for catalyzing these transformations include the gene products ofcatl, cat2, and catS of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci U. S. A 105:2128-2133 (2008); and Sohling et al, JBacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al, J. Biol. Chem. 279:45337- 45346 (2004)).

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium

Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)), substrates similar in structure to 2,3-dehydroadipyl- CoA. The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl- CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckel et al., Eur. J Biochem. 118:315- 321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

Gene name GI# GenBank Accession # Organism

gctA 559392 CAA57199.1 Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcus fermentans A CoA transferase that can utilize acetyl-CoA as the CoA donor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch. Biochem. Biophys. 171 : 14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al, Appl Environ. Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli et al., Eur. J Biochem. 29:553-562 (1972)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol 56: 1576-1583 (1990); and Wiesenborn et al, Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem 71 :58-68 (2007)).

The de-acylation 3-oxopimeloyl-CoA to 3-oxopimelate (Figure 21, Step B) is catalyzed by a 3-oxoacid-CoA transferase (EC 2.8.3.6). Succcinyl-CoA:3-oxoacid-CoA transferase, also known as beta-ketoadipyl-CoA transferase, is encoded by peal and pcaJ in Pseudomonas putida (Kaschabek et al., J Bacteriol. 184:207-215 (2002)). Similar enzymes based on protein sequence homology exist in Acinetobacter sp. ADPl (Kowalchuk et al., Gene 146:23- 30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403 (2007)).

3.1.2.a CoA hydrolase - The hydrolysis of 6-aminopimeloyl-CoA to 6-aminopimelate (Figure 20, Step I) is carried out by an acyl CoA hydrolase enzyme in the 3.1.2 family. An enzyme catalyzing this transformation has not been demonstrated to date. Several eukaryotic acetyl- CoA hydrolases (EC 3.1.2.1) have broad substrate specificity and thus represent suitable candidate enzymes for hydrolyzing 6-aminopimelate. For example, the enzyme from Rattus norvegicus brain (Robinson et al., Res. Commun. 71 :959-965 (1976)) can react with butyryl- CoA, hexanoyl-CoA and malonyl-CoA. Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf also has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl- CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al, Plant. Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al., J Biol. Chem. 278: 17203-17209 (2003)) .

Gene name GI# GenBank Accession # Organism

acotl2 18543355 NP_570103.1 Rattus norvegicus

ACH1 6319456 NP_009538 Saccharomyces cerevisiae Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and

dodecanedioyl-CoA (Westin et al, J Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homo log, tesB, which can also hydro lyze a broad range of CoA thioesters (Naggert et al., J Biol. Chem. 266: 11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana et al., Biochem. Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolases include the gene products of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al, FEMS Microbiol Rev Ι^.Ιβ ^ (2005); and (Zhuang et al, FEBS Lett. 516: 161-163 (2002)), paal (Song et al, J Biol. Chem. 281 : 11028-11038 (2006)), and ybdB (Leduc et al, JBacteriol. 189:7112-7126 (2007)).

Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3- ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this reaction step but would require certain mutations to change their function.

4.1.1.a Carboxy-lyase - The decarboxylation reactions of homo lysine to HMDA (Figure 20, Step H; Figure 21, Step S; Figure 22, Step E; Figure 26, Step H),2-aminopimelate to 6-ACA (Figure 20, Step J, Figure 21, Step AA and Figure 26, Step E), 2,7-diaminosubarate to homolysine (Figure 26, Step L), 2-amino-7-oxoheptanoate to 6-aminohexanal (Figure 26, Step B; Figure 22, Step F) and 2-amino-7-oxosubarate to 2-oxo-7-aminoheptanoate (Figure 26, Step I) are catalyzed by amino acid decarboxylase enzymes. Lysine decarboxylase (EC 4.1.1.18) catalyzes a similar transformation: the decarboxylation of lysine to form cadaverine. Two isozymes of this enzyme are encoded in the E. coli genome by genes cadA and IdcC. CadA is involved in acid resistance and is subject to positive regulation by the cadC gene product (Lemonnier et al, Microbiology 144 (Pt 3):751-760 (1998)). CadC accepts hydroxylysine and S-aminoethylcysteine as alternate substrates, but 2-Aminopimelate and 6- ACA act as competitive inhibitors to this enzyme (Sabo et al., Biochemistry 13:662-670

(1974)). Directed evolution or other enzyme engineering methods may be required for this enzyme to decarboxylate 2-aminopimelate. The constitutively expressed Idc gene product is less active than CadA (Lemonnier et al, Microbiology 144 (Pt 3):751-760 (1998)). A lysine decarboxylase analogous to CadA was recently identified in Vibrio parahaemolyticus (Tanaka, et al., J Appl Microbiol 104: 1283-1293 (2008)). The lysine decarboxylase from Selenomonas ruminantium, encoded by Idc, bears sequence similarity to eukaryotic ornithine decarboxylases, and accepts both L-lysine and L-ornithine as substrates (Takatsuka et al., Biosci. Biotechnol Biochem. 63: 1843-1846 (1999)). Active site residues were identified and engineered to alter the substrate specificity of the enzyme (Takatsuka et al., J Bacteriol.

182:6732-6741 (2000)).

Several ornithine decarboxylase enzymes (EC 4.1.1.17) exhibit activity on lysine and other similar compounds. Such enzymes are found in Nicotiana glutinosa (Lee et al., Biochem. J 360:657-665 (2001)), Lactobacillus sp. 30a (Guirard et al, J Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). The enzymes from Lactobacillus sp. 30a (Momany et al., J Mol. Biol. 252:643-655 (1995)) and V. vulnificus have been crystallized. The V. vulnificus enzyme efficiently catalyzes lysine decarboxylation and the residues involved in substrate specificity have been elucidated (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). A similar enzyme has been characterized in Trichomonas vaginalis but the gene encoding this enzyme is not known (Yarlett et al., Biochem. J 293 (Pt 2):487-493 (1993)).

Keto-acid decarboxylase enzymes are required to convert 2-oxo-7-aminoheptanoate to 6- aminohexanal (Step F of Figure 22;Step G of Figure 26) and 2-amino-7-oxosubarate to 2- amino-7-oxoheptanoate (Step A of Figure 26). The decarboxylation of keto-acids is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2- ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (Henning et al., Appl. Environ.

Microbiol. 72:7510-7517 (2006)). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268: 1698-1704 (2001); Li, H. and F. Jordan, Biochemistry. 38: 10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64: 1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al, Eur. J. Biochem. 268: 1698-1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443- 451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263 (2002)).

Gene name GI# GenBank Accession # Organism

pdc 118391 P06672.1 Zymomonas mobilus pdc I 30923172 P06169 Saccharomyces cerevisiae pdc 20385191 AM21208 Acetobacter pasteurians pdc I 52788279 Q12629 Kluyveromyces lactis

A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US. A 102: 10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (-130 kD) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J Bacteriol. 182:2838- 2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO:) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

A fourth candidate enzyme for catalyzing this reaction is branched chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988); and Smit et al, Appl Environ Microbiol. 71 :303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2- oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate isocaproate (Smit et al., Appl Environ Microbiol. 71 :303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318: 1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase oiZymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

Gene name GI# GenBank Accession # Organism

kdcA 44921617 AAS49166.1 Lactococcus lactis Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the El subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J Biol. Chem. 267: 16601-16606 (1992); Wynn et al., J Biol. Chem. 267: 1881-1887 (1992); and Wynn et al, J Biol. Chem. 267: 12400-12403 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al, J Biol. Chem. 267: 12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.

4.1.2.a The condensation of pyruvate with 4-aminobutanal (Figure 22, Step A) or glutamate- 5-semialdehyde (Figure 27, Step A) is catalyzed by an aldehyde lyase in the EC class 4.1.2. A variety of aldehyde lyase enzymes utilize pyruvate as an acceptor; however, none have been demonstrated to utilize 4-aminobutanal or glutamate-5-semialdehyde as a donor. The enzyme 4-hydroxy-2-oxopimelate (HODH) aldolase (EC 4.1.2.-), condenses succinic semialdehyde and pyruvate to catalyze the formation of 4-hydroxy-2-oxopimelate. This enzyme is a divalent metal ion-dependent class II aldolase, catalyzing the final step of 4- hydroxyphenylacetic acid degradation in E. coli C, E. coli W, and other organisms. In the native context, the enzyme functions in the degradative direction. The reverse (condensation) reaction is thermodynamically unfavorable; however the equilibrium can be shifted through coupling HODH aldolase with downstream pathway enzymes that work efficiently on reaction products. Such strategies have been effective for shifting the equilibrium of other aldolases in the condensation direction (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995); and Pollard et al, Appl Environ. Microbiol 64:4093-4094 (1998)). The E. coli C enzyme, encoded by hpcH is able to condense a range of aldehyde acceptors with pyruvate and has recently been crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007); and Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme is encoded by hpal (Prieto et al., J Bacteriol. 178:111-120 (1996)).

Another pyruvate-utilizing aldehyde lyase is 2-dehydro-3-deoxyglucarate aldolase (DDGA, EC 4.1.2.20), a type II aldolase that participates in the catabolic pathway for D- glucarate/galactarate utilization in E. coli. The natural donor of this enzyme is tartronate semialdehyde, but this enzyme has a broad substrate specificity and has been shown to reversibly condense a wide range of aldehydes with pyruvate (Fish et al., Methods Enzymol. 9:529-534 (1966)). The crystal structure of this enzyme has been determined and a catalytic mechanism proposed (Izard et al, EMBO J 19:3849-3856 (2000)). Additional candidate DDGA enzymes are found in Leptospira interrogans (118) and Sulfolobus solfataricus (Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)). The S. solfataricus enzyme is highly thermostable and was cloned and expressed in E. coli (Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)).

4.2.1. a Hydro-lyase - Two reactions in Figures 20 and 22 employ enzymes in the dehydratase class (EC 4.1.2). The dehydration of 3-hydroxy-6-aminopimeloyl-CoA (Figure 20, Step D) is catalyzed by an enoyl-CoA hydratase. This reaction is not known to occur naturally; however the ability to dehydrate 3-hydroxyacyl-CoA derivatives is widespread. Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydration of a range of 3-hydroxyacyl-CoA substrates

(Agnihotri et al, Bioorg. Med. Chem. 11 :9-20 (2003; Conrad et al, J Bacteriol. 118: 103-111 (1974) and Roberts et al, Arch. Microbiol 117:99-108 (1978)). The enoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P.

fluoresce™ (Olivera et al, Proc. Natl. Acad. Sci U. S. A 95:6419-6424 (1998)). The gene product oipimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation (Harrison et al., Microbiology 151 :727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., J Bacteriol. 185:5391-5397. 2003), paaF (Ismail et al, Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004) and (Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Park et al, JBacteriol. 185:5391-5397. 2003), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl. Biochem. Biotechnol 113- 116:335-346 (2004) and (Park et al, Biotechnol Bioeng 86:681-686 (2004)).

3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase, is an enoyl-CoA hydratase that dehydrates 3-hydroxyisobutyryl-CoA to form crotonyl-CoA. Crotonase enzymes are required for n-butanol formation in some organisms, particularly Clostridial species, and also comprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermo acidophilic Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera.

Exemplary genes encoding crotonase enzymes can be found in C. acetobutylicum (Atsumi et al, Metab Eng. 10:305-311 (2008); and Boynton et al, JBacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer et al., FEBS Lett. 21 :351-354. 1972)), and Metallosphaera sedula (Berg et al., Science. 318: 1782-1786 (2007)) though the sequence of the latter gene is not known. Gene name GI# GenBank Accession # Organism

crt 15895969 NP_349318.1 Clostridium acetobutylicum crtl 153953091 YP 001393856.1 Clostridium kluyveri

Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Nakahigashi et al, Nucleic Acids Res. 18:4937 (1990); Yang et al, J Bacteriol. 173:7405-7406 (1991) and Yang et al., Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci.Bioeng 103:38- 44 (2007)). The fadl and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

2-Oxo-7-aminohept-3-enoate is formed from the dehydration of 2-oxo-4-hydroxy-7- aminoheptanoate (Figure 22, Step B). The dehydration of 2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosubarate (Figure 27, Step B) is a similar transformation. Enzymes catalyzing these exact reactions are not known to occur naturally. A candidate enzyme that catalyzes a similar reaction is OHED hydratase, which naturally dehydrates 2-oxo-4- hydroxy-hepta-l,7-dioate (HODH) to 2-oxo-hept-4-ene-l,7-dioate (OHED) . HODH is similar in structure to the desired substrates. This enzyme requires magnesium as a cofactor (Burks et al., J Am. Chem. Soc. 120 (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007; and Roper et al, Gene 156:47-51 (1995)) and E. coli ^(Prieto et al., J Bacteriol. 178: 111-120 (1996)). Sequence comparison reveals homologs in a range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (91% identity, evalue = 2e-138) and Salmonella enterica (91% identity, evalue = 4e-138), among others.

Gene name GI# GenBank Accession # Organism

hpcG 556840 CAA57202.1 Escherichia coli C hpaH 757830 CAA86044.1 Escherichia coli W hpaH 150958100 ABR80130.1 Klebsiella pneumoniae

Sari_01896 160865156 ABX21779.1 Salmonella enterica

An alternate enzyme candidate for catalyzing this reaction is fumarase, also known as fumarate hydratase (EC 4.2.1.2). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Guest et al., J Gen Microbiol. 131 :2971-2984 (1985); Tseng et al., J Bacteriol 183:461-467 (2001) and Woods et al., Biochim Biophys Acta 954: 14- 26 (1988)). FumC has been shown to dehydrate alternate substrates including tartrate and threo-hydroxyaspartate (Teipel et al, J Biol. Chem. 243:5684-5694 (1968)). A wealth of structural information is available for FumC and researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver et al., D Biol Crystallogr.

61 : 1395-1401 (2005)). Additional fumarate hydratase enzymes are found in Escherichia coli (Estevez et al., Protein Sci 11 : 1552-1557 (2002); Hong,et al., Biotechnol. Bioprocess Eng. 9:252-255 (2005)) and Rose et al, Proc Natl Acad Sci US. A 101 :3393-3397 (2004)), Corynebacterium glutamicum (Genda et al., Biotechnol Biochem. 70: 1102- 1109 (2006)), Campylobacter jejuni (Smith et al., Cell Biol 31 :961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)), and Rattus norvegicus

(Kobayashi et al, J Biochem. 89: 1923-1931 (1981)). The MmcBC fumarase from

Pelotomaculum thermopropionicum is another class of fumarase with two subunits

(Shimoyama et al, FEMS Microbiol Lett 270:207-213 (2007)).

Gene name GI# GenBank Accession # Organism

fumA 81175318 P0AC33 Escherichia coli K12

fumB 33112655 P14407 Escherichia coli K12

fumC 120601 P05042.1 Escherichia coli K12

fumC 39931596 Q8NRN8.1 Corynebacterium glutamicum

fumC 9789756 069294.1 Campylobacter j ejuni

fumC 75427690 P84127 Thermus thermophilus

fumH 120605 P14408.1 Rattus norvegicus

MmcB 147677691 YP 001211906 Pelotomaculum thermopropionicum MmcC 147677692 YP 001211907 Pelotomaculum thermopropionicum

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in

Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium

tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch. Microbiol 168:457-463 1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms.

5.4.3. a Aminomutase - Several reactions in Figure 21 involve shifting a secondary amine from the 3- to the 2- position (Figure 21, Steps P, R, T). A promising enzyme candidate for catalyzing these transformations is lysine 2,3-aminomutase (EC 5.4.3.2), an enzyme that naturally converts lysine to (3S)-3,6-diaminohexanoate, reversibly shifting an amine group from the 2- to the 3- position. The enzyme is found in bacteria that ferment lysine to acetate and butyrate, including Fusobacterium nucleatum (kamA) (Barker et al., J Bacteriol.

152:201-207 (1982)) and Clostridium subterminale (kamA) (Chirpich et al., J Biol. Chem. 245: 1778-1789 (1970)). The enzyme from Clostridium subterminale has been crystallized (117). An enzyme encoding this function is also encoded by yodO in Bacillus subtilis (Chen et al., Biochem. J 348 Pt 3:539-549 (2000)). The enzyme utilizes pyridoxal 5'-phosphate as a cofactor, requires activation by S-adenosylmethoionine, and is stereoselective for L-lysine. The enzyme has not been shown to react with alternate substrates, so directed evolution or other engineering methods may be required for this enzyme to react with the non-natural substrates 3-amino-7-oxohexanoate, 3,7-diaminoheptanoate and 3-aminopimelate. For example, Cargill has developed a novel 2,3-aminomutase enzyme derived from lysine-2,3- aminomutase that converts L-alanine to β-alanine (Liao et al., United States Patent

20050221466 (2005)). Gene name GI# GenBank Accession # Organism

kamA 75423266 Q9XBQ8.1 Clostridium subterminale kamA 81485301 Q8RHX4 Fusobacterium nucleatum yodO 4033499 034676.1 Bacillus subtilis

Other enzymes with 2,3-aminomutase activity include tyrosine 2,3-aminomutase (EC 5.4.3.6) and leucine 2,3-aminomutase (EC 5.4.3.7). Tyrosine 2,3-aminomutase participates in tyrosine biosynthesis, reversibly converting tyrosine to 3-amino-3-(4-hydroxyphenyl)-propionoate by shifting an amine from the 2- to the 3- position. In Streptomyces globisporus the enzyme has also been shown to react with tyrosine derivatives (Christenson et al., Biochemistry

42: 12708-12718 (2003)); however, the sequence of this enzyme is not yet available. Leucine 2,3-aminomutase converts L-leucine to beta-leucine during leucine biosynthesis and degradation. A leucine 2,3-aminomutase-specific assay detected enzyme activity in many organisms (Poston et al., Methods Enzymol. 166:130-135 (1988)) but genes encoding the enzyme have not been identified to date.

6.2.1. a Acid-thiol ligase - The activation of carboxylic acids to acyl-CoA derivatives is catalyzed by Co A acid-thiol ligases or Co A synthetases in the EC class 6.2.1 (the terms ligase, synthetase, and synthase are used herein interchangeably and refer to the same enzyme class). Such enzymes couple the energetic cost of thioester bond formation to the hydrolysis of ATP into ADP or AMP. Several ADP-forming CoA ligases have been demonstrated to react in the reverse direction, removing the CoA moiety from acyl-CoA molecules and concomitantly forming ATP. Reversible CoA ligases are required to de-acylate 6- aminopimeloyl-CoA (Figure 20, Step I) and 3-oxopimeloyl-CoA (Figure 21, Step B), whereas AMP or ADP forming ligases can acylate 3-oxopimelate (Figure 21, Step H), 3- aminopimelate (Figure 21, Step K) and 2-aminopimelate (Figure 21, Step V). Enzymes catalyzing these exact transformations have not been characterized to date; however, several enzymes with broad substrate specificities have been described in the literature.

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al, JBacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on cyclic compounds phenylacetate and indoleacetate (Musfeldt et al., J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al, Arch. Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., Arch. Microbiol 182:277-287 (2004)).

Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H.

marismortui and P. aerophilum have all been cloned, functionally expressed, and

characterized in E. coli (Brasen et al., Arch. Microbiol 182:277-287 (2004); and Musfeldt et al., J Bacteriol. 184:636-644 (2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al, Biochemistry 24:6245-6252 (1985)).

Another candidate enzyme is the AMP-forming pimeloyl-CoA ligase (EC 6.2.1.14) which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)). Other pimeloyl-CoA ligase candidates are found in Bacillus subtilis (Bower et al., J Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al, Biochem. J 287 (Pt 3):685-690 (1992)). Gene name GI# GenBank Accession # Organism

bioW 115012 P22822.1 Lysinibacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem J 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from /⁵, chrysogenum (Lamas-et al., Maceiras, J 395: 147-155 (2006); and Wang et al, Biophys. Res. Commun. 360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al., J Biol. Chem. 265:7084-7090 (1990)). Acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.

Example XXVII

Addtitional Pathways for Production of Hexamethylenediamine from 6-Aminocaproate

Figure 24 provides additional pathways for HMDA production and is further to Figure 13 and Example XX above. Arrows for Steps O and P indicate the direct conversion of 6- aminocaproate and 6-acetamidohexanoate to 6-aminocaproic semialdehyde and 6- acetamidohexanal, respectively. These reactions are catalyzed by a reductase in EC class 1.2. I .e. For a description of enzyme candidates, see Example XXVI (EC 1.2.1.e).

Example XXVIII

Pathways for Production of 6-Aminocaproate from Adipate Figure 25 provides additional pathways for 6-ACA production and is further to Figure 10 and Example XVI above. The conversion of adipate to adipate semialdehyde (Figure 25, Step X) is catalyzed by an enzyme with adipate reductase functionality. Adipate kinase catalyzes the formation of adipylphosphate from adipate (Figure 25, Step Y). Adipate semialdehyde is formed from adipylphosphate by adipylphosphate reductase (Figure 25, Step Z). Enzyme candidates for catalyzing these transformations are described in Example XXVI. Example XXIX

Pathway for Production of Levulinic acid

Levulinic acid (LA), also known as 4-oxopentanoic acid and 4-ketovaleric acid, is a precursor to nylon-like polymers, synthetic rubbers and plastics. It is also a precursor of other commodity chemicals such as methyltetrahydrofuran, valerolactone and ethyl levulinate. Other potential applications include use as a fuel extender and a biodegradable

herbicide/pesticide. It is traditionally prepared by treating cellulosic biomass with strong acids such as hydrochloric and sulfuric acids. This process has the disadvantages of low LA yield and numerous byproducts. More recently, the Biofine Process was developed which converts cellulosic biomass into LA, formic acid and furfural at a yield of 70% the theoretical maximum (Hayes et al., "The biofine process-production of levulinic acid, furfural and formic acid from lignocellulosic feedstock" p. 139-164. In Biorefineries: Industrial Processes and Products. Wiley, Weinheim, Germany (2006)). Described herein is a process for selectively producing LA from sugar or syngas feedstocks using a microbial organism.

The maximum theoretical yield of LA from glucose is 1.45 moles of LA per mole glucose utilized (0.938 g/g), according to the following equation:

Glucose (C₆Hi₂0₂) + 1.27 C0₂ 1.45 LA (C₅H₈0₃) + 0.18 H₂0

LA is produced from the central metabolites succinyl-CoA and acetyl-CoA in three enzymatic steps. In the first step, acetyl-CoA and succinyl-CoA are condensed by a beta- ketothiolase to form 3-oxoadipyl-CoA (Step A of Figure 25). The CoA moiety is

subsequently removed by a CoA hydrolase, transferase or ligase (Steps E/F/G of Figure 25). In the final step of the pathway, 3-oxoadipate is decarboxylated to LA (Step AA of Figure 25).

The decarboxylation of 3-oxoadipate to LA can occur enzymatically or spontaneously. In E. coli, several 3-oxoacids produced during amino acid biosynthesis have been shown to undergo spontaneous decarboxylation (Boylan et al., Biochem. Biophys. Res Commun.

85: 190-197 (1978)). An enzyme catalyzing the decarboxylation of 3-oxoadipate to LA has not been demonstrated to our knowledge. An exemplary enzyme candidate catalyzing a similar reaction is acetoacetate decarboxylase (EC 4.1.1.4). The acetoacetate decarboxylase from Clostridium acetobutylicum, encoded by adc, has a broad substrate specificity and has been shown to decarboxylate 3-oxopentanoate, 2-oxo-3-phenylpropionic acid and 2-methyl- 3-oxobutyrate (Benner et al, J Am. Chem. Soc. 103:993-994 (1981) and Rozzel et al, J Am. Chem. Soc. 106:4937-4941 (1984)). An acetoacetate decarboylase has also been

characterized in Clostridium beijerinckii (Ravagnani et al., Mol. Microbiol 37: 1172-1185 (2000)). The acetoacetate decarboxylase from Bacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylate 3- oxopentanoate (Matiasek et al., Curr. Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence of B. polymyxa is not yet available. Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)).

EXAMPLE XXX

In Silico Knockout Strategies for Production of Adipate, 6- AC A and HMDA

This example describes gene disruption strategies for production of adipate 6-aminocaproic acid (6-ACA) and hexamethylenediamine (HMDA).

Described below in more detail are sets of enzymatic activities that can be reduced by appropriate gene disruptions or deletions in a production host engineered to contain the adipate, 6-aminocaproic acid (6-ACA) and hexamethylenediamine (HMDA) production pathways, for example, pathways using succinyl Co A and acetyl Co A as precursors.

OptKnock is a bilevel computational framework formulated with the overall objective of developing genetically stable overproducing microorganisms. Specifically, the framework examines the complete network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene disruptions or deletions, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, in the case of a gene deletion, there is negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are completely removed from the genome. Growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical production will generally result in sub-maximal growth rates. The knockouts suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point should lie within its calculated solution boundary. Plots such as these allow

visualization of how close strains are to their performance limits or, in other words, how much room is available for improvement. The OptKnock framework has been used to identify promising gene deletion strategies for biochemical overproduction and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks.

Described below are sets of enzyme activities that should be absent, attenuated, or eliminated for creating host organisms that achieve growth-coupled adipate, 6-ACA or HMDA production upon the addition of the biosynthetic pathway that proceeds through succinyl- CoA and acetyl-CoA. To enumerate all potential strategies, an optimization technique, termed integer cuts, has been implemented which entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration.

The OptKnock algorithm was used to identify designs based on a stoichiometric model of Escherichia coli metabolism. Assumptions include (i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic or microaerobic conditions; and (iii) a minimum non-growth associated maintenance requirement of 4 mmol/gdw/hr. Table 12 provides a list of all the reaction stoichiometries and the associated genes known to be associated with the reactions identified for deletion in the strategies. Table 13 provides a list of the metabolite

abbreviations, the corresponding names and locations of all the metabolites that participate in the reactions listed in Table 12. The growth-coupled productions designs for adipic acid, 6 AC A and HMD A are provided in Tables 14-16. The product formation rates shown in

Tables 14-16 are in mmol/gDCW hr. The basis glucose uptake rate is 10 mmol/gDCW hr and the biomass formation rate is shown in units of 1/hr. These tables list the reactions that are knocked out in a particular strategy, the anticipated product and biomass yields. Although the designs were identified using a metabolic model of E. coli metabolism, and the gene names listed are specific to E. coli, the method of choosing the metabolic engineering strategies and also the designs themselves are applicable to any HMDA, 6-ACA or adipate- producing organism. Thus the designs are essentially lists of enzymatic transformations whose activity is to be either eliminated, attenuated, or initially absent from a microorganism to provide the growth coupled production of adipate, 6ACA and HMDA. The key criterion for prioritizing the final selection of designs was the growth-coupled yield of each of the products. To examine this, production cones were constructed for each strategy by first maximizing and, subsequently minimizing the product yields at different rates of biomass formation, as described above. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the product in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs were given a lower priority. The metabolic engineering strategies described below assume that the organism can produce adipate, 6-ACA or HMDA via the succinyl CoA and acetyl-CoA utilizing pathway. The construction of a recombinant host organism capable of producing these products via the pathway is described herein.

Strain construction: In order to validate the computational predictions proposed in this report, the strains are constructed, evolved, and tested. Escherichia coli K-12 MG1655 housing the succinyl-CoA-acetyl-CoA pathway serves as the strain into which the deletions are introduced. The strains are constructed by incorporating in-frame deletions using homologous recombination via the λ Red recombinase system of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97(12):6640-6645 2000)). The approach involves replacing a chromosomal sequence, that is, the gene targeted for removal, with a selectable antibiotic resistance gene, which itself is later removed. The knockouts are integrated one by one into the recipient strain. No antibiotic resistance markers remain after each deletion, allowing accumulation of multiple mutations in each target strain. The deletion technology completely removes the gene targeted for removal so as to substantially reduce the possibility of the constructed mutants reverting back to the wild-type.

Shake flask characterization: As intermediate strains are being constructed, strain

performance is quantified by performing shake flask fermentations. Anaerobic conditions are obtained by sealing the flasks with a rubber septum and then sparging the medium with nitrogen. For strains where growth is not observed under strict anaerobic conditions, microaerobic conditions can be applied by covering the flask with foil and poking a small hole for limited aeration. All experiments are performed using M9 minimal medium supplemented with glucose unless otherwise stated. Pre-cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. The products, ethanol, and organic acids are analyzed by GC-MS or HPLC using routine procedures. Triplicate cultures are grown for each strain.

Batch Fermenter Testing: The performance of select strains is tested in anaerobic, pH- controlled batch fermentations. This allows reliable quantification of the growth, glucose uptake, and formation rates of all products, as well as ensure that the accumulation of acidic fermentation products will not limit cell growth. In addition, it allows accurate determination of volumetric productivity and yield of product formation, two of the most important parameters in benchmarking strain performance. Fermentations are carried out in 1-L bioreactors with 600 mL working volume, equipped with temperature and pH control. The reactor is continuously sparged with N₂ at approximately 0.5 L/min to ensure that dissolved oxygen (DO) levels remain below detection levels. The culture medium is the same as described above, except that the glucose concentration is increased in accordance with the higher cell density achievable in a fermentation vessel. Chemostat Testing: Chemostat experiments are conducted to obtain a direct measure of how the switch in fermentation mode from batch to continuous affects product yield and volumetric productivity. The bioreactors described above using batch mode are operated in chemostat mode through continuous supply of medium and removal of spent culture. The inlet flow rate is set to maintain a constant dilution rate of 80% of the maximum growth rate observed for each strain in batch, and the outlet flow is controlled to maintain level. Glucose is the limiting nutrient in the medium, and set to achieve the desired optical density in the vessel.

Adaptive evolution: The knockout strains are initially expected to exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To allow this adjustment, the strains is adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat. Genet. 36(10):1056-1058 (2004). The OptKnock-generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli (Fong and Palsson, Nat Genet. 36(10): 1056-1058 (2004); Fong et al, J Bacteriol. 185(21):6400- 6408 (2003); Ibarra et al, Nature 420(6912): 186- 189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions are run for a period of 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained. The growth-coupled biochemical production concept behind the OptKnock approach results in the generation of genetically stable overproduces.

Although described as deletion sets, it is understood, as disclosed herein, that gene sets can be deleted or disrupted so that encoded gene product activities are reduced or eliminated. Thus, the gene deletion sets of Tables 14-16 can be used to delete or disrupt a gene set in a host organism in which an increased production of 6-ACA, adipate and/or HMDA is desired. It is understood that any of the disclosed gene deletion sets can be used to generate knockout strains with disrupted or deleted genes that confer increased production of 6-ACA, adipate and/or HMDA. Table 12: A list of all the reaction stoichiometries and the associated genes known to be associated with the reactions identified for deletion in the strategies listed in Tables 1 and 2.

Reaction j

Abbreviation j Reaction Name Reaction Stoichiometry Associated genes

ACKr Acetate kinase [c] : ac + atp <==> actp + adp ( b3115 or b2296 or bl849 )

[c] : accoa + (2) h + (2) nadh

ADHEr Alcohol dehydrogenase ( b0356 or bl478 or bl241 )

<==> coa + etoh + (2) nad

Alpha-ketoglutarate [c] : akg + coa + nad— > co2 +

AKGD ( b0116 and b0726 and b0727 ) dehydrogenase nadh + succoa

ALAR Alanine racemase [c] : ala-L <==> ala-D b4053

[c] : asp-L + atp + nh4— > amp +

ASNS2 Asparagine synthetase b3744

asn-L + h + ppi

ASPT L-aspartase [c] : asp-L— > fum + nh4 b4139

( ( ( b3736 and b3737 and b3738 ) and ( b3731 and b3732 and b3733 adp[c] + (4) h[p] + pi[c] <==> and b3734 and b3735 ) ) or ( ( b3736

ATPS4r ATP synthase

atp[c] + (3) h[c] + h2o[c] and b3737 and b3738 ) and ( b3731 and b3732 and b3733 and b3734 and b3735J) and b3739 ) )

[c] : atp + co2 + nh4— > adp +

CBMK2 Carbamate kinase ( b0521 or b0323 or b2874 )

cbp + (2) h

D-amino acid [c] : ala-D + fad + h2o -> fadh2

DAAD bl l89

dehydrogenase + nh4 + pyr

2-dehydro-3-deoxy-

EDA phosphogluconate [c] : 2ddg6p— > g3p + pyr bl850

aldolase

ENO Enolase [c] : 2pg <==> h2o + pep b2779

Fructose-bis-phosphate

FBA [c] : fdp <==> dhap + g3p ( b2097 or b2925 or bl773 )

aldolase

[c] : fum + mql8— > mqn8 + ( b4151 and b4152 and b4153 and FRD Fumarate reductase

succ b4154 )

FUM Fumarase [c] : fum + h2o <==> mal-L ( bl612 or b4122 or bl611 )

Glutamate-5-

[c] : glu5p + h + nadph— >

G5SD semialdehyde b0243

glu5sa + nadp + pi

dehyrogenase

Glucose-6-phosphate [c] : g6p + nadp <==> 6pgl + h +

G6PDHy bl852

dehydrogenase nadph

( ( b2417 and bl lOl and b2415 and

D-glucose transport via glc-D[e] + pep[c] -> g6p[c] + b2416 ) or ( bl817 and bl818 and GLCpts bl819 and b2415 and b2416 ) or (

PTS mechanism pyr[c] b2417 and bl621 and b2415 and b2416 ) ) GLU5K Gluatmate - 5 -kinase : [c] : atp + glu-L— > adp + glu5p b0242

: [c] : glu-L + h2o + nadp <==>

GLUDy : Glutamate dehydrogenase bl761

: akg + h + nadph + nh4

[c] : atp + glc-D— > adp + g6p +

HEX1 : Hexokinase b2388

: h

' [c] : lac-D + nad <==> h + nadh

LDH D : Lactate dehydrogenase bl380 or b2133

; + Pyr

[c] : mal-L + nad <==> h + nadh

MDH : Malate dehydrogenase b3236

: + oaa

: [c] : mal-L + nad— > co2 + nadh

MElx ; Malic enzyme (NAD) bl479

: + Pyr

^: [c] : mal-L + nadp— > co2 +

ME2 : Malic enzyme (NADP) b2463

■ nadph + pyr

I NADH dehydrogenase [c] : h + nadh + ubq8 -> nad +

NADH12 bl l09

I (ubiquinone-8) ubq8h2

( b2276 and b2277 and b2278 and

^• NADH dehydrogenase (4.5) h[c] + nadh[c] + ubq8[c] - b2279 and b2280 and b2281 and

NADH6 ; (ubiquinone- 8 and 3.5 > (3.5) h[e] + nad[c] + b2282 and b2283 and b2284 and

; protons) ubq8h2[c] b2285 and b2286 and b2287 and b2288 )

^: [c] : coa + nad + pyr— > accoa + ((b0114 and bO 115 and bO 116) or (bO 116

PDH : Pyruvate dehydrogenase and b0726 and b0727) or (bOl 16 and co2 + nadh b2903 and b2904 and b2905))

PFK : Phosphofructokinase ; [c] : atp + f6p -> adp + fdp + h ( b3916 or bl723 )

( ( ( b0902 and b0903 ) and b2579 ) or

PFLi : Pyruvate formate lyase : [c] : coa + pyr— > accoa + for ( b0902 and b0903 ) or ( b0902 and b3114 ) or ( b3951 and b3952 ) )

^: Phosphogluconate ^: [c] : 6pgc + nadp— > co2 +

PGDH b2029

: dehyrogenase : nadph + ru5p-D

■ Phosphogluconate

PGDHY : [c] : 6pgc— > 2ddg6p + h2o bl851

■ dehydratase

; Glucose-6-phosphate

PGI : [c] : g6p <=> ffip b4025

: isomerase

PGL : 6-

: [c] : 6pgl + h2o— > 6pgc + h b0767

^: Phosphogluconolactonase

PGM : Phosphoglycerate mutase : [c] : 3pg <==> 2pg b3612

^: Phosphoenolpyruvate ^'■ [c] : co2 + h2o + pep— > h + oaa

PPC b3956

■ carboxylase ; + pi

: Phosphoenolpyruvate : [c] : atp + oaa— > adp + co2 +

PPCK b3403

; carboxykinase ; Pep

^: [c] : fad + pro-L— > lpyr5c +

PROlz : Proline oxidase bl014

: fadh2 + h PTAr : Phosphotransacetylase : [c] : accoa + pi <==> actp + coa b2297

PYK I Pyruvate kinase [c] : adp + h + pep— > atp + pyr ( bl854 or bl676 )

^: Ribulose-5-phosphate-5-

RPE : [c] : ru5p-D <==> xu5p-D ( b4301 or b3386 )

: epimerase

: [c] : fadh2 + ubq8 fad + • ( b0721 and b0722 and b0723 and

SUCD4 ; Succinate dehydrogenase

: ubq8h2 ; b0724

• [c] : atp + coa + succ <==> adp

SUCOAS : Succinyl-CoA synthetase ( b0728 and b0729 )

: + pi + succoa

TAL : Transaldoalse ; [c] : g3p + s7p <==> e4p + f6p ; ( b2464 or b0008 )

: (2) h[e] + nadh[c] + nadp[c] -->

THD2 : NADP transhydrogenase ( bl602 and bl603 )

; (2) h[c] + nad[c] + nadphfc]

^: [c] : nad + nadph— > nadh +

THD5 : NAD transhydrogenase ; ( b3962 or ( bl602 and bl603 ) ) ■ nadp

; [c] : r5p + xu5p-D <==> g3p +

TKT1 : Transketolase ; ( b2935 or b2465)

: [c] : e4p + xu5p-D <==> f6p +

TKT2 : Transketolase ; (b2935 or b2465)

^': g³P

^: Triosephosphate

TPI : [c] : dhap <==> g3p : b3919

: isomerase

! [c] : akg + val-L <==> 3mob +

YALTA : Valine transaminase b3770

! glu-L

Table 13: List of the metabolite abbreviations, the corresponding names and locations of all the metabolites that participate in the reactions listed in Table 12.

Metabolite

Abbreviation Compartment Metabolite Name

13dpg Cytosol 3-Phospho-D-glyceroyl phosphate

lpyr5c _L Cytosol 1 -Pyrroline-5-carboxylate

2ddg6p Cytosol 2-Dehydro-3 -deoxy-D-gluconate 6-phosphate

2pg Cytosol D-Glycerate 2-phosphate

3 mob Cytosol 3 -Methyl-2-oxobutanoate

„¾g Cytosol 3 -Phospho-D-glycerate

6pgc _L Cytosol _l 6-Phospho-D-gluconate

6pgl Cytosol 6-phospho-D-glucono- 1 ,5-lactone

ac Cytosol Acetate

accoa Cytosol Acetyl-CoA

mql-8 Cytosol Menaquinol-8

mgn-8 Cytosol Menaquinone-8

nad Cytosol Nicotinamide adenine dinucleotide

nadh Cytosol Nicotinamide adenine dinucleotide - reduced

nadp _l Cytosol _l Nicotinamide adenine dinucleotide jghosphate

nadph _l Cytosol _l Nicotinamide adenine dinucleotide jphosphate - reduced nh4 Cytosol Ammonium

o2 Cytosol Oxygen

oaa Cytosol Oxaloacetate

_P_¾ Cytosol Pho spho enolpyruvate

.Pi _μ Cytosol Phosphate

.PPA Cytosol Diphosphate

pyr Cytosol Pyruvate

r5p Cytosol alpha-D-Ribose 5-phosphate

ru5p-D Cytosol D-Ribulose 5-phosphate

s7p Cytosol Sedoheptulose 7-_phosphate

succ _μ Cytosol Succinate

succoa Cytosol Succinyl-CoA

ubq8 Cytosol Ubiquinone- 8

ubq8h2 Cytosol Ubiquinol-8

val-L Cytosol L-valine

xu5p-D Cytosol D-Xylulose 5-phosphate

Table 14. Knockout strain designs for increased production of 6-ACA, showing yields of 6- ACA and biomass.

_iii2

1 184 5 2.958777052 0.228457649 ADHEr,L DH_D,MDH,THD2 and/or GLUDy,TKTl

Table 15. Knockout strain designs for increased production of adipate, showing yields of adipate and biomass.

and/or GLUDy

Table 16. Knockout strain designs for increased production of HMD A, showing yields of HMD A and biomass.

Minimal Gene Deletion Sets for Enhanced Production of 6-ACA. Described above are strain design strategies for improving 6-aminocaproate (6-ACA) production in microorganisms that possess the 6-ACA pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for 6-ACA production described in Table 14, a minimum set of deletions that is required for growth-coupled 6-ACA production has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D) are required for preventing the formation of competing byproducts, ethanol and lactate. Therefore, the minimal deletion set comprises deletion of acetaldehyde

dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D). Additional deletion strains include strains lacking at least one of the following activities in addition to ADHEr and LDH_D: malate dehydrogenase (MDH), aspartase (ASPT), NAD(P) transhydrogenase (THD2), and glutamate dehydrogenase (GLUDy). Such additional deletions result in a much tighter coupling of production with cell growth. Figures 28-31 show calculated 6-ACA versus growth yields for deletion mutants having at least deletion of ADHEr and LDH D (Figure 28). Calculated yields for strains with additional deletions are shown in Figures 29- 31. An additional minimal set of deletions includes phosphoglucoisomerase (PGI). This design focuses on generating reducing equivalents via the pentose phosphate pathway. Additional beneficial deletions include any of: acetaldehyde dehydrogenase (ADHEr), hexokinase (HEX1), 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA) and phosphogluconate dehydratase (PGDHy). Figures 32-34 show calculated 6-ACA versus growth yields for deletion mutants having at least deletion of PGI, with additional exemplary mutants shown Figures 32-34.

Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. For example, succinate dehydrogenase that oxidizes succinate to fumarate and is known to be active only under aerobic conditions can assume significant activity even under anaerobic conditions, and therefore such activity can be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled 6- ACA production strains. Minimal Gene Deletion Sets for Enhanced Production of adipate. Described above are strain design strategies for improving adipate production in microorganisms that possess the adipate pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for adipate production described in Table 15, a minimum set of deletions that is required for growth-coupled adipate production in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.

dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D). Additional deletion strains include a strain lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D): fumarase (FUM),

phosphoglucose isomerase (PGI), PEP carboxykinase (PPCK), hexokinase (HEX1), malate dehydrogenase (MDH), and NADH dehydrogenase (NADH6).

Additional deletions have been identified by the OptKnock framework for improving the growth-coupled formation of adipate. These include one or more of the following: malic enzyme (ME2), aspartate transaminase (ASPT), acetate kinase (ACKr),

phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), transhydrogenase (THD2), and glutamate dehydrogenase (GLUDy), and PTS system of glucose uptake (GLCpts). Further improvements in yields can be achieved by additional deletions in any of the following enzymes: ATP synthase (ATPS4r), phosphogluconate dehydratase (PGDHY), 2-dehydro-3- deoxy-phosphogluconate aldolase (EDA), 6-phosphogluconolactonase (PGL), glucose 6- phosphate dehydrogenase (G6PDHY), and phosphogluconate dehydrogenase (PGDH).

Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled adipate production strains. Minimal Gene Deletion Sets for Enhanced Production of HMDA. Described above are strain design strategies for improving hexamethylene diamine (HMDA) production in

microorganisms that possess the HMDA pathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysis of the strain designs for HMDA production described in Table 16, a minimum set of deletions that is required for growth-coupled HMDA production in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.

dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D). Additional deletion strains include a strain lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D): fumarate reductase (FRD2), fumarase (FUM), phosphoglucose isomerase (PGI), or PEP carboxykinase (PPCK). Additional deletions have been identified by the OptKnock framework for improving the growth-coupled formation of HMDA. These include one or more of the following:

hexokinase (HEX1), malic enzyme (ME2), malate dehydrogenase (MDH), aspartate transaminase (ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), and pyruvate kinase (PYK). The HMDA yields can be further improved by further deleting one or more of the following enzymes: transhydrogenase (THD2), glutamate dehydrogenase (GLUDy), ATP synthase (ATPS4r), GLCpts (PTS system of glucose uptake), PGDHY (phosphogluconate dehydratase) and EDA (2-dehydro-3-deoxy- phosphogluconate aldolase).

Each of these strains can be supplemented with additional deletions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation or for increased efficiency of coupling the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such enzyme activities can also be knocked out. For example, succinate dehydrogenase that oxidizes succinate to fumarate and is known to be active only under aerobic conditions can assume significant activity even under anaerobic conditions, and therefore such activity can be knocked out. However, the list of gene deletion sets provided herein serves as a good starting point for construction of high-yielding growth-coupled HMDA production strains.

Optknock Strain Design for Growth-Coupled Production of Adipic Acid. Described below is a further exemplification of a deletion strategy for engineering a strain to synthesize adipate using a succinyl-CoA pathway. All high-priority growth-coupled designs for adipate synthesis build upon a strain lacking acetylaldehyde-CoA dehydrogenase (ADHEr) and lactate dehydrogenase (LDH D) activities to prevent the formation of fermentation byprouducts, as discussed above. The further deletion of malate dehydrogenase (MDH) also reduces byproduct production. Figure 35 shows growth-coupled adipate production characteristics of high priority strain designs (grey) compared with that of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. A strain deficient in

ADHEr, LDH D and MDH activities (Design 1 in Figure 35) is predicted to achieve an adipate yield of 0.51 grams adipate per gram of glucose utilized (g/g) at the maximum biomass yield. Designs 2-4 build on Design 1 as a base design. Design 2 entails the removal of

phosphoenolpyruvate carboxykinase (PPCK). This design improves the adipate yield to 3.6 g/g at the maximum biomass yield. The additional deletion of pyruvate formate lyase (PFLi) activity in Design 3 further improves yield by preventing secretion of formate as a byproduct. The predicted adipate yield of this design is 5.8 g/g. Design 4 features the deletion of NAD(P) transhydrogenase (THD2) in addition to ADHEr, LDH D, MDH, PPCK and PFLi. This results in an adipate yield of 6.8 g/g at a growth rate of 0.117 1/hr. Design 4 serves to tightly couple adipate production to cell growth while achieving 91% of the theoretical maximum yield.

Example XXXI

Biosynthesis of Adipate Semialdehyde from Adipate and 6-Aminocaproate

Semialdehyde from 6-Aminocaproate

This Example describes the biosynthetic production of adipate semialdehyde from adipate and of 6-aminocaproate semialdehyde from 6-aminocaproate. The transformation from adipate to adipate semialdehyde (Figure 25, step X) can by catalyzed by the carboxylic acid reductases (CAR). This is demonstrated by the following results. Chemically competent cells of E. coli strain ECKh-422 (AadhE, AldhA, ApflB, AlpdA, integrated IpdA from Klebsiella pneumonia::E354K, Amdh, AarcA, gltA-R\63V) with f pKLJ33s were transformed with pZs*13s plasmids harboring various CAR gene (Table 17) or a control plasmid without any CAR gene. Single colonies of the transformations were selected and grown overnight in LB at 37°C with 100 μg/ml carbenecillin and 10 μg/ml chloramphenicol. The cells were subcultured at a ratio of 1 :50 and induced with 200 μΜ IPTG at an OD600 of 0.6. Cells were incubated for 5 hours at 37°C before harvesting. Cell cultures were aliquoted into 15 ml samples and pelleted. The cell pellets were stored at - 80°C until used for the assay.

Table 17. CAR genes used this example

Cell pellets were lysed by addition of 500 μΐ B-PER with 0.5 μΐ lysozyme and benzonase. CAR activity was measured by addition of 2 μΐ of crude lysate to the assay solution of 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mM MgC12, 1 mM DTT, 10% (v/v) glycerol, 1 mM ATP, 0.5 mM NADPH and 20 mM Adipate or 50 mM 6-Aminocaproate in a 96 well format microplate with a total volume of 250 μΐ. The oxidation of NADPH to NADP⁺ was monitored at an absorbance of 340 nm for 30 minutes at room temperature. The rate of NADPH depletion was used to calculate activity of the various CAR proteins. Total protein concentration of each lysate was determined by Bradford, and the activity was normalized to the total protein concentration (Units/mg). Transformation from adipate to adipate semialdehyde (Figure 24, step ). As shown in Figure 36, significant CAR activities using adipate as a substrate was observed with both CAR genes 889 and 891, whereas the control lysate did not show CAR activity.

Furthermore, a reaction was setup consisting of 500 μΐ of 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mM MgCl₂, 1 mM DTT, 10% (v/v) glycerol, 5 mM ATP, 3 mM NADPH and 20 mM Adipate. The reaction was incubated at room temperature for 30 minutes and stopped by addition of 1% formic acid. The sample was then centrifuged and the supernatant was analyzed by LC-MS. Low mM level of adipate semialdehyde was detected that confirmed the transformation from adipate to adipate semialdehyde.

Transformation from 6-aminocaproate to 6-aminocaproate semialdehyde. As shown in Figure 37, significant CAR activities using 6-aminocaproate as a substrate was observed with several CAR genes 720, 889, 890, 891 and 892, whereas the control lysate did not show CAR activity. These results indicate the transformation from 6-aminocaproate to 6-aminocaproate semialdehyde.

EXAMPLE XXXII

Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting Reducing

Equivalents from Syngas and Exemplary Reductive TCA Cycle Enzymes

Enzymes of the reductive TCA cycle useful in the non-naturally occurring microbial organisms of the present invention include one or more of ATP-citrate lyase and three C0₂- fixing enzymes: isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate :ferredoxin oxidoreductase. The presence of ATP-citrate lyase or citrate lyase and alpha-ketoglutarate:ferredoxin oxidoreductase indicates the presence of an active reductive TCA cycle in an organism. Enzymes for each step of the reductive TCA cycle are shown below.

ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase, catalyzes the ATP- dependent cleavage of citrate to oxaloacetate and acetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied in green sulfur bacteria Chlorobium limicola and

Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was cloned and characterized in E. coli (Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, is irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP. A recombinant ACL from Chlorobium tepidum was also expressed in E. coli and the holoenzyme was reconstituted in vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism (Kim and Tabita, J Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in Balnearium lithotrophicum, Sulfu ihydrogenibium subterraneum and other members of the bacterial phylum Aquificae (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungi as well. Exemplary organisms include Sordaria macrospora (Nowrousian et al, Curr. Genet. 37:189-93 (2000), Aspergillus nidulans , Yarrowia lipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010) and Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Other candidates can be found based on sequence homology. Information related to these enzymes is tabulated below:

In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by cc A and ccsB, respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). The citryl-CoA synthetase ofAquifex aeolicus is composed of alpha and beta subunits encoded by sucCl and sucDl (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme is a homotrimer encoded by ccl in Hydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol. 52:763-770 (2004)) and aq_150 in Aquifex aeolicus (Hugler et al., supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA have also been reported recently in

Chlorobium tepidum (Eisen et al, PNAS 99(14): 9509-14 (2002).

Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the forward and reverse direction. S. cerevisiae possesses three copies of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11 :370-380 (1991); Gibson and McAlister-Henn, J Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. E. coli is known to have an active malate dehydrogenase encoded by mdh.

Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygen availability. FumB is oxygen sensitive and is active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is active under aerobic growth conditions (Tseng et al., J Bacteriol. 183:461-467 (2001);Woods et al., Biochim. Biophys. Acta 954: 14-26 (1988); Guest et al, J Gen. Microbiol. 131 :2971-2984 (1985)). S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al., J Biol. Chem.

278:45109-45116 (2003)). Additional fumarase enzymes are found in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell. Biol. 31 :961-975 (1999)), Thermus thermophilus

(Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus

(Kobayashi et al., J Biochem. 89: 1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate reductase of E. coli, composed of four subunits encoded by frdABCD, is membrane-bound and active under anaerobic conditions. The electron donor for this reaction is menaquinone and the two protons produced in this reaction do not contribute to the proton gradient (Iverson et al., Science 284: 1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto et al., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch. Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used during anaerobic growth on glucose (Arikawa et al, FEMS Microbiol. Lett. 165:111-116 (1998)).

The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). The product of the LSCl and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below:

Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also known as 2-oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase (OFOR), forms alpha-ketoglutarate from C02 and succinyl-CoA with concurrent consumption of two reduced ferredoxin equivalents. OFOR and pyruvate: ferredoxin oxidoreductase (PFOR) are members of a diverse family of 2-oxoacid: ferredoxin (flavodoxin) oxidoreductases which utilize thiamine pyrophosphate, CoA and iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in this class are reversible and function in the carboxylation direction in organisms that fix carbon by the RTCA cycle such as Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus and Chlorobium species (Shiba et al. 1985; Evans et al., Proc. Natl. Acad. Scl. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from H.

thermophilus, encoded by korAB, has been cloned and expressed in E. coli (Yun et al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFOR from the same organism with strict substrate specificity for succinyl-CoA, encoded by forDABGE, was recently identified and expressed in E. coli (Yun et al. 2002). The kinetics of C02 fixation of both H. thermophilus OFOR enzymes have been characterized (Yamamoto et al.,

Extremophiles 14:79-85 (2010)). A C02-fixing OFOR from Chlorobium thiosulfatophilum has been purified and characterized but the genes encoding this enzyme have not been identified to date. Enzyme candidates in Chlorobium species can be inferred by sequence similarity to the H. thermophilus genes. For example, the Chlorobium limicola genome encodes two similar proteins. Acetogenic bacteria such as Moorella thermoacetica are predicted to encode two OFOR enzymes. The enzyme encoded by Moth_0034 is predicted to function in the C02-assimilating direction. The genes associated with this enzyme,

Moth_0034 have not been experimentally validated to date but can be inferred by sequence similarity to known OFOR enzymes.

OFOR enzymes that function in the decarboxylation direction under physiological conditions can also catalyze the reverse reaction. The OFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7, encoded by ST2300, has been extensively studied (Zhang et al. 1996. A plasmid-based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74- 80 (2002)). The OFOR encoded by Apel472/Apel473 from Aeropyrum pernix str. Kl was recently cloned into E. coli, characterized, and found to react with 2-oxoglutarate and a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded by oorDABC in Helicobacter pylori (Hughes et al. 1998). An enzyme specific to alpha-ketoglutarate has been reported in Thauera aromatica (Dorner and Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similar enzyme can be found in

Rhodospirillum rubrum by sequence homology. A two subunit enzyme has also been identified in Chlorobium tepidum (Eisen et al, PNAS 99(14): 9509-14 (2002)).

Protein GenBank ID GI Number Organism

korA BAB21494 12583691 Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacter thermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forA BAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.1 14970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997 Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacter thermophilus

Clim_0204 ACD89303.1 189339900 Chlorobium limicola

Clim_0205 ACD89302.1 189339899 Chlorobium limicola

Clim 1123 ACD90192.1 189340789 Chlorobium limicola

Clim_1124 ACD90193.1 189340790 Chlorobium limicola

Moth_1984 YP_430825.1 83590816 Moorella thermoacetica

Moth_1985 YP_430826.1 83590817 Moorella thermoacetica

Moth_0034 YP_428917.1 83588908 Moorella thermoacetica

ST2300 NP_378302.1 15922633 Sulfolobus sp. strain 7

Apel472 BAA80470.1 5105156 Aeropyrum pernix

Apel473 BAA80471.2 116062794 Aeropyrum pernix

oorD NP_207383.1 15645213 Helicobacter pylori

oorA NP_207384.1 15645214 Helicobacter pylori

oorB NP_207385.1 15645215 Helicobacter pylori

oorC NP_207386.1 15645216 Helicobacter pylori

CT0163 NP 661069.1 21673004 Chlorobium tepidum

CT0162 NP 661068.1 21673003 Chlorobium tepidum

korA CAA12243.2 19571179 Thauera aromatica

korB CAD27440.1 19571178 Thauera aromatica

Rru_A2721 YP_427805.1 83594053 Rhodospirillum rubrum

Rru_A2722 YP_427806.1 83594054 Rhodospirillum rubrum

Isocitrate dehydrogenase catalyzes the reversible decarboxylation of isocitrate to 2- oxoglutarate coupled to the reduction of NAD(P)⁺. IDH enzymes in Saccharomyces cerevisiae and Escherichia coli are encoded by IDPl and icd, respectively (Haselbeck and McAlister-Henn, J Biol. Chem. 266:2339-2345 (1991); Nimmo, H.G., Biochem. J. 234:317- 2332 (1986)). The reverse reaction in the reductive TCA cycle, the reductive carboxylation of 2-oxoglutarate to isocitrate, is favored by the NADPH-dependent C0₂-fixing IDH from Chlorobium limicola and was functionally expressed in E. coli (Kanao et al., Eur. J. Biochem. 269: 1926-1931 (2002)). A similar enzyme with 95% sequence identity is found in the C. tepidum genome in addititon to some other candidates listed below.

I H. thermophilus the reductive carboxylation of 2-oxoglutarate to isocitrate is catalyzed by two enzymes: 2-oxoglutarate carboxylase and oxalosuccinate reductase. 2-Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent carboxylation of alpha-ketoglutarate to oxalosuccinate (Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a large complex composed of two subunits. Biotinylation of the large (A) subunit is required for enzyme function (Aoshima et al., Mol. Microbiol. 51 :791-798 (2004)).

Oxalosuccinate reductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate to D-t/zra isocitrate. The enzyme is a homodimer encoded by icd in H. thermophilus. The kinetic parameters of this enzyme indicate that the enzyme only operates in the reductive carboxylation direction in vivo, in contrast to isocitrate dehydrogenase enzymes in other organisms (Aoshima and Igarashi, J Bacteriol. 190:2050-2055 (2008)). Based on sequence homology, gene candidates have also been found in Thiobacillus denitrificans and Thermocrinis albus.

Protein GenBank ID GI Number Organism

cfiA BAF34932.1 116234991 Hydrogenobacter thermophilus ciJB BAF34931.1 116234990 Hydrogenobacter thermophilus led BAD02487.1 38602676 Hydrogenobacter thermophilus

Tbd_1556 YP_315314 74317574 Thiobacillus denitrificans

Tbd_1555 YP_315313 74317573 Thiobacillus denitrificans

Tbd_0854 YP_314612 74316872 Thiobacillus denitrificans

Thal_0268 YP 003473030 289548042 Thermocrinis albus

Thal_0267 YP 003473029 289548041 Thermocrinis albus Protein GenBank ID GI Number Organism

Thal_0646 YP 003473406 289548418 Thermocrinis albus

Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate cz^'s-aconitate. Two aconitase enzymes are encoded in the E. coli genome by acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active under conditions of oxidative or acid stress (Cunningham et al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in Salmonella typhimurium are encoded by acnA and acnB (Horswill and Escalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiae aconitase, encoded by ACOl, is localized to the mitochondria where it participates in the TCA cycle (Gangloff et al, Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (Regev-Rudzki et al, Mol. Biol. Cell. 16:4163-4171 (2005)).

Pyruvate :ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation of pyruvate to form acetyl-CoA. The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. Two cysteine residues in this enzyme form a disulfide bond that prtotects it against inactivation in the form of oxygen. This disulfide bond and the stability in the presence of oxygen has been found in other Desulfovibrio species also (Vita et al., Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also well characterized (Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, J Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, encoding a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)). PFORs have also been described in other organisms, including Rhodobacter capsulatas (Yakunin and

Hallenbeck, Biochimica et Biophysica Acta 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al, PNAS 99(14): 9509-14 (2002)). The five subunit PFOR from H.

thermophilus, encoded by porEDABG, was cloned into E. coli and shown to function in both the decarboxylating and C0₂-assimilating directions (Ikeda et al. 2006; Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs also exist in C. carboxidivorans P7. Several additional PFOR enzymes are described in the following review (Ragsdale, S.W., Chem. Rev. 103:2333-2346 (2003)). Finally, flavodoxin reductases (e.g.,fqrB from Helicobacter pylori or Campylobacter jejuni) (St Maurice et al., J Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below.

Protein GenBank ID GI Number Organism

DesfrDRAFT 0121 ZP 07331646.1 303245362 Desulfovibrio fructosovorans

JJ

Por CAA70873.1 1770208 Desulfovibrio africanus por YP 012236.1 46581428 Desulfovibrio vulgaris str.

Hildenborough

Dde_3237 ABB40031.1 78220682 DesulfoVibrio desulfuricans

G20

Ddes_0298 YP_002478891.1 220903579 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774

Por YP_428946.1 83588937 Moorella thermoacetica

YdbK NP_415896.1 16129339 Escherichia coli

nifj (CT1628) NP_66251 1.1 21674446 Chlorobium tepidum

CJE1649 YP l 79630.1 57238499 Campylobacter jejuni nifj ADE85473.1 294476085 Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacter thermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porA BAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891 Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacter thermophilus

FqrB YP 001482096.1 157414840 Campylobacter jejuni

15645778

HP1 164 NP_207955.1 Helicobacter pylori

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

RnfE EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

RnfB EDK3331 1.1 146346775 Clostridium kluyveri

The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes or their combinations thereof. For example, pyruvate dehydrogenase can transform pyruvate into acetyl-CoA with the concomitant reduction of a molecule of NAD into NADH. It is a multi-enzyme complex that catalyzes a series of partial reactions which results in acylating oxidative decarboxylation of pyruvate. The enzyme comprises of three subunits: the pyruvate decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide

dehydrogenase (E3). This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae. In the E. coli enzyme, specific residues in the El component are responsible for substrate specificity (Bisswanger, H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem. 8:535-540 (1969); Gong et al, J. Biol. Chem. 275: 13645-13653 (2000)). Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858 (2008); Kim et al., Appl.

Environ. Microbiol. 73: 1766-1771 (2007); Zhou et al, Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (5). Crystal structures of the enzyme complex from bovine kidney (18) and the E2 catalytic domain from Azotobacter vinelandii are available (4). Yet another enzyme that can catalyze this conversion is pyruvate formate lyase. This enzyme catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate. Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli encoded by pflB (Knappe and Sawers,

FEMS. Microbiol Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen et al, Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al., Oral. Microbiol Immunol. 18:293-297 (2003)). E. coli possesses an additional pyruvate formate lyase, encoded by tdcE, that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Both pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA. Further, a short protein encoded by yfliD in E. coli can associate with and restore activity to oxygen-cleaved pyruvate formate lyase (Vey et al., Proc.Natl. Acad. Sci. U.S.A. 105: 16137-16141 (2008). Note that pflA and pflB from E. coli were expressed in S. cerevisiae as a means to increase cytosolic acetyl-CoA for butanol production as described in WO/2008/080124]. Additional pyruvate formate lyase and activating enzyme candidates, encoded by pfl and act, respectively, are found in Clostridium pasteurianum (Weidner et al, J Bacteriol. 178:2440-2444 (1996)).

Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde; the latter is oxidized to acetate by acetaldehyde

dehydrogenase and subsequently activated to form acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme in several other organisms including E. coli (Kumari et al., J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology 151 :3793-3801 (2005); Starai et al, J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica (described already). Alternatively, acetate can be activated to form acetyl-CoA by acetate kinase and phosphotransacetylase. Acetate kinase first converts acetate into acetyl-phosphate with the accompanying use of an ATP molecule. Acetyl- phosphate and CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase. Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several Clostridia and Methanosarcina thermophila.

Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor. In E. coli, this activity is encoded by poxB. PoxB has similarity to pyruvate decarboxylase of S.

cerevisiae and Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor (Koland and Gennis, Biochemistry 21 :4438-4442 (1982)); O'Brien et al, Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then be converted into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, as described earlier. Some of these enzymes can also catalyze the reverse reaction from acetyl-CoA to pyruvate.

For enzymes that use reducing equivalents in the form of NADH or NADPH, these reduced carriers can be generated by transferring electrons from reduced ferredoxin. Two enzymes catalyze the reversible transfer of electrons from reduced ferredoxins to NAD(P)⁺, ferredoxin:NAD⁺ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP⁺ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP⁺ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low- potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982); Fujii et al, 1977). The Helicobacter pylori FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacter jejuni (St et al. 2007). A ferredoxin:NADP⁺ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD⁺ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD⁺. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD⁺ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus strain TK-6, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Finally, the energy-conserving membrane- associated Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); Herrmann et al., J Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. Additional ferredoxin:NAD(P)+

oxidoreductases have been annotated in Clostridium carboxydivorans P7.

Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase, pyruvate: ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H. thermophilus gene fdxl encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid: ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N- terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7 and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed in the table below.

Protein GenBank ID GI Number Organism

fdxl BAE02673.1 68163284 Hydrogenobacter thermophilus

M11214.1 AAA83524.1 144806 Clostridium pasteurianum

Zjx AAY79867.1 68566938 Sulfolobus acidocalarius

Fdx AAC75578.1 1788874 Escherichia coli

hp_0277 AAD07340.1 2313367 Helicobacter pylori

fdxA CAL34484.1 112359698 Campylobacter jejuni

Moth_0061 ABC18400.1 83571848 Moorella thermoacetica

Moth_1200 ABC19514.1 83572962 Moorella thermoacetica

Moth J ^'888 ABC20188.1 83573636 Moorella thermoacetica

Moth_2112 ABC20404.1 83573852 Moorella thermoacetica

Moth J 037 ABC19351.1 83572799 Moorella thermoacetica

CcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7

CcarbDRAFT_2958 ZP 05392958.1 255526034 Clostridium carboxidivorans P7

CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7

CcarbDRAFT_5296 ZP 05395295.1 255528511 Clostridium carboxidivorans P7 CcarbDRAFT_1615 ZP 05391615.1 255524662 Clostridium carboxidivorans P7

CcarbDRAFT l 304 ZP 05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus

hydrogenoformans

fdxN CAA35699.1 46143 Rhodobacter capsulatus

Rru_A2264 ABC23064.1 83576513 Rhodospirillum rubrum

Rru_A1916 ABC22716.1 83576165 Rhodospirillum rubrum

Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605 Rhodospirillum rubrum

Alvin_2884 ADC63789.1 288897953 Allochromatium vinosum DSM

180

fdx YP 002801146.1 226946073 Azotobacter vinelandii DJ

CKL 3790 YP 001397146.1 153956381 Clostridium kluyveri DSM 555 ferl NP_949965.1 39937689 Rhodopseudomonas palustris

CGA009

fdx CAA12251.1 3724172 Thauera aromatica

CHY_2405 YP 361202.1 78044690 Carboxydothermus

hydrogenoformans

fer YP 359966.1 78045103 Carboxydothermus

hydrogenoformans

fer AAC83945.1 1146198 Bacillus subtilis

fdxl NP 249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP 003148.1 89109368 Escherichia coli K-12

Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, and butyrate, among others.

The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product oicatl of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and

Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004). The succinyl-Co A: acetate CoA-transferase from Acetobacter aceti, encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al. 2008). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoA transferase encoded by pcal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are identified below.

An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-Co A: 3 :ketoacid-Co A transferase (EC 2.8.3.5). Exemplary succinyl-Co A: 3 :ketoacid-Co A transferases are present in

Helicobacter pylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified below.

Converting succinate to succinyl-CoA by succinyl-Co A: 3 :ketoacid-Co A transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA:acetate:CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa.

Exemplary enzymes include the gene products oiatoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008), and ctfAB from Clostridium

saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)) are shown below.

Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J Bact. 183(14) 4288-4295 (2001)). Homologs can be found m ^' Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below.

Additionally, ygfH encodes a propionyl CoA: succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below.

Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting in the cleavage of citrate to acetate and oxaloacetate. The enzyme is active under anaerobic conditions and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and a acyl lyase (beta). Enzyme activation uses covalent binding and acetylation of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3-'-dephospho-CoA, which is similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme into the active holo enzyme (Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD and the citrate lyase synthetase is encoded by citC (Nilekani and

SivaRaman, Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in E. coli (Bekal et al., J Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and

Klebsiella pneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below.

Protein GenBank ID GI Number Organism

citF AAC73716.1 1786832 Escherichia coli

Cite AAC73717.2 87081764 Escherichia coli

citD AAC73718.1 1786834 Escherichia coli

citC AAC73719.2 87081765 Escherichia coli

citG AAC73714.1 1786830 Escherichia coli Protein GenBank ID GI Number Organism

citX AAC73715.1 1786831 Escherichia coli

citF CAA71633.1 2842397 Leuconostoc mesenteroides

Cite CAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998 Salmonella typhimurium cite AAL 19573.1 16419133 Salmonella typhimurium citD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.1 16764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonella typhimurium citX NP_459612.1 16763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependent phosphorylation of acetate to acetylphosphate. Exemplary acetate kinase enzymes have been characterized in many organisms including E. coli, Clostridium acetobutylicum and Methanosarcina thermophila (Ingram-Smith et al., J Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J Biol. Chem. 261 : 13487-13497 (1986); Winzer et al, Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity has also been demonstrated in the gene product of E. coli purT (Marolewski et al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example bukl and buk2 from Clostridium acetobutylicum, also accept acetate as a substrate (Hartmanis, M.G., J Biol. Chem. 262:617-621 (1987)).

Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231 Escherichia coli

Ack AAB18301.1 1491790 Clostridium acetobutylicum Protein GenBank ID GI Number Organism

Ack AAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155 Escherichia coli

bukl NP_349675 15896326 Clostridium acetobutylicum buk2 Q97II1 20137415 Clostridium acetobutylicum

The formation of acetyl-CoA from acetylphosphate is catalyzed by phosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl- CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191 :559-569 (969)).

Additional acetyltransferase enzymes have been characterized in Bacillus subtilis (Rado and Hoch, Biochim. Biophys. Acta 321 : 114-125 (1973), Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1 :5896-599 (1955), and Thermotoga maritima (Bock et al., J Bacteriol. 181 : 1861-1867 (1999)). This reaction is also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19) including the ptb gene products from Clostridium acetobutylicum (Wiesenborn et al., App. Environ. Microbiol. 55:317-322 (1989); Walter et al., Gene

134: 107-111 (1993)). Additional ptb genes are found in butyrate-producing bacterium L2-50 (Louis et al., J Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al., Curr. Microbiol. 42:345-349 (2001).

The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase activity. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl- CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al., J Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and

Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43: 1425-1431 (2004)). ADP- forming acetyl-CoA synthetases are reversible enzymes with a generally broad substrate range (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). Two isozymes of ADP- forming acetyl-CoA synthetases are encoded in the Archaeoglobus fulgidus genome by are encoded by AF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and reversibility of the enzyme was demonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)). Additional candidates include the succinyl- CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez- Valverde et al., Appl. Environ. Microbiol. 59: 1149-1154 (1993)). The aforementioned proteins are tabulated below.

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichia coli

acoE AAA21945.1 141890 Ralstonia eutropha

acsl ABC87079.1 86169671 Methanothermobacter

thermautotrophicus

acsl AAL23099.1 16422835 Salmonella enterica

ACS1 Q01574.2 257050994 Saccharomyces cerevisiae

AF1211 NP 070039.1 11498810 Archaeoglobus fulgidus

AF1983 NP_070807.1 11499565 Archaeoglobus fulgidus scs YP_135572.1 55377722 Haloarcula marismortui

PAE3250 NP 560604.1 18313937 Pyrobaculum aerophilum str. IM2 Protein GenBank ID GI Number Organism sucC NP_415256.1 16128703 Escherichia coli

sucD AAC73823.1 1786949 Escherichia coli

paaF AAC24333.2 22711873 Pseudomonas putida

The product yields per C-mol of substrate of microbial cells synthesizing reduced

fermentation products such as adipate, 6-aminocaproic acid, caprolactam or

hexamethylenediamine, are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from synthesis gas components such as CO and ¾ using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes, respectively. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, ¾, or water, respectively. Reduced ferredoxin and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway and reductive TCA cycle enzymes.

Here, we show specific examples of how additional redox availability from CO and/or ¾ can improve the yields of reduced products such as adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine.

The maximum theoretical yield to produce adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine from glucose is 0.923 mole adipate, 0.80 mole 6-aminocaproic acid, 0.80 mole caprolactam or 0.706 mole hexamethylenediamine per mole of glucose.

13 C₆H₁₂0₆ = 12 C₆H₁₀O₄ + 6 C0₂ + 18 H₂0 5 C₆H₁₂0₆ + 4 NH₃ = 4 C₆H₁₃N0₂ + 6 C0₂ + 10 H₂0

5 C₆H₁₂0₆ + 4 NH₃ = 4 C₆H_nNO + 6 C0₂ + 14 H₂0

17 C₆H₁₂0₆ + 24 NH₃ = 12 C₆H₁₆N₂ + 30 C0₂ + 42 H₂0

Similar high yields can be obtained on other carbohydrates (e.g., xylose and arabinose).

15.6 C₅H₁₀O₅ = 12 C₆H₁₀O₄ + 6 C0₂ + 18 H₂0 6 C₅H₁₀O₅ + 4 NH₃ = 4 C₆H₁₃N0₂ + 6 C0₂ + 10 H₂0

6 C₅H₁₀O₅ + 4 NH₃ = 4 C₆H_nNO + 6 C0₂ + 14 H₂0

20.4 C₅H₁₀O₅ + 24 NH₃ = 12 C₆H₁₆N₂ + 30 C0₂ + 42 H₂0

Achieving these maximum yields via the pathway described in Figure 39B requires activity of both the reductive and oxidative TCA cycle to form succinyl-CoA. This enables the appropriate balance the intracellular reducing equivalents.

Achieving these maximum yields via the pathway described in Figure 40B requires activity of both oxidative and reductive TCA cycle enzymes. For example, the presence of at least one enzyme to produce alpha-ketoglutarate from succinyl-CoA (e.g., AKG ferredoxin oxidoreductase) is required. Otherwise, the maximum yield is capped at 0.67 mol/mol glucose for all of the nylon intermediates (i.e., 0.5 glucose molecules are required to make one acetyl-CoA molecule, 1.0 glucose molecule is required to make 1.0 alpha-ketoglutarate molecule via the oxidative TCA cycle, and each nylon intermediate requires one acetyl-CoA and one alpha-ketoglutarate molecule).

Achieving the maximum yields via the pathway described in Figure 38B requires the complete reductive TCA cycle, particularly for the conversion of AKG to acetyl-CoA (e.g., AKG ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, citrate lyase, ATP citrate lyase, acetate kinase, phosphotransacetylase, or acetyl-CoA synthetase). Otherwise the maximum yield is capped at 0.67 mol/mol glucose for each of the nylon intermediates (i.e., 0.5 glucose molecules are required per acetyl-CoA molecule produced from glycolysis and 3 acetyl-CoA molecules are required to synthesize each nylon intermediate.).

When both feedstocks of sugar and syngas are available, the syngas components CO and H₂ can be utilized to generate reducing equivalents by employing the hydrogenase and CO dehydrogenase. The reducing equivalents generated from syngas components will be utilized to power the glucose to adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine production pathways. Theoretically, all carbons in glucose can be conserved, thus resulting in a maximal theoretical yield to produce adipate, 6-aminocaproic acid, caprolactam or hexamethylenediamine from glucose at 1.0 mole adipate, 1.0 mole 6-aminocaproic acid, 1.0 mole caprolactam or 1.0 mole hexamethylenediamine per mol of glucose. Achieving these maximum yields via the pathway described in Figure 39B requires activity of the reductive TCA cycle to generate succinyl-CoA but does not require additional reducing equivalents to be synthesized via the oxidative TCA cycle.

Similarly, achieving these maximum yields via the pathway described in Figure 40B requires activity of the reductive TCA cycle to generate alpha-ketoglutarate but does not require additional reducing equivalents to be synthesized via the oxidative TCA cycle. In particular, at least one enzyme must be present to convert succinyl-CoA to alpha-ketoglutarate (e.g., AKG ferredoxin oxidoreductase).

Achieving the maximum yields via the pathway described in Figure 38B requires the complete reductive TCA cycle, particularly for the conversion of AKG to acetyl-CoA (e.g., AKG ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, citrate lyase, ATP citrate lyase, acetate kinase, phosphotransacetylase, or acetyl-CoA synthetase).

In all cases, enzymes for the extraction of reducing equivalents from CO and/or H2 (e.g., carbon monoxide dehydrogenase, hydrogenase, ferredoxin:NAD(P)+ oxidoreductase) are also required.

As shown in above example, a combined feedstock strategy where syngas is combined with a sugar-based feedstock or other carbon substrate can greatly improve the theoretical yields. In this co-feeding appoach, syngas components H₂ and CO can be utilized by the hydrogenase and CO dehydrogenase to generate reducing equivalents, that can be used to power chemical production pathways in which the carbons from sugar or other carbon substrates will be maximally conserved and the theoretical yields improved. Such improvements provide environmental and economic benefits and greatly enhance sustainable chemical production. Alternatively, note that the pathways described in Figures 2-14, 20-27 and 38-40 allow for the direct conversion of carbon dioxide and/or carbon monoxide to adipate, caprolactam, 6- aminocaproate, or hexamethylenediamine assuming an ample supply of reducing equivalents (e.g., CO and/or H2) and at least one enzyme selected from carbon monoxide dehydrogenase and hydrogenase.

Herein below the enzymes and the corresponding genes used for extracting redox from synags components are described. CODH is a reversible enzyme that interconverts CO and C0₂ at the expense or gain of electrons. The natural physiological role of the CODH in

ACS/CODH complexes is to convert C0₂ to CO for incorporation into acetyl-CoA by acetyl- Co A synthase. Nevertheless, such CODH enzymes are suitable for the extraction of reducing equivalents from CO due to the reversible nature of such enzymes. Expressing such CODH enzymes in the absence of ACS allows them to operate in the direction opposite to their natural physiological role (i.e., CO oxidation). In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The M. thermoacetica gene (Genbank Accession Number: YP_430813) is expressed by itself in an operon and is believed to transfer electrons from CO to an external mediator like ferredoxin in a "Ping-pong" reaction. The reduced mediator then couples to other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191 :243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., J Bacteriol.

183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al, Science 293: 1281-1285 (2001)). Similar ACS-free CODH enzymes can be found in a diverse array of organisms including Geobacter metallireducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, and

Campylobacter curvus 525.92.

Protein GenBank ID GI Number Organism

CODH (putative) YP_430813 83590804 Moorella thermoacetica

CODH-II (CooS-II) YP_358957 78044574 Carboxydothermus

hydrogenoformans

CooF YP_358958 78045112 Carboxydothermus

hydrogenoformans

CODH ( putative) ZP 05390164.1 255523193 Clostridium carboxidivorans

P7

CcarbDRAFT_0341 ZP 05390341.1 255523371 Clostridium carboxidivorans

P7 CcarbDRAFT_1756 ZP 05391756.1 255524806 Clostridium carboxidivorans

P7

CcarbDRAFT_2944 ZP 05392944.1 255526020 Clostridium carboxidivorans

P7

CODH YP_384856.1 78223109 Geobacter metallireducens

GS-15

Cpha266_0148 YP 910642.1 119355998 Chlorobium

(cytochrome c) phaeobacteroides DSM266

Cpha266_0149 YP 910643.1 119355999 Chlorobium

phaeobacteroides DSM266 (CODH)

Ccel_0438 YP 002504800.1 220927891 Clostridium cellulolyticum

H10

Ddes_0382 YP_002478973.1 220903661 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC (CODH)

27774

Ddes_0381 (CooC) YP_002478972.1 220903660 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC

27774

Pcar_0057 YP 55490.1 7791767 Pelobacter carbinolicus DSM

2380

(CODH)

Pcar_0058 YP 55491.1 7791766 Pelobacter carbinolicus DSM

2380

(CooC)

Pcar_0058 YP 55492.1 7791765 Pelobacter carbinolicus DSM

2380

(HypA)

CooS (CODH) YP 001407343.1 154175407 Campylobacter curvus 525.92

In some cases, hydrogenase encoding genes are located adjacent to a CODH. In

Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H₂0 to C0₂ and H₂ (Fox et al., J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. I :e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0₂ reduction activities when linked to an electrode (Parkin et al., J Am.Chem.Soc. 129: 10328- 10329 (2007)). The protein sequences of exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers. Protein GenBank ID GI Number Organism

CODH-I YP 360644 78043475 Carboxydothermus (CooS-I) hydrogenoformans

CooF YP 360645 78044791 Carboxydothermus

hydrogenoformans

HypA YP 360646 78044340 Carboxydothermus

hydrogenoformans

CooH YP 360647 78043871 Carboxydothermus

hydrogenoformans

CooU YP 360648 78044023 Carboxydothermus

hydrogenoformans

CooX YP 360649 78043124 Carboxydothermus

hydrogenoformans

CooL YP 360650 78043938 Carboxydothermus

hydrogenoformans

CooK YP 360651 78044700 Carboxydothermus

hydrogenoformans

CooM YP 360652 78043942 Carboxydothermus

hydrogenoformans

CooC YP 360654.1 78043296 Carboxydothermus

hydrogenoformans

CooA-1 YP 360655.1 78044021 Carboxydothermus

hydrogenoformans

CooL AAC45118 1515468 Rhodospirillum rubrum

CooX AAC45119 1515469 Rhodospirillum rubrum

CooU AAC45120 1515470 Rhodospirillum rubrum

CooH AAC45121 1498746 Rhodospirillum rubrum

CooF AAC45122 1498747 Rhodospirillum rubrum

CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum

CooC AAC45124 1498749 Rhodospirillum rubrum

CooT AAC45125 1498750 Rhodospirillum rubrum

CooJ AAC45126 1498751 Rhodospirillum rubrum

Native to E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol. 164: 1324-1331 (1985); Sawers and Boxer, Eur.J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities, E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1 and Hyd-2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters, respectively (Lukey et al., How E. coli is equipped to oxidize hydrogen under different redox conditions, J Biol Chem published online Nov 16, 2009). Hyd-1 is oxygen- tolerant, irreversible, and is coupled to quinone reduction via the hyaC cytochrome. Hyd-2 is sensitive to 0₂, reversible, and transfers electrons to the periplasmic ferredoxin hybA which, in turn, reduces a quinone via the hybB integral membrane protein. Reduced quinones can serve as the source of electrons for fumarate reductase in the reductive branch of the TCA cycle. Reduced ferredoxins can be used by enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPH or NADH. They can alternatively be used as the electron donor for reactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxin oxidoreductase, and 5, 10-methylene-H4 folate reductase.

The hydrogen-lyase systems of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase 3 has been shown to be a reversible enzyme (Maeda et al., Appl Microbiol Biotechnol 76(5): 1035-42 (2007)). Hydrogenase activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al., J BacterioL 190: 1447-1458 (2008)).

Protein GenBank ID GI Number Organism

HypA NP_417206 16130633 Escherichia coli HypB NP_417207 16130634 Escherichia coli

HypC NP_417208 16130635 Escherichia coli

HypD NP_417209 16130636 Escherichia coli

HypE NP_417210 226524740 Escherichia coli

HypF NP_417192 16130619 Escherichia coli

The M. thermoacetica hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica can grow with C0₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Re*. Microbiol. 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)) (see Figure 38A). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. The protein sequences encoded for by these genes are identified by the following GenBank accession numbers.

Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below.

Proteins in M. thermoacetica that are homologous to the E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table.

Protein GenBank ID GI Number Organism

Moth_2182 YP_431014 83591005 Moorella thermoacetica

Moth_2183 YP_431015 83591006 Moorella thermoacetica

Moth_2184 YP 431016 83591007 Moorella thermoacetica Moth_2185 YP_431017 83591008 Moorella thermoacetica

Moth_2186 YP_431018 83591009 Moorella thermoacetica

Moth_2187 YP 431019 83591010 Moorella thermoacetica

Moth_2188 YP 431020 83591011 Moorella thermoacetica

Moth_2189 YP_431021 83591012 Moorella thermoacetica

Moth_2190 YP_431022 83591013 Moorella thermoacetica

Moth_2191 YP_431023 83591014 Moorella thermoacetica

Moth_2192 YP_431024 83591015 Moorella thermoacetica

In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and their corresponding protein sequences are provided below.

Protein GenBank ID GI Number Organism

Moth_0439 YP_429313 83589304 Moorella thermoacetica

Moth_0440 YP_429314 83589305 Moorella thermoacetica

Moth_0441 YP_429315 83589306 Moorella thermoacetica

Moth_0442 YP_429316 83589307 Moorella thermoacetica

Moth_0809 YP 429670 83589661 Moorella thermoacetica

Moth_0810 YP_429671 83589662 Moorella thermoacetica

Moth_0811 YP_429672 83589663 Moorella thermoacetica

Moth_0812 YP_429673 83589664 Moorella thermoacetica

Moth_0814 YP_429674 83589665 Moorella thermoacetica

Moth_0815 YP_429675 83589666 Moorella thermoacetica

Moth_0816 YP 429676 83589667 Moorella thermoacetica

Moth_1193 YP 430050 83590041 Moorella thermoacetica

Moth_1194 YP 430051 83590042 Moorella thermoacetica

Moth_1195 YP_430052 83590043 Moorella thermoacetica

Moth_1196 YP 430053 83590044 Moorella thermoacetica

Moth_1717 YP_430562 83590553 Moorella thermoacetica

Moth_1718 YP_430563 83590554 Moorella thermoacetica

Moth_1719 YP_430564 83590555 Moorella thermoacetica

Moth_1883 YP_430726 83590717 Moorella thermoacetica

Moth_1884 YP_430727 83590718 Moorella thermoacetica

Moth_1885 YP_430728 83590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorella thermoacetica

Moth_1887 YP_430730 83590721 Moorella thermoacetica

Moth_1888 YP_430731 83590722 Moorella thermoacetica

Moth_1452 YP 430305 83590296 Moorella thermoacetica

Moth_1453 YP 430306 83590297 Moorella thermoacetica

Moth_1454 YP_430307 83590298 Moorella thermoacetica

Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an "02-tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0₂-tolerant soluble

hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J Biol. Chem. 284(52), 36462-36472 (2009)).

Protein GenBank ID GI Number Organism

HoxF NP_942727.1 38637753 Ralstonia eutropha HI 6

HoxU NP_942728.1 38637754 Ralstonia eutropha HI 6

HoxY NP_942729.1 38637755 Ralstonia eutropha HI 6

HoxH NP_942730.1 38637756 Ralstonia eutropha HI 6

HoxW NP_942731.1 38637757 Ralstonia eutropha HI 6

Hoxl NP_942732.1 38637758 Ralstonia eutropha HI 6

HoxE NP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815 Geobacter sulfurreducens

HoxU NP_953765.1 39997814 Geobacter sulfurreducens

HoxY NP_953764.1 39997813 Geobacter sulfurreducens

HoxH NP_953763.1 39997812 Geobacter sulfurreducens

GSU2717 NP_953762.1 39997811 Geobacter sulfurreducens

HoxE NP_441418.1 16330690 Synechocystis str. PCC

6803

HoxF NP_441417.1 16330689 Synechocystis str. PCC

6803

Unknown function NP_441416.1 16330688 Synechocystis str. PCC

6803

HoxU NP_441415.1 16330687 Synechocystis str. PCC

6803

HoxY NP_441414.1 16330686 Synechocystis str. PCC

6803

Unknown function NP_441413.1 16330685 Synechocystis str. PCC

6803

Unknown function NP_441412.1 16330684 Synechocystis str. PCC

6803

HoxH NP_441411.1 16330683 Synechocystis str. PCC

6803

HypF NP_484737.1 17228189 Nostoc sp. PCC 7120

HypC NP_484738.1 17228190 Nostoc sp. PCC 7120

HypD NP_484739.1 17228191 Nostoc sp. PCC 7120

Unknown function NP_484740.1 17228192 Nostoc sp. PCC 7120

HypE NP_484741.1 17228193 Nostoc sp. PCC 7120

HypA NP_484742.1 17228194 Nostoc sp. PCC 7120

HypB NP_484743.1 17228195 Nostoc sp. PCC 7120

HoxlE AAP50519.1 37787351 Thiocapsa roseopersicina

HoxlF AAP50520.1 37787352 Thiocapsa roseopersicina

HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina

HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina

HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina Several enzymes and the corresponding genes used for fixing carbon dioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycle intermediates, oxaloacetate or malate are described below.

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by phosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys. 414: 170-179 (2003), ppcA in Methylobacterium extorquens AMI (Arps et al., J Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacterium glutamicum (Eikmanns et al, Mol. Gen. Genet. 218:330-339 (1989).

An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al., FEBS Lett. 258:313- 316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K_m for bicarbonate of PEP carboxykinase (Kim et al., Appl. Environ. Microbiol. 70: 1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al, J Microbiol. Biotechnol. 16: 1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHC0₃ concentrations. Mutant strains of E. coli can adopt Pck as the dominant C02-fixing enzyme following adaptive evolution (Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum succiniciproducens (Laivenieks et al., Appl. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP.

Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate to oxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes are encoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991) and PYC2 (Walker et al, supra) in

Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475: 191-206 (2000)).

Malic enzyme can be applied to convert C0₂ and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent). For example, one of the E. coli malic enzymes (Takeo, J Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion of pyruvate and C0₂ to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport. Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E. coli while restoring the lethal Apfl-AldhA phenotype under anaerobic conditions by operating in the carbon-fixing direction (Stols and Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from Ascaris suum m E. coli (Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al, J. Biochem. 85(5): 1355-65 (1979)).

The enzymes used for converting oxaloacetate (formed from, for example, PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate (formed from, for example, malic enzyme or malate dehydrogenase) to succinyl-CoA via the reductive branch of the TCA cycle are malate dehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, and succinyl-CoA transferase. The genes for each of the enzymes are described herein.

Enzymes, genes and methods for engineering pathways from succinyl-CoA to various products into a microorganism are now known in the art. The additional reducing equivalents obtained from CO and/or H₂, as disclosed herein, improve the yields of adipate, 6- aminocaproic acid, caprolactam or hexamethylenediamine when utilizing carbohydrate-based feedstock.

Enzymes, genes and methods for engineering pathways from glycolysis intermediates to various products into a microorganism are known in the art. The additional reducing equivalents obtained from CO and H₂, as described herein, improve the yields of all these products on carbohydrates.

EXAMPLE XXXIII

Methods for Handling CO and Anaerobic Cultures This example describes methods used in handling CO and anaerobic cultures. A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures and assays that utilized CO required special handling. Several assays, including CO oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization in small batch cultures, called for small quantities of the CO gas that were dispensed and handled within a fume hood.

Biochemical assays called for saturating very small quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing the assay. All of the CO handling steps were performed in a fume hood with the sash set at the proper height and blower turned on; CO was dispensed from a compressed gas cylinder and the regulator connected to a Schlenk line. The latter ensures that equal concentrations of CO were dispensed to each of several possible cuvettes or vials. The Schlenk line was set up containing an oxygen scrubber on the input side and an oil pressure release bubbler and vent on the other side. Assay cuvettes were both anaerobic and CO -containing. Threfore, the assay cuvettes were tightly sealed with a rubber stopper and reagents were added or removed using gas-tight needles and syringes. Secondly, small (-50 mL) cultures were grown with saturating CO in tightly stoppered serum bottles. As with the biochemical assays, the CO-saturated microbial cultures were equilibrated in the fume hood using the Schlenk line setup. Both the biochemical assays and microbial cultures were in portable, sealed containers and in small volumes making for safe handling outside of the fume hood. The compressed CO tank was adjacent to the fume hood.

Typically, a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber. The glass or quartz spectrophotometer cuvettes have a circular hole on top into which a Kontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned proximal to the fume hood.

B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and ¾ to simulate syngas as a feedstock in fermentative production. Therefore, quantities of cells ranging from 1 liter to several liters can include the addition of CO gas to increase the dissolved concentration of CO in the medium. In these circumstances, fairly large and continuously administered quantities of CO gas are added to the cultures. At different points, the cultures are harvested or samples removed. Alternatively, cells are harvested with an integrated continuous flow centrifuge that is part of the fermenter.

The fermentative processes are carried out under anaerobic conditions. In some cases, it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen saturation to provide a respiratory environment. In addition, the reducing power generated during anaerobic fermentation may be needed in product formation rather than respiration.

Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying degrees. Classic acetogens such as M. thermoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive to irreversible inactivation by molecular oxygen. While there are oxygen-tolerant acetogens, the repertoire of enzymes in the Wood- Ljungdahl pathway might be incompatible in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins, and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize energy acquisition. At the same time, cells in culture act as oxygen scavengers that moderate the need for extreme measures in the presence of large cell growth.

C. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an 0₂ concentration of 1 ppm or less and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 0₂ electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N₂ prior to

introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels. The chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve.

The anaerobic chambers achieved levels of 0₂ that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based 0₂ monitoring, test strips can be used instead.

D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for -30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCl is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B12 (10 μΜ cyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 μΜ— made as 100-lOOOx stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pAl-lacOl promoter in the vectors was performed by addition of isopropyl β-D-l- thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs. Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles.

EXAMPLE XXXIV CO oxidation (CODH) Assay

This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH).

The 7 gene CODH/ ACS operon of Moorella thermoacetica was cloned into E. coli expression vectors. The intact -10 kbp DNA fragment was cloned, and it is likely that some of the genes in this region are expressed from their own endogenous promoters and all contain endogenous ribosomal binding sites. These clones were assayed for CO oxidation, using an assay that quantitatively measures CODH activity. Antisera to the M. thermoacetica gene products was used for Western blots to estimate specific activity. M. thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in E. coli. This activity, described below in more detail, was estimated to be ~l/50th of the M. thermoacetica specific activity. It is possible that CODH activity of recombinant E. coli cells could be limited by the fact that M. thermoacetica enzymes have temperature optima around 55°C. Therefore, a mesophilic CODH/ ACS pathway could be advantageous such as the close relative of Moorella that is mesophilic and does have an apparently intact CODH/ ACS operon and a Wood-Ljungdahl pathway, Desulfitobacterium hafniense. Acetogens as potential host organisms include, but are not limited to, Rhodospirillum rubrum, Moorella thermoacetica and Desulfitobacterium hafniense.

CO oxidation is both the most sensitive and most robust of the CODH/ ACS assays. It is likely that an E. co/z^'-based syngas using system will ultimately need to be about as anaerobic as Clostridial (i.e., Moorella) systems, especially for maximal activity. Improvement in CODH should be possible but will ultimately be limited by the solubility of CO gas in water. Initially, each of the genes was cloned individually into expression vectors. Combined expression units for multiple subunits/1 complex were generated. Expression in E. coli at the protein level was determined. Both combined M. thermoacetica CODH/ACS operons and individual expression clones were made. CO oxidation assay. This assay is one of the simpler, reliable, and more versatile assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955 (2004)). A typical activity of M. thermoacetica CODH specific activity is 500 U at 55°C or ~60U at 25°C. This assay employs reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes. In more detail, glass rubber stoppered cuvettes were prepared after first washing the cuvette four times in deionized water and one time with acetone. A small amount of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen (CH₃ viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration. When methyl viologen was added, an 18 Ga needle (partial) was used as a jacket to facilitate use of a Hamilton syringe to withdraw the CH₃ viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up the CH₃ viologen stock to slightly reduce the CH₃ viologen. The temperature was equilibrated to 55°C in a heated Olis spectrophotometer (Bogart GA). A blank reaction (CH₃ viologen + buffer) was run first to measure the base rate of CH₃ viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91 (CODH-ACS operon of M. thermoacetica with and without, respectively, the first cooC). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH₃ viologen turns purple. The results of an assay are shown in Table I. Table I. Crude extract CO Oxidation Activities.

ACS90 7.7 mg/ml ACS91 11.8 mg/ml

: Mta98 9.8 mg/ml : Mta99 11.2 mg/ml

Extract Vol OD/ U/ml U/me

ACS90 lO microliters 0.073 0.376 0.049

ACS91 lO microliters 0.096 0.494 0.042

Mta99 lO microliters 0.0031 0.016 0.0014

ACS90 lO microliters 0.099 0.51 0.066

Mta99 25 microliters 0.012 0.025 0.0022

ACS91 25 microliters 0.215 0.443 0.037

Mta98 25 microliters 0.019 0.039 0.004

ACS91 lO microliters 0.129 0.66 0.056

Averages

ACS90 0.057 U/mg

ACS91 0.045 U/mg

Mta99 0.0018 U/mg

Mta98/Mta99 are E. coli MG1655 strains that express methanol methyltransferase genes from M. thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons.

If ~ 1% of the cellular protein is CODH, then these figures would be approximately 100X less than the 500 U/mg activity of pure M. thermoacetica CODH. Actual estimates based on Western blots are 0.5% of the cellular protein, so the activity is about 50X less than for M. thermoacetica CODH. Nevertheless, this experiment demonstrates CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation (C¾ viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce C¾ viologen.

To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe-S assays. The antisera used were polyclonal to purified M. thermoacetica CODH-ACS and Mtr proteins and were visualized using an alkaline phosphatase-linked goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown in Figure 41. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes. Expression of CODH-ACS operon genes including 2 CODH subunits and the methyltransferase were confirmed via Western blot analysis. Therefore, the recombinant E. coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase and corrinoid iron sulfur protein were active in the same recombinant E. coli cells. These proteins are part of the same operon cloned into the same cells.

The CO oxidation assays were repeated using extracts of Moorella thermoacetica cells for the positive controls. Though CODH activity in E. coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the M. thermoacetica control. The results of the assay are shown in Figure 42. Briefly, cells ( thermoacetica or E. coli with the CODH/ ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared as described herein. Assays were performed as described above at 55°C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. These results describe the CO oxidation (CODH) assay and results. Recombinant E. coli cells expressed CO oxidation activity as measured by the methyl viologen reduction assay.

EXAMPLE XXXV

E. coli CO Tolerance Experiment and CO Concentration Assay (myoglobin assay) This example describes the tolerance of E. coli for high concentrations of CO.

To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing solution, NiCl₂, Fe(II)NH₄S0₄, cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology in small volumes. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33) was used as a control, and cultures containing the pZA33 empty vector as well as both ACS90 and ACS91 were tested with both N₂ and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37°C. At the end of the 36 hour period, examination of the flasks showed high amounts of growth in all. The bulk of the observed growth occurred overnight with a long lag.

Given that all cultures appeared to grow well in the presence of CO, the final CO

concentrations were confirmed. This was performed using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20°C and 1 atm.

For the myoglobin test of CO concentration, cuvettes were washed 10X with water, IX with acetone, and then stoppered as with the CODH assay. N₂ was blown into the cuvettes for -10 min. A volume of 1 ml of anaerobic buffer (HEPES, pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton syringe. A volume of 10 microliter myoglobin (-1 mM— can be varied, just need a fairly large amount) and 1 microliter dithionite (20 mM stock) were added. A CO standard curve was made using CO saturated buffer added at 1 microliter increments. Peak height and shift was recorded for each increment. The cultures tested were pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in 1 microliter increments to the same cuvette. Midway through the experiment a second cuvette was set up and used. The results are shown in Table II.

Table II. Carbon Monoxide Concentrations, 36 hrs.

Strain and Growth Conditions Final CO concentration (micromolar)

pZA33-CO 930

ACS90-CO 638

494

734

883

ave 687

SD 164

ACS91-CO 728

812

760

611

ave. 728

SD 85 The results shown in Table II indicate that the cultures grew whether or not a strain was cultured in the presence of CO or not. These results indicate that E. coli can tolerate exposure to CO under anaerobic conditions and that E. coli cells expressing the CODH-ACS operon can metabolize some of the CO. These results demonstrate that E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in place of CO. This experiment demonstrated that laboratory strains of E. coli are insensitive to CO at the levels achievable in a syngas project performed at normal atmospheric pressure. In addition, preliminary experiments indicated that the recombinant E. coli cells expressing CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide.

EXAMPLE XXXVI

The following example provides various gene candidates for enzymes disclosed herein for convertion of alpha-ketoglutarate to alpha-ketoadipate as depicted in Figure 40B.

Enzyme Gene Organis Accession No, GI Number

A. Homocitrate Methanocaldococcus

synthase aksA j annas chii Q57926.3 GL327478593 lies Thermus thermophilus 2ZYF A GL281500682

Saccharomyces

LYS20 cerevisiae S288c NP 010099.1 GL6320019

Saccharomyces

I..YS21 cerevisiae S288c NP 010151.1 GL6320071 iiiiV Klebsiella pneumoniae P05345.2 GI: 128323

B. Homocitrate Methanocaldococcus

dehydratase aksE j annas chii Q58667.1 GI:3122344

C. cis-

Homoaconitate Methanocaldococcus

hydratase aksD j annas chii Q58409.1 GI:3122347

(candidates do

both steps)

Methanococcus

MMP1480 maripaludis S2 NP 988600.1 GL45359043

Methanococcus

MMP0381 maripaludis S2 YP 001097769.1 GI: 134046284

Saccharomyces

LYS4 cerevisiae S288c NP 010520.1 GL6320440 Enzyme Gene Organis Accessiosi No. GI Number

D. Threo- isohomocitrate Methanocaldococcus

dehydrogenase aksF j annas chii ACA28837.1 GL 168805636

Saccharomyces

LYS12 cerevisiae S288c NP 012172.1 GL6322097 hicdh Thermus thermophilus 1X0L B GI:78100866

EXAMPLE XXXVII

The following example provides various gene candidates for enzymes disclosed herein for convertion of acetyl-CoA to pimelyl-acp as depicted in 38B.

Gene

Name Organism Accession Number GI Number bioC Escherichia coli NP 415298 16128745

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

bioC ATCC 9150 YP 151177 56414102 bioC Klebsiella pneumoniae 342 YP 002239590 206578655 fabH Escherichia coli NP 415609 16129054

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

fabH ATCC 9150 YP 150896 56413821 fabH Klebsiella pneumoniae 342 YP 002239290 206579577 fabG Escherichia coli NP 415611 16129056

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

fabG ATCC 9150 YP 150894 56413819 fabG Klebsiella pneumoniae 342 YP 002238066 206579712 fabZ Escherichia coli NP 414722 16128173

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

fabZ ATCC 9150 YP 149575 56412500 fabZ Klebsiella pneumoniae 342 YP 002240332 206576644 fabl Escherichia coli NP 415804 16129249

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

fabl ATCC 9150 YP 150450 56413375 fabl Klebsiella pneumoniae 342 YP 002238974 206581008 fabB Escherichia coli NP 416826 16130258

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

fabB ATCC 9150 YP 149800 56412725 Gene

Name Organism Accession Number GI Number fabB Klebsiella pneumoniae 342 YP 002237279 206577113 bioH Escherichia coli NP 417871 16131288

Salmonella enterica subsp.

enterica serovar Paratyphi A str.

bioH ATCC 9150 YP 152499 56415424 bioH Klebsiella pneumoniae 342 YP 002236215 206580545

EXAMPLE XXXVIII

The following example provides various gene candidates for enzymes disclosed herein for convertion of pimelyl-acp to alpha-ketopimelate as depicted in 38B. Moreover, enzymes and gene candidates for conversion of pimelyl-acp to alpha-ketopimelate (AKP) are well known in the art. For example, WO 2010/068944, which is herein incorporatred by reference, discloses several enzymes that can be used for the pathways disclosed herein.

Pimelate is known as intermediate in biotin biosynthesis and as such, the inventors consider that organisms capable of de-novo synthesis of biotin are expected to also contain a synthetic pathway for pimelate. Pimelate has been described to be produced from fatty acids (via oxidation thereof). This results in a break of the carbon chain and yields the second carboxylic acid functionality (W. R. Streit, P. Entcheva. Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production. Appl Microbiol Biotechnol (2003) 61 :21- 31 ; Max J. Cryle, lime Schlichting. Structural insights from a P450 Carrier Protein complex reveal how specificity is achieved in the P450Biol ACP complex. PNAS (2008) 105 (41 ): 15696-15701 ).

Further organisms providing the enzyme system for pimelate synthesis may be selected from genera of the Bacillus sensu lato group, Geobacillus, Brevibacillus and the like (see Table 1 in Zeigler and Perkins, 2008, Practical Handbook of Microbiology", Second Edition (E.

Goldman and L. Green, eds.), pp 301 - 329, CRC Press, Boca Raton, FL). In particular from Bacillus species represented by the Bacillus sensu stricto group, in particular Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus anthracis, Bacillus firmus, Bacillus

pantothenticus, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus megaterium, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus halodurans (Zeigler and Perkins, 2008, Ibid). More in particular, from Bacillus subtilis 168 and its strain derivatives. Further, organisms providing the enzyme system for pimelate synthesis may also be selected from genera of e.g. Corynebacterium, Lactobacillus, Lactococci, Streptomyces, and Pseudomonas. In particular, a host cell comprising an enzyme system for synthesising pimelate may be selected from the group of gram-positive bacteria (Streit and Entcheva, Appl Microbiol Biotechnol (2003) 61 :21 -31 ) For instance, Bacillus sphaericus has been reported to comprise an enzyme system for synthesising pimelate (Gloeckler et al., Gene 87:63-70, 1990). Further, Bacillus subtilis is an example of an organism comprising enzymes for a pimelate synthesis pathway (see e.g. EP-A 635 572).

Gram negative bacteria may also provide pimelic acid. These microbes usually also comprise an enzyme system to prepare pimeloyl-CoA, see for instance for Escherichia coll Otsuka et al, J. Biol. Chem . 263: 19577-19585 (1988); O'Regan et al. Nucleic Acids Res . 17:8004

(1989))). Even in case wild-type strains of these bacteria are not capable of producing pimelic acid, by their capacity to prepare pimeloyl-CoA, they may provide a source for pimelate, in that upon hydrolysis of pimeloyl-CoA, pimelate is formed.

Thioesterase gene candidates

Pimelate-2-monooxygenase

In a further embodiment, the enzyme catalysing the oxidation of heptane dioic acid into 2- hydroxyheptanedioic acid is a hydroxylase with pimelate hydroxylase activity. In a further embodiment, the enzyme catalysing the oxidation of heptane dioic acid into 2- hydroxyheptanedioic acid is a hydroxylase with pimelate-2- monooxygenase activity. An enzyme catalysing the oxidation of heptane dioic acid into 2- hydroxyheptanedioic acid may in principle be selected from any organism having a nucleic acid sequence encoding such enzyme. In particular the enzyme may originate from an organism selected from the group of Corynebacterium, Escherichia (e.g. EC 1.1 .3.3 - malate oxidase: from Escherichia coli or an enzyme activity from E. coli referred to in the list of sequences herein below) Bacillus, Pichia, Pseudomonas, Vibrio, Zymonas, Aspergillus, Rattus (e.g. EC 1 .1 .1 .98: (R)-2- hydroxy-fatty-acid dehydrogenases or EC 1.1 .1 .99: (S)-2-hydroxy-fatty-acid

dehydrogenases from rat kidney), Primates (e.g. EC 1 .1 .1.172 : 2-oxoadipate reductases from human placenta), Saccharomyces (e.g. EC 1 .1 .99.6: D-2-hydroxy-acid dehydrogenase or an enzyme activity from Saccharomyces referred to in the list of sequences herein below), Mirococcus (e.g. EC 1.1 .3.3 - malate oxidase from Micrococcus lysodeikticus),

Gluconobacter, Caenorhabditis, Drosophila, Leporidae (e.g. EC 1 .1 .99.6: D-2-hydroxy-acid dehydrogenase from rabbit kidney)

In a specific embodiment, the enzyme catalysing the oxidation of heptane dioic acid into 2- hydroxyheptanedioic acid is selected from the group of enzymes comprising an amino acid sequence as shown Seq ID 1-19 or a homologue of any of these sequences.

Gluconobacter, Caenorhabditis, Drosophila, Leporidae (e.g. EC 1 .1 .99.6: D-2-hydroxy-acid dehydrogenase from rabbit kidney) In a specific embodiment, the enzyme catalysing the oxidation of heptane dioic acid into 2- hydroxyheptanedioic acid is selected from the group of enzymes comprising an amino acid sequence as shown Seq ID 1-19 or a homologue of any of these sequences.

EC 1.1.1.27 - L-lactate dehydrogenases >Q 8NLN0_Corynebacterium glutamicum (SEQ ID NO:)

MKETVGNKIVLIGAGDVGVAYAYALINQGMADHLAIIDIDEKKLEGNVMDLNHGW WADSRTRVTKGTYADCEDAAMWICAGAAQKPGETRLQLVDKNVKIMKSIVGDVM D S GFDGIFL VASNP VDILT Y AVWKF S GLE WNRVIGS GT VLD S ARFRYMLGEL YE VAP SSVHAYIIGEHGDTELPVLSSATIAGVSLSRMLDKDPELEGRLEKIFEDTRDAAYHIID AKGSTSYGIGMGLARITRAILQNQDVAVPVSALLHGEYGEEDIYIGTPAVVNRRGIRR WELEITDHEMERFKHSANTLREIQKQFF

EC 1.1.1.28 - D-lactate dehydrogenases >P52643_Escherichia coli (SEQ ID NO:)

MKLAVYSTKQYDKKYLQQVNESFGFELEFFDFLLTEKTAKTANGCEAVCIFVNDDG SRPVLEELKKHGVKYIALRCAGFNNVDLDAAKELGLKWRVPAYDPEAVAEHAIGM MMTLNRRIHRAYQRTRDANFSLEGLTGFTMYGKTAGVIGTGKIGVAMLRILKGFGM RLLAFDPYPSAAALELGVEYVDLPTLFSESDVISLHCPLTPENYHLLNEAAFEQMKNG VMIVNTSRGALIDSQAAIEALKNQKIGSLGMDVYENERDLFFEDKSNDVIQDDVFRR LSACHNVLFTGHQAFLTAEALTSISQTTLQNLSNLEK GETCPNELV Gene Accession number GI number Organism

Mdh NP 417703, 1 16131126 Escherichia coli K-12

EC 1.1.1.37 - malate dehydrogenases >P61889_Escherichia coli (SEQ ID NO:) MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGF SGEDATPALEGADVVLISAGVARKPGMDRSDLFNVNAGIVKNLVQQVAKTCPKACI GIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELKGKQPGEVEVP VIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEWEAKAGGGSATLSMGQ AAARFGLSLVRALQGEQGWECAYVEGDGQYARFFSQPLLLGKNGVEERKSIGTLS AFEQNALEGMLDTLKKDIALGEEFVNK

>P49814_Bacillus subtilis (SEQ ID NO:)

MGNTRKKVSVIGAGFTGATTAFLIAQKELADWLVDIPQLENPTKGKALDMLEASPV QGFDAKITGTSNYEDTAGSDIWITAGIARKPGMSRDDLVSTNEKIMRSVTQEIVKYS PD SII WLTNP VD AMT YAV YKE S GFPKERVIGQ S G VLD T ARFRTF VAEELNL S VKD VT GFVLGGHGDDMVPLVRYSYAGGIPLETLIPKERIDAIVERTRKGGGEIVNLLGNGSAY YAPAASLTEMVEAILKDQRRVLPTIAYLEGEYGYEGIYLGVPTIVGGNGLEQIIELELT DYERAQLNKSVESVKNVMKVLS

EC 1.1.1.81 - hydroxypyruvate reductase >A3LRN9_Pichia stipitis (SEQ ID NO:)

MTLKQQVLFVGKPNTNTEAYKKFSANFEVINYKITSKSQLIEDFEGRLRYIEAIYAGW GGFDGVGGFQGEVLRHCPPNVKWAICSIGHDGYDTEGMSKRGITLTNVPSVIASEA VADLVLYNTLSSFRNFKMFEKNLGGKLTNTGALRTALVRGEFDQFNGVPVIKPTVG GAFASSCCGRDILSPRGHNVVIVGFGSIGKLIGERLACIGMNIHYVKRSKLSEQEEASL GYKVTYHATLKDTKNIADLWIACPGTAHTRHMVNEEMINDFAKPFRLINIGRGYW DEKALVNGLQSGKILFAGLDVFENEPSINPDLLNRQDWLTPHIGSSTTENFNYTAAA AMFNIETVLYDREDTITRVN

>Q 88F00_Pseudomonas putida

(SEQ ID NO:)

MSVDPQKLLRELFDTAIAAAHPRQVLEPYLPADRSGRVIVIGAGKAAAAMAEWEK SWQGEVSGLWTRYGHGANCQKIEWEAAHPVPDAAGLAVAKRVLELVSNLNEED RVIFLLSGGGSALLALPAEGLTLADKQQINKALLKSGATIGEMNCVRKHLSAIKGGRL AKACWPATVYT YAISDVPGDLATVIASGPTVADPSTS ADALAILKRYNIEAPKAVID WLNNPASETVKADDPALARSHFQLIAKPQQSLEAAAVKARQAGFSPLILGDLEGESR EVAKVHAGIARQIVQHGQPLKAPCVILSGGETTVTVRGNGRGGRNAEFLLSLTESLK GLPGVYALAGDTDGIDGSEENAGAFMTPASYASAEALGLSASDELDNNNGYGYFAA LDALIVTEPTRTNVNDFRAILILETAQS

EC 1.1.1.82 - malate dehydrogenases [NADP+] >Q 8NSK9_Corynebacterium glutamicum (SEQ ID NO:) MPEVTVNAQQLTVLCTDILTKTGVPAADAHLVGDSLVQADLWGHPSHGVLRLPWY VRRLHSGAMTTHAHVEVLNDLGAVLALDGHNGIGQVLADHARKEAVTRAMMFGI GAVSVRNSNHFGTAMYYTRKAAAQGCVSILTTNASPAMAPWGGREKRIGTNPWSIA APFGETATWDIANTAVARGKIYHARQTNMPIPETWAITSEGAPTTDPAEAINGWLP MAGHKGYAISFMMDVLSGVLTGSQHSTKVHGPYDPTPPGGAGHLFIALDVAAFRDP QDFDDALSDLVGEVKSTPKAQNTEEIFYPGESEDRAHRKNSAHGISLPEKTWMELQE LAIENHWTHR

>Q5E5E9_Vibrio fischeri (SEQ ID NO:)

MKVSYYEVKERLIRKFIASGLAWDDANWVTDVLISSEQRGDKSHGIKHAKNIFDVIN SECYIAQAPIIHDERSITILDGQNSIGPIVAKQAIDIAIKKAKKYGTAAISLRSSNHLFSLS HYVRYIA NMIGFICSSSSPAMAAPNSLNATIGTNPFAFGAPSSKDPIVIDMSSTNVA RGKIKEYKDAELDIPVSWALDEYGNPTTCAIEALKGTLSPLGGYKGFALGCMIDIFSS VLSGS AFSTQITGTSLHMEEAD VNKKGDFLFVLDISKFIQLSEFKIRMDEFIHIIESNGG YIPGTNYINNQFADIEILN

EC 1.1.1.85 - 3-isopropylmalate dehydrogenases >A9VLG8_Bacillus weihenstephanensis (SEQ ID NO:)

MEKRIVCLAGDGVGPEIMESAKEVLHMVERLYGHHFHLQDEYFGGAAIDLNGQPLP QRTLAACLASDAVLLGAVGGPRWDDAKERPEKGLLALRKGLGVFANVRPVTVESA TAHLSPLKNADEIDFVWRELTGGIYFSYPKERTEESATDTLTYHRHEIERIVSYAFQL ASKREKKVTSIDKANVLESSKLWRAVTEEVALRYPNVELEHILVDAAAMELIRNPRR FDVIVTENLFGDILSDEASVLAGSLGMLPSASHAENGPSLYEPIHGSAPDIAGKNKAN

PIAMMRSVAMMLGQSFGLTREGYAIEEAISAVLQSGKCTADIGGNETTTSFTRAVIQE

MEEQALVGRGR

>Q5NPQ9_Zymomonas mobilis

(SEQ ID NO:)

MRIALLAGDGIGPEITAEAVKILKAWGQEIEFDEALIGGAAWKVTGSPLPEETLKLC KNSDAILFGSVGDPECDHLERALRPEQAILGLRKELDLFANLRPARLFPELQAESPLK ENIVTGTDLMIVRELTGDVYFGTPRGQRKDDQNRREGFDTMRYNEDEVKRIARIGFE TARSRSGNLCSIDKSNVLETSQLWRT WLEIAQEYPDVELSHMYVDNAAMQLVRAP DQFDVIVTGNLFGDILSDLASACVGSIGLLPSASLNSEGKGLYEPIHGSAPDIAGLGKA NPLATILSGAMMLRYSLKREADADRIEKAVSTALE KGARTADLGGKMTTSEMGNAVLAALN

EC 1.1.1.93 - tartrate dehydrogenases >P76251 Escherichia coli (SEQ ID NO:)

MMKTMRIAAIPGDGIGKEVLPEGIRVLQAAAERWGFALSFEQMEWASCEYYSHHGK MMPDDWHEQLSRFDAIYFGAVGWPDTVPDHISLWGSLLKFRREFDQYVNLRPVRLF PGVPCPLAGKQPGDIDFYWRENTEGEYSSLGGRVNEGTEHEWIQESVFTRRGVDRI LRYAFELAQSRPRKTLTSATKSNGLAISMPYWDERVEAMAENYPEIRWDKQHIDILC ARFVMQPERFDVWASNLFGDILSDLGPACTGTIGIAPSANLNPERTFPSLFEPVHGSA PDIYGKNIANPIATIWAGAMMLDFLGNGDERFQQAHNGILAAIEEVIAHGPKTPDMK GNATTPQVADAICKIILR Gene Accession number GI number Organism

Aspergillus niger CBS

AN! 1 2398014 XP 001388737. 1 145228857 5 13.88

>A2Q846_Aspergillus niger (SEQ ID NO:) MTTETTTYRIASIPGDGIGEEWRATIEVINKLAQTLNTFNIEFTHLPWGTEYYKQHGR YVSEGYLDTLRQFDAGLFGSVGHPDVPDHVSLWGLLLALRSPLQLYANVRPVRTFP GTKSPLTTAVNGIDWVLVRENSEGEYCGQGGRSHTGQPWEAATEVAIFTRVGVERI MRFAFETARSRPRRHLTWTKSNAMRHGMVLWDEVAEEVAKDFPDVTWDKMLVD AMTLRMISKPESLDTIVGTNLHMDILSDLAAGLAGSIGVAPSSNLDPTRKNPSLFEPV HGS AFDIMGKGVANPVATFWS AAEMLAWLGEKDAAKKLMDCVEKVCAAGILTPD LGGSANTQGWDAVCKEIEQQLASS

EC 1.1.2.3 - L-lactate dehydrogenase (cytochrome)

>P00175_Saccharomyces cerevisiae (SEQ ID NO:)

MLKYKPLLKISKNCEAAILRASKTRLNTIRAYGSTVPKSKSFEQDSRKRTQSWTALR VGAILAATSSVAYLNWHNGQIDNEPKLDMNKQKISPAEVAKHNKPDDCWWINGY VYDLTRFLPNHPGGQDVIKFNAGKDVTAIFEPLHAPNVIDKYIAPEKKLGPLQGSMPP ELVCPPYAPGETKEDIARKEQLKSLLPPLDNIINLYDFEYLASQTLTKQAWAYYSSGA NDEVTHRENHNAYHRIFFKPKILVDVRKVDISTDMLGSHVDVPFYVSATALCKLGNP LEGEKDVARGCGQGVTKVPQMISTLASCSPEEIIEAAPSDKQIQWYQLYVNSDRKITD DLVKNVEKLGVKALFVTVDAPSLGQREKDMKLKFSNTKAGPKAMKKTNVEESQGA SRALSKFIDPSLTWKDIEELKKKTKLPIVIKGVQRTEDVIKAAEIGVSGVVLSNHGGR QLDFSRAPIEVLAETMPILEQRNLKDKLEVFVDGGVRRGTDVLKALCLGAKGVGLG RPFLYANSCYGRNGVEKAIEILRDEIEMSMRLLGVTSIAELKPDLLDLSTLKARTVGV PNDVLYNE VYEGPTLTEFEDA

>P33232_Escherichia coli (SEQ ID NO:)

MIISAASDYRAAAQRILPPFLFHYMDGGAYSEYTLRRNVEDLSEVALRQRILKNMSD LSLETTLFNEKLSMPVALAPVGLCGMYARRGEVQAAKAADAHGIPFTLSTVSVCPIE EVAPAIKRPMWFQLYVLRDRGFMRNALERAKAAGCSTLVFTVDMPTPGARYRDAH SGMSGPNAAMRRYLQAVTHPQWAWDVGLNGRPHDLGNISAYLGKPTGLEDYIGWL GNNFDPSIS WKDLEWIRDFWDGPMVIKGILDPEDARDAVRFGADGIWSNHGGRQL DGVLSSARALPAIADAVKGDIAILADSGIRNGLDWRMIALGADTVLLGRAFLYALA TAGQAGVANLLNLIEKEMKVAMTLTGAKSISEITQDSLVQGLGKELPAALAPMAKG NAA

EC 1.1.2.4 - D-lactate dehydrogenase (cytochrome)

>P32891_Saccharomyces cerevisiae (SEQ ID NO:)

MLWKRTCTRLIKPIAQPRGRLVRRSCYRYASTGTGSTDSSSQWLKYSVIASSATLFG YLFAKNLYSRETKEDLIEKLEMVKKIDPVNSTLKLSSLDSPDYLHDPVKIDKWEDLK QVLGNKPENYSDAKSDLDAHSDTYFNTHHPSPEQRPRIILFPHTTEEVSKILKICHDNN MPWPFSGGTSLEGHFLPTRIGDTITVDLSKFMNNWKFDKLDLDITVQAGLPWEDL NDYLSDHGLMFGCDPGPGAQIGGCIANSCSGTNAYRYGTMKENIINMTIVLPDGTIV KTKKRPRKSSAGYNLNGLFVGSEGTLGIVTEATVKCHVKPKAETVAWSFDTIKDAA ACASNLTQSGIHLNAMELLDENMMKLINASESTDRCDWVEKPTMFFKIGGRSPNIVN ALVDEVKAVAQLNHCNSFQFAKDDDEKLELWEARKVALWSVLDADKSKDKSAKI WTTDVAVPVSQFDKVIHETKKDMQASKLINAIVGHAGDGNFHAFIVYRTPEEHETCS

QLVDRMVKRALNAEGTCTGEHGVGIGKREYLLEELGEAPVDLMRKIKLAIDPKRIM

NPDKIFKTDPNEPANDYR

>Q5FP89_Gluconobacter oxydans

(SEQ ID NO:)

MPEPVMTASSASAPDRLQAVLKALQPVMGERISTAPSVREEHSHGEAMNASNLPEA WFAESTQDVATVLRHCHEWRVPWAFGAGTSVEGHWPPEQAISLDLSRMTGIVD LNAEDLDCRVQAGITRQTLNVEIRDTGLFFPVDPGGEATIGGMCATRASGTAAVRYG TMKENVLGLTWLATGEIIRTGGRVRKSSTGYDLTSLFVGSEGTLGIITEVQLRLHGR PDSVSAAICQFESLHDAIQTAMEIIQCGIPITRVELMDSVQMAASIQYSGLNEYQPLTT LFFEFTGSPAAVREQVETTEAIASGNNGLGFAWAESPEDRTRLWKARHDAYWAAKA IVPDARVISTDCIVPISRLGELIEGVHRDIEASGLRAPLLGHVGDGNFHTLIITDDTPEG HQQALDLDRKIVARALSLNGSCSGEHGVGMGKLEFLETEHGPGSLSVMRALKNTMD PHHILNPGKLLPPGAVYTG

EC 1.1.99.22-hydroxyglutarate dehydrogenase >Q9N4Z0 Caenorhabditis elegans (SEQ ID NO:) MLNRGTFQVFRGISGPPKKSVDLPKYDLVIVGGGIVGCATARQLLIEKPQLKVALIEK EKELAVHQSGHNSGVIHAGIYYTPGSLKAKLCVEGLDLSYEFFDKEKVPYKKTGKLI VAVEPEEVPRLDALFSRAQTNGCRDIEMIDSSKITELEPHCRGLKALWSPHTGIVDWG YVTKRFGEDFEKRGGKIYTSYPLEKISDNHDPGYPIRVSSGPALAEFETKNLITCAGLQ SDRVAALSGCSTDPKIVPFRGEYLLLKPEKRHLVKTNIYPVPDPRFPFLGVHFTPRMN GDIWLGPNAVLAYKREGYSYFSISPSDLLESLSYSGMQKLVKKHFTFGIKELYRGVWI AAQ VKQLQRFIPELKL SD VTRGP AG VRAQ AMD S AGNL VDDF VFD S GTGKL SPLLMH VRNAPSPAATSSLAIAKMITSEAINRFKL

>Q9VJ28_Drosophila melanogaster (SEQ ID NO:)

MAQVRLLVQGLRRSLLNVGVAAPNESTATHKRSQHSSSSCGDYDLVWGGGIVGA ASAREIVLRHPSLKVAVLEKECKLAKHQSGHNSGVIHAGIYYKPGTLKARLCVEGM HLAYAYLDEKKI PYKK TGKLIVATDEKEVKLLKDLEKRGIANNVPDLRMIEGSEIQEI

EPYCQGVMALHSPHTGIVDWGLVTEHYGQDFKQCGGDIYLDFNVSKFTETKEGTDY PVTIHGAKPGQTVRTKNVLTCGGLQSDLLAEKTGCPRDPRIVPFRGEYLLLTKEKQH MVKGNIYPVPDPRFPFLGVHFTPRMDGSIWLGPNAVLALKREGYTWGDINLFELFDA LRYPGFVKMASKYIGFGLSEMSKSWFINLQIKALQKYIPDITEYDIQRGPAGVRAQA MDLDGNLVDDFVFDRGQGSGALAKRVLHCRNAPSPGATSSLAIAKMIADKIENEFSI GK

>P13714_Bacillus subtilis (SEQ ID NO:)

MMNKHVNKVALIGAGFVGSSYAFALINQGITDELVVIDVNKEKAMGDVMDLPHGK

AFGLQPVKTSYGTYEDCKDADIVCICAGANQKPGETRLELVEKNLKIFKGIVSEVMA

SGFDGIFLVATNPVDILTYATWKFSGLPKERVIGSGTTLDSARFRFMLSEYFGAAPQN

VHAHIIGEHGDTELPVWSHANVGGVPVSELVEKNDAYKQEELDQIVDDVKNAAYHI

IEKKGATYYGVAMSLARITKAILHNENSILTVSTYLDGQYGADDVYIGVPAWNRGG

IAGITELNLN EKEKEQFLH S AG VLKN ILKPHFAEQKVN Gene Accession number GI number Organism

IdliA NP 743806, 1 26988381 Pseudomonas putida KT2440

>Q88MC4_Pseudomonas putida

(SEQ ID NO:)

MTHPRHALQRSSTMRALLFSSQHYDQESFTKAAGGTALELHFQPARLTLDTAALAD GFEWCAFINDELDAPVLQRLAAAGTRLIALRSAGYNHVDLAAAQRLGLAWRVPA YSPHAVAEHAVALILALNRRLHRAYNRTREGDFTLHGLTGFDLHGKTVGWGTGQI GVAFARIMAGFGCQLLAYDPYPNPELLALGARYLPLPELLREARIISLHCPLTEHTRH LINAQSLAQLQPGAMLINTGRGALVDTPALIDALKSGQLGYLGLDVYEEEAQLFFED RSDLPLQDDVLARLLTFPNVI ITAHQAFLTREALDAIAATTLDNINRWAAGNPQNLVMG

2-hydroxyheptanedioic acid dehydrogenase

As disclosed herein 2- hydroxyheptanedioic acid is biocatalytically converted into AKP (alpha-ketopimelate). The biocatalyst may in particular comprise an enzyme for catalysing the conversion of hydroxyheptanedioic acid into AKP selected from the group of

oxidoreductases acting on the CH-OH group of donors (EC 1 .1 ), in particular such an oxidoreductase selected from the group of EC 1 .1 .1 (with NAD+ or NADP+ as acceptor), EC 1 .1 .2 (with a cytochrome as acceptor), EC 1.1 .3 (with oxygen as acceptor), EC 1 .1 .4 (with a disulphide as acceptor), EC 1 .1.5 (with a quinone or similar compound as acceptor), EC 1 .1 .7 (with an iron sulphur protein as acceptor), and EC 1 .1.99 (with other acceptors); - oxidoreductases acting on the aldehyde or oxo group of donors (EC 1 .2);

- enzymes with 2-hydroxypimelate dehydrogenase activity enzymes with 2- hydroxypimelate oxidase activity;

- oxidoreductases classified under EC 1 .97; and

- oxidoreductases classified under EC 1 .98. An oxidoreductase classifiable under EC 1 .1.1 catalysing the conversion of

hydroxyheptanedioic acid into AKP may in particular be selected from alcohol dehydrogenases with NAD+ as acceptor of EC 1 .1 .1.1 ; alcohol dehydrogenases with NADP+ as acceptor of EC 1 .1 .1.2; glyoxylate reductases of EC 1 .1 .1.26, L-lactate dehydrogenases of EC 1 .1 .1 .27, D- lactate dehydrogenases of EC 1 .1 .1 .28, glycerate dehydrogenases of EC 1 .1 .1.29, 3- hydroxybutyrate dehydrogenases of EC 1.1 .1.30, 3- hydroxyisobutyrate dehydrogenases of EC 1 .1 .1 .31 , malate dehydrogenase of EC 1.1 .1.37, 3- hydroxypropionate dehydrogenase of EC 1 .1.1 .59, 2-hydroxy-3-oxopropionate reductase of EC 1.1 .1.60, alcohol dehydrogenase [NAD(P)+] of EC 1 .1 .1 .71 , glyoxylate reductase [NADP+] of EC 1 .1 .1.79, hydroxypyruvate reductases of EC 1 .1.1 .81 , malate

dehydrogenases [NADP+] of EC 1 .1 .1 .82, 3-isopropylmalate dehydrogenases of EC 1.1 .1.85, tartrate dehydrogenases of EC 1 .1 .1 .93, (R)-2-hydroxy-fatty-acid dehydrogenases of EC 1 .1 .1 .98, (S)-2-hydroxy-fatty-acid dehydrogenases of EC 1.1 .1.99, hydroxymalonate dehydrogenase of EC 1 .1 .1 .167, 2-oxoadipate reductases of EC 1.1 .1.172,

hydroxyphenylpyruvate reductases of EC 1 .1 .1.237, and 3- hydroxypimeloyl-CoA dehydrogenases of EC 1.1 .1 .259 An enzyme classifiable under EC 1.1 .2 catalysing the conversion of hydroxyheptanedioic acid into AKP may in particular be selected from D-lactate dehydrogenases (EC 1.1.2.4 and EC 1 .1 .2.5).

An enzyme classifiable under EC 1.1 .3 catalysing the conversion of hydroxyheptanedioic acid into AKP may in particular be selected from the group of lactate oxidases and other hydroxy acid oxidases; malate oxidases (EC 1 .1 .3.3), (S)-2- hydroxy-acid oxidase (EC 1 .1 .3.15); secondary-alcohol oxidases (EC 1 .1 .3.18); hydroxyphytanate oxidases (EC 1 .1 .3.27).

An enzyme classifiable under EC 1 .1 .99 catalysing the conversion of hydroxyheptanedioic acid into AKP may in particular be selected from 2- hydroxyglutarate dehydrogenases (EC 1 .1.99.2); D-2-hydroxy-acid dehydrogenases (EC 1 .1 .99.6); glycolate dehydrogenase (EC 1 .1 .99.14), malate dehydrogenase (EC 1 .1 .99.16), and 2-oxo-acid reductases (EC 1.1 .99.30).

In a particularly preferred method, an enzyme catalysing the preparation of AKP is selected from the group of

- oxidoreductases with oxygen as acceptor (EC 1 .1 .3), such as a lactate oxidase or another hydroxy acid oxidase; such as hydroxy acid oxidase HA01 from Hominidae, in particular from Homo sapiens (EC 1.1 .3.15) or lactate oxidase from Aerococci, in particular from Aerococcus vi dans;

- L-lactate dehydrogenases (EC 1 .1 .1 .27);

- D-lactate dehydrogenases (EC 1 .1 .1 .28); - malate dehydrogenase [NAD+] (EC 1.1.1.37);

- hydroxypyruvate reductases (ECl .1 .1 .81 );

- malate dehydrogenases [NADP+] (EC 1 .1 .1 .82);

- 3-isopropylmalate dehydrogenases (EC 1 .1 .1 .85);

- tartrate dehydrogenases (EC 1.1 .1.93); - (R)-2-hydroxy-fatty-acid dehydrogenases (ECl .1 .1 .98);

- (S)-2-hydroxy-fatty-acid dehydrogenases (EC 1 .1 .1 .99);

- 2-oxoadipate reductases (ECl .1 .1 .172);

- 2-hydroxyglutarate dehydrogenase (EC 1 .1 .99.2); and

- D-2-hydroxy-acid dehydrogenase (EC 1 .1 .99.6). Most preferably, the enzyme catalysing the preparation of AKP is selected from the group of 2-oxoadipate reductases (ECl .1.1 .172). In a specifically preferred the enzyme comprises an amino acid sequence according to SEQ ID NO: 21, SEQ ID NO: 24, or a homologue of any of these sequences. Suitable nucleic acids encoding an enzyme catalysing the preparation of AKP may in particular comprise a nucleic acid sequence represented by SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25 and functional analogues thereof.

wt DNA-sequence (from NCBI) >gi| 1 1 1 84232|ref]NM_01 7545.2| Homo sapiens hydroxyacid oxidase (glycolate oxidase) 1 (HA01 ), mRNA

(SEQ ID NO:)

ATGCTCCCCCGGCTAATTTGTATCAATGATTATGAACAACATGCTAAATCAGTAC TTCCAAAGTCTATATATGACTATTACAGGTCTGGGGCAAATGATGAAGAAACTTT GGCTGATAATATTGCAGCATTTTCCAGATGGAAGCTGTATCCAAGGATGCTCCGG AATGTTGCTGAAACAGATCTGTCGACTTCTGTTTTAGGACAGAGGGTCAGCATGC CAATATGTGTGGGGGCTACGGCCATGCAGCGCATGGCTCATGTGGACGGCGAGC TTGCCACTGTGAGAGCCTGTCAGTCCCTGGGAACGGGCATGATGTTGAGTTCCTG GGCCACCTCCTCAATTGAAGAAGTGGCGGAAGCTGGTCCTGAGGCACTTCGTTGG CTGCAACTGTATATCTACAAGGACCGAGAAGTCACCAAGAAGCTAGTGCGGCAG GCAGAGAAGATGGGCTACAAGGCCATATTTGTGACAGTGGACACACCTTACCTG GGCAACCGTCTGGATGATGTGCGTAACAGATTCAAACTGCCGCCACAACTCAGG ATGAAAAATTTTGAAACCAGTACTTTATCATTTTCTCCTGAGGAAAATTTTGGAG ACGACAGTGGACTTGCTGCATATGTGGCTAAAGCAATAGACCCATCTATCAGCTG GGAAGATATCAAATGGCTGAGAAGACTGACATCATTGCCAATTGTTGCAAAGGG CATTTTGAGAGGTGATGATGCCAGGGAGGCTGTTAAACATGGCTTGAATGGGAT CTTGGTGTCGAATCATGGGGCTCGACAACTCGATGGGGTGCCAGCCACTATTGAT GTTCTGCCAGAAATTGTGGAGGCTGTGGAAGGGAAGGTGGAAGTCTTCCTGGAC GGGGGTGTGCGGAAAGGCACTGATGTTCTGAAAGCTCTGGCTCTTGGCGCCAAG GCTGTGTTTGTGGGGAGACCAATCGTTTGGGGCTTAGCTTTCCAGGGGGAGAAAG GTGTTCAAGATGTCCTCGAGATACTAAAGGAAGAATTCCGGTTGGCCATGGCTCT GAGTGGGTGCCAGAATGTGAAAGTCATCGACAAGACATTGGTGAGGAAAAA TCCTTTGGCCGTTTCCAAGATCTGA

HAOX-5B

>Q9UJM8| HAOX1 HUMAN Hydroxyacid oxidase 1 - Homo sapiens (Human). Protein sequence (SEQ ID NO:)

MLPRLICINDYEQHAKSVLPKSIYDYYRSGANDEETLADNIAAFSRWKLYPRMLRNV AETDLSTSVLGQRVSMPICVGATAMQRMAHVDGELATVRACQSLGTGMMLSSWAT SSIEEVAEAGPEALRWLQLYIYKDREVTKKLVRQAEKMGYKAIFVTVDTPYLGNRL DDVRNRFKLPPQLRMKNFETSTLSFSPEENFGDDSGLAAYVAKAIDPSISWEDIKWLR RLTSLPIVAKGILRGDDAREAVKHGLNGILVSNHGARQLDGVPATIDVLPEIVEAVEG KVEVFLDGGVRKGTDVLKALALGAKAVFVGRPIVWGLAFQGEKGVQDVLEILKEEF RLAMALSGCQNVKVIDKTLVRKNPLAVSKI

Optimized DNA sequence: (optimization done by DNA2.0)

(SEQ ID NO:)

ATGCTGCCACGTCTGATTTGTATTAACGATTACGAACAACACGCGAAGAGCGTAC TGCCGAAATCCATTTACGATTATTACCGTTCTGGTGCAAACGATGAAGAAACGCT GGCTGATAACATCGCCGCTTTTTCCCGTTGGAAACTGTACCCACGTATGCTGCGT AACGTTGCCGAAACCGACCTGTCCACCAGCGTCCTGGGTCAGCGTGTGTCCATGC CAATCTGCGTGGGTGCAACCGCAATGCAGCGTATGGCACACGTTGACGGCGAAC TGGCAACCGTCCGTGCGTGCCAGAGCCTGGGTACCGGTATGATGCTGAGCAGCT GGGCTACCTCTAGCATCGAGGAAGTGGCAGAAGCTGGTCCGGAAGCACTGCGCT GGCTGCAGCTGTACATCTACAAAGATCGCGAAGTCACTAAGAAACTGGTGCGCC AGGCGGAAAAGATGGGTTACAAGGCAATCTTTGTGACTGTTGACACCCCGTACCT GGGTAACCGCCTGGATGACGTTCGTAACCGCTTCAAGCTGCCGCCGCAGCTGCGT ATGAAGAACTTTGAAACCAGCACCCTGTCCTTTTCCCCAGAAGAAAATTTCGGTG ATGACTCTGGTCTGGCCGCGTACGTCGCGAAAGCTATCGATCCGTCCATCTCCTG GGAAGATATCAAATGGCTGCGTCGTCTGACTTCCCTGCCGATCGTTGCTAAGGGT ATTCTGCGTGGTGACGACGCGCGTGAAGCTGTTAAACATGGTCTGAACGGCATTC TGGTAAGCAACCATGGCGCACGCCAGCTGGATGGTGTACCTGCTACTATTGATGT GCTGCCGGAAATCGTGGAAGCGGTTGAAGGTAAAGTTGAAGTGTTCCTGGACGG TGGTGTGCGCAAAGGCACCGATGTACTGAAAGCACTGGCGCTGGGTGCGAAAGC CGTCTTTGTTGGCCGTCCTATTGTTTGGGGTCTGGCATTCCAGGGTGAGAAAGGT GTACAGGACGTTCTGGAGATCCTGAAAGAGGAGTTCCGCCTGGCTATGGCGCTGT CTGGTTGTCAAAACGTGAAAGTAATCGATAAAACCCTGGTACGTAAAAACCCTCT GGCAGTAAGCAAGATCTAA

LAOX-8C wt DNA-sequence (from NCBI, Acc. D5061 1 ) (SEQ ID NO:) atgaa taacaatgac attgaatata atgcacctag tgaaatcaag tacattgatg ttgtcaatac ttacgactta gaagaagaag caagtaaagt ggtaccacat ggtggtttta actatattgc cggtgcatct ggtgatgagt ggactaaacg cgctaatgac cgtgcttgga aacataaatt actataccca cgtctagcgc aagatgttga agcgcccgat acaagtactg aaattttagg tcataaaatt aaagccccat tcatcatggc accaattgct gcacatggtt tagcccacac tactaaagaa gctggtactg cacgtgcagt ttcagaattt ggtacaatta tgtccatctc agcttattct ggtgcaacat ttgaagaaat ttctgaaggc ttaaatggcg gaccccgttg gttccaaatc tatatggcta aagatgacca acaaaaccgt gatatcttag acgaagctaa atctgatggt gcaactgcta tcatccttac agctgactca actgtttctg gaaaccgtga ccgtgatgtg aagaataaat tcgtttaccc atttggtatg ccaattgttc aacgttactt acgtggtaca gcagaaggta tgtcattaaa caatatctac ggtgcttcaa aacaaaaaat ctcaccaaga gatattgagg aaatcgccgg tcattctgga ttaccagtat tcgttaaagg tattcaacac ccagaagatg cagatatggc aatcaaacgt ggtgcatcag gtatctgggt atctaaccac ggtgctcgtc aactatatga agctccaggt tcatttgaca cccttccagc tattgctgaa cgtgtaaaca aacgtgtacc aatcgtcttt gattcaggtg tacgtcgtgg tgaacacgtt gccaaagcgc tagcttcagg ggcagacgtt gttgctttag gacgcccagt cttatttggt ttagctttag gtggctggca aggtgcttac tcagtacttg actacttcca aaaagactta acacgcgtaa tgcaattaac aggttcacaa aatgtggaag acttgaaggg tctagattta ttcgataacc catacggtta tgaatactag

LAOX-8C Q44467 9LACT

>Q44467|Q44467_9LACT Lactate oxidase - Aerococcus viridans. Protein sequence (SEQ ID NO:) MN NDIEYNAPSEIKYIDVVNTYDLEEEASKWPHGGFNYIAGASGDEWTKRANDR AWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVS EFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILT ADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEI AGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGSFDTLPAIAERV NKRVPIVFDSGVRRGEHVAKALASGADWALGRPVLFGLALGGWQGAYSVLDYFQ KDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY

LAOX-8C Optimized DNA sequence: (optimization done by DNA2.0) (SEQ ID NO:)

ATGAACAACAACGACATCGAATATAACGCTCCTTCTGAAATCAAATATATCGAC GTGGTTAACACCTATGACCTGGAGGAAGAAGCGTCTAAGGTCGTACCGCACGGT GGTTTCAATTACATTGCAGGTGCCTCTGGTGATGAATGGACCAAACGCGCAAACG ATCGTGCATGGAAACACAAACTGCTGTATCCGCGCCTGGCCCAGGATGTGGAAG CACCGGATACTTCCACTGAAATCCTGGGTCACAAAATCAAGGCACCGTTTATTAT GGCTCCGATCGCAGCGCACGGCCTGGCACACACCACCAAAGAAGCTGGCACCGC TCGTGCGGTTTCTGAGTTCGGCACCATTATGTCTATCTCTGCGTATAGCGGTGCCA CTTTCGAGGAAATTTCCGAGGGCCTGAACGGTGGCCCGCGTTGGTTTCAGATTTA CATGGCGAAAGATGACCAGCAGAACCGCGATATCCTGGATGAAGCCAAATCTGA CGGCGCGACTGCTATCATCCTGACCGCGGACTCTACCGTATCCGGTAACCGTGAC CGTGATGTGAAGAACAAGTTCGTCTATCCTTTCGGTATGCCGATTGTTCAGCGCT ATCTGCGCGGTACCGCTGAGGGTATGAGCCTGAACAACATCTATGGTGCGTCCAA ACAGAAAATCAGCCCACGTGACATCGAAGAAATTGCTGGTCATAGCGGTCTGCC GGTGTTTGTGAAAGGTATCCAGCATCCAGAAGATGCGGACATGGCAATCAAACG TGGTGCGTCTGGCATCTGGGTTAGCAACCACGGTGCGCGTCAGCTGTACGAAGCT CCGGGTAGCTTCGATACCCTGCCGGCCATCGCGGAACGTGTGAATAAACGCGTG CCGATCGTTTTCGATTCCGGTGTGCGTCGTGGTGAACATGTGGCAAAAGCACTGG CGTCTGGCGCTGATGTCGTAGCACTGGGCCGTCCAGTGCTGTTCGGTCTGGCTCT GGGTGGCTGGCAGGGCGCTTACTCCGTCCTGGATTACTTTCAGAAAGACCTGACC CGTGTTATGCAGCTGACCGGTTCCCAGAACGTAGAGGACCTGAAAGGCCTGGAC CTGTTCGACAACCCTTACGGTTACGAATACTAA

EXAMPLE XXXIX

Exemplary Carboxylic Acid Reductases

This example describes the use of carboxylic acid reductases to carry out the conversion of a caroboxylic acid to an aldehyde.

1.2.1. e Acid reductase .

The conversion of unactivated acids to aldehydes can be carried out by an acid reductase. Examples of such conversions include, but are not limited, the conversion of 4- hydroxybutyrate, succinate, alpha-ketoglutarate, and 4-aminobutyrate to 4-hydroxybutanal, succinate semialdehyde, 2,5-dioxopentanoate, and 4-aminobutanal, respectively. One notable carboxylic acid reductase can be found in Nocardia iowensis which catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). This enzyme is encoded by the car gene and was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in

Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL. (2006)).

Gene Accession No. GI No. Organism

Nocardia iowensis (sp. NRRL car AAR91681.1 40796035 5646)

Nocardia iowensis (sp. NRRL npt ABI83656.1 114848891 5646)

Additional car and npt genes can be identified based on sequence homology.

Gene Accession No. GI No. Organism fadD9 YP 978699.1 121638475 Mycobacterium bovis BCG

BCG 2812c YP 978898.1 121638674 Mycobacterium bovis BCG nfa20150 YP 118225.1 54023983 Nocardia farcinica IFM 10152 nfa40540 YP 120266.1 54026024 Nocardia farcinica IFM 10152

Streptomyces griseus subsp.

SGR 6790 YP 001828302.1 182440583 griseus NBRC 13350

Streptomyces griseus subsp.

SGR 665 YP 001822177.1 182434458 griseus NBRC 13350

An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino- 4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J Antibiot. 60(6):380-387 (2007)). Co-expression ofgriC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial.

Gene Accession No. GI No. Organism

Streptomyces griseus subsp. griC 182438036 YP_001825755.1

griseus NBRC 13350

Streptomyces griseus subsp. griD 182438037 YP_001825756.1

griseus NBRC 13350

Mycobacterium smegmatis

MSMEG 2956 YP_887275.1 YP_887275.1

MC2 155

Mycobacterium smegmatis

MSMEG 5739 YP_889972.1 118469671

MC2 155

Mycobacterium smegmatis

MSMEG_2648 YP_886985.1 118471293

MC2 155

Mycobacterium avium subsp.

MAP 1040c NP_959974.1 41407138

paratuberculosis K-10

Mycobacterium avium subsp.

MAP2899c NP_961833.1 41408997

paratuberculosis K-10

MMAR_2117 YP_001850422.1 183982131 Mycobacterium marinum M

MMAR 2936 YP 001851230.1 183982939 Mycobacterium marinum M

MMAR 1916 YP 001850220.1 183981929 Mycobacterium marinum M

TpauDRAFT 33 Tsukamurella paurometabola

ZP 04027864.1 227980601

060 DSM 20162

TpauDRAFT 20 Tsukamurella paurometabola

ZP 04026660.1 227979396

920 DSM 20162

CPCC7001 132 ZP 05045132.1 254431429 Cyanobium PCC7001 0

DDBDRAFT 01 Dictyostelium discoideum

XP 636931.1 66806417

87729 AX4

An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by

NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al, Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21 : 1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date.

Cloning and Expression of Carboxylic Acid Reductase. Escherichia coli is used as a target organism to engineer the pathway for adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine . E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine . E. coli is amenable to genetic manipulation and is known to be capable of producing various intermediates and products effectively under various oxygenation conditions.

To generate a microbial organism strain such as an E. coli strain engineered to produce adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine , nucleic acids encoding a carboxylic acid reductase and phosphopantetheine transferase are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, car genes from Nocardia iowensis (designated 720), Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K-10 (designated 891) and Mycobacterium marinum M

(designated 892) were cloned into pZS*13 vectors (Expressys, Ruelzheim, Germany) under control of PAl/lacO promoters. The npt (ABI83656.1) gene (i.e., 721) was cloned into the pKJL33S vector, a derivative of the original mini-F plasmid vector PML31 under control of promoters and ribosomal binding sites similar to those used in pZS* 13. The car gene (GNM_720) was cloned by PCR from Nocardia genomic DNA. Its nucleic acid and protein sequences are shown in Figures 43A and 43B, respectively. A codon- optimized version of the npt gene (GNM_721) was synthesized by GeneArt (Regensburg, Germany). Its nucleic acid and protein sequences are shown in Figures 44 A and 44B, respectively. The nucleic acid and protein sequences for the Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K-10

(designated 891) and Mycobacterium marinum M (designated 892) genes and enzymes can be found in Figures 45, 46, and 47, respectively. The plasmids are transformed into a host cell to express the proteins and enzymes required for adipate, 6-aminocaproic acid, caprolactam or hexametheylenediamine production. Additional CAR variants were generated. A codon optimized version of CAR 891 was generated and designated 891GA. The nucleic acid and amino acid sequences of CAR 891GA are shown in Figures 48 A and 48B, respectively. Over 2000 CAR variants were generated. In particular, all 20 amino acid combinations were made at positions V295, M296, G297, G391, G421, D413, G414, Y415, G416, and S417, and additional variants were tested as well. Exemplary CAR variants include: E16K; Q95L; L100M; A101 IT; K823E; T941S; H15Q; D198E; G446C; S392N; F699L; V883I; F467S; T987S; R12H; V295G;

V295A; V295S; V295T; V295C; V295V; V295L; V295I; V295M; V295P; V295F; V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K; V295R; M296G; M296A;

M296S; M296T; M296C; M296V; M296L; M296I; M296M; M296P; M296F; M296Y; M296W; M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G; G297A; G297S; G297T; G297C; G297V; G297L; G297I; G297M; G297P; G297F; G297Y; G297W; G297D; G297E; G297N; G297Q; G297H; G297K; G297R; G391G; G391A; G391S; G391T; G391C; G391V; G391L; G391I; G391M; G391P; G391F; G391Y; G391W; G391D; G391E; G391N; G391Q; G391H; G391K; G391R; G421G; G421A; G421S; G421T; G421C; G421V; G421L; G421I G421M; G421P; G421F; G421Y; G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R; D413G; D413A; D413S; D413T; D413C; D413V; D413L; D413I; D413M; D413P; D413F; D413Y; D413W; D413D; D413E; D413N; D413Q; D413H;

D413K; D413R; G414G; G414A; G414S; G414T; G414C; G414V; G414L; G414I; G414M; G414P; G414F; G414Y; G414W; G414D; G414E; G414N; G414Q; G414H; G414K;

G414R; Y415G; Y415A; Y415S; Y415T; Y415C; Y415V; Y415L; Y415I; Y415M; Y415P; Y415F; Y415Y; Y415W; Y415D; Y415E; Y415N; Y415Q; Y415H; Y415K; Y415R;

G416G; G416A; G416S; G416T; G416C; G416V; G416L; G416I; G416M; G416P; G416F; G416Y; G416W; G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G;

S417A; S417S; S417T; S417C; S417V S417L; S417I; S417M; S417P; S417F; S417Y;

S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and S417R.

The CAR variants were screened for activity, and numerous CAR variants were found to exhibit CAR activity.

This example describes the use of CAR for converting carboxylic acids to aldehydes.

EXAMPLE XL

The following example provides various gene candidates for enzymes as disclosed herein for convertion of a-ketopimelate to adipate, 6-aminocaproate and hexamethylenediamine as depicted in 38B. a-ketopimelate dehydrogenase (aminating)

Conversion of α-ketopimelate to 2-aminopimelate requires the conversion of a ketone to an amine group. Such a transformation can be accomplished by aminating oxidoreductases in the EC class 1.4.1. Enzymes in this EC class catalyze the oxidative deamination of amino groups with NAD+ or NADP+ as acceptor, and the reactions are typically reversible.

Exemplary enzymes for catalyzing these reactions include glutamate dehydrogenase (EC 1.4.1.2), leucine dehydrogenase (EC 1.4.1.9), and aspartate dehydrogenase (EC 1.4.1.21). The gdhA gene product from Escherichia coli (Korber, .et al., JMoI. Biol. 234: 1270-1273. (1993)), gdh from Thermotoga maritime (Kort et al., Extremophiles 1 :52-60. 1997); Lebbink et al, J. Mol. Biol. 280:287-296 (1998) and Lebbink et al, JMoI. Biol. 289:357-369 (1999))), and gdhAl from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversible conversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)); and Stoyan et al., J Biotechnol 54:77-80 (1997)). The nadX gene from Thermotoga maritima encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al, J Biol.

Chem. 278:8804-8808 (2003)).

a-ketopimelate aminotransferase

Several aminotransferases transaminate the amino groups of 2-oxo acids to form amino acids. Such an enzyme is required for the transamination of -ketopimelate to 2-aminopimelate. A promising enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39), an enzyme that participates in lysine biosynthesis and degradation in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadipate, using alpha-ketoglutarate as the amino acceptor. Gene candidates are found in Homo sapiens (Okuno et al., Enzyme Protein 47: 136-148 (1993)) and Thermus thermophilus (Miyazaki et al, Microbiology 150:2327- 2334 (2004)). The Thermus thermophilus enzyme, encoded by lysN, is active with several alternate substrates including oxaloacetate, 2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3- methylvalerate.

Another candidate is aspartate aminotransferase, an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al, FEBS Lett. 100:81-84 (1979); and Yagi et al, Methods

Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (de la et al., Plant J 46:414-425 (2006); Kwok et al., J Exp. Bot. 55:595-604 (2004) and Wilkie et al, Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583- 4589 (1980)). Aminotransferases that work on other amino-acid substrates may also be able to catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen et al., /. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the transamination of a-ketobutyrate to generate a-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen et al., J. Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase andp hosphohydroxythreonine aminotransferase (Lam, J. et al., Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et al., FEBS. Lett. 390: 179-182 (1996)).

2-aminopimelate decarboxylase

The decarboxylation of 2-AHD (or 2-aminopimelate) to 6-aminocaproate (Figure 12, Step I) is catalyzed by an amino acid decarboxylase such as aspartate decarboxylase. Aspartate decarboxylase participates in pantothenate biosynthesis and is encoded by gene panD in Escherichia coli (Dusch et al., Appl. Environ. Microbiol 65: 1530-1539 (1999); Merke and Nichols, FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al, Biochem. J 323 (Pt 3):661-669 (1997); and Schmitzberger et al, EMBO J 22:6193-6204 (2003)). Similar enzymes from Mycobacterium tuberculosis (Chopra et al., Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum (Dusch et al., Appl. Environ. Microbiol 65: 1530-1539 (1999)) have been expressed and characterized in E. coli. Glutamate decarboxylase (e.g., gadA, gadB), diaminopimelate decarboxylase (lysA), arginine decarboxylase (adiA, speA), ornithine decarboxylase (speF, speC) and lysine decarboxylase enzymes (e.g., cadA) are additional candidates to catalyze the decarboxylation of 2-aminopimelate.

a-ketopimelate decarboxylase

Keto-acid decarboxylase enzymes are required to alpha-ketopimelate to 6-oxohexanoate. The decarboxylation of keto-acids is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to

acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2- phenylpyruvate (Henning et ah, Appl. Environ. Microbiol. 72:7510-7517 (2006)). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268: 1698-1704 (2001); Li, H. and

F. Jordan, Biochemistry. 38: 10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64: 1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., Eur. J. Biochem. 268: 1698-1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al, Eur. J. Biochem. 269:3256-3263 (2002)).

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al, Biochemistry 42: 1820- 1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

Gene name GI No. GenBank Accession # Organism

mdlC 3915757 P20906.2 Pseudomonas putida

mdlC 81539678 Q9HUR2.1 Pseudomonas aeruginosa dpgB 126202187 ABN80423.1 Pseudomonas stutzeri

ilvB-1 70730840 YP 260581.1 Pseudomonas fluorescens A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US. A 102: 10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (-130 kD) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J Bacteriol. 182:2838- 2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

6-oxohexanoate dehydrogenase (aminating) - rest of text similar to old application

Conversion of 6-oxohexanoate to 6-aminocaproate is catalyzed by aminating oxidoreductase enzymes that transform aldehydes to their corresponding primary amines. An enzyme that catalyzes a similar reaction is lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH genes. This enzyme catalyzes the reversible oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde (Misono et al., J Bacteriol. 150:398-401 (1982)). Exemplary enzyme candidates are found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al, J Biochem 106:76-80 (1989); and Misono et al, J Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMP Rep. 41 :790-795 (2008)).

6-oxohexanoate aminotransferase - rest of text similar to old application

Transamination of 6-oxohexanoate to form 6-aminocaproate is catalyzed by an

aminotransferase enzyme. An exemplary enzyme for converting aldehydes to primary amines is lysine-6-aminotransferase (EC 2.6.1.36). This enzyme function, converting lysine to alpha- aminoadipate semialdehyde, has been demonstrated in yeast and bacteria. Candidates from Candida utilis (Hammer et al., J Basic Microbiol 32:21-27 (1992)), Flavobacterium lutescens (Fujii et al., J Biochem. 128:391-397 (2000)) and Streptomyces clavuligenus (Romero et al., Microbiol Biotechnol 18:241-246 (1997)) have been characterized. A recombinant lysine-6- aminotransferase from S. clavuligenus was functionally expressed in E. coli (Tobin et al., J Bacteriol. 173:6223-6229 (1991)). The F. lutescens enzyme is specific to alpha-ketoglutarate as the amino acceptor (Soda et al., Biochemistry 7:4110-4119 (1968)). Other enzymes which convert aldehydes to terminal amines include the dat gene product in Acinetobacter baumanii encoding 2,4-diaminobutanoate:2-ketoglutarate 4-transaminase (Ikai et al., J Bacteriol.

179:5118-5125 (1997)). In addition to its natural substrate, 2,4-diaminobutyrate, DAT transaminates the terminal amines of lysine, 4-aminobutyrate and ornithine.

Additional enzyme candidates include putrescine aminotransferases or other diamine aminotransferases. The E. coli putrescine aminotransferase is encoded by the yg ^'G gene and the purified enzyme was also able to transaminate cadaverine and spermidine (Samsonova et al., Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim et al., J Biol. Chem. 239:783-786 (1964); and Samsonova et al., Microbiol 3:2 (2003)). The spuC gene of Pseudomonas aeruginosa encodes a putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate (Lu et al., J Bacteriol. 184:3765-3773 (2002)).

The conversion of an aldehyde to a terminal amine can also be catalyzed by gamma- aminobutyrate transaminase (GABA transaminase). This enzyme naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Liu et al., Biochemistry 43: 10896-10905 (2004); and Schulz et al., Appl Environ Microbiol 56: 1-6 (1990)). The two GABA transaminases in E. coli are encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al, J Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with a range of alternate substrates including 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985)); and Scott et al., J Biol. Chem. 234:932-936 (1959)).

6-oxohexanoate dehydrogenase (described in previous app + new text in yellow)

1.2.1.a Oxidoreductase (aldehyde to acid) Two transformations in Figure 26 require conversion of an aldehyde to an acid: conversion of 2-amino-7-oxoheptanoate to 2- aminopimelate (Step D) and 6-aminohexanal to 6-aminocaproate (Step F). Conversion of 6- oxohexanoate to adipate also requires such a transformation. Such reactions are catalyzed by NAD(P)+-dependent oxidoreductases that convert aldehydes to acids in the EC class 1.2.1. A candidate enzyme is the NAD+-dependent aldehyde dehydrogenase (EC 1.2.1.3). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes (Klyosov et al., Biochemistry 35:4457-4467 (1996)). Active ALDH-2 has been efficiently expressed in E. coli using the GroEL proteins as chaperonins (Lee et al., Biochem. Biophys. Res. Commun.

298:216-224 (2002)). The rat mitochondrial aldehyde dehydrogenase also has a broad substrate range that includes the enoyl-aldehyde crotonaldehyde (Siew et al., Arch. Biochem. Biophys. 176:638-649 (1976)). The E. coli gene astD also encodes an NAD+-dependent aldehyde dehydrogenase that converts succinic semialdehyde to succinate (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005)). Additional aldehyde dehydrogenase candidates include ydcW, astD, aldA, aldB, feaB, sad, gabD, and betB.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims

What is claimed is:

1. A non-naturally occurring microbial organism, comprising a microbial organism having a 6-aminocaproic acid (6-ACA) pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient amount to produce 6- ACA; said non-naturally occurring microbial organism further comprising:

(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;

(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or

(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said 6-ACA pathway comprises a pathway selected from:

(A) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6-aminocaproyl-CoA/acyl-CoA transferase, a 6-aminocaproyl-CoA synthase, or a 6-aminocaproyl-CoA hydrolase;

(B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl- CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase;

(C) an 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase; a 2-oxohept-4-ene-l,7- dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7-dioate (OHED) reductase; a 2-oxoheptane- 1,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex- 4-enoate (6-OHE) reductase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(E) an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-aminohept-4-ene-l,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase;

(F) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase;

(G) an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3- dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(H) an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(I) an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate- lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl- CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate

semialdehyde oxidoreductase (aminating);

(J) a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3- oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate decarboxylase;

(K) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6- aminocaproate dehydrogenase;

(L) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6-oxohexanoate aminotransferase;

(M) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate

dehydrogenase (aminating) or α-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase;

(N) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase;

(O) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase;

(P) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; (Q) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase;

(R) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and

(S) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase.

2. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

3. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

4. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP-citrate lyase or a citrate lyase; a fumarate reductase; and an alpha- ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate :ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H₂ hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

5. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 6-ACA pathway enzyme.

6. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises:

(A) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; and a 6-aminocaproyl-CoA/acyl-CoA transferase, a 6- aminocaproyl-CoA synthase, or a 6-aminocaproyl-CoA hydrolase;

(B) five exogounous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase;

(C) five exogounous nucleic acids encoding an 4-hydroxy-2-oxoheptane-l,7-dioate (HODH) aldolase; a 2-oxohept-4-ene-l,7-dioate (OHED) hydratase; a 2-oxohept-4-ene-l,7- dioate (OHED) reductase; a 2-oxoheptane-l,7-dioate (2-OHD) decarboxylase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (D) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-oxohex-4-enoate (6-OHE) reductase; and an adipate

semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(E) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-aminohept-4-ene- 1,7-dioate (2-AHE) reductase; and a 2-aminoheptane-l,7-dioate (2-AHD) decarboxylase;

(F) five exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase;

(G) seven exogounous nucleic acids encoding an HODH aldolase; an HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(H) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(I) seven exogounous nucleic acids encoding an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating);

(J) seven exogounous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate;

(K) two exogounous nucleic acids encoding a CoA-dependent aldehyde

dehydrogenase; and a transaminase or a 6-aminocaproate dehydrogenase; (L) sixteen exogounous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and a 6-oxohexanoate dehydrogenase (aminating) or a 6-oxohexanoate aminotransferase;

(M) sixteen exogounous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate dehydrogenase (aminating) or a-ketopimelate amino transferase; and a 2-aminopimelate decarboxylase;

(N) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase;

(O) twelve exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase;

(P) ten exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase;

(Q) nine exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; and an adipate reductase;

(R) eleven exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an adipate kinase; and an adipylphosphate reductase; or

(S) nine exogounous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); and a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase.

7. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

8. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (ii) comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

9. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprising (K) further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from:

(A) succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase;

(B) adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl- CoA:acetyl-CoA transferase and adipyl-CoA hydrolase; and

(C) succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase, 3- oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.

10. The non-naturally occurring microbial organism of claim 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

11. The non-naturally occurring microbial organism of claim 1 , wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

12. A method for producing 6-aminocaproic acid, comprising culturing the non- naturally occurring microbial organism of any one of claims 1-10 under conditions and for a sufficient period of time to produce 6-ACA.

13. The method of claim 12, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

14. A non-naturally occurring microbial organism, comprising a microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in a sufficient amount to produce caprolactam; said non-naturally occurring microbial organism further comprising:

(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said caprolactam pathway comprises a pathway selected from:

(A) a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase;

(B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and a 6-aminohex-2-enoyl-CoA reductase;

(C) a CoA-dependent aldehyde dehydrogenase; a transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase;

(D) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an adipate reductase;

(E) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase;

(F) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase;

(G) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase; (H) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase;

(I) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; (J) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase;

(K) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase;

(L) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; and an amidohydrolase; (M) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-

CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase; (N) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; and

(O) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; and a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase.

15. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

16. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

17. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP-citrate lyase or a citrate lyase; a fumarate reductase; and an alpha- ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate :ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H₂ hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

18. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprises two, three or four exogenous nucleic acids each encoding a caprolactam pathway enzyme.

19. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprises:

(A) one exogenous nucleic acid enconding a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase;

(B) four exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and a 6-aminohex-2-enoyl-CoA reductase;

(C) three exogenous nucleic acids encoding a CoA-dependent aldehyde

dehydrogenase; a transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase;

(D) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

(E) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; an amidohydrolase; an adipate kinase; and an adipylphosphate reductase;

(F) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase;

(G) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl- CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; and an adipate reductase;

(H) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase;

(I) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; an amidohydrolase; and an adipate reductase;

(K) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

adipylphosphate reductase;

(L) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; and an amidohydrolase;

(M) eleven exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; and an adipate reductase;

(N) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; an adipate kinase; and an adipylphosphate reductase; or

(O) ten exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

20. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

21. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

22. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (A) further comprises a 6-aminocaproic acid pathway, wherein said 6-aminocaproic acid pathway is selected from:

(A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6- aminocaproate dehydrogenase; and (B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl- CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase.

23. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprising (C) further comprises an adipyl-CoA pathway, wherein said adipyl-CoA pathway is selected from:

24. The non-naturally occurring microbial organism of claim 14, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

25. The non-naturally occurring microbial organism of claim 14, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

26. A method for producing caprolactam, comprising culturing the non-naturally occurring microbial organism of any one of claims 14 - 24 under conditions and for a sufficient period of time to produce caprolactam.

27. The method of claim 26, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

28. The method for producing caprolactam of claim 24, wherein said microbial organism comprises a caprolactam pathway and wherein said caprolactam is produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam.

29. The method for producing caprolactam of claim 28, wherein said caprolactam pathway is selected from the group consisting of (G), (H), (M), (N) and (O).

30. A non-naturally occurring microbial organism, comprising a microbial organism having a hexamethylenediamine (HMD A) pathway comprising at least one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a sufficient amount to produce HMD A; said non-naturally occurring microbial organism further comprising:

(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said HMDA pathway comprises a pathway selected from:

(A) a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase;

(B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a

hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase;

(C) a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde

aminotransferase;

(D) a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase; (E) a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6- aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase;

(F) a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6- AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide);

(G) a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6- acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide);

(H) a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6- AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or a 6- acetamidohexanamine hydrolase (amide);

(I) a glutamyl-CoA transferase or a glutamyl-CoA ligase; a beta-ketothiolase; a 3- oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA

dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine decarboxylase;

(J) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(K) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a oxo-l-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3- aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(L) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate Co A transferase or 3-oxopimelate Co A ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3 -oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l-carboxyheptanal 7- aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(M) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(N) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5- oxopimeloylphosphonate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3- oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(O) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate Co A transferase or a 3-oxopimelate Co A ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3- aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (P) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7- aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase; (Q) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(R) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7- aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(S) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(T) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (U) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-amino transferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase;

(V) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(W) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2- amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(X) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3- oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate

aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3-aminopimelate 2,3- aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6- aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7-oxoheptanoate 7- aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(Y) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7- aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7- aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(Z) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7- aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7- aminoheptanoate decarboxylase; and a 6-aminohexanal aminotransferase or 6-aminohexanal aminating oxidoreductase; (AA) a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase (aminating);

(AB) a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase; a 6- acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide);

(AC) a 2-amino-7-oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase;

(AD) a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7- aminoheptanoate aminating oxidoreductase or a 2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase;

(AE) a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase;

(AF) a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase;

(AG) a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a homolysine decarboxylase;

(AH) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo- glutaryl-[acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate

decarboxylase; a 6-oxohexanoate dehydrogenase (aminating) or 6-oxohexanoate

aminotransferase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase; (AI) an acetyl-CoA carboxylase; a malonyl-CoA methy transferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an a-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6-aminohexanal dehydrogenase

(aminating) or a 6-aminohexanal aminotransferase;

(AJ) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an α-ketopimelate amino transferase; a 2-aminopimelate; a 2- amino-7-oxoheptanoate dehydrogenase; and a 2,7-diaminoheptanoate decarboxylase; (AK) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase;

(AL) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl- CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6- aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6- aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase;

(AM) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase;

(AN) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase;

(AO) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6- aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl- CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; and (AP) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase.

31. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

32. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

33. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP-citrate lyase or a citrate lyase; a fumarate reductase; and an alpha- ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate: ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H₂ hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

34. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen exogenous nucleic acids each encoding a HMDA pathway enzyme.

35. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprises:

(A) three exogenous nucleic acids encoding a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase;

(B) six exogenous nucleic acids encoding a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA reductase; a 3-hydroxy-6-aminohexanoyl-CoA dehydratase; a 6-aminohex-2-enoyl-CoA reductase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a hexamethylenediamine transaminase or a hexamethylenediamine dehydrogenase;

(C) three exogenous nucleic acids encoding a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6- aminocaproic acid semialdehyde aminotransferase;

(D) four exogenous nucleic acids encoding a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase;

(E) three exogenous nucleic acids encoding a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde aminotransferase;

(F) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide);

(G) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA ligase; a 6- acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or a 6-acetamidohexanamine hydrolase (amide);

(H) five exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and a 6- acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide);

(I) eight exogenous nucleic acids encoding a glutamyl-CoA transferase or a glutamyl- CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3- hydroxy-6-aminopimeloyl-CoA dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate aminotransferase or aminating oxidoreductase; and a homolysine

decarboxylase;

(J) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 7-aminotransferase or a 3-oxo- 1-carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3- aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(K) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l- carboxyheptanal 7-aminotransferase or a 3-oxo-l-carboxyheptanal 7-aminating

oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7- aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(L) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate Co A transferase or 3-oxopimelate Co A ligase; a 5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 7-aminotransferase or 3-oxo-l- carboxyheptanal 7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3 -aminotransferase or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3- aminomutase; and a homolysine decarboxylase;

(M) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3-oxo- 1-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(N) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a 3-oxo-l- carboxyheptanal 3 -aminotransferase or a 3-oxo-l-carboxyheptanal 3-aminating

oxidoreductase; a 3-amino-7-oxoheptanoate 7-amino transferase or a 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(O) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate Co A transferase or a 3-oxopimelate Co A ligase; a 5-oxopimeloyl- CoA reductase (aldehyde forming); a 3-oxo-l-carboxyheptanal 3 -aminotransferase or a 3- oxo-l-carboxyheptanal 3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7- aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(P) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7- oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(Q) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7- oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(R) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate CoA transferase or a 3 -aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(S) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate 7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(T) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate CoA transferase or a 3 -aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7- oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;

(U) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7- diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase; (V) seven exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(W) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(X) eight exogenous nucleic acids encoding a glutaryl-CoA beta-ketothiolase; a 3- oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating oxidoreductase; a 3- aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a 2-amino-7- oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(Y) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase; and a homolysine decarboxylase;

(Z) five exogenous nucleic acids encoding a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a 2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal

aminotransferase or 6-aminohexanal aminating oxidoreductase;

(AA) two exogenous nucleic acids encoding a 6-aminocaproate reductase; and a 6- aminocaproic semialdehyde aminotransferase or a 6-aminocaproic semialdehyde

oxidoreductase (aminating);

(AB) four exogenous nucleic acids encoding a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase; a 6-acetamidohexanal aminotransferase or 6- acetamidohexanal oxidoreductase (aminating); and a 6-acetamidohexanamine N- acetyltransferase or 6-acetamidohexanamine hydrolase (amide);

(AC) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase;

(AD) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a 2-oxo-7- aminoheptanoate aminotransferase; and a homolysine decarboxylase;

(AE) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate amino acid decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase;

(AF) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanal aminating oxidoreductase or a 6-aminohexanal aminotransferase;

(AG) three exogenous nucleic acids encoding a 2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating oxidoreductase or a 2-amino-7- oxoheptanoate aminotransferase; and a homolysine decarboxylase;

(AH) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; a a-ketopimelate decarboxylase; a 6-oxohexanoate dehydrogenase

(aminating) or 6-oxohexanoate aminotransferase; a 6-aminocaproate reductase; and a 6- aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase;

(AI) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate dehydrogenase (aminating) or an a-ketopimelate amino transferase; a 2-aminopimelate decarboxylase; a 6-aminocaproate reductase; and a 6- aminohexanal dehydrogenase (aminating) or a 6-aminohexanal aminotransferase;

(AJ) eighteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an α-ketopimelate dehydrogenase (aminating) or an α-ketopimelate amino transferase; a 2-aminopimelate; a 2-amino-7-oxoheptanoate dehydrogenase; and a 2,7- diaminoheptanoate decarboxylase;

(AK) fourteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase;

(AL) fifteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase;

(AM) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate

dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA reductase (aldehyde forming); a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); and a HMDA transaminase or a HMDA dehydrogenase;

(AN) thirteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; and an adipate reductase;

(AO) fourteen exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate

dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; an adipyl-CoA transferase, an adipyl- CoA synthase or an adipyl-CoA hydrolase; a 6-aminocaproate transaminase or a 6- aminocaproate dehydrogenase; a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6- aminocaproyl-CoA synthase; a 6-aminocaproyl-CoA reductase (aldehyde forming); a HMDA transaminase or a HMDA dehydrogenase; an adipate kinase; and an adipylphosphate reductase; or

(AP) twelve exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA

36. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

37. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

38. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (A) further comprises a 6-aminocaproic acid pathway, wherein said 6-aminocaproic acid pathway is selected from:

(A) a CoA-dependent aldehyde dehydrogenase; and a transaminase or a 6- aminocaproate dehydrogenase; and

(B) a 3-oxo-6-aminohexanoyl-CoA thiolase; a 3-oxo-6-aminohexanoyl-CoA/acyl- CoA transferase, a 3-oxo-6-aminohexanoyl-CoA synthase, or a 3-oxo-6-aminohexanoyl-CoA hydrolase; a 3-oxo-6-aminohexanoate reductase; a 3-hydroxy-6-aminohexanoate dehydratase; and a 6-aminohex-2-enoate reductase.

39. The non-naturally occurring microbial organism of claim 30, wherein said microbial organism comprising (AC), (AD), (AE), (AF) or (AG) further comprises at least one exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme expressed in a sufficient amount to produce 2-amino-7-oxosubarate, wherein said 2-amino-7-oxosubarate pathway comprises a 2-amino-5-hydroxy-7-oxosubarate aldolase; a 2-amino-5-hydroxy-7- oxosubarate dehydratase; and a 2-amino-5-ene-7-oxosubarate reductase.

40. The non-naturally occurring microbial organism of claim 30, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

41. The non-naturally occurring microbial organism of claim 30, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

42. A method for producing HMD A, comprising culturing the non-naturally occurring microbial organism of any one of claims 30-40 under conditions and for a sufficient period of time to produce HMD A.

43. The method of claim 42, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

44. A non-naturally occurring microbial organism, comprising a microbial organism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate; said non- naturally occurring microbial organism further comprising:

(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein said adipate pathway comprises a pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyl transferase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase; (B) a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3- oxoadipate reductase; a 3-hydroxyadipate dehydratase; and a 2-enoate reductase;

(C) an alpha-ketoadipyl-CoA synthetase, a phosphotransketoadipylase/alpha- ketoadipate kinase or an alpha-ketoadipyl-CoA:acetyl-CoA transferase; a 2-hydroxyadipyl- CoA dehydrogenase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase;

(D) a 2-hydroxyadipate dehydrogenase; a 2-hydroxyadipyl-CoA synthetase, a phosphotranshydroxyadipylase/2-hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl- CoA transferase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl- CoA:acetyl-CoA transferase or an adipyl-CoA hydrolase;

(E) an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl- [acp] mthylester synthase; a 3-oxo-glutaryl-[acp] methyl ester reductase; a 3- hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2- monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2- pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; and (G) a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2-Hydroxyadipyl-CoA synthtase, a 2- Hydroxyadipyl-CoA transferase, or a phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase.

45. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

46. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

47. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP-citrate lyase or a citrate lyase; a fumarate reductase; and an alpha- ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate: ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H₂ hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

48. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen exogenous nucleic acids each encoding an adipate pathway enzyme.

49. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprises:

(A) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 5- carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a

phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase or an adipyl- CoA hydrolase;

(B) five exogenous nucleic acids encoding a succinyl-CoA:acetyl-CoA acyl transferase; a 3-oxoadipyl-CoA transferase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydratase; and a 2-enoate reductase;

(C) five exogenous nucleic acids encoding an alpha-ketoadipyl-CoA synthetase, a phosphotransketoadipylase/alpha-ketoadipate kinase or an alpha-ketoadipyl-CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydrogenase; a 2-hydroxyadipyl-CoA dehydratase; a 5-carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a

(D) five exogenous nucleic acids encoding a 2-hydroxyadipate dehydrogenase; a 2- hydroxyadipyl-CoA synthetase, a phosphotranshydroxyadipylase/2-hydroxyadipate kinase or a 2-hydroxyadipyl-CoA:acetyl-CoA transferase; a 2-hydroxyadipyl-CoA dehydratase; a 5- carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA synthetase, a

(E) fourteen exogenous nucleic acids encoding an acetyl-CoA carboxylase; a malonyl-CoA methyltransferase; a 3-oxo-glutaryl-[acp] mthylester synthase; a 3-oxo- glutaryl-[acp] methyl ester reductase; a 3-hydroxyglutaryl-[acp] methylester dehydratase; an enoylglutaryl-[acp] methyl ester reductase; a 3-oxo-pimoloyl-[acp] methyl ester synthase; a 3-oxo-pimeloyl-[acp] methyl ester reductase; a 3-hydroxypimeloyl-[acp] methyl ester dehydratase; an enoylpimeloyl-[acp] methyl ester reductase; a pimeloyl-[acp] methyl ester esterase; a thioesterase; a pimelate-2-monooxygenase; a 2-hydroxyheptanedioic acid dehydrogenase; an a-ketopimelate decarboxylase; and an 6-oxohexanoate dehydrogenase; (F) nine exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a threo-isohomocitrate dehydrogenase; an alpha-ketoadipyl-CoA synthetase, an alpha-ketoadipyl-CoA transferase or a

phosphotransketoadipylase/alpha-ketoadipate kinase; an alpha-ketoadipyl-CoA reductase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl- CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase; or

(G) eight exogenous nucleic acids encoding a homocitrate synthase; a homocitrate dehydratase; a cis-Homoaconitate hydratase; a 2-Hydroxyadipate dehydrogenase; a 2- Hydroxyadipyl-CoA synthtase, a 2-Hydroxyadipyl-CoA transferase, or a

phosphotranshydroxyadipylase/2-hydroxyadipate kinase; a 2-Hydroxyadipyl-CoA dehydratase; a 5-Carboxy-2-pentenoyl-CoA reductase; and an adipyl-CoA transferase, an adipyl-CoA synthase or an adipyl-CoA hydrolase.

50. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

51. The non-naturally occurring microbial organism of claim 44, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

52. The non-naturally occurring microbial organism of claim 44, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

53. The non-naturally occurring microbial organism of claim 44, wherein said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.

54. A method for producing adipate, comprising culturing the non-naturally occurring microbial organism of any one of claims 44-52 under conditions and for a sufficient period of time to produce adipate.

55. The method of claim 54, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.