Genetically modified microorganisms and methods for improving aldehyde dehydrogenase activity

Abstract

Biosynthetic methods and genetically modified microorganisms that enhance or improve the biosynthesis of hexamethylenediamine, caproic acid, or caprolactam are disclosed. [Solution] Genetically modified microorganisms contain selected aldehyde dehydrogenase activity.

Description

[Technical Field]

[0001] (Cross-reference to related applications) This application claims the interests of U.S. Provisional Patent Application No. 62 / 837,888 filed on 24 April 2019, No. 62 / 860,123 filed on 11 June 2019, and No. 62 / 860,160 filed on 11 June 2019, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence lists This application includes a sequence listing titled "GNO0099WO Sequence Listing2.txt", which was created on April 23, 2020, and is 319 kilobytes in size. This sequence listing is incorporated herein by reference. [Background technology]

[0003] Nylon is a polyamide that can be synthesized by condensation polymerization of diamines and dicarboxylic acids or by condensation polymerization of lactams. Nylon 6,6 is produced by the reaction of hexamethylenediamine (HMD) and adipic acid, and nylon 6 is produced by ring-opening polymerization of caprolactam. Therefore, adipic acid, hexamethylenediamine, and caprolactam are important intermediates in nylon production. [Overview of the project] [Problems that the invention aims to solve]

[0004] Some microorganisms have been genetically engineered to produce several nylon intermediates. However, these genetically engineered microorganisms may produce undesirable byproducts as a result of undesirable enzymatic activity in the pathway intermediates or final products. Thus, such byproducts and impurities can increase the cost and complexity of compound biosynthesis and reduce the efficiency or yield of the desired product. [Means for solving the problem]

[0005] (summary) This specification provides non-spontaneously occurring microorganisms having the 6-aminocaproic acid pathway, the caprolactam pathway, the hexamethylenediamine pathway, the caprolactone pathway, the 1,6-hexaxanediol pathway, or one or more combinations thereof. These microorganisms comprise at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde. The aldehyde dehydrogenase has a greater turnover rate, greater catalytic efficiency, or a combination thereof, with respect to the adipyl-CoA substrate compared to succinyl-CoA, acetyl-CoA, or both succinyl-CoA and acetyl-CoA substrates. These non-spontaneously occurring microorganisms may further comprise additional exogenous nucleic acids encoding enzymes necessary to produce sufficient amounts of the respective products of 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine. In some cases, one or more of these exogenous nucleic acids may be heterologous to the microorganism.

[0006] Methods for producing 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine are also disclosed. These methods may involve culturing a non-spontaneously occurring microorganism that produces 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and / or hexamethylenediamine, wherein the microorganism expresses at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde. These methods involve culturing the non-spontaneously occurring microorganism for a sufficient time under conditions that produce 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine.

[0007] In one embodiment, a non-spontaneously occurring microorganism is provided comprising at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde, wherein the aldehyde dehydrogenase has greater catalytic efficiency with respect to adipyl-CoA as a substrate compared to succinyl-CoA, acetyl-CoA, or both succinyl-CoA and acetyl-CoA as substrates, and / or the aldehyde dehydrogenase has a greater turnover rate with respect to an adipyl-CoA substrate compared to succinyl-CoA, acetyl-CoA, or both succinyl-CoA and acetyl-CoA substrates.

[0008] In one embodiment, a method for producing adipic acid semialdehyde is provided, comprising culturing a non-spontaneously occurring microorganism of any one of the above embodiments and embodiments for a time and under conditions sufficient to produce adipic acid semialdehyde.

[0009] In one embodiment, a method for producing 6-aminocaproic acid (6ACA) is provided, comprising culturing a non-spontaneously occurring microorganism of any one of the above embodiments and models for a sufficient time and under conditions to produce 6-aminocaproic acid (6ACA). In some embodiments, the method further comprises recovering 6ACA from the microorganism, a fermentation broth, or both.

[0010] In one embodiment, a method for producing hexamethylenediamine is provided, comprising culturing a non-spontaneously occurring microorganism of any one of the above embodiments and models for a time and under conditions sufficient to produce hexamethylenediamine. In some embodiments, the method further comprises recovering hexamethylenediamine from the microorganism, a fermentation broth, or both. In some embodiments, the non-spontaneously occurring microorganism comprises 2, 3, 4, 5, 6, or 7 exogenous nucleic acid sequences, each encoding a hexamethylenediamine pathway enzyme.

[0011] In one embodiment, a method is provided for producing 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine, comprising culturing a non-spontaneously occurring microorganism of any one of the above embodiments and models for a time and under conditions sufficient to produce 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine. In some embodiments, the method further comprises recovering 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine from the microorganism, a fermentation broth, or both. In some embodiments, the non-spontaneously occurring microorganism comprises 2, 3, 4, 5, 6, or 7 exogenous nucleic acid sequences, each encoding enzymes for the 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, and hexamethylenediamine pathways.

[0012] In one embodiment, a bio-derived 6-aminocaproic acid, hexamethylenediamine, or caprolactam synthesized using the disclosed method is provided.

[0013] In some embodiments, the aldehyde dehydrogenase from a non-spontaneously occurring microorganism does not contain the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0014] In some embodiments, non-spontaneously occurring microbial aldehyde dehydrogenases are SEQ ID NOs: 4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107 It includes an amino acid sequence having at least 60% amino acid sequence identity with any of the following consecutive amino acids: 109, 111, 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159, 164-167, 176, 187, and 188, comprising at least 25, 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids.

[0015] Non-naturally occurring microbial aldehyde dehydrogenases include SEQ ID NOs: 4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 109, 111, 112, 117, 134, 135 The amino acid sequence comprises at least 25, 50, 75, 100, 150, 200, 250, 300 or more consecutive amino acids from any of 137, 145, 146, 148-150, 152, 157-159, 164-167, 176, 187, and 188, and an amino acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity. In some embodiments, the aldehyde dehydrogenase uses NADH as a cofactor.

[0016] In some embodiments, the aldehyde dehydrogenase of a non-naturally occurring microorganism comprises an amino acid sequence having at least about 60% amino acid sequence identity with at least 25, 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of any of SEQ ID NOs: 7, 28, 60, and 107. In some embodiments, the aldehyde dehydrogenase comprises an amino acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity with at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acid sequences of any of SEQ ID NOs: 7, 28, 60, and 188. In some embodiments, the aldehyde dehydrogenase uses NADH as a cofactor.

[0017] In some embodiments, the aldehyde dehydrogenase of a non-naturally occurring microorganism comprises an amino acid sequence having at least about 60% amino acid sequence identity with at least 25, 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of any of SEQ ID NOs: 53, 77, 82, 94, and 152. In some embodiments, the aldehyde dehydrogenase uses NADH, NADPH, or both as cofactors.

[0018] In some embodiments, the aldehyde dehydrogenase of a non-naturally occurring microorganism has a greater catalytic efficiency for adipyl-CoA substrate compared to succinyl-CoA substrate. In some embodiments, the catalytic efficiency of the aldehyde dehydrogenase for adipyl-CoA substrate is at least 2-fold higher than that for succinyl-CoA substrate.

[0019] In some embodiments, the aldehyde dehydrogenase of the non-naturally occurring microorganism has a greater catalytic efficiency for adipyl-CoA substrate compared to the acetyl-CoA substrate. In some embodiments, the catalytic efficiency of the aldehyde dehydrogenase for the adipyl-CoA substrate is at least 5-fold higher than that for the acetyl-CoA substrate. In some embodiments, the aldehyde dehydrogenase has a greater metabolic turnover number for the adipyl-CoA substrate compared to the acetyl-CoA substrate.

[0020] In some embodiments, the aldehyde dehydrogenase of the non-naturally occurring microorganism further reacts with 6-aminocaproyl-CoA to produce 6-aminocaproic acid semialdehyde.

[0021] In some embodiments, a non-naturally occurring microorganism containing at least one exogenous nucleic acid encoding aldehyde dehydrogenase converts more adipyl-CoA to adipic acid semialdehyde than a control microorganism that is substantially identical to the non-naturally occurring microorganism except that it does not contain the exogenous nucleic acid encoding aldehyde dehydrogenase.

[0022] In some embodiments, at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde is heterologous to the microorganism.

[0023] In some embodiments, the non-naturally occurring microorganism includes a 6-aminocaproic acid pathway. In some embodiments, the 6-aminocaproic acid pathway includes (i) an aminotransferase, (ii) a 6-aminocaproic acid dehydrogenase, or (iii) both an aminotransferase and a 6-aminocaproic acid dehydrogenase. In some embodiments, the non-naturally occurring microorganism further includes one or more additional exogenous nucleic acids encoding one or more 6-aminocaproic acid pathway enzymes. In some embodiments, the exogenous nucleic acids encoding one or more 6-aminocaproic acid pathway enzymes are heterologous to the microorganism.

[0024] In some embodiments, non-spontaneously occurring microorganisms include a hexamethylenediamine pathway. In some embodiments, the hexamethylenediamine pathway includes (i) 6-aminocaproyl-CoA transferase, (ii) 6-aminocaproyl-CoA synthase, (iii) 6-aminocaproyl-CoA reductase, (iv) hexamethylenediamine aminotransferase, (v) hexamethylenediamine dehydrogenase, or (vi) a combination of one or more enzymes (i) to (v). In some embodiments, the microorganism further includes one or more additional exogenous nucleic acids encoding one or more hexamethylenediamine pathway enzymes, such as a carboxylate reductase (CAR) that converts 6-aminocaproate to 6-aminocaproic acid semialdehyde. 6-aminocaproic acid semialdehyde can then be converted to hexamethylenediamine. In some embodiments, the exogenous nucleic acids encoding one or more hexamethylenediamine pathway enzymes are heterogeneous to the microorganism.

[0025] In some embodiments, the non-spontaneously occurring microorganism includes a caprolactam pathway. In some embodiments, the caprolactam pathway includes an aminohydrolase. In some embodiments, the microorganism further includes one or more additional exogenous nucleic acids encoding an aminohydrolase. In some embodiments, the exogenous nucleic acid encoding the aminohydrolase is heterogeneous to the microorganism.

[0026] In some embodiments, non-spontaneously occurring microorganisms include the 1,6-hexanediol pathway. In some embodiments, the 1,6-hexanediol pathway includes the following enzymes: 6-aminocaproyl-CoA transferase or synthase catalyzing the conversion of 6ACA to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA reductase catalyzing the conversion of 6-aminocaproyl-CoA to 6-aminocaproic acid semialdehyde; 6-aminocaproic acid semialdehyde reductase catalyzing the conversion of 6-aminocaproic acid semialdehyde to 6-aminohexanol; 6-aminocaproic acid reductase catalyzing the conversion of 6ACA to 6-aminocaproic acid semialdehyde; adipyl-CoA reductase catalyzing the conversion of adipyl-CoA to adipic acid semialdehyde; adipic acid semialdehyde reductase catalyzing the conversion of adipic acid semialdehyde to 6-hydroxyhexanoic acid; 6-hydroxyhexanoic acid 6-hydroxyhexanoyl-CoA transferase or synthase that catalyzes the conversion of acid to 6-hydroxyhexanoyl-CoA; 6-hydroxyhexanoyl-CoA reductase that catalyzes the conversion of 6-hydroxyhexanoyl-CoA to 6-hydroxyhexanal; 6-hydroxyhexanal reductase that catalyzes the conversion of 6-hydroxyhexanal to HDO; 6-aminohexanol aminotransferase or oxidoreductase that catalyzes the conversion of 6-aminohexanol to 6-hydroxyhexanal; 6-hydroxyhexanoate reductase that catalyzes the conversion of 6-hydroxyhexanoic acid to 6-hydroxyhexanal; adipic acid reductase that catalyzes the conversion of ADA to adipic acid semialdehyde; and adipic-CoA transferase, hydrolase, or synthase that catalyzes the conversion of adipyl-CoA to ADA.

[0027] In some embodiments, non-spontaneously occurring microorganisms include a pathway from adipine or adipyl-CoA to caprolactone. In some embodiments, the pathway from adipine or adipyl-CoA to caprolactone includes the following enzymes: adipyl-CoA reductase, adipate semialdehyde reductase, 6-hydroxyhexanoyl-CoA transferase or synthase, 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, adipate reductase, adipyl-CoA transferase, synthase, or hydrolase, 6-hydroxyhexanoate cyclase, 6-hydroxyhexanoate kinase, 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, phosphotrans-6-hydroxyhexanoylase.

[0028] In some embodiments, the aldehyde dehydrogenases of non-spontaneously occurring microorganisms are derived from prokaryotic species. In some embodiments, the aldehyde dehydrogenases are derived from Acidaminococcus, Collinsella, Peptostreptococcaceae, or Romboustsia.

[0029] In some embodiments, non-spontaneously occurring microorganisms include species of Acinetobacter, Actinobacillus, Anaerobiospirillum, Aspergillus, Bacillus, Clostridium, Corynebacterium, Escherichia, Gluconobacter, Klebsiella, Kluyveromyces, Lactococcus, Lactobacillus, Mannheimia, Pichia, Pseudomonas, Rhizobium, Rhizopus, Saccharomyces, Schizosaccharomyces, Streptomyces, and Zymomonas. In some embodiments, the non-spontaneously occurring microorganism is a strain of Escherichia coli.

[0030] In some embodiments, cultivation is carried out in a fermentation broth containing sugar. [Brief explanation of the drawing]

[0031] [Figure 1] Exemplary pathways from succinyl-CoA and acetyl-CoA to 6-aminocaproates, hexamethylenediamine (HMDA), and caprolactam are shown. The enzymes are designated as follows: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydrase, 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 dehydrase, J) 5-carboxy-2-pentenoate reductase, K) adipyl-CoA / acyl-CoA transferase, L) M) Adipyl-CoA synthase, N) Adipyl-CoA hydrolase, O) 6-aminocaproyl aminotransferase, P) 6-aminocaproyl dehydrogenase, Q) 6-aminocaproyl-CoA / acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase, S) Amide hydrolase, T) Spontaneous cyclization, U) 6-aminocaproyl-CoA reductase (aldehyde production), V) HMDA aminotransferase, W) HMDA dehydrogenase, X) Adipic acid reductase, Y) Adipic acid kinase, Z) Adipyl phosphate reductase.

[0032] [Figure 2] This graph displays data on aldehyde dehydrogenase lysates that show greater activity with adipyl-CoA than with succinyl-CoA.

[0033] [Figure 3]This graph displays reaction rate data showing purified aldehyde dehydrogenases exhibiting higher preference for adipyl-CoA than for acetyl-CoA and succinyl-CoA. Figure 3A shows the catalytic efficiency of various aldehyde dehydrogenases, indicated by their sequence numbers, for succinyl-CoA, acetyl-CoA, and adipyl-CoA substrates. Figure 3B shows the ratio of catalytic efficiency of these various aldehyde dehydrogenases for adipyl-CoA substrates to succinyl-CoA substrates. Figure 3C shows the ratio of catalytic efficiency of these various aldehyde dehydrogenases for adipyl-CoA substrates to acetyl-CoA substrates.

[0034] [Figure 4] An exemplary pathway for the synthesis of 6-aminocaproic acid and adipine salts using lysine as a starting point is shown.

[0035] [Figure 5] This shows an exemplary caprolactam synthesis pathway using azipyrus-CoA as a starting point.

[0036] [Figure 6]Exemplary pathways from pyruvate and succinate semialdehyde to 6-aminocaproate are shown. The enzymes are as follows: A) HODH aldolase, B) OHED hydrolase, C) OHED reductase, D) 2-OHD decarboxylase, E) adipic acid semialdehyde aminotransferase and / or adipic acid semialdehyde oxidoreductase (amination), F) OHED decarboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferase and / or 2-OHD oxidoreductase (amination), I) 2-AHD decarboxylase, J )OHED aminotransferase and / or OHED oxidoreductase (amination), K) 2-AHE reductase, L) HODH formate lyase and / or HODH dehydrogenase, M) 3-hydroxyadipyl-CoA dehydrogenase, 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. The abbreviations are as follows: HODH = 4-hydroxy-2-oxoheptane-1,7-dioxide, OHED = 2-oxohepta-4-ene-1,7-dioxide, 2-OHD = 2-oxoheptane-1,7-dioxide, 2-AHE = 2-aminohepta-4-ene-1,7-dioxide, 2-AHD = 2-aminoheptane-1,7-dioxide, and 6-OHE = 6-oxohexa-4-enoic acid.

[0037] [Figure 7]An exemplary pathway from 6-aminocaproic acid to hexamethylenediamine is shown. The enzymes are as follows: A) 6-aminocaproic acid kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproic acid semialdehyde aminotransferase and / or 6-aminocaproic acid semialdehyde oxidoreductase (amination), D) 6-aminocaproic acid N-acetyltransferase, E) 6-acetamidohexanoic acid kinase, F) 6-AAHOP oxidoreductase, G) 6-acetamidohexanal aminotransferase and / or 6-acetamidohexanal oxidoreductase (amination), H) 6 -Acetamidehexanamine N-acetyltransferase and / or 6-acetamidehexanamine hydrolase (amide), I) 6-acetamidehexanoate CoA transferase and / or 6-acetamidehexanoate CoA ligase, J) 6-acetamidehexanoyl 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. The abbreviations are as follows: 6-AAHOP = [(6-acetamidehexanoyl)oxy]phosphonic acid and 6-AHOP = [(6-aminohexanoyl)oxy]phosphonic acid.

[0038] [Figure 8]An exemplary biosynthetic pathway leading to 1,6-hexanediol is shown. A) is a 6-aminocaproyl-CoA transferase or synthase that catalyzes the conversion of 6ACA to 6-aminocaproyl-CoA; B) is a 6-aminocaproyl-CoA reductase that catalyzes the conversion of 6-aminocaproyl-CoA to 6-aminocaproic acid semialdehyde; C) is a 6-aminocaproic acid semialdehyde reductase that catalyzes the conversion of 6-aminocaproic acid semialdehyde to 6-aminohexanol; D) E) is a 6-aminocaproic acid reductase that catalyzes the conversion of 6ACA to 6-aminocaproic acid semialdehyde; F) is an adipyrus-CoA reductase that catalyzes the conversion of adipyl-CoA to adipic acid semialdehyde; G) is an adipic acid semialdehyde reductase that catalyzes the conversion of adipic acid semialdehyde to 6-hydroxyhexanoic acid; H) is a 6-hydroxyhexanoyl-CoA transferase or synthase that catalyzes the conversion to A; I) is a 6-hydroxyhexanoyl-CoA reductase that catalyzes the conversion from 6-hydroxyhexanoyl-CoA to 6-hydroxyhexanal; J) is a 6-aminohexanol aminotransferase or oxidoreductase that catalyzes the conversion from 6-aminohexanol to 6-hydroxyhexanal; K) is a 6-hydroxyhexanoate reductase that catalyzes the conversion from 6-hydroxyhexanoic acid to 6-hydroxyhexanal; L) is an adipicate reductase that catalyzes the conversion from ADA to adipic acid semialdehyde; and M) is an adipic-CoA transferase, hydrolase, or synthase that catalyzes the conversion from adipyl-CoA to ADA.

[0039] [Figure 9]An exemplary pathway from adipic acid or adipyl-CoA to caprolactone is shown. The enzymes are as follows: A. adipyl-CoA reductase, B. adipate semialdehyde reductase, C. 6-hydroxyhexanoyl-CoA transferase or synthase, D. 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, E. adipate reductase, F. adipyl-CoA transferase, synthase, or hydrolase, G. 6-hydroxyhexanoyl cyclase, H. 6-hydroxyhexanoyl kinase, I. 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, J. phosphotrans-6-hydroxyhexanoylase. [Modes for carrying out the invention]

[0040] (Detailed explanation) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods, apparatus, and materials similar or equivalent to those described herein may be used in the practice of the present invention. The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure. All documents referenced herein are incorporated herein by reference in their entirety.

[0041] Disclosed herein are non-naturally occurring microorganisms genetically engineered to express exogenous aldehyde dehydrogenases (ALDs) that have greater catalytic efficiency and turnover rates for azipyrus-CoA substrates compared to succinyl-CoA, acetyl-CoA, or both substrates. Adipyrus-CoA is an intermediate (referred herein to as nylon intermediate) in pathways leading to the biosynthetic production of 6-aminocaproic acid, caprolactam, and hexamethylenediamine. Several different pathways can be used to produce these nylon intermediates. In some embodiments, nylon intermediates may be produced from a pathway as shown in Figure 1. Details of other pathways to nylon intermediates via azipyrus-CoA are described, for example, in U.S. Patent No. 8,377,680, which is incorporated herein by reference in whole.

[0042] Acyl-CoA dehydrogenases capable of reducing acyl-CoA to the corresponding aldehyde in various pathways leading to nylon intermediates can convert adipyl-CoA to adipic semialdehyde (step N, Figure 1). However, some acyl-CoA dehydrogenases may also react with succinyl-CoA and acetyl-CoA. In some embodiments, acyl-CoA dehydrogenases (aldehyde production) are disclosed that have higher catalytic efficiency, a higher turnover rate, or both, with adipyl-CoA substrates than with succinyl-CoA, acetyl-CoA, or both substrates. This improves efficiency and therefore enhances the production of nylon intermediates.

[0043] To identify enzymes with greater catalytic efficiency, higher turnover, or both, for adipyl-CoA substrates than succinyl-CoA, acetyl-CoA, or both substrates, other aldehyde dehydrogenases were identified using the exemplary sequence of Clostridium kluyveri DSM555 encoded by the gene adh (SEQ ID NO: 1). Homologous enzymes were identified as listed in Table 1 (amino acid sequences are shown in the sequence list).

[0044] In some embodiments, the aldehyde dehydrogenase or sequence is identified by BLAST. In some embodiments, the aldehyde dehydrogenase shares at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of the amino acid sequence of ALD in Table 1.

[0045] These aldehyde dehydrogenases, which exhibit greater catalytic efficiency, higher turnover, or both, for adipyl-CoA substrates than succinyl-CoA, acetyl-CoA, or both substrates, originate from genetically diverse organisms. Often, simple amino acid sequence identity between sequences does not indicate their common function. For example, the results of paired sequence alignments of some exemplary aldehyde dehydrogenases disclosed in Table 1 below are shown below. [Table 1]

[0046] These aldehyde dehydrogenases possess multiple conserved domains, such as an N-terminal domain, a C-terminal domain, and cysteine ​​residues in their active site. Aldehyde dehydrogenases contain a cofactor-binding domain with a Rossmann fold-type nucleotide-binding structure. The Rossmann fold, also called the βαβ fold, is a hypersecondary structure characterized by alternating motifs of a beta chain-alpha helix-beta chain secondary structure. The β chain is involved in the formation of the β sheet. The βαβ fold structure is commonly observed in enzymes with dinucleotide coenzymes such as FAD, NAD, and NADP. The βαβ fold structure was associated with a specific Gly-rich sequence of (GxGxxG) in the tight-loop region between the first β chain and the α helix. Furthermore, the cofactor-binding domain is the same domain that binds to the substrate CoA. This is a typical characteristic of Ald, where the substrate CoA first binds to form an intermediate, then a cofactor binds to complete the chemical reaction and perform hydride transfer.

[0047] Based on multiple sequence alignment and Hidden Markov (HMM) models, aldehyde dehydrogenases are grouped into Pfam PF00171, ClanCL0099 in the Pfam database from the European Bioinformatics Institute (pfam.xfam.org). These enzymes are classified as EC1.2.1 according to the Enzyme Commission nomenclature.

[0048] In some embodiments, the ALD enzyme has greater catalytic efficiency and / or turnover rate when adipyl-CoA is the substrate compared to succinyl-CoA, acetyl-CoA, or both. In some embodiments, the aldehyde dehydrogenase is SEQ ID NOs: 4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 10 It contains an amino acid sequence having at least 50, 75, 100, 150, 200, 250, 300 or more consecutive amino acids from any of the following: 9, 111, 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159, 164-167, 176, 187, or 188, and an amino acid sequence having at least approximately 60% amino acid sequence identity. In some embodiments, the amino acid sequence of the aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde is selected from the amino acid sequences of SEQ ID NOs: 1-4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 109, 111, 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159, 164-167, 176, 187, or 188.

[0049] In some embodiments, the amino acid sequences of aldehyde dehydrogenases having greater catalytic efficiency, greater turnover rate, or a combination thereof compared to succinyl-CoA, acetyl-CoA, or both substrates with respect to adipyl-CoA substrates have at least about 60% amino acid sequence identity with at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of any of SEQ ID NOs: 7, 28, 60, and 107. In some embodiments, the amino acid sequences of aldehyde dehydrogenases having greater catalytic efficiency, greater turnover rate, or a combination thereof with respect to adipyl-CoA substrates compared to succinyl-CoA, acetyl-CoA, or both substrates, have at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of any of SEQ ID NOs: 7, 28, 60, and 107.

[0050] In some embodiments, the ALD enzyme has a catalytic efficiency of at least 5 times, at least 10 times, at least 25 times, or 5 to 25 times higher with respect to an adipyl-CoA substrate compared to succinyl-CoA, acetyl-CoA, or both substrates.

[0051] In some embodiments, the enzymatic conversion of the enzyme from a specified substrate (e.g., adipyl-CoA) to a specified product (e.g., adipic acid semialdehyde) under known standard conditions is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent greater than the enzymatic activity of an enzyme that is not specific to adipyl-CoA only.

[0052] In some embodiments, the aldehyde dehydrogenase further reacts with 6-aminocaproyl-CoA to produce 6-aminocaproic acid semialdehyde.

[0053] Cells with reduced enzyme activity can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells with reduced enzyme activity. See, for example, *Enzyme Nomenclature*, Academic Press, Inc., New York 2007. Other assays that can be used to determine reduced ADH include GC / MS analysis. In another example, NADH / NADPH levels may be monitored. For example, NADH / NADPH can be monitored by colorimetric analysis or spectroscopy using an NADP / NADPH assay kit (e.g., ab65349, available from ABCAM®).

[0054] The disclosed ALD enzyme can be used in pathways for the production of nylon intermediates. In some embodiments, non-spontaneously occurring microorganisms may be used for the production of adipic acid semialdehyde, or for the production of other nylon intermediates produced using adipic acid semialdehyde as an intermediate.

[0055] In some embodiments, genetically modified cells (e.g., non-spontaneously occurring microorganisms) can produce nylon intermediates such as 6-aminocaproic acid, caprolactam, and hexamethylenediamine.

[0056] In some embodiments, the nylon intermediate is biosynthesized using the pathway shown in Figure 1. In some embodiments, the pathway of Figure 1 is provided in genetically modified cells (e.g., non-spontaneously occurring microorganisms) as described herein, where the pathway comprises at least one exogenous nucleic acid encoding a pathway enzyme expressed in an amount sufficient to produce 6-aminocaproic acid, caprolactam, and hexamethylenediamine.

[0057] In some embodiments, the pathway is the HMD pathway as shown in Figure 1. The HMD pathway is provided in genetically modified cells (e.g., non-spontaneously occurring microorganisms) described herein, where the HMD pathway comprises at least one exogenous nucleic acid encoding an HMD pathway enzyme expressed in an amount sufficient to produce HMD. The enzymes are as follows: 1A is 3-oxoadipyl-CoA thiolase; 1B is 3-oxoadipyl-CoA reductase; 1C is 3-hydroxyadipyl-CoA dehydrase; 1D is 5-carboxy-2-pentenoyl-CoA reductase; 1E is 3-oxoadipyl-CoA / acyl-CoA transferase; 1F is 3-oxoadipyl-CoA synthase; 1G is 3-oxoadipyl-CoA hydrolase; 1H is 3-oxoadipate reductase; 1I is 3-hydroxyadipate dehydrase; 1J is 5-carboxy-2-pentenoate reductase; 1K is adipyl-CoA / acyl-CoA transferase. These are transfer enzymes; 1L is adipyl-CoA synthase; 1M is adipyl-CoA hydrolase; 1N is adipyl-CoA reductase (aldehyde production); 1O is 6-aminocaproyl aminotransferase; 1P is 6-aminocaproyl dehydrogenase; 1Q is 6-aminocaproyl-CoA / acyl-CoA transferase; 1R is 6-aminocaproyl-CoA synthase; 1S is amide hydrolase; 1T is spontaneous cyclization; 1U is 6-aminocaproyl-CoA reductase (aldehyde production); 1V is HMDA aminotransferase; and 1W is HMDA dehydrogenase.

[0058] Referring to Figure 1, in some embodiments, non-spontaneously occurring microorganisms have one or more of the following pathways: ABCDNOPQRUVW; ABCDNOPQRT; or ABCDNOPS. Other exemplary pathways involving the ALD enzyme for producing adipic acid semialdehyde include those described in U.S. Patent No. 8,377,680, which is incorporated herein by reference in its entirety.

[0059] Figure 1 also shows the pathway from 6-aminocaproic acid to 6-aminocaproyl-CoA by a transferase or synthase (Figure 1, step Q or R), followed by the formation of a caprolactam by the spontaneous cyclization of 6-aminocaproyl-CoA (Figure 1, step T). In other embodiments, 6-aminocaproic acid is activated to 6-aminocaproyl-CoA (Figure 1, step Q or R), followed by reduction (Figure 1, step U) and amination (Figure 1, step V or W) to produce HMDA. 6-aminocaproic acid can also be activated to 6-aminocaproyl-phosphate instead of 6-aminocaproyl-CoA. 6-aminocaproyl-phosphate can spontaneously cyclize to produce a caprolactam. In some embodiments, 6-aminocaproyl-phosphate can be reduced to 6-aminocaproic acid semialdehyde, which can then be converted to HMDA as shown in Figure 1. In some embodiments, 6-aminocaproic acid is converted to 6-aminocaproic acid semialdehyde by aminocaproic acid reductase (CAR). Although not shown in Figure 1, aminocaproic acid reductase can catalyze the conversion from aminocaproic acid to 6-aminocaproic acid semialdehyde as shown in Figure 1. In some embodiments, non-spontaneously occurring microorganisms have a hexamethylenediamine pathway, which comprises (i) 6-aminoacaproyl-CoA transferase, (ii) 6-aminocaproyl-CoA synthase, (iii) 6-aminocaproyl-CoA reductase, (iv) hexamethylenediamine aminotransferase, (v) hexamethylenediamine dehydrogenase, (v) or one or more combinations of enzymes (i) to (v). In other embodiments, non-spontaneously occurring microorganisms have a hexamethylenediamine pathway, which comprises 3-oxoadipyl-CoA thiolase (Thl), 3-oxoadipyl-CoA dehydrogenase (Hbd), and 3-oxoadipyl-CoA dehydrogenase ("crotonase" or Crt), 5-carboxy-2-pentenoyl-CoA reductase (Ter), aminotransferase (HMD TA), and carboxylate reductase (CAR).

[0060] As used herein, the term “non-naturally occurring,” when used in relation to a microbial organism or microorganism, is intended to mean that the microorganism has at least one genetic modification not normally found in naturally occurring strains of the referenced species (including wild-type strains of the referenced species). Genetic modifications include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, additions of other nucleic acids, deletions of nucleic acids, and / or other functional disruptions of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof of heterologous, homologous, or heterologous and homologous polypeptides of the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modification alters the expression of a gene or operon. Exemplary metabolic polypeptides include 6-aminocaproic acid, caprolactam, hexamethylenediamine, or enzymes in the levulinic acid biosynthesis pathway.

[0061] Metabolic modification refers to a biochemical reaction that alters a microorganism's natural state. Therefore, non-naturally occurring microorganisms can genetically modify nucleic acids that encode metabolic lipeptides or their functional fragments. Exemplary metabolic modifications are disclosed herein.

[0062] As used herein, the terms “microbial,” “microbial organism,” and “microorganism” are interchangeable and are intended to mean any organism that exists as a microscopic cell belonging to the group of archaea, bacteria, or eukaryotes. Therefore, this term is intended to encompass prokaryotic or eukaryotic cells, or organisms of microscopic size, including all species of bacteria, archaea, and bacteria, as well as eukaryotic microorganisms such as yeasts and fungi. This term also includes cell cultures of any species that can be cultured for the production of biochemicals.

[0063] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (the non-protein portion of an enzyme) whose presence is required for the activity of many enzymes (apoenzymes) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes and is involved in the transfer of acetyl or other acyl groups, the synthesis and oxidation of fatty acids, the oxidation of pyruvate, and other acetylation processes.

[0064] As used herein, “adipate” having the chemical formula -OOC-(CH2)4-COO-(see Figure 1) (IUPAC name hexanediolate) is the ionized form of adipic acid (IUPAC name hexanediolate), and it is understood that adipicate and adipic acid are interchangeable throughout to refer to either its neutral or ionized form (including its salt form) of the compound. Those skilled in the art will understand that the specific form is pH-dependent.

[0065] As used herein, “6-aminocaproate” having the chemical formula -OOC-(CH2)5-NH2 (see Figure 1, abbreviated as 6-ACA) is the ionized form of 6-aminocaproic acid (IUPAC name 6-aminohexanoic acid), and it is understood that 6-aminocaproate and 6-aminocaproic acid are interchangeable throughout to refer to any of its neutral or ionized forms (including its salt forms) of the compound. Those skilled in the art will understand that certain forms are pH-dependent.

[0066] As used herein, “caprolactam” (IUPAC name azepan-2-one) is a lactam of 6-aminohexanoic acid (see Figure 1, abbreviated as CPO).

[0067] As used herein, "hexamethylenediamine" (also known as 1,6-diaminohexane or 1,6-hexanediamine) has the chemical formula H2N(CH2)6NH2 (see Figure 1, abbreviated as HMD).

[0068] As used herein, the term “substantially anaerobic,” when used in reference to culture or growth conditions, is intended to mean that the amount of oxygen is less than approximately 10% of the saturation of dissolved oxygen in the liquid medium. This term is also intended to include a sealed chamber of a liquid or solid medium maintained in an atmosphere of less than approximately 1% oxygen.

[0069] As used herein, the term “osmotic protective agent,” when used in relation to culture or growth conditions, is intended to mean a compound that acts as an osmotic regulator and helps the microorganisms described herein to withstand osmotic stress. Osmotic protective agents include, for example, betaines, amino acids, and sugar trehaloses. Such non-limiting examples include glycine betaine, praline betaine, dimethyltethine, dimethyl sulfoniopropionate, 3-dimethyl sulfonio-2-methylpropionate, pipecolic acid, dimethyl sulfonioacetate, choline, L-carnitine, and ectoin.

[0070] As used herein, the term “growth-coupled” is intended to mean, when used in relation to the production of a biochemical, that the biosynthesis of the referenced biochemical takes place during the growth phase of the microbial organism. In certain embodiments, growth-coupled production may be mandatory, meaning that the biosynthesis of the referenced biochemical is an mandatory product produced during the growth phase of the microbial organism.

[0071] As used herein, “metabolic modification” is intended to refer to a biochemical reaction that has been altered from its natural state. Metabolic modification may include, for example, the elimination of biochemical reaction activity by functionally disrupting one or more genes encoding enzymes involved in the reaction.

[0072] As used herein, the term “gene disruption” or its grammatical equivalent is intended to mean a genetic modification that inactivates an encoded gene product. Genetic modifications may include, for example, the deletion of an entire gene, the deletion of a regulatory sequence necessary for transcription or translation, the deletion of a portion of a gene resulting in a truncated end of the gene product, or any various mutational strategies that inactivate an encoded gene product. One particularly useful method of gene disruption is complete gene deletion, which reduces or eliminates the occurrence of gene reversion in non-spontaneously occurring microorganisms.

[0073] As used herein, “exogenous” is intended to mean that the reference molecule or activity is introduced into the host microorganism. The molecule may be introduced, for example, by introducing the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as non-chromosomal genetic material such as a plasmid. Therefore, the term used in relation to the expression of coding nucleic acid refers to the introduction of an expressible form of coding nucleic acid into the microorganism. When used in relation to biosynthetic activity, the term refers to the activity introduced into the host reference organism. The source may be, for example, a homogeneous or heterogeneous coding nucleic acid that expresses the reference activity after introduction into the host microorganism. Therefore, the term “endogenous” refers to the reference molecule or activity present in the host. Similarly, the term used in relation to the expression of coding nucleic acid refers to the expression of coding nucleic acid contained within the microorganism.

[0074] The term "heterogeneous" refers to molecules or activities derived from a source other than the referenced species, while "homogeneous" refers to molecules or activities derived from the host microorganism. Therefore, exogenous expression of coding nucleic acids can utilize either heterogeneous or homogeneous coding nucleic acids, or both.

[0075] As used herein, the term "approximately" means within ±10% of the stated value. The term "approximately" may also mean rounded to the nearest significant figure. Therefore, approximately 5% means between 4.5% and 5.5%. Furthermore, when referring to a specific number, the exact number is also included. For example, approximately 5% includes the exact 5%.

[0076] The term "metabolic turnover number" (also called k cat ), as used herein, is defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site performs for a given enzyme concentration [E T . This can be calculated from the maximum reaction rate Vmax and the catalytic site concentration [E T as follows.

[0077] Kcat = Vmax / [E T . The unit is s -1 .

[0078] The term "catalytic efficiency" as used herein is a measure of how efficiently an enzyme converts a substrate to a product. Comparison of catalytic efficiencies can also be used as a measure of the preference (i.e., substrate specificity) of an enzyme for different substrates. The higher the catalytic efficiency, the more an enzyme "prefers" its substrate. This can be calculated from the following equation: k cat / K M , where k cat is the metabolic turnover number and K M is the Michaelis constant, and K M is the substrate concentration at which the reaction rate is half of Vmax. The unit of catalytic efficiency can be expressed as s -1 M -1 .

[0079] The term "biologically derived" as used herein, in the context of 6-aminocaproic acid, 1,6-hexanediol, caprolactone, caprolactam, or hexamethylenediamine, means that these compounds were synthesized within a microorganism.

[0080] When multiple exogenous nucleic acids are present in a microorganism, as described above, it is understood that the exogenous nucleic acids refer to the coding nucleic acid or biosynthetic activity being referenced. As disclosed herein, it is further understood that such exogenous nucleic acids may be introduced into a host microorganism as separate nucleic acid molecules, polycistronic nucleic acid molecules, or a combination thereof, and may still be considered as multiple exogenous nucleic acids. For example, as disclosed herein, a microorganism can be genetically engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. When two exogenous nucleic acids encoding a desired activity are introduced into a host microorganism, it is understood that the two exogenous nucleic acids may be introduced, for example, as single nucleic acids on a single plasmid or on separate plasmids, and may be integrated into the host chromosome at one or more sites, and may still be considered as two exogenous nucleic acids. Similarly, it is understood that three or more exogenous nucleic acids may be introduced into a host organism in any desired combination, for example, on separate plasmids not integrated into the host chromosome, on a single plasmid, and the plasmids remain extrachromosomal elements, and may still be considered as two or more exogenous nucleic acids. The number of exogenous nucleic acids or biosynthetic activities referenced refers to the number of coding nucleic acids or biosynthetic activities, and not the number of separate nucleic acids introduced into the host organism.

[0081] Non-naturally occurring microorganisms can contain stable genetic modifications, which refer to microorganisms that can be cultured for more than five generations without losing their modifications. Generally, stable genetic modifications include modifications that last for more than 10 generations, particularly stable modifications that last for about 25 generations, and more specifically, stable genetic modifications will last for more than 50 generations (including indefinitely).

[0082] In the case of gene disruption, a particularly useful and stable genetic modification is gene deletion. The use of gene deletion to introduce stable genetic modifications is particularly useful in reducing the possibility of reversion to the phenotype prior to the genetic modification. For example, stable proliferation-coupled synthesis of biochemicals can be achieved by the deletion of a gene encoding an enzyme that catalyzes one or more reactions within a series of metabolic modifications. The stability of proliferation-coupled synthesis of biochemicals can be further enhanced by multiple deletions, significantly reducing the possibility of multiple compensatory reversions occurring for each disrupted activity.

[0083] Those skilled in the art will understand that the genetic modifications, including metabolic modifications, exemplified herein are described in relation to suitable host organisms such as Escherichia coli, and suitable source organisms for the desired genetic material, such as genes for their corresponding metabolic reactions or desirable metabolic pathways. However, given the complete genome sequencing of a wide variety of organisms and the advanced skills in the field of genomics, those skilled in the art will be able to readily apply the teachings and guidelines provided herein to essentially all other organisms. For example, the metabolic modifications of Escherichia coli exemplified herein can be readily applied to other species by incorporating the same or similar coding nucleic acids from species other than the referenced species. Such genetic modifications include, for example, genetic modifications of species homologs in general, particularly orthologous, paralogous, or non-orthologous gene substitutions.

[0084] Orthologs are one or more genes that are related by vertical descent and are substantially the same or identical in function in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs of the biological function of epoxide hydrolysis. Genes are related by vertical descent if they share a sufficient amount of sequence similarity to indicate homology, or if they are related by evolution from a common ancestor. Genes can also be considered orthologs if they share a three-dimensional structure, even if the amount of sequence similarity is not sufficient to indicate evolution from a common ancestor to the extent that primary sequence similarity cannot be identified. Ortholog genes can encode proteins with sequence similarity of approximately 25% to 100% amino acid sequence identity. Furthermore, even genes encoding proteins that share less than 25% amino acid similarity can be considered to arise by vertical descent if their three-dimensional structures show similarity. Members of the serine protease family, including tissue plasminogen activator and elastase, are thought to have originated from vertical transmission from a common ancestor.

[0085] Orthologues include genes or gene products that encode them whose structure or overall activity has diverged, for example, through evolution. For example, if one species encodes a gene product exhibiting two functions, and such functions are separated into distinct genes in a second species, then the three genes and their corresponding products are considered orthologues. Regarding the production of biochemical products, those skilled in the art will understand that orthologous genes having metabolic activity to be introduced or disrupted should be selected for the construction of non-spontaneously occurring microorganisms. An example of an orthologue exhibiting separable activity is when distinct activity is separated into distinct gene products between two or more species or within a single species. A specific example is the separation of two serine protease activities, elastase proteolysis and plasminogen proteolysis, 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. DNA polymerase from the first species can be considered an ortholog for either or both of the exonuclease or polymerase from the second species, and vice versa.

[0086] In contrast, paralogs are homologs related, for example, through replication and subsequent evolutionary divergence, and possess similar or common but not identical functions. Paralogs may originate from or be derived from the same or 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 different enzymes that co-evolved from a common ancestor, catalyze different reactions in the same species, and have different functions. Paralogs are proteins from the same species that exhibit significant sequence similarities to each other, suggesting that they are homologous or related through co-evolution from a common ancestor. The paralog protein family includes HipA homologs, luciferase genes, and peptidases.

[0087] A non-orthologous gene substitution is a non-orthologous gene from one species that can substitute for a referenced gene function in a different species. Substitutions include, for example, the ability to perform substantially the same or similar function in the species of origin compared to the referenced function in a different species. Generally, non-orthologous gene substitutions are identifiable as structurally related to a known gene encoding the referenced function; however, structurally unrelated but functionally similar genes and their corresponding gene products are still included in the meaning of the term used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of the non-orthologous gene product compared to the gene encoding the function to be substituted. Therefore, non-orthologous genes include, for example, paralogs or unrelated genes.

[0088] Accordingly, when identifying and constructing non-spontaneously occurring microorganisms capable of biosynthesizing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, those skilled in the art will understand, by applying the teachings and guidelines provided herein to specific species, that the identification of metabolic modifications may include the identification and inclusion or inactivation of orthologs. Those skilled in the art may also utilize these evolutionarily related genes, provided that paralogs and / or non-orthologous gene substitutions exist in reference microorganisms encoding enzymes that catalyze similar or substantially similar metabolic reactions. Gene disruption strategies may also involve disrupting or deleting evolutionarily related genes in host microorganisms, paralogs, or orthologs to reduce or eliminate their activity, ensuring that functional redundancy of the enzyme activity targeted for disruption does not short-circuit the designed metabolic modifications.

[0089] Substitutions of orthologous, paralogous, and non-orthologous genes can be determined by methods well known to those skilled in the art. For example, examining the nucleic acid or amino acid sequences of two polypeptides will reveal sequence identity and similarity between the compared sequences. Based on such similarity, those skilled in the art can determine whether the similarity is high enough to indicate that the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, and Clustal W, compare and determine the similarity or identity of raw sequences and determine the presence or importance of gaps within the sequences to which weights or scores can be assigned. Such algorithms are also known in the art and are similarly applicable to determining the similarity or identity of nucleotide sequences. Parameters for similarity sufficient to determine relevance are calculated based on statistical similarity, or known methods for calculating the probability of finding similar matches within random polypeptides, and the importance of the match is determined. Computer comparison of two or more sequences can also be visually optimized by those skilled in the art, if desired. Related gene products or proteins can be expected to have high similarity, e.g., 25% to 100% sequence identity. Unrelated proteins may have essentially the same identity as those expected to occur by chance when scanning a sufficiently sized (approximately 5%) database. Between 5% and 24% of sequences may or may not show sufficient homology to conclude that the compared sequences are related. Given the size of the dataset, additional statistical analysis can be performed to determine the significance of such coincidences and thus determine the relevance of these sequences.

[0090] For example, exemplary parameters for determining the relationship between two or more sequences using the BLAST algorithm may be as shown below. Briefly, amino acid sequence alignment can be performed using BLASTP version 2.2.29+ (January 14, 2014) and the following parameters: matrix: 0 BLOSUM62; gap open: 11; gap stretch: 1; x_dropoff: 50; expect: 10.0; string size: 3; filter: on. Nucleic acid sequence alignment can be performed using BLASTN version 2.0.6 (September 16, 1998) and the following parameters: match: 1; mismatch: -2; gap open: 5; gap stretch: 2; x_dropoff: 50; expect: 10.0; string size: 11; filter: off. Those skilled in the art will know what changes can be made to the above parameters, for example, to increase or decrease the strictness of the comparison and to determine the relationship between two or more sequences.

[0091] It is understood that, if desired, non-spontaneous microorganisms that produce any pathway intermediate or product can be generated using any of the pathways disclosed herein (including those shown in the figures). As disclosed herein, such intermediate-producing microorganisms can be used in combination with another microorganism expressing an enzyme in a downstream pathway to produce the desired product. However, it is understood that non-spontaneous microorganisms that produce intermediates in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathways can be used to produce the intermediate as the desired product.

[0092] This specification generally refers to metabolic reactions, their reactants, or products, or more specifically, to one or more nucleic acids or genes encoding enzymes related to or catalyzing the metabolic reactions, reactants, or products mentioned herein. Unless otherwise expressly stated herein, those skilled in the art will understand that a reference to a reaction also constitutes a reference to reactants or products of a reaction. Similarly, unless otherwise expressly stated herein, a reference to reactants or products also refers to a reaction, and a reference to any of these metabolic components also refers to the gene encoding the enzyme that catalyzes the reaction, reactant, or product mentioned herein. Likewise, given the well-known fields of metabolic biochemistry, enzymology, and genomics, a reference to a gene or coding nucleic acid herein also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes, as well as the reactants and products of the reaction.

[0093] Non-spontaneously occurring microorganisms can be generated by introducing expressible nucleic acids encoding one or more enzymes involved in the biosynthesis pathways of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Depending on the host microorganism selected for biosynthesis, nucleic acids can be expressed for part or all of the specific 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathways. For example, if the selected host lacks one or more enzymes for the desired biosynthesis pathway, expressible nucleic acids for the deficient enzymes are introduced into the host for subsequent exogenous expression. Alternatively, if the selected host exhibits endogenous expression of some pathway genes but lacks others, the encoding nucleic acids are required for the deficient enzymes to achieve the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Therefore, non-naturally occurring microorganisms can be produced by introducing exogenous enzyme activity to obtain a desired biosynthetic pathway, or the desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme activities, along with one or more endogenous enzymes, that produce desired products such as 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0094] Depending on the components of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway of the selected host microorganism, non-spontaneously occurring microorganisms may include at least one exogenously expressed nucleic acid encoding the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, and even all nucleic acids encoding one or more adipic acid, 6-aminocaproic acid, or caprolactam biosynthesis pathways. For example, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be established in a host lacking the pathway enzymes via the exogenous expression of the corresponding encoding nucleic acid. In a host lacking all enzymes of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, this may include the exogenous expression of all enzymes in the pathway, although it is understood that all enzymes of the pathway can be expressed even if the host contains at least one of the pathway enzymes.

[0095] With regard to the teachings and guidance provided herein, those skilled in the art will understand that the number of coding nucleic acids introduced in an expressible form is at least equivalent to a deficiency in the acid pathway adipine, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway of a selected host microorganism. Thus, a non-spontaneously occurring microorganism may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleic acids, up to all of the above-mentioned enzymes constituting the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway. In some embodiments, the non-spontaneously occurring microorganism may also include other genetic modifications that promote or optimize 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis, or confer other useful functions to the host microorganism. One such other function may be, for example, enhancement of the synthesis of one or more precursors of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, such precursors include succinyl-CoA and / or acetyl-CoA in the case of adipic acid synthesis, or adipyl-CoA or adipic acid in the case of 6-aminocaproic acid or caprolactam synthesis (including enzymes of the adipic acid pathway disclosed herein), pyruvate and succinic acid semialdehyde, glutamic acid, glutaryl-CoA, homolysine, or 2-amino-7-oxosvalate in the case of 6-aminocaproic acid synthesis, or 6-aminocaproic acid, glutamic acid, glutaryl-CoA, pyruvate, and 4-aminobutanal, or 2-amino-7-oxosvalate in the case of hexamethylenediamine synthesis.

[0096] In some embodiments, non-spontaneously occurring microorganisms possess at least one exogenous nucleic acid encoding an aldehyde dehydrogenase, which reacts with adipyl-CoA to produce adipic acid semialdehyde, and are represented by SEQ ID NOs: 1-4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 109, 111, 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159. The aldehyde dehydrogenase is selected from an amino acid sequence having at least 50, 75, 100, 150, 200, 250, 300 or more consecutive amino acids from 164-167, 176, 187, or 188, and having at least approximately 60% amino acid sequence identity. In some embodiments, non-spontaneously occurring microorganisms possess at least one exogenous nucleic acid encoding an aldehyde dehydrogenase, which reacts with adipyl-CoA to produce adipic acid semialdehyde, and are represented by SEQ ID NOs: 1-4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 109, 111, 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159. The aldehyde dehydrogenase is selected from aldehyde dehydrogenases comprising an amino acid sequence having at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids from any of 164-167, 176, 187, or 188, and an amino acid sequence having at least approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid sequence identity.

[0097] In another embodiment, a non-spontaneously occurring microorganism has at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce an adipic acid semialdehyde containing at least 50, 75, 100, 150, 200, 250, 300 or more consecutive amino acids of any of the amino acid sequences of SEQ ID NOs. 7, 28, 60, or 107, and an amino acid sequence having at least 60% amino acid sequence identity. In another embodiment, a non-spontaneously occurring microorganism has at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl-CoA to produce adipic acid semialdehyde comprising at least 50, 75, 100, 150, 200, 250, 300, or more consecutive amino acids of any of the amino acid sequences of SEQ ID NOs. 7, 28, 60, or 107, and an amino acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid sequence identity.

[0098] Generally, host microorganisms are selected to produce precursors of the 6-aminocaproic acid, caprolactam, or hexamethylenediamine pathway, either as naturally occurring molecules or as genetically engineered products that provide de novo production of a desired precursor or increased production of a naturally occurring precursor by the host microorganism. Host organisms can be genetically engineered to increase precursor production as disclosed herein. Furthermore, a microorganism genetically engineered to produce a desired precursor can be used as a host organism and can be further genetically engineered to express enzymes or proteins of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway.

[0099] In some embodiments, non-spontaneously occurring microorganisms are generated from a host possessing the enzymatic ability to synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. In this particular embodiment, it may be useful to increase the synthesis or accumulation of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway products to drive reactions in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway toward the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Increased synthesis or accumulation can be achieved, for example, by overexpression of nucleic acids encoding one or more of the above-mentioned 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzymes. Overexpression of enzymes in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway can occur, for example, through the exogenous expression of endogenous genes or through the exogenous expression of heterologous genes. Therefore, naturally occurring organisms can be easily created as non-spontaneously occurring microorganisms that produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid by overexpressing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, i.e., up to all nucleic acids, which encode enzymes in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway. Furthermore, non-spontaneously occurring organisms can be created by mutagenesis of endogenous genes resulting in increased enzyme activity in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway.

[0100] In particularly useful embodiments, exogenous expression of coding nucleic acids is used. Exogenous expression confers the ability to customize expression and / or regulatory elements to suit the host and application, thereby achieving a desired expression level controlled by the user. However, endogenous expression can also be utilized in other embodiments, such as by removing negative regulatory effectors or inducing the promoter of a gene when linked to an inductive promoter or other regulatory elements. Thus, endogenous genes with naturally occurring inductive promoters can be upregulated by providing a suitable inducer, or the regulatory region of an endogenous gene can be genetically engineered to incorporate an inductive regulatory element, thereby enabling the regulation of increased expression of the endogenous gene over a desired period of time. Similarly, inductive promoters can be included as regulatory elements of exogenous genes introduced into non-naturally occurring microorganisms.

[0101] In some embodiments, a non-spontaneously occurring microorganism comprises one or more gene disruptions, and this organism produces 6-ACA, adipic acid, and / or HMDA. The disruption occurs in a gene encoding an enzyme that couples the production of adipic acid, 6-ACA, and / or HMDA to the growth of the organism, when the gene disruption reduces the activity of the enzyme, and as a result, the gene disruption confers increased production of adipic acid, 6-ACA, and / or HMDA to the non-spontaneously occurring organism. Thus, in some embodiments, a non-spontaneously occurring microorganism is provided comprising one or more gene disruptions, where this one or more gene disruption occurs in a gene encoding a protein or enzyme, and where the one or more gene disruptions result in increased production of adipic acid, 6-ACA, and / or HMDA to the organism. As disclosed herein, such an organism comprises a pathway for the production of adipic acid, 6-ACA, and / or HMDA.

[0102] In the method, it is understood that non-spontaneously occurring microorganisms can be generated by introducing one or more exogenous nucleic acids into a microorganism. By introducing nucleic acids, for example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway can be conferred to the microorganism. Alternatively, by introducing coding nucleic acids, an intermediate microorganism can be generated that has the biosynthetic ability to catalyze some of the reactions necessary to confer the ability to synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. For example, a non-spontaneously occurring microorganism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway may contain at least two exogenous nucleic acids encoding the desired enzymes. In the case of adipic acid production, at least two exogenous nucleic acids can encode enzymes such as succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyadipyl-CoA dehydrogenase, or 3-hydroxyadipyl-CoA and 5-carboxy-2-pentenoyl-CoA reductase, or a combination of 3-hydroxyacyl-CoA and adipyl-CoA synthase. In the case of caprolactam production, at least two exogenous nucleic acids can encode enzymes such as CoA-dependent aldehyde dehydrogenase and aminotransferase, or CoA-dependent aldehyde dehydrogenase and amide hydrolase, or a combination of aminotransferase and amide hydrolase. In the case of 6-aminocaproic acid production, at least two exogenous nucleic acids can encode enzymes such as 4-hydroxy-2-oxoheptane-1,7-dioxide (HODH) aldolase and 2-oxohepta-4-ene-1,7-dioxide (OHED) hydrolase, or 2-oxohepta-4-ene-1,7-dioxide (OHED) hydrolase and 2-aminoheptane-1,7-dioxide (2-AHD) decarboxylase, 3-hydroxyadipyl-CoA dehydrogenase and adipyl-CoA dehydrogenase, glutamyl-CoA transferase and 6-aminopimeloyl-CoA hydrolase, or glutaryl-CoA beta-ketothiolase and 3-aminopimerate 2,3-aminomutase.In the case of hexamethylenediamine production, at least two exogenous nucleic acids can encode enzymes such as 6-aminocaproate kinase and [(6-aminohexanoyl)oxy]phosphonic acid (6-AHOP) oxidoreductase, or 6-acetamidohexanoate kinase and [(6-acetamidohexanoyl)oxy]phosphonic acid (6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydrase and 2-amino-7-oxoheptanoate aminotransferase, or 3-oxopimeloyl-CoA ligase and homolysine decarboxylase. Therefore, it is understood that any combination of two or more enzymes in the biosynthetic pathway may be present in non-naturally occurring microorganisms.

[0103] Similarly, it is understood that, if desired, any combination of three or more enzymes in a biosynthetic pathway may be present in non-naturally occurring microorganisms, insofar as the combination of enzymes in the desired biosynthetic pathway results in the production of the corresponding desired product. For example, in the case of adipic acid production, this may include the enzymes succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydrogenase; or succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 5-carboxy-2-pentenoyl-CoA reductase; or succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and adipyl-CoA synthase; or a combination of 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydrogenase, and adipyl-CoA:acetyl-CoA transferase. In the case of 6-aminocaproic acid production, at least three exogenous nucleic acids are involved: 4-hydroxy-2-oxoheptane-1,7-dioxide (HODH) aldolase, 2-oxohepta-4-ene-1,7-dioxide (OHED) hydrolase, and 2-oxoheptane-1,7-dioxide (2-OHD) decarboxylase, or 2-oxohepta-4-ene-1,7-dioxide (OHED) hydrolase, 2-aminohepta-4-ene-1,7-dioxide (2-AHE) reductase, and 2-aminoheptane-1,7-dioxide (2-AHD) decarboxylase, or 3-hydroxyadipyl Enzymes such as CoA dehydrase, 2,3-dehydroadipyl-CoA reductase, and adipyl-CoA dehydrogenase, or 6-amino-7-carboxyhepta-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA hydrolase, and 2-aminopimerate decarboxylase, or glutaryl-CoA beta-ketothiolase, 3-aminationoxidoreductase, and 2-aminopimerate decarboxylase, or a combination of 3-oxoadipyl-CoA thiolase, 5-carboxy-2-pentenoate reductase, and adipicate reductase can be encoded.In the case of hexamethylenediamine production, at least three exogenous nucleic acids are involved in the oxidative transfer of 6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonic acid (6-AHOP) oxidoreductase, and 6-aminocaproate semialdehyde aminotransferase, or 6-aminocaproate N-acetyltransferase, 6-acetamidohexanoate kinase, and [(6-acetamidohexanoyl)oxy]phosphonic acid (6-AAHOP) oxidoreductase, or 6-aminocaproate N-acetyltransferase, and [(6-acetamidohexanoyl)oxy]phosphonic acid (6-AAHOP) acyl transferase. The enzymes can be encoded as transfer enzymes, and enzymes such as 6-acetamidehexanoyl-CoA oxidoreductase, or 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydrase, and homolysine decarboxylase, or 2-oxo-4-hydroxy-7-aminoheptanoic acid aldolase, 2-oxo-7-aminohepta-3-enoic acid reductase, and homolysine decarboxylase, or combinations of 6-acetamidehexanoic acid reductase, 6-acetamidehexanal aminotransferase, and 6-acetamidehexanamine N-acetyltransferase. Similarly, any combination of four or more enzymes of the biosynthetic pathways disclosed herein can be included in non-spontaneously occurring microorganisms, if desired, as long as the combination of enzymes of the desired biosynthetic pathway results in the production of the corresponding desired product.

[0104] In addition to the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid described herein, non-spontaneously occurring microorganisms and methods can also be used in various combinations with other microorganisms and methods well known in the art to achieve the biosynthesis of products via other pathways. For example, one alternative method for producing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, other than the use of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing substances, is by adding another microorganism capable of converting an intermediate of the adipic acid, 6-aminocaproic acid, or caprolactam pathway to 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. One such method involves, for example, fermentation of a microorganism that produces an intermediate of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway. Next, the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediate can be used as a substrate for a second microorganism that converts the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediate to 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. The 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediate can be directly added to another culture of the second organism, or the final product can be produced without an intermediate purification step by depleting these microorganisms from the original culture of the product of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediate, for example by cell isolation, and then adding the second organism to the subsequent fermentation broth.

[0105] In other embodiments, non-spontaneously occurring microorganisms and methods can be assembled in a wide variety of subpathways to achieve, for example, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. In these embodiments, the biosynthetic pathway of the desired product can be isolated in different microorganisms, and the different microorganisms can be co-cultured to produce the final product. In such biosynthetic schemes, the product of one microorganism becomes a substrate for a second microorganism until the final product is synthesized. For example, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be achieved by constructing a microorganism that includes a biosynthetic pathway for converting one pathway intermediate into another pathway intermediate or product. Alternatively, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can also be biosynthetically produced from microorganisms by co-culture or co-fermentation using two organisms in the same vessel, where the first microorganism produces a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid intermediate, and the second microorganism converts the intermediate into 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0106] In consideration of the teachings and guidance provided herein, those skilled in the art will know about non-naturally occurring microorganisms and methods, together with other microorganisms, together with co-culturing of other non-naturally occurring microorganisms having subpathways, and It will be understood that a wide variety of combinations and rearrangements exist, along with other chemical and / or biochemical methods known in the art for producing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0107] Similarly, it will be understood by those skilled in the art that host organisms can be selected based on desired characteristics for the introduction of one or more gene disruptions to increase the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Therefore, it will be understood that when introducing genetic modifications into a host organism to disrupt a gene, homologs, orthologues, or paralogs that catalyze similar but not identical metabolic reactions can also be disrupted to ensure that the desired metabolic reaction is sufficiently disrupted. Because certain differences exist in metabolic networks between different organisms, those skilled in the art will understand that the actual gene disrupted in a given organism may differ between organisms. However, considering the teachings and guidelines provided herein, those skilled in the art will also understand that the methods can be applied to any suitable host microorganism to identify the genus metabolic modifications necessary to construct an organism of the desired species that increases the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. In certain embodiments, the increased production can, if desired, couple the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid to the growth of the organism, as disclosed herein, and can be obligatoryly coupled to the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid to the growth of the organism.

[0108] Sources of the coding nucleic acids for 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzymes may include, for example, any species capable of catalyzing the reaction referenced by the encoded gene product. Such species include, but are not limited to, both prokaryotes and eukaryotes, as well as bacteria, including archaea and bacteria, and yeasts, plants, insects, animals, and mammals (including humans). In some embodiments, sources of the coding nucleic acids for 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzymes are shown in Table 1. In some embodiments, sources of the coding nucleic acids for aldehyde dehydrogenases are shown in Table 1. In other embodiments, sources of the coding nucleic acids for aldehyde dehydrogenases are Acidaminococcus, Collinsella, Peptostreptococcaceae, or Romboustsia. In some embodiments, the source of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or the nucleic acid encoding the levulinic acid pathway enzyme is Escherichia coli, Escherichia coli str. K12, Escherichia coli C, Escherichia coli W, Pseudomonas sp, Pseudomonas knackmussii, Pseudomonas sp. Strain B13, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas mendocina, Rhodopseudomonas palustris, Mycobacterium tuberculosis, Vibrio cholera, Heliobacter pylori, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces sp.2065、Pseudomonas aeruginosa、Pseudomonas aeruginosa PAO1、Ralstonia eutropha、Ralstonia eutropha H16、Clostridium acetobutylicum、Euglena gracilis、Treponema denticola、Clostridium kluyveri、Homo sapiens、Rattus norvegicus、Acinetobacter sp. ADP1、Acinetobacter sp. Strain M-1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium botulinum A3 str, Clostridium tyrobutyricum, Clostridium pasteurianum, Clostridium thermoaceticum (Moorella thermoaceticum), Moorella thermoacetica Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp.M62 / 1, Fusobacterium nucleatum, Bos taurus, Zoogloea ramigera, Rhodobacter sphaeroides, Clostridium beijerinckii, Metallosphaera sedula, Thermoanaerobacter species, Thermoanaerobacter brockii, Acinetobacter baylyi, Porphyromonas gingivalis, Leuconostoc mesenteroides, Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sulfolobus solfataricus, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Salmonella typhimurium, Salmonella enterica, Thermotoga maritima, Halobacterium salinarum, Bacillus cereus, Clostridium difficile, Alkaliphilus metalliredigenes, Thermoanaerobacter tengcongensis, Saccharomyces kluyveri, Helicobacter pylori, Corynebacterium glutamicum, Clostridium 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, Methanosarcina mazei, Methanosarcina 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.Species such as 30a, Leptospira interrogans, Moorella thermoacetica, Myxococcus xanthus, Nicotiana glutinosa, Nocardia iowensis (sp. NRRL 5646), Pseudomonas reinekei MT1, Ralstonia eutropha JMP134, Ralstonia metallidurans, Rhodococcus jostii, Schizosaccharomyces pombe, Selenomonas ruminantium, Streptomyces clavuligenus, Syntrophus aciditrophicus, Vibrio parahaemolyticus, Vibrio vulnificus, and other exemplary species available as source organisms for genes disclosed herein or corresponding to them (see Examples). However, with the availability of complete genome sequences for over 550 species, including 395 microbial genomes and various yeast, fungal, plant, and mammalian genomes (more than half of which are available in public databases such as NCBI), the identification of genes encoding the necessary 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis activity (e.g., including homologous, orthologous, paralogous, and non-orthologous gene substitutions of known genes) for one or more genes in related or distant species, and the exchange of genetic modifications between organisms, are routine and well known in the art. Therefore, metabolic modifications enabling the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid described herein for specific organisms such as Escherichia coli can be readily applied to other microorganisms, including prokaryotes and eukaryotes as well. Given the teachings and guidance provided herein, those skilled in the art will be aware that metabolic modifications exemplified in one organism can be equally applied to other organisms.

[0109] In some cases, such as when the biosynthetic pathways for 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid are present in unrelated species, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid may be conferred to a host species by the exogenous expression of paralogs from unrelated species that catalyze similar but not identical metabolic reactions to replace the referenced reaction. Because certain differences exist between metabolic networks in different organisms, those skilled in the art will understand that the actual gene usage may differ between different organisms. However, given the teachings and guidelines provided herein, those skilled in the art will also understand that such teachings and methods can be applied to all microorganisms using metabolic modifications of the same genus as those exemplified herein to construct microorganisms in the target species for the synthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0110] For example, a host microorganism can be selected from bacteria, yeasts, fungi, or various other microorganisms applicable to fermentation processes, and non-spontaneously occurring microorganisms can be created from bacteria, yeasts, fungi, or various 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, and Rhizobus oryzae. For example, Escherichia coli is a particularly useful host organism because it is a well-characterized microorganism suitable for genetic engineering. Other particularly useful host organisms include yeasts such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and / or genetic modifications to produce the desired product.

[0111] Methods for constructing and testing the expression levels of non-spontaneously occurring 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing hosts can be carried out, for example, by recombinant and detection methods well known in the art. Such methods are described, for example, in 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).

[0112] Exogenous nucleic acid sequences involved in the production pathways of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, but not limited to those mentioned above, can be stably or transiently introduced into host cells using techniques well known in the art, including conjugation, electroporation, chemical transformation, transduction, transfection, and sonic transformation. In the case of exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNA of the eukaryotic nucleic acid may encode N-terminal mitochondrial target signals or other target signals, which can be removed if desired before transformation into prokaryotic host cells. For example, removal of a mitochondrial leader sequence resulted in increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). In the case of exogenous expression in yeast or other eukaryotic cells, the gene can be expressed in the cytosol without the addition of a leader sequence, or it can target mitochondria or other organelles, or target secretion, by adding appropriate targeting sequences such as mitochondrial targeting or secretory signals appropriate for the host cell. In other words, it is understood that desirable properties can be conferred by incorporating appropriate modifications to the nucleic acid sequence to remove or include targeting sequences into the exogenous nucleic acid sequence. Furthermore, the gene can be subjected to codon optimization using techniques well known in the art to achieve optimized protein expression.

[0113] One or more expression vectors can be constructed containing nucleic acids, such as those exemplified herein, operably ligated to an expression regulatory sequence functional in a host organism, and comprising one or more nucleic acids encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway. Expression vectors applicable to use in microbial host organisms include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, and include vectors and selectable sequences or markers functional for stable integration into host chromosomes. Furthermore, an expression vector may include one or more selectable marker genes and appropriate expression regulatory sequences. For example, it may include selectable marker genes that provide resistance to antibiotics or toxins, complement nutritional deficiencies, or supply essential nutrients not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcriptional enhancers, transcriptional terminators, etc., which are well known in the art. When two or more exogenous encoding nucleic acids are co-expressed, both nucleic acids may be inserted, for example, into a single expression vector or separate expression vectors. In the case of single-vector expression, the coding nucleic acid may be operably ligated to one common expression regulatory sequence or to different expression regulatory sequences such as one inductive promoter or one constitutive promoter. Transformation of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the exogenous nucleic acid is expressed in an amount sufficient to produce the desired product, and further understand that the expression level can be optimized to obtain sufficient expression using methods well known to those skilled in the art as disclosed herein.

[0114] In some embodiments, methods are provided for producing desired intermediates or products such as adipic acid, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. For example, a method for producing an adipic acid salt may involve culturing a non-spontaneously occurring microorganism having an adipic acid pathway, which comprises at least one exogenous nucleic acid encoding an enzyme of the adipic acid pathway, expressed for a sufficient time and in a sufficient amount to produce adipic acid under conditions that produce adipic acid, wherein the adipic acid pathway comprises succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydrogenase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthase, or phosphotransadipylase / adipate kinase, or adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Furthermore, a method for producing adipic acid salts may include culturing a non-spontaneously occurring microorganism having an adipic acid pathway, which comprises at least one exogenous nucleic acid encoding an adipic acid pathway enzyme expressed for a sufficient time and in sufficient quantities to produce adipic acid under conditions that produce adipic acid, wherein the adipic acid pathway comprises succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipicate dehydrogenase, and 2-enoic acid reductase.

[0115] Furthermore, a method for producing 6-aminocaproic acid may include culturing a non-spontaneously occurring microorganism having a 6-aminocaproic acid pathway, the pathway comprising at least one exogenous nucleic acid encoding an enzyme of the 6-aminocaproic acid pathway, expressed for a sufficient time and in a sufficient amount to produce 6-aminocaproic acid under conditions for producing 6-aminocaproic acid, the 6-aminocaproic acid pathway comprising a CoA-dependent aldehyde dehydrogenase and an aminotransferase or 6-aminocaproic acid dehydrogenase. Furthermore, a method for producing caprolactam may include culturing a non-spontaneously occurring microorganism having a caprolactam pathway, the pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme, expressed for a sufficient time and in a sufficient amount to produce caprolactam under conditions for producing caprolactam, the caprolactam pathway comprising a CoA-dependent aldehyde dehydrogenase, an aminotransferase or 6-aminocaproic acid dehydrogenase, and an amide hydrolase.

[0116] Appropriate purification and / or assays for testing the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be carried out using well-known methods. Appropriate duplicate cultures, such as triple cultures, can be grown for each genetically modified strain to be tested. For example, the formation of products and by-products in the genetically modified production host can be tracked. Final products, intermediates, and other organic compounds can be analyzed by methods such as HPLC (high-performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), LC-MS (liquid chromatography-mass spectrometry), or other appropriate analytical methods using routine methods well known in the art. The release of products in the fermentation broth can also be tested in the culture supernatant. By-products and residual glucose can be quantified by HPLC using, for example, refractive index detectors for glucose and alcohol, and UV detectors for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other appropriate assays and detection methods well known in the art. The activity of individual enzymes from exogenous DNA sequences can also be assayed using methods well known in the art.

[0117] 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be separated from other components in a culture using various methods known in the art. Such separation methods include, for example, extraction methods, as well as methods including continuous liquid-liquid extraction, permeation vaporization, 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.

[0118] Any of the non-naturally occurring microorganisms described herein can be cultured to produce and / or secrete biosynthetic products. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing substances can be cultured for the biosynthesis and production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0119] For the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, recombinant strains are cultured in a medium containing a carbon source and other essential nutrients. To reduce the overall cost of the process, it is desirable, and very desirable, to maintain anaerobic conditions in the fermenter. Such conditions can be obtained, for example, by first spraying nitrogen onto the medium and then sealing the flask with a diaphragm and crimp cap. For strains in which growth is not observed under anaerobic conditions, microaerophilic or substantially anaerobic conditions can be applied by making small holes in the diaphragm for limited aeration. Exemplary anaerobic conditions have been previously described and are well known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in U.S. Patent No. 7,947,483 issued on 24 May 2011. Fermentation can be carried out in batch, feed-batch, or continuous manner as disclosed herein.

[0120] If desired, the pH of the culture medium can be maintained at a neutral pH, such as approximately pH 7, by adding a base or acid, such as NaOH or other bases, as needed to maintain the medium at the desired pH. The growth rate can be determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate can be determined by tracking the depletion of the carbon source over time.

[0121] The growth medium may contain any carbohydrate source that can supply a carbon source to, for example, non-spontaneously occurring microorganisms. Such sources include sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch. Other sources of carbohydrates include, for example, renewable raw materials and biomass. Exemplary types of biomass that can be used as raw materials in this method include cellulosic biomass, hemicellar biomass, and lignin raw materials, or parts of raw materials. Such biomass raw materials include carbohydrate substrates that are useful as carbon sources, such as glucose, xylose, arabinose, galactose, mannose, fructose, and starch. In consideration of the teachings and guidance provided herein, those skilled in the art will understand that renewable raw materials and biomass other than those exemplified above can also be used for culturing microorganisms for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

[0122] In addition to the renewable raw materials exemplified above, microorganisms producing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can also be modified for growth on syngas as their carbon source. In this particular embodiment, one or more proteins or enzymes are expressed in 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing organisms to provide metabolic pathways for the utilization of syngas or other gaseous carbon sources.

[0123] Syngas, also known as syngas or generator gas, is the primary product of the gasification of coal and carbonaceous materials (such as biomass materials including crops and residues). Syngas is primarily a mixture of H2 and CO, but can be obtained from the gasification of any organic raw material, including coal, coal oil, natural gas, biomass, and waste organic matter, though not particularly limited. Gasification is generally carried out under a high fuel-to-oxygen ratio. While mainly H2 and CO, syngas may also contain small amounts of CO2 and other gases. Thus, synthesis gas provides a cost-effective gaseous carbon source, including CO and even CO2.

[0124] The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl-CoA and other products (e.g., acetic acid). Organisms that can utilize CO and syngas also generally possess the ability to utilize CO2 and CO2 / H2 mixtures through the same basic set of enzymes and conversions encompassed by the Wood-Ljungdahl pathway. The H2-dependent conversion of CO2 to acetic acid by microorganisms was recognized long before it became clear that CO could also be utilized by the same organisms and that the same pathway was involved. Many acetogens have been shown to grow in the presence of CO2 and produce compounds such as acetic acid, as long as hydrogen is available to supply the necessary reducing equivalents (see, e.g., Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized in the following equation:

[0125] 2CO2+4H2+nADP+nPi→CH3COOH+2H2O+nATP.

[0126] Therefore, non-spontaneously occurring microorganisms possessing the Wood-Ljungdahl pathway can also utilize a mixture of CO2 and H2 for the production of acetyl-CoA and other desired products.

[0127] The Wood-Lyngdahl pathway is well known in the art and consists of 12 reactions that can be separated into two branches: (1) the methyl branch and (2) the carbonyl branch. The methyl branch converts syngas to methyltetrahydrofolate (methyl-THF), while the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthase, methenyltetrahydrofolate cyclodehydrase, methylenetetrahydrofolate dehydrogenase, and methylenetetrahydrofolate reductase. Carbonyl branching reactions are catalyzed in order by the following enzymes or proteins: cobalamidocorinoid / iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase, and hydromenase, and these enzymes are also called methyltetrahydrofolate:corinoid protein methyltransferase (e.g., AcsE), corinoid iron-sulfur protein, nickel-protein assembly protein (e.g., AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase, and nickel-protein assembly protein (e.g., CooC). Following the teachings and guidance provided herein for introducing a sufficient number of coding nucleic acids to generate the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, those skilled in the art will understand that the same genetic engineering designs can be carried out with respect to introducing nucleic acids that code for Wood-Lungdahl enzymes or proteins that are not present in the host organism. Therefore, introducing one or more coding nucleic acids into a microorganism confers syngas utilization ability to the modified organism, such that the modified organism contains the complete Wood-Lyungdahl pathway.

[0128] Furthermore, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and / or hydrogenase activity can also be used for the conversion of CO, CO2 and / or H2 to other products such as acetyl-CoA and acetates. 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, α-ketoglutarate; ferredoxin oxidoreductase, succinyl-CoA synthase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P) ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reduced equivalents extracted from CO and / or H2 by carbon monoxide dehydrogenase and hydrogenase can be used to fix CO2 to acetyl-CoA or acetates via the reductive TCA cycle. Acetates can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase / phosphotransacetylase, and acetyl-CoA synthase. Acetyl-CoA can be converted to p-toluate, terephthalate, or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonic acid precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvic acid, and pyruvate by pyruvate:ferredoxin oxidoreductase and gluconeogenic enzymes. Following the teachings and guidance provided herein for introducing a sufficient number of coding nucleic acids to generate the p-toluate, terephthalate, or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonic acid pathway, those skilled in the art will understand that the same genetic engineering designs can also be carried out with respect to introducing nucleic acids encoding reductive TCA pathway enzymes or proteins that are not present in the host organism. Thus, introducing one or more coding nucleic acids into a microorganism so that the modified organism contains a complete reductive TCA pathway confers syngas utilization capacity.

[0129] In consideration of the teachings and guidelines provided herein, those skilled in the art will understand that unnaturally occurring microorganisms that secrete biosynthesized compounds can be produced when grown on a carbon source such as carbohydrates. Such compounds include, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, and any of the intermediate metabolites of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway. What is needed is to genetically engineer one or more of the enzyme activities necessary to achieve the biosynthesis of the desired compound or intermediate, for example, including the inclusion of part or all of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis pathway. Accordingly, some embodiments provide non-spontaneously occurring microorganisms that, when grown on carbohydrates, produce and / or secrete 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, and that, when grown on carbohydrates, produce and / or secrete any of the intermediate metabolites represented in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway. For example, a microorganism producing adipic acid may, if necessary, initiate synthesis from an intermediate, such as 3-oxoadipyl-CoA, 3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipic-CoA (see Figure 1). Furthermore, a microorganism producing adipic acid may initiate synthesis from an intermediate, such as 3-oxoadipyl-CoA, 3-oxoadipic acid, 3-hydroxyadipic acid, or hexa-2-enedioic acid. Microorganisms producing 6-aminocaproic acid can initiate synthesis from an intermediate, such as adipic acid semialdehyde. Microorganisms producing caprolactam can, if necessary, initiate synthesis from an intermediate, such as adipic acid semialdehyde or 6-aminocaproic acid (see Figure 1).

[0130] In some embodiments, the non-spontaneously occurring microorganism further comprises an exogenously expressed nucleic acid encoding trans-enoyl-CoA reductase (TER). TER reacts with 5-carboxy-2-pentenoyl-CoA to produce adipyl-CoA. In some embodiments, TER may be a known TER, and in other embodiments, the TER enzyme is genetically engineered. In some embodiments, the genetically engineered trans-enoyl-CoA reductase has an amino acid sequence having at least 50% identity with the amino acid sequence of SEQ ID NO: 189, where the genetically engineered trans-enoyl-CoA reductase includes any of the variant amino acid sequence changes shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0131] In some embodiments, non-spontaneously occurring microorganisms have a hexamethylenediamine pathway, which includes (i) 6-aminoacaproyl-CoA transferase, (ii) 6-aminocaproyl-CoA synthase, (iii) 6-aminocaproyl-CoA reductase, (iv) hexamethylenediamine aminotransferase, (v) hexamethylenediamine dehydrogenase, (v) or a combination of one or more enzymes (i) to (v). In other embodiments, non-spontaneously occurring microorganisms have a hexamethylenediamine pathway, which includes 3-oxoadipyl-CoA thiolase (Thl), 3-oxoadipyl-CoA dehydrogenase (Hbd), and 3-oxoadipyl-CoA dehydrogenase ("crotonase", or Crt), 5-carboxy-2-pentenoyl-CoA reductase (Ter), aminotransferase (HMD TA), and carboxylate reductase (CAR).

[0132] Non-spontaneously occurring microorganisms are constructed using methods well known in the art, such as those exemplified herein, and exogenously express at least one nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzyme in an amount sufficient to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. It is understood that the microorganisms are cultured under conditions sufficient to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Following the teachings and guidelines provided herein, non-spontaneously occurring microorganisms can achieve biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, resulting in intracellular concentrations of approximately 0.1 to 200 mM or higher. In general, intracellular concentrations of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid range from approximately 3 to 150 mM, more specifically about 5 to 125 mM, and more specifically about 8 to 100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or higher. Intracellular concentrations between and above each of these exemplary ranges can also be achieved from non-spontaneously occurring microorganisms.

[0133] In some embodiments, the culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been previously described and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein, for example, in U.S. Patent No. 7,947,483 issued on May 24, 2011. Any of these conditions can be used with non-spontaneously occurring microorganisms and with other anaerobic conditions well known in the art. Under such anaerobic conditions, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid products can be synthesized at intracellular concentrations of 5–10 mM or higher, and all other concentrations exemplified herein. Even if the above description refers to intracellular concentrations, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing microorganisms can produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid intracellularly and / or secrete the products into the culture medium.

[0134] Culture conditions may include, for example, liquid culture methods, as well as fermentation and other large-scale culture methods. As described herein, particularly useful yields of biosynthetic products can be obtained under anaerobic or substantially anaerobic culture conditions.

[0135] As described herein, one exemplary growth condition for achieving the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid includes anaerobic culture or fermentation conditions. In certain embodiments, non-spontaneously occurring microorganisms may be maintained, cultured, or fermented under anaerobic or substantially anaerobic conditions. Simply put, anaerobic conditions refer to an environment without oxygen. Substantially anaerobic conditions include, for example, culture, batch fermentation, or continuous fermentation such that the dissolved oxygen concentration in the culture medium is maintained at 0–10% saturation. Substantially anaerobic conditions also include growing or quiescent cells in a liquid medium or on solid agar in a sealed chamber maintained in an atmosphere of less than 1% oxygen. The percentage of oxygen can be maintained, for example, by spraying the culture with an N2 / CO2 mixture or one or more other suitable non-oxygen gases.

[0136] The culture conditions described herein can be scaled up and continuously propagated for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Exemplary propagation methods include, for example, fed-batch batch fermentation and batch separation; fed-batch batch fermentation and continuous separation; or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation methods are particularly useful for the biosynthesis and production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid in commercial quantities. Generally, and as with discontinuous culture methods, continuous and / or nearly continuous production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid involves culturing non-spontaneously occurring 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing organisms in a culture medium with sufficient nutrients and nutrients to maintain and / or nearly maintain exponential growth. Continuous culture under such conditions may include, for example, one day, two, three, four, five, six, or seven days or more. Furthermore, continuous culture may include one week, two weeks, three weeks, four weeks, or five weeks or more, up to several months. Alternatively, if suitable for a particular application, the organism can be cultured for several hours. It should be understood that continuous and / or nearly continuous culture conditions may also include all time intervals between these exemplary periods. Furthermore, it should be understood that the time spent culturing the microorganism is sufficient to produce a sufficient amount of product for the desired purpose.

[0137] Fermentation methods are well known in the art. Simply put, fermentation for the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be used, for example, in fed-batch batch fermentation and batch separation; fed-batch batch fermentation and continuous separation; or continuous fermentation and continuous separation. Examples of batch and continuous fermentation methods are well known in the art.

[0138] In addition to the above fermentation method using 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing substances for the continuous production of a substantial amount of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid-producing substances can also, if desired, be subjected to simultaneous chemical synthesis methods to convert the product to other compounds, for example, or the product can be separated from the fermentation culture and subjected to continuous chemical conversion to convert the product to other compounds. As described herein, intermediates in the adipic acid pathway utilizing 3-oxoadipate, hexa-2-enediic acid, can be converted to adipic acid, for example, by chemical hydrogenation on a platinum catalyst.

[0139] As described herein, exemplary growth conditions for achieving the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid include the addition of an osmotic protective agent to the culture conditions. In certain embodiments, non-spontaneously occurring microorganisms may be maintained, cultured, or fermented as described above in the presence of an osmotic protective agent. Briefly, an osmotic protective agent means a compound that acts as an osmoregulator and helps the microorganisms described herein to withstand osmotic stress. Osmotic protective agents include, but are not limited to, betaines, amino acids, and sugar trehaloses. Such non-limiting examples are glycine betaine, praline betaine, dimethyltethine, dimethylthroniopropionate, 3-dimethylsulfonio-2-methylpropionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine, and ectoin. In one embodiment, the osmotic protective agent is glycine betaine. Those skilled in the art will understand that the amount and type of osmotic protective agent suitable for protecting the microorganisms described herein from osmotic stress depends on the microorganisms used. For example, as described in Example XXII, Escherichia coli in the presence of various amounts of 6-aminocaproic acid grows well in the presence of 2 mM glycine betaine. The amount of osmotic protective agent in the culture conditions may be, for example, about 0.1 mM or less, about 0.5 mM or less, about 1.0 mM or less, about 1.5 mM or less, about 2.0 mM or less, about 2.5 mM or less, about 3.0 mM or less, about 5.0 mM or less, about 7.0 mM or less, about 10 mM or less, about 50 mM or less, about 100 mM or less, or about 500 mM or less.

[0140] Successfully genetically engineering the pathway involves identifying an appropriate set of enzymes with sufficient activity and specificity. This includes identifying the appropriate enzyme set, cloning the corresponding genes into a generative host, optimizing fermentation conditions, and assaying product formation after fermentation. To genetically engineer a generative host for the production of 6-aminocaproic acid or caprolactam, one or more exogenous DNA sequences can be expressed in the host microorganism. Furthermore, endogenous genes can be functionally deleted from the microorganism. These modifications enable the production of 6-aminocaproic acid or caprolactam using renewable raw materials.

[0141] In some embodiments, minimizing or even eliminating the formation of cyclic imines or caprolactams during the conversion of 6-aminocaproic acid to HMDA involves adding functional groups (e.g., acetyl, succinyl) to the amine group of 6-aminocaproic acid to protect it from cyclization. This is similar to the production of ornithine from L-glutamic acid in E. coli. Specifically, glutamic acid is first converted to N-acetyl-L-glutamic acid by N-acetylglutamic acid synthase. Next, N-acetyl-L-glutamic acid is activated to N-acetylglutamyl phosphate, which is reduced and transaminated to produce N-acetyl-L-ornithine. Then, the acetyl group is removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase to produce L-ornithine. This pathway is necessary because, after glutamic acid is converted to glutamic acid-5-phosphate, it is reduced to glutamic acid-5-semialdehyde, which produces (S)-1-pyrroline-5-carboxylic acid (a cyclic imine spontaneously generated from glutamic acid-5-semialdehyde). When producing HMDA from 6-aminocaproic acid, this step may involve acetylating 6-aminocaproic acid to acetyl-6-aminocaproic acid, activating the carboxylic acid group with CoA or a phosphate group, reducing it, aminating it, and then deacetylating it. [Examples]

[0142] (experiment) Example 1. Screening of aldehyde dehydrogenase candidates for activity against adipyr-CoA Genes encoding candidate aldehyde dehydrogenases (Ald) were bioinformatically identified in the genomes of multiple species (Table 1). The genes encoding each aldehyde dehydrogenase were synthesized, expressed in E. coli, and their Ald activity was evaluated.

[0143] The genes encoding candidate Ald enzymes from Table 1 were cloned into low-copy vectors under constitutive promoters, and the constructs were transformed into E. coli using standard techniques. The transformants were cultured overnight in LB medium at 35°C in the presence of antibiotics, and the cells were harvested at room temperature at 15,000 rpm. To prepare lysates, the cells were resuspended in a chemical lysis solution containing lysozyme, nuclease, and 10 mM DTT, and incubated at room temperature for at least 30 minutes. The resulting lysates were used to test aldehyde dehydrogenase activity.

[0144] The lysate (5 μl) was added to the assay mixture to obtain a total volume of 20 μL with a final concentration of 0.1 M Tris-HCl, pH 7.5, 2.5 mM azipyrus-CoA (AdCoA), and either 0.5 mM NADH or 0.5 mM NADPH. This assay was used to screen all Ald enzyme candidates. Some Ald candidates were assayed using succinyl-CoA (SuCoA) or acetyl-CoA (AcCoA) as substrates. AdCoA, SuCoA, and AcCoA were obtained from commercial suppliers. Activity was monitored by a linear decrease in fluorescence of NADH or NADPH in the presence of the CoA substrate. Alds that were significantly active against azipyrus-CoA using either NADH or NADPH were shown as positive (+) in Table 3, while those with little or no activity were shown as negative (-). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0145] Example 2. Aldehyde dehydrogenase assay for determining substrate specificity To determine the substrate preference of several aldehyde dehydrogenases, a substrate CoA depletion assay was performed using succinyl CoA and azipyrus CoA substrates. In this assay, the substrate solution contained 0.1 M Tris-HCl, pH 7.5, 1 mM azipyrus CoA, 0.2 mM succinyl CoA, and 0.2 mM acetyl CoA, with an excess of 1.5 mM NADH or NADPH cofactor. The reaction was initiated by adding the lysate to the assay buffer and incubated at room temperature for 2 hours. The reaction was quenched with 1% formic acid, and the residual substrate CoA of each was quantified by LC / MS analysis. Ald activity was measured as % depletion of each CoA substrate. A higher % depletion of a particular CoA substrate compared to another CoA substrate present in the assay indicated preference for that particular substrate CoA. Figure 2 indicates that oral aldehyde dehydrogenases from Peptostreptococcaceae bacteria (SEQ ID NO: 7), aldehyde dehydrogenase from Acidaminococcus massiliensis (SEQ ID NO: 28), GD7 aldehyde dehydrogenase from Collinsella sp. (SEQ ID NO: 60), and DSM aldehyde dehydrogenase from Romboutsia lituseburensis (SEQ ID NO: 107) depleted far more azipyl-CoA than succinyl-CoA from the assay mixture, thus demonstrating azipyl-CoA preference. Aldehyde dehydrogenase from Porphyromonas gingivalis W83 (SEQ ID NO: 2) was found to exhibit succinyl-CoA preference.

[0146] Example 3. In vivo assay of aldehyde dehydrogenase Aldehyde dehydrogenases demonstrated to exhibit adipyrus-CoA substrate preference were also tested in vivo, where E. coli strains expressing genes encoding 3-oxoadipyl-CoA thiolase (Thl), 3-oxoadipyl-CoA dehydrogenase (Hbd), 3-oxoadipyl-CoA dehydrogenase ("crotonase" or Crt), 5-carboxy-2-pentenoyl-CoA reductase (Ter), and aminotransferase (TA) were transformed using constructs containing the aldehyde dehydrogenase (Ald) gene. The Thl, Hbd, Crt, Ter, and TA E. coli strains contained all the pathway enzymes necessary for 6-aminocaproic acid (6ACA) production, except for the Ald enzyme. The genes encoding Porphyromonas gingivalis W83Ald (SEQ ID NO: 2), Peptostreptococcaceae bacterium oral Ald (SEQ ID NO: 7), Acidaminococcus massiliensis Ald (SEQ ID NO: 28), Collinsella sp. GD7Ald (SEQ ID NO: 60), and Romboutsia lituseburensis DSMAld (SEQ ID NO: 107) were separately cloned into low copy number plasmid vectors under constitutive promoters. Plasmids for expressing the Ald genes were transformed into Thl / Hbd / Crt / Ter / TA strains using standard techniques. Transformants containing any of the Ald genes were then tested for 6-aminocaproic acid (6ACA) production. Genetically engineered E. coli cells were added to minimal medium with 2% glucose and incubated at 35°C for 18 hours. Cells were then harvested, and the supernatant was evaluated for 6ACA by analytical HPLC or standard LS / MS analysis. As shown in Table 4, the expression of genes encoding Ald enzymes in E. coli, including the Thl, Hbd, Crt, Ter, and TA genes, resulted in 6ACA production in these strains. [Table 4]

[0147] Example 4. Analysis of reaction kinetics of aldehyde dehydrogenase The reaction rate characteristics were analyzed under the same conditions as the lysate screening described in Example 1. However, in this case, purified protein was used instead of cell lysates. Acidaminococcus massiliensis Ald (SEQ ID NO: 28), Collinsella sp. GD7Ald (SEQ ID NO: 60), and Romboutsia lituseburensis DSMAld (SEQ ID NO: 107) were each purified using affinity chromatography. In these assays, the concentration of each substrate CoA was varied to determine the turnover rate (k cat ), the affinity of the enzyme for its substrate (K M ), catalytic efficiency of each selected Ald enzyme for each substrate (k cat / K M The following was determined and is shown in Table 5. [Table 5]

[0148] The catalytic efficiency (k) of various aldehyde dehydrogenases using various substrates cat / K M ) were plotted as a bar graph for comparison (Figure 3A). The catalytic efficiency (k) of the Ald homolog of adipyl-CoA relative to succinyl-CoA. cat / K M ) to the k of Adipil CoA cat / K M k against succinyl CoA cat / K M The ratio was calculated. Figure 3B shows that all three Ald enzymes assayed have greater catalytic efficiency with respect to adipyl-CoA than with succinyl-CoA. Figure 3C shows that all three Ald enzymes assayed have greater catalytic efficiency with respect to adipyl-CoA than with respect to acetyl-CoA.

[0149] Example 5: In vivo assay of aldehyde dehydrogenase

[0150] Aldehyde dehydrogenases that have been demonstrated to exhibit adipyrus-CoA substrate preference were tested in vivo using an in vivo assay. This assay involved transforming E. coli strains expressing genes encoding 3-oxoadipyl-CoA thiolase (Thl), 3-oxoadipyl-CoA dehydrogenase (Hbd), 3-oxoadipyl-CoA dehydrogenase ("crotonase" or Crt), 5-carboxy-2-pentenoyl-CoA reductase (Ter), and aminotransferase (TA), as described in Example 3, along with two additional genes, carboxylate reductase (CAR) (CAR-WP_003872682.1) and another TA gene (HMD-TA WP_001301395.1), together with the Ald gene integrated into the E. coli chromosome. The genes encoding Porphyromonas gingivalis W83Ald (SEQ ID NO: 2), Peptostreptococcaceae bacterium oral Ald (SEQ ID NO: 7), Acidaminococcus massiliensis Ald (SEQ ID NO: 28), Collinsella sp. GD7Ald (SEQ ID NO: 60), and Romboutsia lituseburensis DSMAld (SEQ ID NO: 107) were separately cloned into low copy number plasmid vectors under constitutive promoters. Plasmids for expressing the Ald genes were transformed into Thl / Hbd / Crt / Ter / TA strains using standard techniques. These constructs were subjected to the same conditions and tests as described for 6ACA generation in Example 3. The constructs were shown to produce HMD when detected by the LC / MS analysis method described in Example 3.

Claims

1. A non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl CoA to produce adipic acid semialdehyde, wherein the aldehyde dehydrogenase has greater catalytic efficiency with respect to adipyl CoA as a substrate compared to succinyl CoA, acetyl CoA, or both succinyl CoA and acetyl CoA as substrates, and / or the aldehyde dehydrogenase has a greater turnover rate with respect to an adipyl CoA substrate compared to succinyl CoA, acetyl CoA, or both succinyl CoA and acetyl CoA substrates.

2. The non-naturally occurring microorganism according to claim 1, wherein the aldehyde dehydrogenase does not contain the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

3. The non-naturally occurring microorganism according to claim 2, wherein the aldehyde dehydrogenase has greater catalytic efficiency with respect to an adipyl CoA substrate compared to a succinyl CoA substrate.

4. A non-naturally occurring microorganism according to any one of claims 1 to 3, wherein the catalytic efficiency of the aldehyde dehydrogenase for an adipyl-CoA substrate is at least twice as high as its specificity for a succinyl-CoA substrate.

5. The non-naturally occurring microorganism according to any one of claims 1 to 4, wherein the aldehyde dehydrogenase has a greater turnover rate with respect to an adipyl CoA substrate compared to a succinyl CoA substrate.

6. The non-naturally occurring microorganism according to any one of claims 1 to 5, wherein the aldehyde dehydrogenase has greater catalytic efficiency with respect to an adipyl-CoA substrate compared to an acetyl-CoA substrate.

7. A non-naturally occurring microorganism according to any one of claims 1 to 6, wherein the catalytic efficiency of the aldehyde dehydrogenase for an adipyl-CoA substrate is at least five times higher than the catalytic efficiency for an acetyl-CoA substrate.

8. The non-spontaneously occurring microorganism according to any one of claims 1 to 7, wherein the aldehyde dehydrogenase has a greater turnover rate with an adipyl-CoA substrate compared with an acetyl-CoA substrate.

9. An unnaturally occurring microorganism according to any one of claims 1 to 8, wherein the unnaturally occurring microorganism converts more adipyl CoA to adipic acid semialdehyde than a control microorganism that is substantially identical to the unnaturally occurring microorganism, except that the unnaturally occurring microorganism does not contain an exogenous nucleic acid encoding an aldehyde dehydrogenase.

10. The aldehyde dehydrogenases mentioned above are sequence numbers: 4, 7, 11, 15, 17, 19, 24, 25, 27, 28, 31-33, 36, 38, 40-42, 44, 45, 47, 53, 58-60, 63, 65-67, 74, 75, 77, 80, 82, 84, 86-88, 90, 91, 94, 95, 97, 100, 101, 103, 107, 109, 111 A non-naturally occurring microorganism according to any one of claims 1 to 9, comprising at least 25 consecutive amino acids, one of 112, 117, 134, 135, 137, 145, 146, 148-150, 152, 157-159, 164-167, 176, 187, and 188, and an amino acid sequence having at least about 60% amino acid sequence identity.

11. The non-naturally occurring microorganism according to any one of claims 1 to 10, wherein the aldehyde dehydrogenase comprises an amino acid sequence having at least 25 consecutive amino acids of any one of sequence numbers 7, 28, 60, or 107 and at least about 60% amino acid sequence identity.

12. The non-naturally occurring microorganism according to any one of claims 1 to 11, wherein the aldehyde dehydrogenase comprises the amino acid sequence of SEQ ID NO: 7, 28, 60, or 107.

13. The non-naturally occurring microorganism according to any one of claims 1 to 12, wherein the aldehyde dehydrogenase uses NADH as a cofactor.

14. The non-naturally occurring microorganism according to any one of claims 1 to 10, wherein the aldehyde dehydrogenase comprises an amino acid sequence having at least 25 consecutive amino acids from one of sequence numbers 53, 77, 82, 94, and 152 and at least about 60% amino acid sequence identity.

15. The non-naturally occurring microorganism according to claim 14, wherein the aldehyde dehydrogenase uses NADH, NADPH, or both as cofactors.

16. A non-naturally occurring microorganism according to any one of claims 1 to 15, wherein at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that reacts with adipyl CoA to produce adipic acid semialdehyde is heterogeneous to the microorganism.

17. The non-spontaneously occurring microorganism according to any one of claims 1 to 16, wherein the non-spontaneously occurring microorganism includes the 6-aminocaproic acid pathway.

18. The non-spontaneously occurring microorganism according to claim 17, wherein the 6-aminocaproic acid pathway comprises (i) an aminotransferase, (ii) a 6-aminocaproic acid dehydrogenase, or (iii) both an aminotransferase and a 6-aminocaproic acid dehydrogenase.

19. The non-spontaneously occurring microorganism according to any one of claims 17 to 18, wherein the microorganism further comprises one or more additional exogenous nucleic acids encoding one or more 6-aminocaproic acid pathway enzymes.

20. The non-naturally occurring microorganism according to claim 19, wherein one or more exogenous nucleic acids encoding the 6-aminocaproic acid pathway enzymes are heterogeneous to the microorganism.

21. The non-spontaneously occurring microorganism according to any one of claims 1 to 20, wherein the non-spontaneously occurring microorganism includes a hexamethylenediamine pathway.

22. The non-naturally occurring microorganism according to claim 21, wherein the hexamethylenediamine pathway comprises (i) 6-aminoacaproylCoA transferase, (ii) 6-aminocaproylCoA synthase, (iii) 6-aminocaproylCoA reductase, (iv) hexamethylenediamine aminotransferase, (v) hexamethylenediamine dehydrogenase, or (vi) a combination of one or more enzymes (i) to (v).

23. The non-spontaneously occurring microorganism according to any one of claims 21 to 22, wherein the microorganism further comprises one or more additional exogenous nucleic acids encoding one or more hexamethylenediamine pathway enzymes.

24. The non-naturally occurring microorganism according to claim 23, wherein the exogenous nucleic acid encoding one or more hexamethylenediamine pathway enzymes is heterogeneous to the microorganism.

25. The non-spontaneously occurring microorganism according to any one of claims 1 to 24, wherein the non-spontaneously occurring microorganism includes a caprolactam pathway.

26. The non-spontaneously occurring microorganism according to claim 25, wherein the caprolactam pathway comprises an aminohydrolase.

27. The non-naturally occurring microorganism according to any one of claims 25 to 26, wherein the microorganism further comprises one or more additional exogenous nucleic acids encoding aminohydrolases.

28. The non-naturally occurring microorganism according to claim 27, wherein the exogenous nucleic acid encoding an aminohydrolase is heterogeneous to the microorganism.

29. The non-naturally occurring microorganism according to any one of claims 1 to 28, wherein the aldehyde dehydrogenase is derived from a prokaryotic species.

30. The non-naturally occurring microorganism according to claim 29, wherein the aldehyde dehydrogenase is derived from Acidaminococcus, Collinsella, Peptostreptococcaceae, or Romboustsia.

31. The non-naturally occurring microorganism according to any one of claims 1 to 30, wherein the non-naturally occurring microorganism includes the species Acinetobacter, Actinobacillus, Anaerobiospirillum, Aspergillus, Bacillus, Clostridium, Corynebacterium, Escherichia, Gluconobacter, Klebsiella, Kluyveromyces, Lactococcus, Lactobacillus, Mannheimia, Pichia, Pseudomonas, Rhizobium, Rhizopus, Saccharomyces, Schizosaccharomyces, Streptomyces, and Zymomonas.

32. The non-naturally occurring microorganism according to any one of claims 1 to 31, wherein the non-naturally occurring microorganism comprises two, three, four, five, six, or seven exogenous nucleic acids that encode enzymes of the 6-aminocaproic acid pathway, the hexamethylenediamine pathway, the caprolactam pathway, the 1,6-hexanediol pathway, the caprolactone pathway, or a combination of two or more pathways.

33. The non-naturally occurring microorganism according to any one of claims 1 to 31, wherein the aldehyde dehydrogenase further reacts with 6-aminocaproyl CoA to produce 6-aminocaproic acid semialdehyde.

34. A method for producing adipic acid semialdehyde, comprising culturing a non-naturally occurring microorganism described in any one of claims 1 to 33 for a sufficient time and under conditions to produce adipic acid semialdehyde.

35. A method for producing 6-aminocaproic acid (6ACA), comprising culturing a non-naturally occurring microorganism according to any one of claims 1 to 33 for a sufficient time and under conditions to produce 6-aminocaproic acid (6ACA).

36. The method according to claim 35, further comprising recovering 6ACA from the microorganisms, fermentation broth, or both.

37. A method for producing hexamethylenediamine, comprising culturing a non-naturally occurring microorganism according to any one of claims 1 to 33 for a sufficient time and under conditions to produce hexamethylenediamine.

38. The method according to claim 37, further comprising recovering hexamethylenediamine from the microorganism, the fermentation broth, or both.

39. The method according to any one of claims 34 to 38, wherein the non-naturally occurring microorganism comprises two, three, four, five, six, or seven exogenous nucleic acid sequences, each encoding a hexamethylenediamine pathway enzyme.

40. A method for producing caprolactam, comprising culturing a non-naturally occurring microorganism according to any one of claims 1 to 33 for a sufficient time and under conditions to produce caprolactone, 1,6-hexanediol, or caprolactam.

41. The method according to claim 40, further comprising recovering caprolactam from the non-naturally occurring microorganism, fermentation broth, or both.

42. The method according to any one of claims 34 to 41, wherein the non-naturally occurring microorganism comprises two, three, four, five, six, or seven exogenous nucleic acids, each encoding a caprolactam pathway enzyme.

43. The method according to any one of claims 34 to 42, wherein the culture is carried out in a fermentation broth containing sugar.

44. The method according to any one of claims 34 to 43, wherein the non-naturally occurring microorganisms include the species Acinetobacter, Actinobacillus, Anaerobiospirillum, Aspergillus, Bacillus, Clostridium, Corynebacterium, Escherichia, Gluconobacter, Klebsiella, Kluyveromyces, Lactococcus, Lactobacillus, Mannheimia, Pichia, Pseudomonas, Rhizobium, Rhizopus, Saccharomyces, Schizosaccharomyces, Streptomyces, and Zymomonas.

45. A biologically derived 6-aminocaproic acid, hexamethylenediamine, 1,6-hexanediol, caprolactone, or caprolactam synthesized by the method described in any one of claims 34 to 44.

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