Carboxylic acid reductase and alcohol dehydrogenase variants and methods of use

Abstract

The disclosure provides polypeptides and encoding nucleic acids of engineered carboxylic acid reductases and / or engineered alcohol dehydrogenases. The disclosure also provides cells expressing an engineered form of the carboxylic acid reductases and / or engineered alcohol dehydrogenases. The disclosure further provides methods for producing a bioderived compound, such as 1,6 hexanediol (HDO), hexamethylenediamine (HMD), upstream precursors (e.g., adipate semi-aldehyde, 6-aminocaproic acid, 6-aminocaproate semialdehyde, 6-hydroxyhexanoic acid, 6-aminohexanol, caprolactone, caprolactam, and / or 6- hydroxyhexanal), and products derived therefrom comprising culturing cells expressing an engineered carboxylic acid reductase and / or engineered alcohol dehydrogenases.

Description

CARBOXYLIC ACID REDUCTASE AND ALCOHOL DEHYDROGENASE VARIANTS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 667,653, filed July 3, 2024, the entire contents of which are incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 199683-813002_PCT_SL.xml, which was created on May 28, 2025 and is 106,532 bytes in size, is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to carboxylic acid reductase variants and alcohol dehydrogenase variants and methods of using such enzymes, and more specifically to carboxylic acid reductase variants encoded by recombinant nucleic acids that have been introduced to a non-naturally occurring microbial organism to produce a bioderived compound such as adipate semi-aldehyde, 6-aminocaproic acid, 6-aminocaproate semialdehyde, 6- hydroxyhexanoic acid, caprolactone, caprolactam, 6-aminohexanol, 6-hydroxyhexanal, 1,6 hexanediol, hexamethylenediamine, and products derived therefrom.BACKGROUND OF THE INVENTION

[0004] Various commodity chemicals are used to make desired products for commercial use. Many of the commodity chemicals are derived from petroleum. Such commodity chemicals have various uses, including use as solvents, resins, polymer precursors, and specialty chemicals. Desired commodity chemicals include 6-carbon molecules, such as, 1,6 hexanediol (HDO), and hexamethylenediamine (HMD), upstream precursors (e.g., adipate semi-aldehyde, 6-aminocaproate semialdehyde, 6-hydroxyhexanoic acid, 6-aminocaproic acid, 6- aminohexanol, caprolactone, caprolactam, and / or 6-hydroxyhexanal) and downstream products. HDO can be an intermediate in the production of HMD and nylon 6,6. (O'Neil, M.J. (ed.). The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Cambridge, UK: Royal Society of Chemistry, 2013., p. 868). Nylons are polyamides that can be synthesized by the condensation polymerization of a diamine with a dicarboxylic acid or the condensationpolymerization of lactams. Nylon 6,6 can also be produced by reaction of HMD and adipic acid, while nylon 6 is produced by a ring opening polymerization of caprolactam. Therefore, HDO, HMD and upstream precursors are commodity chemicals.

[0005] Microorganisms have been engineered to produce some of the nylon intermediates. However, engineered microorganisms can produce undesirable byproducts as a result of undesired enzymatic activity on pathway intermediates and final products. Such byproducts and impurities therefore increase cost and complexity of biosynthesizing compounds and can decrease efficiency or yield of the desired products.SUMMARY

[0006] Provided herein is an engineered carboxylic acid reductase (CAR) comprising a variant of amino acid sequence SEQ ID NO: 1 or a functional fragment thereof, wherein the engineered CAR comprises one or more alterations at a position described in TABLE 3B. In some embodiments, the engineered CAR is capable of: catalyzing the conversion of 6- hydroxycaproic acid (6-HCA) to 6-hydroxyhexanal (6-HH); catalyzing the conversion of 6- HCA to 6-HH at a greater rate compared to wild-type CAR; having a higher specificity for the 6-HCA substrate as compared to the wild type CAR; or catalyzing the conversion of 6- aminocaproic acid (6ACA) to 6-aminocaproate semialdehyde (6ACA-SAld); catalyzing the conversion of 6ACA to 6ACA-SAld at a greater rate compared to wild-type CAR; having a higher specificity for the 6ACA substrate as compared to the wild type CAR; or any combination thereof. In some embodiments, the engineered CAR has higher specificity for conversion of 6-HCA to 6-HH or 6ACA to 6ACA-SAld, or both, over conversion of: propanoic acid to propanoate semialdehyde; hexanoic acid to hexanoate semialdehyde; adipic acid to adipate semialdehyde; acetic acid to acetaldehyde; succinic acid to succinate semialdehyde; benzoate to benzaldehyde; 4HB to 4HBAld; 3HB to the (R) or (S) isomer of 3HBAld; butyric acid to butyraldehyde; or any combination thereof. In some embodiments, the engineered CAR has higher specificity for conversion of 6-HCA to 6-HH over conversion of 6ACA to 6ACA-SAld. In some embodiments, the engineered CAR comprises an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a CAR consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid alterations comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110 or more alterations. In some embodiments, the one or more amino acid alterations are conservative or non-conservative amino acid substitutions, deletions, or insertions, or any combination thereof. In some embodiments, the CAR comprises one or more amino acid alterations at a position corresponding to position 48, 98, 134, 179, 238, 299, 354, 379, 389, 937, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the CAR comprises one or more amino acid alterations at a position corresponding to position 379, 354, 937, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the one or more amino acid alterations result in an engineered CAR comprising: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1 ; T at a residue corresponding to position 937 in SEQ ID NO: 1 ; W at a residue corresponding to position 299 in SEQ ID NO: 1; G at a residue corresponding to position 238 in SEQ ID NO: 1; E or R at a residue corresponding to position 134 in SEQ ID NO: 1; R at a residue corresponding to position 134 in SEQ ID NO: 1; S at a residue corresponding to position 389 in SEQ ID NO: 1; V at a residue corresponding to position 48 in SEQ ID NO: 1; D at a residue corresponding to position 179 in SEQ ID NO: 1; E at a residue corresponding to position 98 in SEQ ID NO: 1; or a combination thereof. In some embodiments, the one or more amino acid alterations result in an engineered CAR comprising: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; or a combination thereof. In some embodiments, the engineered CAR comprises one or more amino acid alterations selected from the group consisting of: Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, and any combination thereof. In some embodiments, the engineered CAR comprises one or more amino acid alterations selected from the group consisting of: Y379R, R354H, and / or Q937T, and any combination thereof. In some embodiments, the engineered CAR comprises a single alteration or a combination of alterations described in TABLE 3B or TABLE 4A. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence referenced in SEQ ID NO: 1.

[0007] Also provided herein is an engineered alcohol dehydrogenase (ADH) comprising a variant of amino acid sequence SEQ ID NO: 15 or a functional fragment thereof, wherein the engineered ADH comprises one or more alterations at a position described in TABLE 4C. In some embodiments, the engineered ADH is capable of: catalyzing the conversion of Ad-SAld to HHA; catalyzing the conversion of 6-HH to HDO; catalyzing the conversion of 6ACA- SAld to 6-AHL; converting NAD(P) to NAD(P)H; converting NAD to NADH; having a higher specificity for the NAD(P) substrate as compared to the wild type ADH; or any combination thereof. In some embodiments, the engineered ADH has higher specificity for NAD(P), over NAD. In some embodiments, the engineered ADH comprises an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a ADH consisting of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the one or more amino acid alterations comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 or more alterations. In some embodiments, the one or more amino acid alterations are conservative or non-conservative amino acid substitutions, deletions, or insertions, or any combination thereof. In some embodiments, the ADH comprises one or more amino acid alterations at a position corresponding to position 215, 216, or a combination thereof, in SEQ ID NO: 15. In some embodiments, the one or more amino acid alterations result in an engineered ADH comprising: A, S, or T at a residue corresponding to position 215 in SEQ ID NO: 15; H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; or a combination thereof. In some embodiments, the one or more amino acid alterations result in an engineered ADH comprising: A or S at a residue corresponding to position 215 in SEQ ID NO: 15; H or Q at a residue corresponding to position 216 in SEQ ID NO: 15; or a combination thereof. In some embodiments, the engineered ADH comprises one or more amino acid alterations selected from the group consisting of: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, and any combination thereof. In some embodiments, the engineered ADH comprises one or more amino acid alterations selected from the group consisting of: D215A, D215S, L216H, or L216Q, and any combination thereof. In some embodiments, the engineered ADH comprises one or more amino acid alterations selected fromthe group consisting of: D215A, L216H, and any combination thereof. In some embodiments, the engineered ADH comprises a single alteration or a combination of alterations described in TABLE 4C or TABLE 3B. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence referenced in SEQ ID NO: 15.

[0008] Also provided herein is a recombinant nucleic acid encoding: the engineered CAR provided herein; the engineered ADH provided herein; or both. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the engineered CAR and / or the engineered ADH operatively linked to a promoter.

[0009] Also provided herein is a vector comprising the recombinant nucleic acid provided herein.

[0010] Also provided herein non-naturally occurring microbial organism comprising: a recombinant nucleic acid encoding an engineered CAR provided herein; a recombinant nucleic acid encoding an engineered ADH provided herein; or a recombinant nucleic acid encoding both the engineered CAR and the engineered ADH. In some embodiments, the non-naturally occurring microbial organism further comprises a pathway that produces adipate semialdehyde (Ad-SAld), 6-aminocaproic acid (6ACA), 6-hydroxycaproic acid (HHA), 6-hydroxyhexanal (6-HH), 6-aminocaproate semialdehyde (6ACA-SAld), caprolactam (CPL), caprolactone (CPO), 6-aminohexanol (6-AHL), hexamethylenediamine (HMD), and / or 1,6 hexanediol (HDO), or an ester or amide thereof. In some embodiments, the non-naturally occurring microbial organism is capable of producing at least 10% more Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof compared to a control microbial organism that does not comprise any one of the nucleic acids. In some embodiments, the microbial organism produces a decreased amount of one or more by-product as compared to a control microbial organism that does not comprise the recombinant nucleic acid. In some embodiments, the one or more by-product comprises propanoate semialdehyde, hexanoate semialdehyde, acetaldehyde, succinate semialdehyde, 4HBAld, either isomer of 3HBAld, butyraldehyde, 4HB, BDO, or combinations thereof. In some embodiments, the microbial organism is capable of producing at least 10% less by-product compared to a control microbial organism that does not comprise the recombinant nucleic acid. In some embodiments, the one or more enzymes of the pathway are encoded by an exogenous nucleicacid. In some embodiments, the exogenous nucleic acid is heterologous or homologous. In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the microbial organism is a species of bacteria, yeast, or fungus.

[0011] Also provided herein is a method for producing Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof, comprising culturing the non-naturally occurring microbial organism provided herein under conditions and for a sufficient period of time to produce the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof. In some embodiments, the method further comprises separating the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6- AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof from other components in the culture. In some embodiments, the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

[0012] Also provided herein is a culture medium comprising the Ad-SAld, 6ACA, 6ACA- SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof produced by the method provided herein, wherein the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof has a carbon-12, carbon- 13 and carbon- 14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.

[0013] Also provided herein is an Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof produced according to the method provided herein. In some embodiments, the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof, wherein the Ad-SAld, 6ACA, 6ACA-S Aid, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

[0014] Also provided herein is a composition comprising the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof provided herein and a compound other than the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6- AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof. In some embodiments, thecompound other than the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a pathway that produces Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof.

[0015] Also provided herein is composition comprising the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof provided herein or a cell lysate or culture supernatant thereof.

[0016] Also provided herein is a use of the engineered CAR provided herein as a biocatalyst.

[0017] Also provided herein is a composition comprising the engineered CAR provided herein and at least one substrate for the engineered CAR. In some embodiments, the engineered CAR can react with the substrate under in vitro conditions. In some embodiments, the substrate is adipate or adipic acid. In some embodiments, the substrate is 6-hydroxycaproic acid. In some embodiments, the substrate is 6-hydroxyhexanoate. In some embodiments, the substrate is 6- aminocaproate.

[0018] Also provided herein is a use of the engineered ADH provided herein as a biocatalyst.

[0019] Also provided herein is a composition comprising the engineered ADH probided herein and at least one substrate for the engineered ADH. In some embodiments, the engineered ADH can react with the substrate under in vitro conditions. In some embodiments, the substrate is Ad-SAld. In some embodiments, the substrate is 6-HH. In some embodiments, the substrate is 6ACA-SAld. In some embodiments, the composition provided herein comprises NAD(P) or NAD(P)H.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows exemplary pathways from succinyl-CoA and acetyl-CoA or adipic acid to HDO. The enzymes are designated as follows: A) P-ketoadipyl-CoA thiolase, B) 3- hydroxyadipyl-CoA dehydrogenase, C) 2,3-dehydroadipyl-CoA hydratase, D) trans-enoyl CoA reductase (TER), E) aldehyde dehydrogenase (ALDH) or acyl-CoA reductase / dehydrogenase, G) ACL or acyl-CoA transferase, H) ALDH, I) alcohol dehydrogenase (ADH), J) alcohol -forming fatty acyl-CoA reductase (FAR), K) carboxylic acid reductase (CAR) or CAR and phosphopantetheinyl transferase (PPTase), L) ACL or acyl-CoAtransferase, M) carboxylic acid reductase (CAR) or CAR and phosphopantetheinyl transferase (PPTase), N) FAR, and O) ADH.

[0021] FIG. 2 shows exemplary pathways from succinyl-CoA and acetyl-CoA to HDO. The enzymes are designated as follows: A) thiolase (THL), B) a hydroxyadipyl-CoA dehydrogenase (HBD), C) a crotonase (CRT), D) TER, E) a 6ACA-aldehyde dehydrogenase (6ACA-ALD), F) an ADH, G) ACL or acyl-CoA transferase, H) ALDH or acyl-CoA reductase, I) an ADH, and K) CAR or CAR and phosphopantetheinyl transferase (PPTase).

[0022] FIG. 3 shows exemplary pathways from succinyl-CoA and acetyl-CoA to 6- aminocaproate (6ACA), 6-aminocaproate semi-aldehyde (6ACA-SAM), caprolactam (CPL), and hexamethylenediamine (HMD). The enzymes are designated as follows: A) 3-oxoadipyl- CoA thiolase, succinyl-CoA:acetyl-CoA transferase or a thiolase, B) 3-oxoadipyl-CoA reductase or 3-hydroxyacyl-CoA dehydrogenase, C) 3-hydroxyadipyl-CoA dehydratase, D) adipate semialdehyde reductase or 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 -hydroxy adipate dehydratase, J) 5-carboxy-2-pentenoate reductase, K) adipyl-CoA / acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoA hydrolase, N) adipyl-CoA reductase (aldehyde forming) or 6-ACA-aldehyde dehydrogenase (ALD), O) 6- aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q) 6-aminocaproyl- CoA / acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase or acyl-CoA synthetase (ACS), S) amidohydrolase, T) spontaneous and / or chemical cyclization, U) 6-aminocaproyl-CoA reductase (aldehyde forming) or ALDH, V) HMDA transaminase, W) HMDA dehydrogenase, X) adipate reductase or CAR, Y) adipate kinase, Z) adipylphosphate reductase, and AB) CAR.

[0023] FIG. 4 shows exemplary pathways to hexamethylenediamine from 6-aminocapropate. The enzymes are designated as follows: A) 6-aminocaproate kinase, B) [(6- aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, C) 6-aminocaproic semialdehyde aminotransferase and / or 6-aminocaproic semialdehyde oxidoreductase (aminating), D) 6-aminocaproate N-acetyltransferase, E) 6-acetamidohexanoate kinase, F) [(6- acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, G) 6-acetamidohexanal aminotransferase and / or 6-acetamidohexanal oxidoreductase (aminating), H) 6- acetamidohexanamine N-acetyltransferase and / or 6-acetamidohexanamine hydrolase (amide), I) 6-acetamidohexanoate CoA transferase and / or 6-acetamidohexanoate CoA ligase, J) 6- acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L) 6-AHOPacyltransferase, M) 6-aminocaproate CoA transferase, 6-aminocaproate CoA ligase and / or ACL, N) 6-aminocaproyl-CoA oxidoreductase and / or ALDH, O) CAR, and P) CAR.

[0024] FIG. 5 shows exemplary biosynthetic pathways leading to 1,6-hexanediol. The enzymes are designated as follows: A) 6-aminocaproyl-CoA transferase or synthetase or ACL, B) 6-aminocaproyl-CoA reductase or ALDH, C) 6-aminocaproate semialdehyde reductase, D) 6-aminocaproate reductase or CAR, E) adipyl-CoA reductase, F) adipate semialdehyde reductase, G) 6-hydroxyhexanoyl-CoA transferase or synthetase or ACL, H) 6- hydroxyhexanoyl-CoA reductase or ALDH, I) 6-hydroxyhexanal reductase, J) 6-aminohexanol aminotransferase or oxidoreductases, K) 6-hydroxyhexanoate reductase or CAR, L) adipate reductase, M) adipyl-CoA transferase, N) adipyl-CoA hydrolase, and O) adipyl-CoA synthase.

[0025] FIG. 6 shows exemplary pathways from adipate or adipyl-CoA to caprolactone. Enzymes are A) adipyl-CoA reductase, B) adipate semialdehyde reductase, C) 6- hydroxyhexanoyl-CoA transferase or synthetase, D) 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization, E) adipate reductase or CAR, F) adipyl-CoA transferase, synthetase or hydrolase, G) 6-hydroxyhexanoate cyclase, H) 6-hydroxyhexanoate kinase, I) 6- hydroxyhexanoyl phosphate cyclase or spontaneous cyclization, J) phosphotrans-6- hydroxyhexanoylase.

[0026] FIG. 7 shows an exemplary hexamethylenediamine (HMD) biosynthetic pathway. Starting from succinyl-CoA and acetyl-CoA the enzymes are designated as follows: (A) thiolase; (B) hydroxyadipyl-CoA dehydrogenase (HBD); (C) crotonase; (D) trans-enoyl-CoA reductase (Ter); (E) 6ACA-aldehyde dehydrogenase (ALD); (F) 6ACA-transaminase (TA); (G) CoA transferase / CoA ligase; (H) HMD-aldehyde dehydrogenase (ALD); (I) carboxylic acid reductase (CAR) and PPTase, and (J) HMD-transaminase (TA2). PPTase corresponds to a phosphopantetheinyl transferase.

[0027] FIG. 8 shows exemplary pathways to 6-aminocaproate from pyruvate and succinic semialdehyde. Enzymes are A) HODH aldolase, B) OHED hydratase, C) OHED reductase, D) 2-OHD decarboxylase, E) adipate semialdehyde aminotransferase and / or adipate semialdehyde oxidoreductase (aminating), F) OHED decarboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferase and / or 2-OHD oxidoreductase (aminating),!) 2-AHD decarboxylase, J) OHED aminotransferase and / or OHED oxidoreductase (aminating), K) 2-AHE reductase, L) HODH formate-lyase and / or HODH dehydrogenase, M) 3-hydroxyadipyl-CoAdehydratase, N) 2,3-dehydroadipyl-CoA reductase, O) adipyl-CoA dehydrogenase, P) OHED formate-lyase and / or OHED dehydrogenase, Q) 2-OHD formate-lyase and / or 2-OHD dehydrogenase. Abbreviations are: HODH = 4-hydroxy-2-oxoheptane-l,7-dioate, OHED = 2- oxohept-4-ene-l,7-dioate, 2-OHD = 2-oxoheptane-l,7-dioate, 2-AHE = 2-aminohept-4-ene- 1,7-dioate, 2-AHD = 2-aminoheptane-l,7-dioate, and 6-OHE = 6-oxohex-4-enoate.

[0028] FIG. 9A depicts kinetic parameters of CAR enzymes (SEQ ID NO: 1-4), Step K of FIG. 1, with different substrates.

[0029] FIG. 9B depicts activity of HiBiT tagged CAR enzymes in a lysate assay with 6-HCA as substrate.

[0030] FIG. 10A-10B depicts the results from the MaCAR protein engineering library primary screen. Ranking of the library by rate (FIG. 10A) and by fold improvement (FIG. 10B).

[0031] FIG. 11A-11B depicts results from the MaCAR Variant library secondary screen. FIG. HA depicts library ranking by rate. The dark gray dots mark the selected hits for secondary screening. FIG. 11B depicts the correlation between activity rate and protein concentration as quantified by HiBiT.

[0032] FIG. 12 depicts 1,6-HDO production titers of strains carrying wild-type MaCAR (SEQ ID NO: 1) and engineered variants expressed from a pTet inducible promoter. Strains were screened in a QSS assay and CAR enzymes were induced by the addition of aTc.

[0033] FIG. 13 depicts 1,6-HDO production titers of wild-type MaCAR and engineered variants expressed from constitutive promoters of different strength. Strains were screened in a QSS assay.

[0034] FIG. 14 depicts the performance of 1,6-HDO producing strains carrying MaCAR variants induced with varying concentrations of aTc.

[0035] FIG. 15 depicts HDO titers produced from strains constitutively expressing MaCAR variants constitutively in Ambr250.

[0036] FIGS. 16A-16B depicts results from alcohol dehydrogenase library primary screen.FIG. 16A depicts relative activity of the library and controls per plate. FIG. 16B depictsselection of top 44 strains based on activity above background and activity relative to the high positive control.

[0037] FIG. 17 depicts results from alcohol dehydrogenase library secondary screen, measuring activity on 6 hydroxyhexanal using NADPH or NADH.

[0038] FIG. 18 depicts ADH cofactor engineering primary screen results. Strain ranking is based on activity rate with NADPH as cofactor.

[0039] FIG. 19 depicts ADH Variants cofactor engineering secondary screen results. Strain ranking is based on activity rate with NADPH as cofactor.

[0040] FIG. 20 depicts a correlation plot comparing the activity rate of each glycerol stock between the primary and secondary screens of the ADH variants.

[0041] FIGS. 21A-21B depict hit selection based on NADPH specificity. FIG. 21A depicts the comparison between various protein engineering strategies. The box highlights hit variants that maintained activity on NADPH while significantly lowering the activity rate on NADH. FIG. 21B depicts strain ranking based on NADPH / NADH ratio.

[0042] FIG. 22 depicts NADH / NAD+ and NADPH / NADP+ levels from Ambr250 samples.

[0043] FIG. 23 depicts results from the Ambr250 fermentation run for the overexpression of pntAB.DETAILED DESCRIPTION OF THE INVENTION

[0044] The subject matter described herein relates to enzyme variants that have desirable properties and are useful for producing desired products, e.g., 6-hydroxy caproic acid (interchangeably referred to herein as “6-hydroxyhexanoic acid” or “HHA”, “6-HCA”, or “6- HHA”), 6-hydroxyhexanal, (interchangeably referred to herein as “6-HH”), 1,6 hexanediol (interchangeably referred to herein as “1,6-HDO” or “HDO”) , 6-aminocaproate semialdehyde (interchangeably referred to herein as “6-aminocaproic semialdehyde” or “6ACA-SAld”), adipate semialdehyde (interchangeably referred to herein as “Ad-SAld”), caprolactone (interchangeably referred to herein as “CPO” or “CLO”), caprolactam (interchangeably referred to herein as “CPL”), 6-aminohexanol (interchangeably referred to herein as “6- AHL”), and hexamethylenediamine (interchangeably referred to herein as “HMD” or “HMD A”), or an ester or amide thereof.

[0045] In some embodiments, the subject matter described herein relates to engineered carboxylic acid reductases (interchangeably referred herein as an “CARs”), which are enzyme variants that have markedly different structural and / or functional characteristics compared to a wild-type CARs that occurs in nature, respectively. Thus, the engineered CARs provided herein are not naturally occurring enzymes. Such engineered CARs provided are useful in an engineered cell, such as a microbial organism, that has been engineered to produce a desired product (e.g., Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof). In some embodiments, the subject matter described herein relates to engineered alcohol dehydrogenases (interchangeably referred herein as an “ADHs”), which are enzyme variants that have markedly different structural and / or functional characteristics compared to a wild-type ADHs that occurs in nature, respectively. Thus, the engineered ADHs provided herein are not naturally occurring enzymes. Such engineered ADHs provided are useful in an engineered cell, such as a microbial organism, that has been engineered to produce a desired product (e.g., Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6- AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof).

[0046] For example, as disclosed herein, a cell, such as a microbial organism, having a metabolic pathway can produce a desired product (e.g., Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof). Engineered CARs and / or engineered ADHs having desirable characteristics as described herein can be introduced into a cell, such as microbial organism, that has a metabolic pathway that uses carboxylic acid reductase activity to produce a desired product (e.g., Ad-SAld, 6 AC A, 6ACA- SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof). Thus, the engineered CARs and / or engineered ADHs provided herein can be utilized in engineered cells, such as microbial organisms, to produce a desired product. Such engineered CARs and / or engineered ADHs are additionally useful as biocatalysts for carrying out desired reactions in vitro. Thus, the engineered CARs and / or engineered ADHs provided herein can be utilized in engineered cells, such as microbial organisms, to produce a desired product or as an in vitro biocatalyst to produce a desired product.Conventions and Abbreviations

[0047] As used herein, “1,6-hexanediol” also referred to as 1,6 HDO, hexane- 1,6-diol, hexamethylene glycol, 1,6 -dihydroxyhexane, hexamethylenediol, 1,6-hexylene glycol, and 6- hydroxy-1 -hexanol; has the chemical structure CeHuCh (see FIG. 1 and 2 and abbreviated herein as HDO).

[0048] As used herein, “6-aminocaproate,” having the chemical formula -OOC- (CH2)s-NH2 (see FIG. 3, and abbreviated herein 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 can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on the pH.

[0049] As used herein the term “about” means ± 10% of the stated value. The term “about” can mean rounded to the nearest significant digit. Thus, about 5% means 4.5% to 5.5%.Additionally, about in reference to a specific number also includes that exact number. For example, about 5% also includes exact 5%.

[0050] As used herein, the term “alteration” or grammatical equivalents thereof when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a change in structure of an amino acid residue or nucleic acid base relative to the starting or reference residue or base. An alteration of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a nonconservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. An alteration of a nucleic acid base includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. An alteration of a nucleic acid base may result in an alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue or function of the peptide, polypeptide or protein. An alteration of a nucleic acid base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[0051] As used herein, the term “bioderived” means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the non-naturally occurring microbial organism disclosed herein, can utilize feedstock or biomass, such as, sugars (e.g., cellobiose, glucose, fructose, xylose, galactose (e.g., galactose from marine plant biomass), and sucrose), carbohydrates obtained from an agricultural, plant, bacterial, or animal source, and glycerol (e.g., crude glycerol by-product from biodiesel manufacturing) for synthesis of a desired bioderived compound.

[0052] As used herein, “caprolactam” (IUPAC name azepan-2-one) is a lactam of 6- aminohexanoic acid (see FIG. 3 and abbreviated herein as CPL).

[0053] As used herein, “caprolactone” (IUPAC name Oxepan-2-one) is a lactone of 6- hydroxyhexanoic acid (see FIG. 3 and abbreviated herein as CPO).

[0054] As used herein, the term “conservative substitution” refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acidsthat are related in their side chains. Alternatively, the term “non-conservative substitution” refers to the replacement of one amino acid residue for another such that the replaced residue is going from one family of amino acids to a different family of residues. Genetically encoded amino acids can be divided into four families: (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic) = Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic = Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar = Asn (N), Gin (Q), Ser (S), Thr (T). In alternative fashion, the amino acid repertoire can be grouped as (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H), and (3) aliphatic = Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (Q); and (6) sulfur-containing = Cys (C) and Met (M) (see, for example, Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co., 1995, which is incorporated by reference herein in its entirety).

[0055] As used herein, the term “culture medium,” “medium,” “growth medium” or grammatical equivalents thereof refers to a liquid or solid (e.g., gelatinous) substance containing nutrients that support the growth of a cell, including a microbial organism, such as the microbial organism described herein. Nutrients that support growth include, but are not limited to, the following: a substrate that supplies carbon, such as, but are not limited to, cellobiose, galactose, glucose, xylose, ethanol, acetate, arabinose, arabitol, sorbitol and glycerol; salts that provide essential elements including magnesium, nitrogen, phosphorus, and sulfur; a source for amino acids, such as peptone or tryptone; and a source for vitamin content, such as yeast extract. Culture medium can be a defined medium, in which quantities of all ingredients are known, or an undefined medium, in which the quantities of all ingredients are not known. Culture medium can also include substances other than nutrients needed for growth, such as a substance that only allows select cells to grow e.g., antibiotic or antifungal), which are generally found in selective medium, or a substance that allows for differentiation of one microbial organism over another when grown on the same medium, which are generally found in differential or indicator medium. Such substances are well known to a person skilled in the art.

[0056] As used herein, the term “engineered” or “variant” when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a sequence of amino acids or nucleic acids having at least one alteration at an amino acid residue or nucleic acid base as compared to a parent sequence. Such a sequence of amino acids or nucleic acids is not naturally occurring. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a modified variant of a wild-type sequence or homolog thereof.

[0057] “Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid described herein can utilize either or both a heterologous or homologous encoding nucleic acid.

[0058] It is understood that, when more than one recombinant nucleic acid and / or exogenous nucleic acid is included into a microbial organism, the more than one recombinant nucleic acid and / or exogenous nucleic acid refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed herein. It is further understood, as disclosed herein, that such more than one recombinant nucleic acids or exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one recombinant nucleic acid and / or exogenous nucleic acid. For example, as disclosed herein a microbialorganism can be engineered to express two or more recombinant and / or exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two recombinant and / or exogenous nucleic acids encoding an enzyme or protein having a desired activity are introduced into a host microbial organism, it is understood that the two recombinant and / or exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two recombinant and / or exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more recombinant or exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced recombinant or exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

[0059] The term “Fm value” or “Fraction Modern value” when used in reference to a compound is a ratio of carbon-14 (14C) to carbon-12 (12C). Specifically, Fm value is computed from the expression: Fm = (S-B) / (M-B), where B, S and M represent the14C / 12C ratios of the blank, the sample and the modem reference, respectively. Fm value is a measurement of the deviation of the14C / 12C ratio of a sample from “Modern.” Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (z.e., standard reference materials (SRM) 4990b) normalized to 513CVPDB=-19 per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 513CVPDB=- 19 per mil. This is equivalent to an absolute (AD 1950)14C / 12C ratio of 1.176 ± 0.010 x IO’12(Karlen et al., Arkiv Geofysik. 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C12over C13over C14, and these corrections are reflected as a Fm corrected for 513. An Fm = 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source, whereas a Fm = 100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modem carbon source. The percent modern carbon(pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon- 14 in the atmosphere. Because all sample carbon-14 activities are referenced to a “pre-bomb” standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old. Applications of carbon-14 dating techniques to quantify biobased content of materials are well known in the art (see, e.g., Currie etal., Nuclear Instruments andMethods in Physics Research B, 172:281-287 (2000), and Colonna etal., Green Chemistry, 13:2543-2548 (2011)).

[0060] As used herein, the term “functional fragment” when used in reference to a peptide, polypeptide or protein is intended to refer to a portion of the peptide, polypeptide or protein that retains some or all of the activity (e.g., catalyzing the conversion of HHA to 6-HH, the conversion of ADA to Ad-SAld, and / or the conversion of 6-ACA to 6ACA-SAld) of the original peptide, polypeptide or protein from which the fragment was derived. Such functional fragments include amino acid sequences that are about 200 to about 460, about 200 to about 450, about 200 to about 440, about 200 to about 430, about 200 to about 420, about 200 to about 410, about 200 to about 400, about 200 to about 390, about 200 to about 380, about 200 to about 370, about 200 to about 360, about 200 to about 350, about 300 to about 460, about 300 to about 450, about 300 to about 440, about 300 to about 430, about 300 to about 420, about 300 to about 410, about 300 to about 400, about 300 to about 390, about 300 to about 380, about 300 to about 370, about 300 to about 350, about 300 to about 340, about 300 to about 330, about 300 to about 320, about 300 to about 310, about 400 to about 460, about 400 to about 450, about 400 to about 440, about 400 to about 430, about 400 to about 420, about 400 to about 410, about 450 to about 460 amino acids in length. These functional fragments can, for example, be truncations (e.g., C-terminal or N-terminal truncations) of a peptide, polypeptide, or protein. Functional fragments can also include one or more amino acid alteration described herein, such as an amino acid alteration of an engineered peptide described herein.

[0061] As used herein, the term “isolated” when used in reference to a molecule (e.g, peptide, polypeptide, protein, nucleic acid, polynucleotide, vector) or a cell (e.g., a yeast cell) refers toa molecule or cell that is substantially free of at least one component with which the referenced molecule or cell is found in nature. The term includes a molecule or cell that is removed from some or all components with which it is found in its natural environment. Therefore, an isolated molecule or cell can be partly or completely separated from other substances with which it is found in nature or with which it is grown, stored or subsisted in non-naturally occurring environments.

[0062] As used herein, the terms “microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

[0063] As used herein, the term “non-naturally occurring” when used in reference to a microbial organism described herein is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and / or other functional disruption of the microbial organism’s genetic material. Such modifications include, for example, genetic alterations within coding regions and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a 1,6-HDO, HMD or other bioderived compound pathway described herein.

[0064] As use herein, the term “operatively linked” when used in reference to a nucleic acid encoding an engineered CAR and / or an engineered ADH refers to connection of a nucleotide sequence encoding an engineered CAR and / or an engineered ADH described herein to another nucleotide sequence (e.g., a promoter) is such a way as to allow for the connected nucleotide sequences to function (e.g., express the engineered CAR and / or an engineered ADH in the microbial organism).

[0065] As used herein, the term “pathway” when used in reference to production of a desired product (e.g., Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof) refers to one or more polypeptides (e.g., proteins or enzymes) that catalyze the conversion of a substrate compound to a product compound and / or produce a co-substrate for the conversion of a substrate compound to a product compound. Such a product compound can be one of the bioderived compounds described herein, or an intermediate compound that can lead to the bioderived compound upon further conversion by other proteins or enzymes of the metabolic pathway. Accordingly, a metabolic pathway can be comprised of a series of metabolic polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten or more) that act upon a substrate compound to convert it to a given product compound through a series of intermediate compounds. The metabolic polypeptides of a metabolic pathway can be encoded by an exogenous nucleic acid as described herein or produced naturally by the host microbial organism.

[0066] As used herein, the term “recombinant” with respect to a nucleic acid, such as a nucleic acid comprising a gene that encodes a protein or polypeptide (e.g., an engineered CAR or an engineered ADH described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. The recombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a microbial organism, such as by integration into a microbial organism chromosome, or as non-chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a microbial organism may be a nucleic acid that comes from a different organism or species from the microbial organism, or may be a synthetic nucleic acid, or may be a nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the microbial organism relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the microbial organism relative to the endogenously expressed sequence, and / or the nucleicacid is expressed as non-chromosomal genetic material such as a plasmid in the microbial organism.

[0067] As used herein, the term “promoter” when used in reference to a nucleic acid encoding an engineered CAR and / or an engineered ADH refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding an engineered CAR and / or an engineered ADH) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, i.e. the sense strand.

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

[0069] As used herein, the term “vector” refers to a compound and / or composition that transduces, transforms, or infects a microbial organism, thereby causing the microbial organism to express nucleic acids and / or proteins other than those native to the microbial organism, or in a manner not native to the cell. Vectors can be constructed to include one or more biosynthetic pathway enzyme or protein, such as an engineered CAR and / or an engineered ADH described herein, encoded by a nucleotide sequence operably linked to expression control sequences (e.g., promoter) that are functional in the microbial organism (“expression vector”). Expression vectors applicable for use in the microbial organisms described herein include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more recombinant or exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of a recombinant or exogenous nucleic acid encoding an enzyme or protein involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid or its corresponding gene product (e.g., enzyme or protein). It is understood by those skilled in the art that the recombinant or exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0070] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable microbial organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

[0071] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequencesimilarity of about 25% to 100% amino acid sequence identity. For example, genes that are orthologs can encode proteins with sequence similarity of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less than 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

[0072] Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microbial organism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 ’-3’ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease and the polymerase from the second species and vice versa.

[0073] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolutionfrom a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

[0074] A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

[0075] Therefore, in identifying and constructing the non-naturally occurring microbial organisms described herein having biosynthetic capability for a desired product, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and / or nonorthologous gene displacements are present in the referenced microbial organism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly, for a gene disruption, evolutionally related genes can also be disrupted or deleted in a microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.

[0076] Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well-known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. For example, related gene products or proteins can be expected to have about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

[0077] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleotide sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.Carboxylic Acid Reductase (CAR)

[0078] An engineered carboxylic acid reductase described herein refers to an enzyme that belongs in the oxidoreductase family of enzymes and may also be referred to as “CAR.” An engineered CAR described herein converts a carboxylic acid to into its corresponding aldehyde. This reaction can require ATP and NAD(P)H to catalyze the reduction. Such an engineered CAR described herein can be classified as a reaction 1.2.1.30, where the first three digits correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity (e.g., EC 1.2.1.-). Exemplary enzymatic conversions of an engineered CAR provided herein include, but are not limited to, theconversion of HHA to 6-HH, the conversion of ADA to Ad-SAld, and / or the conversion of 6- ACA to 6ACA-SAld. A CAR described herein can be used to produce desired products, such as Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or other desired products such as a downstream product, including an ester or amide thereof, in a cell, such as a microbial organism, containing a suitable metabolic pathway, or in vitro. CARs described herein are particularly useful to provide an improved enzymatic route and microorganism to provide an improved composition of HDO and / or HMD, and further having improved purity qualities with respect to by-products.

[0079] In some embodiments, provided herein is an engineered CAR that is a variant of a wildtype CAR (e.g, SEQ ID NO: 1). Such an engineered CAR includes one or more alterations at a position described in TABLE 3B, and, in some embodiments, a combination of alterations described in TABLE 3B, and has higher activity relative to the WT CAR (e.g., SEQ ID NO: 1) as described herein. In certain embodiments, provided herein is an engineered CAR that is a variant of a WT CAR (e.g., SEQ ID NO: 1). Such an engineered CAR includes one or more alterations at a position described in TABLE 3B, and, in some embodiments, a combination of alterations described in TABLE 3B, and has higher activity relative to the WT CAR (e.g., SEQ ID NO: 1) as described herein. In some embodiments, the term “activity” as used herein to describe enzymatic activity, refers to catalytic activity, substrate specificity, substrate affinity, or any combination thereof.

[0080] In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of: 1) HHA to 6-HH; 2) 6-ACA to 6ACA-SAld; 3) ADA to Ad-SAld; 4) propanoic acid to propanoate semialdehyde; 5) hexanoic acid to hexanoate semialdehyde; 6) acetic acid to acetaldehyde; 7) succinic acid to succinate semialdehyde; 8) benzoate to benzaldehyde; 9) 4-hydroxybutyrate (4HB) to 4-hydroxybutyraldehyde (4-HBAld); 10) 3 -hydroxybutyrate (3HB) to either isomer of 3 -hydroxybutyraldehyde (3HBAld); 11) butyric acid to butyraldehyde, or combinations thereof. Accordingly, in some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of HHA to 6-HH. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of 6-ACA to 6ACA-SAld. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of ADA to Ad-SAld. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of propanoic acid to propanoate semialdehyde. In some embodiments, an engineered CAR provided herein is capable ofcatalyzing the conversion of hexanoic acid to hexanoate semialdehyde. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of acetic acid to acetaldehyde. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of succinic acid to succinate semialdehyde. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of benzoate to benzaldehyde. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of 4HB to 4-HBAld. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of 3HB to either isomer of 3HB Aid. In some embodiments, an engineered CAR provided herein is capable of catalyzing the conversion of butyric acid to butyraldehyde.

[0081] In some embodiments, an engineered CAR as described herein has higher catalytic activity in the conversion of select substrates over other substrates. For example, in some embodiments, an engineered CAR as described herein has: 1) higher specificity for conversion of HHA to 6-HH over conversion of 6-ACA to 6ACA-SAM; 2) higher specificity for conversion of HHA to 6-HH over conversion of ADA to Ad-SAld; 3) higher specificity for conversion of HHA to 6-HH over conversion of propanoic acid to propanoate semialdehyde; 4) higher specificity for conversion of HHA to 6-HH over conversion of hexanoic acid to hexanoate semialdehyde; 5) higher specificity for conversion of HHA to 6-HH over conversion of acetic acid to acetaldehyde; 6) higher specificity for conversion of HHA to 6-HH over conversion of succinic acid to succinate semialdehyde; 7) higher specificity for conversion of HHA to 6-HH over conversion of benzoate to benzaldehyde; 8) higher specificity for conversion of HHA to 6-HH over conversion of 4HB to 4-HBAld; 9) higher specificity for conversion of HHA to 6-HH over conversion of 3HB to either isomer of 3HBAld; 10) higher specificity for conversion of HHA to 6-HH over conversion of butyric acid to butyraldehyde; or any combination thereof.

[0082] In another example, in some embodiments, an engineered CAR as described herein has: 1) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of ADA to Ad- SAld; 2) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of propanoic acid to propanoate semialdehyde; 3) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of hexanoic acid to hexanoate semialdehyde; 4) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of acetic acid to acetaldehyde; 5) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of succinic acid tosuccinate semialdehyde; 6) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of benzoate to benzaldehyde; 7) higher specificity for conversion of 6-ACA to 6ACA-SAld over conversion of 4HB to 4-HBAld; 8) higher specificity for conversion of 6- ACA to 6ACA-SAld over conversion of 3HB to either isomer of 3HBAld; 9) higher specificity for conversion of 6-ACA to 6ACA-SAM over conversion of butyric acid to butyraldehyde; or any combination thereof.

[0083] Accordingly, in some embodiments, an engineered CAR as described herein has: 1) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of ADA to Ad-SAld; 2) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of propanoic acid to propanoate semialdehyde; 3) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of hexanoic acid to hexanoate semialdehyde; 4) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAM over conversion of acetic acid to acetaldehyde; 5) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAM over conversion of succinic acid to succinate semialdehyde; 6) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of benzoate to benzaldehyde; 7) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of 4HB to 4-HBAld; 8) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAM over conversion of 3HB to either isomer of 3HBAld; 9) higher specificity for conversion of HHA to 6-HH and / or 6-ACA to 6ACA-SAld over conversion of butyric acid to butyraldehyde, or any combination thereof.

[0084] In some embodiments, provided herein is an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1, wherein the engineered CAR comprises one or more alterations at a position described in TABLE 3B. In some embodiments, such an engineered CAR is capable of catalyzing the conversion of 1) HHA to 6-HH; 2) 6-ACA to 6ACA-SAM; 3) ADA to Ad-SAld; 4) propanoic acid to propanoate semialdehyde; 5) hexanoic acid to hexanoate semialdehyde; 6) acetic acid to acetaldehyde; 7) succinic acid to succinate semialdehyde; 8) benzoate to benzaldehyde; 9) 4HB to 4-HBAld; 10) 3HB to either isomer of 3HBAld; 11) butyric acid to butyraldehyde, or combinations thereof. Accordingly, in some embodiments, such an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of HHA to 6-HH. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable ofcatalyzing the conversion of 6-ACA to 6ACA-SAM. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of ADA to Ad-SAld.

[0085] In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of propanoic acid to propanoate semialdehyde. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of hexanoic acid to hexanoate semialdehyde. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of acetic acid to acetaldehyde. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of succinic acid to succinate semialdehyde. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of benzoate to benzaldehyde. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of 4HB to 4-HBAld. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of 3HB to either isomer of 3HBAld. In some embodiments, an engineered CAR comprising a variant of amino acid sequence SEQ ID NO: 1 is capable of catalyzing the conversion of butyric acid to butyraldehyde.

[0086] It is understood that the engineered CARs as described herein can carry out a similar enzymatic reaction as the wild-type CARs (e.g., SEQ ID NO: 1) as discussed above. It is further understood that the variants of the CAR enzyme can include alterations that provide a beneficial characteristic to the engineered CAR, including but not limited to, increased activity (e.g., ability to catalyze a reaction described herein and / or selectivity for a substrate, such as HHA or 6ACA) as described herein (see, e.g., EXAMPLES 1 and 2). In some embodiments, the engineered CAR can exhibit an activity that is at least the same or higher than the wildtype CAR (e.g., SEQ ID NO: 1), that is, it has activity that is the same or higher than a CAR without the variant at the same amino acid position(s). In some embodiments, the engineered CAR can exhibit two or more activities (e.g, ability to catalyze a reaction described herein and selectivity for a substrate, such as HHA or 6ACA) that are at least the same or higher than the wild-type CAR (e.g., SEQ ID NO: 1), that is, it has two or more activities that are the same or higher than an CAR without the variant at the same amino acid position(s). For example, theengineered CARs provided here can have one or more activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher over a wild-type CAR (see, e.g., EXAMPLES 1 and 2). In some embodiments, an engineered CAR provided herein has an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of an CAR consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an activity that is at least 10% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 20% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 30% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 40% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 50% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 60% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 70% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 80% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 90% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 100% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 110% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 120% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 130% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 140% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 150% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 160% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 170% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 180% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 190% higher. In some embodiments, an engineered CAR provided herein has an activity that is at least 200% higher. It is understood that activity refers to the ability of an engineered CAR described herein to convert a substrate to a product relative to a wild-typeCAR e.g., SEQ ID NO: 1) under the same assay conditions, such as those described herein (see, e.g., EXAMPLES 1 and 2).

[0087] In some embodiments, the activity of a CAR described herein is measured as the catalytic constant (kcat) value or turnover number. In some embodiments, the kcat is at least 0.1 s’1, at least 0.2 s’1, at least 0.3 s’1, at least 0.4 s’1, at least 0.5 s’1, at least 0.6 s’1, at least 0.7 s’1, at least 0.8 s’1, at least 0.9 s’1, at least 1 s’1, at least 2 s’1, at least 3 s’1, at least 4 s’1, at least 5 s’ at least 6 s’1, at least 7 s’1, at least 8 s’1, at least 9 s’1, at least 10 s’1, at least 11 s’1, at least 12 s’1, at least 13 s’1, at least 14 s’1, at least 15 s’1, at least 16 s’1, at least 17 s’1, at least 18 s’1, at least 19 s’1, at least 20 s’1, at least 21 s’1, at least 22 s’1, at least 23 s’1, at least 24 s’1, at least 25 s’1, at least 26 s’1, at least 27 s’1, at least 28 s’1, at least 29 s’1, at least 30 s’1, at least 31 s’1, at least 32 s’1, at least 33 s’1, at least 34 s’1, at least 35 s’1, at least 36 s’1, at least 37 s’1, at least 38 s’1, at least 39 s’1, at least 40 s’1, at least 41 s’1, at least 42 s’1, at least 43 s’1, at least 44 s’1, at least 45 s’1, at least 46 s’1, at least 47 s’1, at least 48 s’1, at least 49 s’1, at least 50 s’1, at least 51 s’1, at least 52 s’1, at least 53 s’1, at least 54 s’1, at least 55 s’1, at least 56 s’1, at least 57 s’1, at least 58 s’1, at least 59 s’1, at least 60 s’1, at least 61 s’1, at least 62 s’1, at least 63 s’1, at least 64 s’1, at least 65 s’1, at least 66 s’1, at least 67 s’1, at least 68 s’1, at least 69 s’1, at least 70 s’1, at least 71 s’1, at least 72 s’1, at least 73 s’1, at least 74 s’1, at least 75 s’1, at least 76 s’1, at least 77 s’1, at least 78 s’1, at least 79 s’1, at least 80 s’1, at least 81 s’1, at least 82 s’1, at least 83 s’1, at least 84 s’1, at least 85 s’1, at least 86 s’1, at least 87 s’1, at least 88 s’1, at least 89 s’1, at least 90 s’1, at least 91 s’1, at least 92 s’1, at least 93 s’1, at least 94 s’1, at least 95 s’1, at least 96 s’1, at least 97 s’1, at least 98 s’1, at least 99 s’1, at least 100 s’1, at least 500 s’1, or at least 1000 s’1. In some embodiments, the Kcat is between 1 s’1and 100 s’1, between 5 s’1and 50 s’1, or between 10 s’1and 50 s’1.

[0088] In some embodiments, the activity of a CAR described herein is measured as the Michaelis constant (Km). In some embodiments, the Kmis less than 0.1 |tM, less than 0.2 |tM, less than 0.3 jtM, less than 0.4 jtM, less than 0.5 jtM, less than 0.6 jtM, less than 0.7 jtM, less than 0.8 |tM, less than 0.9 |tM, less than 1 |tM, less than 2 |tM, less than 3 |tM, less than 4 |tM, less than 5 jtM, less than 6 jtM, less than 7 jtM, less than 8 jtM, less than 9 jtM, less than 10 |rM, less than 11 jtM, less than 12 jtM, less than 13 jtM, less than 14 jtM, less than 15 jtM, less than 16 |tM, less than 17 |tM, less than 18 |tM, less than 19 |tM, less than 20 |tM, less than 21 |rM, less than 22 |tM, less than 23 |tM, less than 24 |tM, less than 25 |tM, less than 26 |tM, less than 27 |tM, less than 28 |tM, less than 29 |tM, less than 30 |tM, less than 31 |tM, less than 32gM, less than 33 gM, less than 34 gM, less than 35 gM, less than 36 gM, less than 37 gM, less than 38 gM, less than 39 gM, less than 40 gM, less than 41 gM, less than 42 gM, less than 43 gM, less than 44 gM, less than 45 gM, less than 46 gM, less than 47 gM, less than 48 gM, less than 49 gM, less than 50 gM, less than 51 gM, less than 52 gM, less than 53 gM, less than 54 gM, less than 55 gM, less than 56 gM, less than 57 gM, less than 58 gM, less than 59 gM, less than 60 gM, less than 61 gM, less than 62 gM, less than 63 gM, less than 64 gM, less than 65 gM, less than 66 gM, less than 67 gM, less than 68 gM, less than 69 gM, less than 70 gM, less than 71 gM, less than 72 gM, less than 73 gM, less than 74 gM, less than 75 gM, less than 76 gM, less than 77 gM, less than 78 gM, less than 79 gM, less than 80 gM, less than 81 gM, less than 82 gM, less than 83 gM, less than 84 gM, less than 85 gM, less than 86 gM, less than 87 gM, less than 88 gM, less than 89 gM, less than 90 gM, less than 91 gM, less than 92 gM, less than 93 gM, less than 94 gM, less than 95 gM, less than 96 gM, less than 97 gM, less than 98 gM, less than 99 gM, less than 100 gM, less than 200 gM, less than 300 gM, less than 400 gM, less than 500 gM, less than 600 gM, less than 700 gM, less than 800 gM, less than 900 gM, less than 1000 gM, less than 1100 gM, less than 1200 gM, less than 1300 gM, less than 1400 gM, less than 1500 gM, less than 1600 gM, less than 1700 gM, less than 1800 gM, less than 1900 gM, or less than 2000 gM. In some embodiments, the Kmis between 0.1 gM and 2000 gM, between 1 gM and 1000 gM, between 1 gM and 100 gM, between 0.1 gM and 1000 |iM, between 100 gM and 2000 gM, between 100 gM and 1000 gM, between 1000 gM and 2000 gM, between 500 gM and 1500 gM, between 500 gM and 1500 gM, or between 0.1 gM and 1000 gM.

[0089] In some embodiments, the activity of a CAR described herein is measured as the catalytic efficiency (kcat / km). In some embodiments, the catalytic efficiency is measured in units of s'1mM-1. In some embodiments, the catalytic efficiency is greater than 0.1, greater than 0.2, greater than 0.3, greater than 0.4, greater than 0.5, greater than 0.6, greater than 0.7, greater than 0.8, greater than 0.9, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 26, greater than 27, greater than 28, greater than 29, greater than 30, greater than 31, greater than 32, greater than 33, greater than 34, greater than 35, greater than 36, greater than 37, greater than 38, greater than 39, greater than 40, greater than 41, greater than 42, greater than 43, greater than 44, greater than45, greater than 46, greater than 47, greater than 48, greater than 49, greater than 50, greater than 51, greater than 52, greater than 53, greater than 54, greater than 55, greater than 56, greater than 57, greater than 58, greater than 59, greater than 60, greater than 61, greater than 62, greater than 63, greater than 64, greater than 65, greater than 66, greater than 67, greater than 68, greater than 69, greater than 70, greater than 71, greater than 72, greater than 73, greater than 74, greater than 75, greater than 76, greater than 77, greater than 78, greater than 79, greater than 80, greater than 81, greater than 82, greater than 83, greater than 84, greater than 85, greater than 86, greater than 87, greater than 88, greater than 89, greater than 90, greater than 91, greater than 92, greater than 93, greater than 94, greater than 95, greater than 96, greater than 97, greater than 98, greater than 99, greater than 100, greater than 500, greater than 1000 s'1mM-1. In some embodiments, the catalytic efficiency (kcat / km) is between 1 and 30 s'1mM-1, between 5 and 30 s'1mM-1, between 1 and 10 s'1mM-1, between 10 and 30 s'1mM-1, or between 20 and 30 s'1mM-1.

[0090] In some embodiments, an engineered CAR provided herein is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, and the engineered CAR has one or more alterations at a position described in TABLE 3B relative to SEQ ID NO: 1. Accordingly, in some embodiments, an engineered CAR provided herein includes one or more amino acid alterations at a residue corresponding to position 48, 98, 134, 179, 238, 299, 354, 379, 389, 937, more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein includes one or more amino acid alterations at a residue corresponding to position 379, 354, 937, more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1.

[0091] In some embodiments, an engineered CAR provided herein includes one or more alterations at a position described in TABLE 3B, wherein the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, an engineered CAR provided herein includes one or more conservative amino acid substitutions relative to an alteration described in TABLE 3B. As a non-limiting example, a conservative amino acid substitution relative to the Y379R substitution in SEQ ID NO: 1 may include substitution of Y379R for another basic (positively charged) amino acid (e.g., Lys (K) or His (H)). In some embodiments, an engineered CAR provided herein includes one or more alterations at a position described in TABLE 3B, wherein the one or more amino acid alterations are nonconservative amino acid substitutions. In some embodiments, an engineered CAR providedherein includes a conservative amino acid substitution and / or non-conservative amino acid substitution in 1 to 10 amino acid positions as set forth in TABLE 3B.

[0092] In some embodiments, an engineered CAR provided herein can further include a conservative amino acid substitution in from 1 to 50 amino acid positions, or alternatively from 2 to 50 amino acid positions, or alternatively from 3 to 50 amino acid positions, or alternatively from 4 to 50 amino acid positions, or alternatively from 5 to 50 amino acid positions, or alternatively from 6 to 50 amino acid positions, or alternatively from 7 to 50 amino acid positions, or alternatively from 8 to 50 amino acid positions, or alternatively from 9 to 50 amino acid positions, or alternatively from 10 to 50 amino acid positions, or alternatively from 15 to 50 amino acid positions, or alternatively from 20 to 50 amino acid positions, or alternatively from 30 to 50 amino acid positions, or alternatively from 40 to 50 amino acid positions, or alternatively from 45 to 50 amino acid positions, or any integer therein, wherein the positions are other than the variant amino acid positions set forth in TABLE 3B. In some aspects, such a conservative amino acid sequence is a chemically conservative or an evolutionary conservative amino acid substitution. Methods of identifying conservative amino acids are well known to one of skill in the art, any one of which can be used to generate the isolated engineered CARs described herein.

[0093] An engineered CAR provided herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, or 250 alterations relative to a wild-type CAR (e.g., SEQ ID NO: 1) An engineered CAR provided herein may comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most84, at most 85, at most 86, at most 87, at most 88, at most 89, at most 90, at most 91, at most92, at most 93, at most 94, at most 95, at most 96, at most 97, at most 98, at most 99, at most100, at most 101, at most 102, at most 103, at most 104, at most 105, at most 106, at most 107, at most 108, at most 109, at most 110, at most 111, at most 112, at most 113, at most 114, at most 115, at most 116, at most 117, at most 118, at most 119, at most 120, at most 121, at most 122, at most 123, at most 124, at most 125, at most 126, at most 127, at most 128, at most 129, at most 130, at most 131, at most 132, at most 133, at most 134, at most 135, at most 136, at most 137, at most 138, at most 139, at most 140, at most 141, at most 142, at most 143, at most 144, at most 145, at most 146, at most 147, at most 148, at most 149, at most 150, at most 151, at most 152, at most 153, at most 154, at most 155, at most 156, at most 157, at most 158, at most 159, at most 160, at most 161, at most 162, at most 163, at most 164, at most 165, at most 166, at most 167, at most 168, at most 169, at most 170, at most 171, at most 172, at most 173, at most 174, at most 175, at most 176, at most 177, at most 178, at most 179, at most 180, at most 181, at most 182, at most 183, at most 184, at most 185, at most 186, at most 187, at most 188, at most 189, at most 190, at most 191, at most 192, at most 193, at most 194, at most 195, at most 196, at most 197, at most 198, at most 199, at most 200, at most 201, at most 202, at most 203, at most 204, at most 205, at most 206, at most 207, at most 208, at most 209, at most 210, at most 211, at most 212, at most 213, at most 214, at most 215, at most 216, at most 217, at most 218, at most 219, at most 220, at most 221, at most 222, at most 223, at most 224, at most 225, at most 226, at most 227, at most 228, at most 229, at most 230, at most 231, at most 232, at most 233, at most 234, at most 235, at most 236, at most 237, at most 238, at most 239, at most 240, at most 241, at most 242, at most 243, at most 244, at most 245, at most 246, at most 247, at most 248, at most 249, or at most 250 alterations relative to a wild-type CAR (e.g., SEQ ID NO: 1) The one or more alterations may be located at one or more positions corresponding to the one or more positions described in TABLE 3B. The one or morealterations may be located at one or more positions corresponding to one or more positions in SEQ ID NO: 1. As used herein, the phrase “a residue corresponding to position X in SEQ ID NO: Y” refers to a residue at a corresponding position following an alignment of two sequences. For example, the residue in SEQ ID NO: 2 corresponding to position 379 in SEQ ID NO: 1 is the residue at position 379 in SEQ ID NO: 2. In some embodiments, a reference sequence is an CAR that is not SEQ ID NO: 1.

[0094] An engineered CAR provided herein can include any combination of the alterations set forth in TABLE 3B. One alteration alone, or in combination, can produce an engineered CAR that retains or improves the activity as described herein relative to a reference polypeptide, for example, the wild-type CAR (e.g., SEQ ID NO: 1). In some embodiments, an engineered CAR provided herein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9 alterations as set forth in TABLE 3B, including up to an alteration at all of the positions identified in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 2 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 3 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 4 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 5 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 6 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 7 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 8 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 9 alterations as set forth in TABLE 3B. In some embodiments, an engineered CAR provided herein includes at least 10 alterations as set forth in TABLE 3B.

[0095] In certain embodiments, the one or more amino acid alterations of the engineered CAR is an alteration described in TABLE 3B. For example, in some embodiments, the one or more amino acid alterations result in an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; W at a residue corresponding to position 299 in SEQ ID NO: 1; G at a residue corresponding to position 238 in SEQ ID NO: 1; E or R at a residue corresponding to position 134 in SEQ ID NO: 1; R at a residue corresponding to position 134 in SEQ ID NO: 1; S at a residue corresponding to position 389in SEQ ID NO: 1; V at a residue corresponding to position 48 in SEQ ID NO: 1; D at a residue corresponding to position 179 in SEQ ID NO: 1; E at a residue corresponding to position 98 in SEQ ID NO: 1 ; more than one of the foregoing; or a combination thereof.

[0096] In some embodiments, the one or more amino acid alterations of the engineered CAR is an alteration described in TABLE 3B, which results in the engineered CAR having greater or similar activity on HHA, as compared to the WT, when the engineered CAR is expressed in an organism having a pathway for production of HHA and assayed under conditions as described in EXAMPLE 2. For example, in some embodiments, the one or more amino acid alterations result in an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; W at a residue corresponding to position 299 in SEQ ID NO: 1; G at a residue corresponding to position 238 in SEQ ID NO: 1; E or R at a residue corresponding to position 134 in SEQ ID NO: 1; R at a residue corresponding to position 134 in SEQ ID NO: 1; S at a residue corresponding to position 389 in SEQ ID NO: 1; V at a residue corresponding to position 48 in SEQ ID NO: 1; D at a residue corresponding to position 179 in SEQ ID NO: 1; E at a residue corresponding to position 98 in SEQ ID NO: 1 ; more than one of the foregoing; or a combination thereof.

[0097] In certain embodiments, the one or more amino acid alterations of the engineered CAR is an alteration described in TABLE 3B, which results in the engineered CAR having about or greater than 1 ,5x the amount of activity on HHA, as compared to the WT, when the engineered CAR is expressed in an organism having a pathway for production of HHA and assayed under conditions as described in EXAMPLE 1 or EXAMPLE 2. For example, in some embodiments, the one or more amino acid alterations result in an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; more than one of the foregoing; or a combination thereof.

[0098] In some embodiments, an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes a combination of alterations described in TABLE 3B. Such an engineered CAR can include one or more alterations at a position described in TABLE 3B in addition to one or more alteration as described in TABLE 4C. Alternatively, in some embodiments, such an engineered CAR can include one or more alteration as described in TABLE 4C. Accordingly, in some embodiments, an engineered CARprovided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alteration as described in TABLE 4C.

[0099] In some embodiments, an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations at a position described in TABLE 3B, and wherein the engineered CAR further includes one or more alteration described in TABLE 4C. Such alterations, in some embodiments, results in an engineered CAR having: Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, more than one of the foregoing, or a combination thereof.

[0100] In some embodiments, an engineered CAR described herein has one or more amino acid alterations comprising: Y379R, R354H, and / or Q937T in reference to SEQ ID NO: 1.

[0101] In some embodiments, an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 3B, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 65% identical to SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 70% identical to SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 75% identical to SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 80% identical to SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 85% identical to SEQ ID NO: 1 In some embodiments, an engineered CARprovided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 90% identical to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 95% identical to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 98% identical to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 99% identical to SEQ ID NO: 1 In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR is identical to SEQ ID NO: 1

[0102] In some embodiments, an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C, wherein the portion, other than the one or more alterations described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, anengineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineeredCAR has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, an engineered CAR provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, as described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, as described in TABLE 4C, of the engineered CAR is identical to SEQ ID NO: 1.Alcohol dehydrogenase (ADH)

[0103] An engineered alcohol dehydrogenase described herein refers to an enzyme that belongs in the oxidoreductase family of enzymes and may also be referred to as “ADH.” An engineered ADH described herein reversibly converts an alcohol into its corresponding aldehyde. This reaction can require cofactors NADH or NAD(P)H to catalyze the conversion. Such an engineered ADH described herein can be classified as a reaction 1.1.1.1, where the first three digits correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity (e.g., EC 1.1.1.-). Exemplary enzymatic conversions of an engineered ADH provided herein include, but are not limited to, the conversion of Ad-SAld to HHA, 6-HH to HDO, and / or the conversion of 6ACA- SAld to 6-AHL. An ADH described herein can be used to produce desired products, such as CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or other desired products such as a downstream product, including an ester or amide thereof, in a cell, such as a microbial organism, containing a suitable metabolic pathway, or in vitro. ADHs described herein are particularly useful to provide an improved enzymatic route and microorganism to provide an improved composition of HDO and / or HMD, and further having improved purity qualities with respect to by-products.

[0104] In some embodiments, provided herein is an engineered ADH that is a variant of a wildtype ADH (e.g., SEQ ID NO: 15). Such an engineered ADH includes one or more alterations at a position described in TABLE 4C, and, in some embodiments, a combination of alterations described in TABLE 4C, and has higher activity relative to the WT ADH (e.g., SEQ ID NO: 15) as described herein. In certain embodiments, provided herein is an engineered ADH that is a variant of a WT ADH (e.g., SEQ ID NO: 15). Such an engineered ADH includes one or more alterations at a position described in TABLE 4C, and, in some embodiments, a combination of alterations described in TABLE 4C, and has higher activity relative to the WT ADH (e.g., SEQ ID NO: 15) as described herein. In some embodiments, the term “activity”as used herein to describe enzymatic activity, refers to catalytic activity, cofactor specificity, substrate specificity, substrate affinity, or any combination thereof.

[0105] In some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of: 1) Ad-SAld to HHA; 2) 6-HH to HDO; 3) 6ACA-SAM to 6-AHL; 4) 4-HBAld to 1,4 butanediol (BDO); and / or 5) 4-HBAld to 4HB; or combinations thereof. Accordingly, in some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of Ad-SAld to HHA. In some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of 6-HH to HDO. In some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of 6ACA-SAld to 6-AHL. In some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of 4-HBAld to 1,4 butanediol (BDO). In some embodiments, an engineered ADH provided herein is capable of catalyzing the conversion of 4-HBAld to 4HB.

[0106] In some embodiments, an engineered ADH as described herein has higher catalytic activity in the conversion of select substrates over other substrates. For example, in some embodiments, an engineered ADH as described herein has: 1) higher specificity for conversion of 6-HH to HDO over conversion of Ad-SAld to HHA; 2) higher specificity for conversion of 6-HH to HDO over conversion of 6ACA-SAld to 6-AHL; 3) higher specificity for conversion of 6-HH to HDO over conversion of 4-HBAld to BDO; and / or 4) higher specificity for conversion of 6-HH to HDO over conversion of 4-HBAld to 4HB; or any combination thereof.

[0107] In another example, in some embodiments, an engineered ADH as described herein has: 1) higher specificity for conversion of 6ACA-SAld to 6-AHL over conversion of Ad-SAld to HHA; 2) higher specificity for conversion of 6ACA-SAld to 6-AHL over conversion of 4- HBAld to 1,4 butanediol (BDO); and / or 3) higher specificity for conversion of 6ACA-SAld to 6-AHL over conversion of 4-HBAld to 4HB; or any combination thereof.

[0108] Accordingly, in some embodiments, an engineered ADH as described herein has: 1) higher specificity for conversion of 6-HH to HDO and / or 6ACA-SAld to 6-AHL over conversion of Ad-SAld to HHA; 2) higher specificity for conversion of 6-HH to HDO and / or 6ACA-SAld to 6-AHL over conversion of 4-HBAld to BDO; 3) higher specificity for conversion of 6-HH to HDO and / or 6ACA-SAld to 6-AHL over conversion of 4-HBAld to 4HB; or any combination thereof.

[0109] In some embodiments, an engineered ADH provided utilizes a cofactor for catalytic conversion of a substrate (for example a substrate described above). In some embodiments, an engineered ADH provided is capable of converting 1) NAD(P) to NAD(P)H and / or 2) NAD to NADH. In some embodiments, an engineered ADH provided has higher specificity for the conversion of NAD(P) to NAD(P)H over NAD to NADH.

[0110] In some embodiments, provided herein is an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15, wherein the engineered ADH comprises one or more alterations at a position described in TABLE 4C. In some embodiments, such an engineered ADH is capable of catalyzing the conversion of: 1) Ad-SAld to HHA; 2) 6-HH to HDO; 3) 6ACA-SAld to 6-AHL; 4) 4-HBAld to 1,4 butanediol (BDO); 5) 4-HBAld to 4HB; or combinations thereof. Moreover, in some embodiments, such an engineered ADH is capable of converting 1) NAD(P) to NAD(P)H and / or 2) NAD to NADH.

[0111] Accordingly, in some embodiments, such an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of catalyzing the conversion of Ad-SAld to HHA. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of catalyzing the conversion of 6-HH to HDO. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of catalyzing the conversion of 6ACA-SAld to 6-AHL. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of catalyzing the conversion of 4-HBAld to BDO. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of catalyzing the conversion of 4-HBAld to 4HB. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of converting NAD(P) to NAD(P)H. In some embodiments, an engineered ADH comprising a variant of amino acid sequence SEQ ID NO: 15 is capable of converting NAD to NADH.

[0112] It is understood that the engineered ADHs as described herein can carry out a similar enzymatic reaction as the wild-type ADHs (e.g., SEQ ID NO: 15) as discussed above. It is further understood that the variants of the ADH enzyme can include alterations that provide a beneficial characteristic to the engineered ADH, including but not limited to, increased activity (e.g., ability to catalyze a reaction described herein and / or selectivity for a substrate, such as HHA or 6ACA) as described herein (see, e.g., EXAMPLE 3). In some embodiments, the engineered ADH can exhibit an activity that is at least the same or higher than the wild-typeADH (e.g., SEQ ID NO: 15), that is, it has activity that is the same or higher than a ADH without the variant at the same amino acid position(s). In some embodiments, the engineered ADH can exhibit two or more activities (e.g., ability to catalyze a reaction described herein and selectivity for a substrate, such as HHA or 6ACA) that are at least the same or higher than the wild-type ADH (e.g., SEQ ID NO: 15), that is, it has two or more activities that are the same or higher than an ADH without the variant at the same amino acid position(s). For example, the engineered ADHs provided here can have one or more activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher over a wildtype ADH (see, e.g., EXAMPLE 3). In some embodiments, an engineered ADH provided herein has an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of an ADH consisting of the amino acid sequence of SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an activity that is at least 10% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 20% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 30% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 40% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 50% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 60% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 70% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 80% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 90% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 100% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 110% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 120% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 130% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 140% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 150% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 160% higher. In some embodiments, an engineered ADH provided herein has an activitythat is at least 170% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 180% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 190% higher. In some embodiments, an engineered ADH provided herein has an activity that is at least 200% higher. It is understood that activity refers to the ability of an engineered ADH described herein to convert a substrate to a product relative to a wild-type ADH e.g., SEQ ID NO: 15) under the same assay conditions, such as those described herein (see, e.g., EXAMPLE 3).

[0113] In some embodiments, the activity of a ADH described herein is measured as the catalytic constant (kcat) value or turnover number. In some embodiments, the kcat is at least 0.1 s’1, at least 0.2 s’1, at least 0.3 s’1, at least 0.4 s’1, at least 0.5 s’1, at least 0.6 s’1, at least 0.7 s’1, at least 0.8 s’1, at least 0.9 s’1, at least 1 s’1, at least 2 s’1, at least 3 s’1, at least 4 s’1, at least 5 s’ at least 6 s’1, at least 7 s’1, at least 8 s’1, at least 9 s’1, at least 10 s’1, at least 11 s’1, at least 12 s’1, at least 13 s’1, at least 14 s’1, at least 15 s’1, at least 16 s’1, at least 17 s’1, at least 18 s’1, at least 19 s’1, at least 20 s’1, at least 21 s’1, at least 22 s’1, at least 23 s’1, at least 24 s’1, at least 25 s’1, at least 26 s’1, at least 27 s’1, at least 28 s’1, at least 29 s’1, at least 30 s’1, at least 31 s’1, at least 32 s’1, at least 33 s’1, at least 34 s’1, at least 35 s’1, at least 36 s’1, at least 37 s’1, at least 38 s’1, at least 39 s’1, at least 40 s’1, at least 41 s’1, at least 42 s’1, at least 43 s’1, at least 44 s’1, at least 45 s’1, at least 46 s’1, at least 47 s’1, at least 48 s’1, at least 49 s’1, at least 50 s’1, at least 51 s’1, at least 52 s’1, at least 53 s’1, at least 54 s’1, at least 55 s’1, at least 56 s’1, at least 57 s’1, at least 58 s’1, at least 59 s’1, at least 60 s’1, at least 61 s’1, at least 62 s’1, at least 63 s’1, at least 64 s’1, at least 65 s’1, at least 66 s’1, at least 67 s’1, at least 68 s’1, at least 69 s’1, at least 70 s’1, at least 71 s’1, at least 72 s’1, at least 73 s’1, at least 74 s’1, at least 75 s’1, at least 76 s’1, at least 77 s’1, at least 78 s’1, at least 79 s’1, at least 80 s’1, at least 81 s’1, at least 82 s’1, at least 83 s’1, at least 84 s’1, at least 85 s’1, at least 86 s’1, at least 87 s’1, at least 88 s’1, at least 89 s’1, at least 90 s’1, at least 91 s’1, at least 92 s’1, at least 93 s’1, at least 94 s’1, at least 95 s’1, at least 96 s’1, at least 97 s’1, at least 98 s’1, at least 99 s’1, at least 100 s’1, at least 500 s’1, or at least 1000 s’1. In some embodiments, the Kcat is between 1 s’1and 100 s’1, between 5 s’1and 50 s’1, or between 10 s’1and 50 s’1.

[0114] In some embodiments, the activity of a ADH described herein is measured as the Michaelis constant (Km). In some embodiments, the Kmis less than 0.1 pM, less than 0.2 pM, less than 0.3 pM, less than 0.4 pM, less than 0.5 pM, less than 0.6 pM, less than 0.7 pM, less than 0.8 pM, less than 0.9 pM, less than 1 pM, less than 2 pM, less than 3 pM, less than 4 pM,less than 5 gM, less than 6 gM, less than 7 gM, less than 8 gM, less than 9 gM, less than 10 gM, less than 11 gM, less than 12 gM, less than 13 gM, less than 14 gM, less than 15 gM, less than 16 gM, less than 17 gM, less than 18 gM, less than 19 gM, less than 20 gM, less than 21 gM, less than 22 gM, less than 23 gM, less than 24 gM, less than 25 gM, less than 26 gM, less than 27 gM, less than 28 gM, less than 29 gM, less than 30 gM, less than 31 gM, less than 32 gM, less than 33 gM, less than 34 gM, less than 35 gM, less than 36 gM, less than 37 gM, less than 38 gM, less than 39 gM, less than 40 gM, less than 41 gM, less than 42 gM, less than 43 gM, less than 44 gM, less than 45 gM, less than 46 gM, less than 47 gM, less than 48 gM, less than 49 gM, less than 50 gM, less than 51 gM, less than 52 gM, less than 53 gM, less than 54 gM, less than 55 gM, less than 56 gM, less than 57 gM, less than 58 gM, less than 59 gM, less than 60 gM, less than 61 gM, less than 62 gM, less than 63 gM, less than 64 gM, less than 65 gM, less than 66 gM, less than 67 gM, less than 68 gM, less than 69 gM, less than 70 gM, less than 71 gM, less than 72 gM, less than 73 gM, less than 74 gM, less than 75 gM, less than 76 gM, less than 77 gM, less than 78 gM, less than 79 gM, less than 80 gM, less than 81 gM, less than 82 gM, less than 83 gM, less than 84 gM, less than 85 gM, less than 86 gM, less than 87 gM, less than 88 gM, less than 89 gM, less than 90 gM, less than 91 gM, less than 92 gM, less than 93 gM, less than 94 gM, less than 95 gM, less than 96 gM, less than 97 gM, less than 98 gM, less than 99 gM, less than 100 gM, less than 200 gM, less than 300 gM, less than 400 gM, less than 500 gM, less than 600 gM, less than 700 gM, less than 800 gM, less than 900 gM, less than 1000 gM, less than 1100 gM, less than 1200 gM, less than 1300 gM, less than 1400 gM, less than 1500 gM, less than 1600 gM, less than 1700 gM, less than 1800 gM, less than 1900 gM, or less than 2000 gM. In some embodiments, the Kmis between 0.1 gM and 2000 gM, between 1 gM and 1000 gM, between 1 gM and 100 gM, between 0.1 gM and 1000 |iM, between 100 gM and 2000 gM, between 100 gM and 1000 gM, between 1000 gM and 2000 gM, between 500 gM and 1500 gM, between 500 gM and 1500 gM, or between 0.1 gM and 1000 gM.

[0115] In some embodiments, the activity of a ADH described herein is measured as the catalytic efficiency (kcat / km). In some embodiments, the catalytic efficiency is measured in units of s'1mM-1. In some embodiments, the catalytic efficiency is greater than 0.1, greater than 0.2, greater than 0.3, greater than 0.4, greater than 0.5, greater than 0.6, greater than 0.7, greater than 0.8, greater than 0.9, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, greater than 13, greater than 14, greater than 15, greater than 16, greaterthan 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 26, greater than 27, greater than 28, greater than 29, greater than 30, greater than 31, greater than 32, greater than 33, greater than 34, greater than 35, greater than 36, greater than 37, greater than 38, greater than 39, greater than 40, greater than 41, greater than 42, greater than 43, greater than 44, greater than 45, greater than 46, greater than 47, greater than 48, greater than 49, greater than 50, greater than 51, greater than 52, greater than 53, greater than 54, greater than 55, greater than 56, greater than 57, greater than 58, greater than 59, greater than 60, greater than 61, greater than 62, greater than 63, greater than 64, greater than 65, greater than 66, greater than 67, greater than 68, greater than 69, greater than 70, greater than 71, greater than 72, greater than 73, greater than 74, greater than 75, greater than 76, greater than 77, greater than 78, greater than 79, greater than 80, greater than 81, greater than 82, greater than 83, greater than 84, greater than 85, greater than 86, greater than 87, greater than 88, greater than 89, greater than 90, greater than 91, greater than 92, greater than 93, greater than 94, greater than 95, greater than 96, greater than 97, greater than 98, greater than 99, greater than 100, greater than 500, greater than 1000 s'1mM-1. In some embodiments, the catalytic efficiency (kcat / km) is between 1 and 30 s'1mM-1, between 5 and 30 s'1mM-1, between 1 and 10 s'1mM-1, between 10 and 30 s'1mM-1, or between 20 and 30 s'1mM-1.

[0116] In some embodiments, an engineered ADH provided herein is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 15, and the engineered ADH has one or more alterations at a position described in TABLE 4C relative to SEQ ID NO: 15. Accordingly, in some embodiments, an engineered ADH provided herein includes one or more amino acid alterations at a residue corresponding to position 215, 216, or both, in SEQ ID NO: 15.

[0117] In some embodiments, an engineered ADH provided herein includes one or more alterations at a position described in TABLE 4C, wherein the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, an engineered ADH provided herein includes one or more conservative amino acid substitutions relative to an alteration described in TABLE 4C. As a non-limiting example, a conservative amino acid substitution relative to the D215A substitution in SEQ ID NO: 15 may include substitution of D215A for another aliphatic amino acid (e.g., Gly (G), Vai (V), Leu (L), He (I), Ser (S), Thr (T)). In some embodiments, an engineered ADH provided herein includes one or morealterations at a position described in TABLE 4C, wherein the one or more amino acid alterations are non-conservative amino acid substitutions. In some embodiments, an engineered ADH provided herein includes a conservative amino acid substitution and / or non-conservative amino acid substitution in 1 to 2 amino acid positions as set forth in TABLE 4C.

[0118] In some embodiments, an engineered ADH provided herein can further include a conservative amino acid substitution in from 1 to 50 amino acid positions, or alternatively from 2 to 50 amino acid positions, or alternatively from 3 to 50 amino acid positions, or alternatively from 4 to 50 amino acid positions, or alternatively from 5 to 50 amino acid positions, or alternatively from 6 to 50 amino acid positions, or alternatively from 7 to 50 amino acid positions, or alternatively from 8 to 50 amino acid positions, or alternatively from 9 to 50 amino acid positions, or alternatively from 10 to 50 amino acid positions, or alternatively from 15 to 50 amino acid positions, or alternatively from 20 to 50 amino acid positions, or alternatively from 30 to 50 amino acid positions, or alternatively from 40 to 50 amino acid positions, or alternatively from 45 to 50 amino acid positions, or any integer therein, wherein the positions are other than the variant amino acid positions set forth in TABLE 4C. In some aspects, such a conservative amino acid sequence is a chemically conservative or an evolutionary conservative amino acid substitution. Methods of identifying conservative amino acids are well known to one of skill in the art, any one of which can be used to generate the isolated engineered ADHs described herein.

[0119] n engineered ADH provided herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, or 250 alterations relative to a wild-type ADH (e.g., SEQ IDNO: 15). An engineered ADH provided herein may comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most84, at most 85, at most 86, at most 87, at most 88, at most 89, at most 90, at most 91, at most92, at most 93, at most 94, at most 95, at most 96, at most 97, at most 98, at most 99, at most100, at most 101, at most 102, at most 103, at most 104, at most 105, at most 106, at most 107, at most 108, at most 109, at most 110, at most 111, at most 112, at most 113, at most 114, at most 115, at most 116, at most 117, at most 118, at most 119, at most 120, at most 121, at most 122, at most 123, at most 124, at most 125, at most 126, at most 127, at most 128, at most 129, at most 130, at most 131, at most 132, at most 133, at most 134, at most 135, at most 136, at most 137, at most 138, at most 139, at most 140, at most 141, at most 142, at most 143, at most 144, at most 145, at most 146, at most 147, at most 148, at most 149, at most 150, at most 151, at most 152, at most 153, at most 154, at most 155, at most 156, at most 157, at most 158, at most 159, at most 160, at most 161, at most 162, at most 163, at most 164, at most 165, at most 166, at most 167, at most 168, at most 169, at most 170, at most 171, at most 172, at most 173, at most 174, at most 175, at most 176, at most 177, at most 178, at most 179, at most 180, at most 181, at most 182, at most 183, at most 184, at most 185, at most 186, at most 187, at most 188, at most 189, at most 190, at most 191, at most 192, at most 193, at most 194, at most 195, at most 196, at most 197, at most 198, at most 199, at most 200, at most 201, at most 202, at most 203, at most 204, at most 205, at most 206, at most 207, at most 208, at most 209, at most 210, at most 211, at most 212, at most 213, at most 214, at most 215, at most 216, at most 217, at most 218, at most 219, at most 220, at most 221, at most 222, at most 223, at most 224, at most 225, at most 226, at most 227, at most 228, at most 229, at most 230, at most 231, at most 232, at most 233, at most 234, at most 235, at most 236, at most 237, at most 238, at most 239, at most 240, at most 241, at most 242, at most 243, at most 244, at most 245, at most 246, atmost 247, at most 248, at most 249, or at most 250 alterations relative to a wild-type ADH(e.g., SEQ ID NO: 15)

[0120] The one or more alterations may be located at one or more positions corresponding to the one or more positions described in TABLE 4C. The one or more alterations may be located at one or more positions corresponding to one or more positions in SEQ ID NO: 15. As used herein, the phrase “a residue corresponding to position X in SEQ ID NO: Y” refers to a residue at a corresponding position following an alignment of two sequences. For example, the residue in SEQ ID NO: 16 corresponding to position 215 in SEQ ID NO: 15 is the residue at position 215 in SEQ ID NO: 16. In some embodiments, a reference sequence is an ADH that is not SEQ ID NO: 15.

[0121] An engineered ADH provided herein can include any combination of the alterations set forth in TABLE 4C. One alteration alone, or in combination, can produce an engineered ADH that retains or improves the activity as described herein relative to a reference polypeptide, for example, the wild-type ADH (e.g., SEQ ID NO: 15). In some embodiments, an engineered ADH provided herein includes at least 1 alteration as set forth in TABLE 4C. In some embodiments, an engineered ADH provided herein includes at least 2 alterations as set forth in TABLE 4C.

[0122] In certain embodiments, the one or more amino acid alterations of the engineered CAR is an alteration described in TABLE 4C. For example, in some embodiments, the one or more amino acid alterations result in an engineered CAR having: A, S, or T at a residue corresponding to position 215 in SEQ ID NO: 15; H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0123] In some embodiments, the one or more amino acid alterations of the engineered ADH is an alteration described in TABLE 4C, which results in the engineered ADH having greater or similar activity on NAD(P), as compared to the WT, when the engineered ADH is expressed in an organism and assayed with NAD(P) and / or NAD cofactors under conditions as described in EXAMPLE 2. For example, in some embodiments, the one or more amino acid alterations result in an engineered ADH having: A, S, T at a residue corresponding to position 215 in SEQ ID NO: 15; H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0124] In certain embodiments, the one or more amino acid alterations of the engineered ADH is an alteration described in TABLE 4C, which results in the engineered ADH having about or greater than 1.5x, 2. Ox, 2.5x, 3. Ox the amount of activity on NAD(P), as compared to the WT, when the engineered ADH is expressed in an organism having a pathway for production of NAD(P) and assayed under conditions as described in EXAMPLE 3. For example, in some embodiments, the one or more amino acid alterations result in an engineered ADH having: A or S at a residue corresponding to position 215 in SEQ ID NO: 15; H or Q at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0125] In some embodiments, an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes a combination of alterations described in TABLE 4C. Such an engineered ADH can include one or more alterations at a position described in TABLE 4C in addition to one or more alteration as described in TABLE 4C. Alternatively, in some embodiments, such an engineered ADH can include one or more alteration as described in TABLE 3B. Accordingly, in some embodiments, an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alteration as described in TABLE 3B.

[0126] In some embodiments, an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations at a position described in TABLE 4C, and wherein the engineered ADH further includes one or more alteration described in TABLE 3B. Such alterations, in some embodiments, results in an engineered ADH having: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, more than one of the foregoing, or a combination thereof.

[0127] In some embodiments, an engineered ADH described herein has one or more amino acid alterations comprising: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15.

[0128] In some embodiments, an engineered ADH described herein has one or more amino acid alterations comprising: D215A, D215S, L216H, L216Q, more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15

[0129] In some embodiments, an engineered ADH described herein has one or more amino acid alterations comprising: D215A, L216H, more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15.

[0130] In some embodiments, an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C, wherein the portion, other than the one or more alterations described in TABLE 4C, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 65% identical to SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 70% identical to SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 75% identical to SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 80% identical to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 85% identical to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 90% identical to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 95% identical to SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 98%identical to SEQ ID NO: 15 In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 99% identical to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH is identical to SEQ ID NO: 15.

[0131] In some embodiments, an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 65% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 70% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 75% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineeredADH has at least 80% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 85% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 95% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 98% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH has at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered ADH provided herein has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations, as described in TABLE 3B and the portion, other than the alteration described in TABLE 4C or the one or more, or combination of alterations, as described in TABLE 3B, of the engineered ADH is identical to SEQ ID NO: 15.

[0132] Sequence identity, homology or similarity refers to sequence similarity between two polypeptides or between two nucleic acid molecules. Identity can be determined by comparinga position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polypeptide or polypeptide region (or a polynucleotide or polynucleotide region) has a certain percentage (for example, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity" to another sequence means that, when aligned, that percentage of amino acids (or nucleotide bases) are the same in comparing the two sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information (see also Altschul et al., " J. Mol. Biol. 215:403-410 (1990)).

[0133] Methods of generating and assaying the engineered CARs and / or engineered ADHs described herein are well known to one of skill in the art. Examples of such methods are described in the Examples provided herein. Any of a variety of methods can be used to generate an engineered CAR and / or an engineered ADH disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing an engineered CAR or an engineered ADH is to express recombinant nucleic acids encoding the engineered CAR or an engineered ADH in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.

[0134] In some embodiments, an engineered CAR provided herein is an isolated CAR. In some embodiments, an engineered ADH provided herein is an isolated ADH. An isolated engineered CAR and / or an isolated engineered ADH provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., 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)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.Recombinants

[0135] In some embodiments, the provided herein is a recombinant nucleic acid that has a nucleotide sequence encoding an engineered CAR and / or an engineered ADH described herein. Accordingly, in some embodiments, provided herein is a recombinant nucleic acid selected from (a) a nucleic acid molecule encoding an engineered CAR that is a variant of a wild-type CAR (e.g, SEQ ID NO: 1), such as an engineered CAR having one or more alterations at a position described in TABLE 3B and, in some embodiments, a combination of alterations described in TABLE 3B and / or TABLE 4C (b) a recombinant nucleic acid that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a recombinant nucleic acid that is complementary to (a) or (b).

[0136] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered CAR that is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, and the engineered CAR has one or more alterations at a position described in TABLE 3B relative to SEQ ID NO: 1. It is understood that the positions identified the Tables (e.g, TABLE 3B) correspond to codon positions in the encoding nucleic acid. An alteration in the nucleic acid would alter the corresponding codon at that position to encode a variant amino acid as described herein. In some embodiments, the recombinant nucleic acid encodes an engineered CAR that includes one or more amino acid alterations at a residue corresponding to position 48, 98, 134, 179, 238, 299, 354, 379, 389, 937more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered CAR that includes one or more amino acid alterations at a residue corresponding to position 379, 354, 937, more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1.

[0137] In some embodiments, the recombinant nucleic acid encodes an engineered CAR having one or more alterations described in TABLE 3B. Accordingly, in some embodiments, the recombinant nucleic acid encodes an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; W at a residue corresponding to position 299 in SEQ ID NO: 1; G at a residue corresponding to position 238 in SEQ ID NO: 1; E or R at a residue corresponding to position 134 in SEQ ID NO: 1; R at a residue corresponding to position 134 in SEQ ID NO: 1; S at a residue corresponding to position 389 in SEQ ID NO: 1; V at a residue corresponding to position 48 in SEQ ID NO: 1; D at a residue corresponding to position 179 in SEQ ID NO: 1; E at a residue corresponding to position 98 in SEQ ID NO: 1; more than one of the foregoing; or a combination thereof.

[0138] In some embodiments, the recombinant nucleic acid encodes an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1 ; more than one of the foregoing; or a combination thereof.

[0139] In some embodiments, the recombinant nucleic acid encodes an engineered CAR having one or more amino acid alterations comprising: Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, more than one of the foregoing; or a combination thereof, in reference to SEQ ID NO: 1

[0140] In some embodiments, the recombinant nucleic acid encodes an engineered CAR having one or more amino acid alterations comprising: Y379R, R354H, and / or Q937T, more than one of the foregoing; or a combination thereof, in reference to SEQ ID NO: 1.

[0141] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered CAR described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered CAR that is a variant of a wild-typeCAR (e.g., SEQ ID NO: 1), such as an engineered CAR having one or more alterations at a position described in TABLE 3B. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered CAR having one or more alterations at a position described in TABLE 3B. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered CAR having a combination of alterations described in TABLE 3B

[0142] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 3B, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 65% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 70% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1 In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 75% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 80% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 85% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1 In other embodiments, a recombinant nucleic acid encodes an engineeredCAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 90% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 95% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 96% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1 In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 97% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 98% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1. In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR has at least 99% sequence identity to an amino acid sequence referenced as SEQ ID NO: 1 In other embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B, other than the alteration described in TABLE 3B, of the engineered CAR is identical to an amino acid sequence referenced as SEQ ID NO: 1.

[0143] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered CAR described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered CAR that is a variant of a wild-type or parent CAR (e.g., SEQ ID NO: 1), such as an engineered CAR having one or more alterations at a position described in TABLE 3B, and / or, in some embodiments, a one or more, or a combination of alterations described in TABLE 4C. In some embodiments, the recombinantnucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered CAR having one or more alterations at a position described in TABLE 3B and / or TABLE 4C.

[0144] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations described in TABLE 4C, wherein the portion, other than the one or more alterations described in TABLE 3B and / or the one or more, or a combination of alterations described in TABLE 4C, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 65% identical to SEQ ID NO: 1 In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 70% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 75% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 80% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / orone or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 85% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 90% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 95% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 98% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR has at least 99% identical to SEQ ID NO: 1. In some embodiments, a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more, or a combination of alterations, described in TABLE 4C and the portion, other than the alteration described in TABLE 3B and / or the one or more, or combination of alterations, described in TABLE 4C, of the engineered CAR is identical to SEQ ID NO: 1.

[0145] In some embodiments, provided herein is a recombinant nucleic acid that includes a nucleotide sequence encoding an engineered CAR described herein that is operatively linked to a promoter. Such a promoter can express the engineered CAR in a microbial organism as described herein.

[0146] In some embodiments, provided herein is a recombinant nucleic acid selected from (a) a nucleic acid molecule encoding an engineered ADH that is a variant of a wild-type ADH (e.g., SEQ ID NO: 15), such as an engineered ADH having one or more alterations at a position described in TABLE 4C and, in some embodiments, a combination of alterations described in TABLE 4C and / or TABLE 3B (b) a recombinant nucleic acid that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a recombinant nucleic acid that is complementary to (a) or (b).

[0147] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered ADH that is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 15, and the engineered ADH has one or more alterations at a position described in TABLE 4C relative to SEQ ID NO: 15. It is understood that the positions identified the Tables (e.g., TABLE 4C) correspond to codon positions in the encoding nucleic acid. An alteration in the nucleic acid would alter the corresponding codon at that position to encode a variant amino acid as described herein. In some embodiments, the recombinant nucleic acid encodes an engineered ADH that includes one or more amino acid alterations at a residue corresponding to position 215, 216 or both, in SEQ ID NO: 15.

[0148] In some embodiments, the recombinant nucleic acid encodes an engineered ADH having one or more alterations described in TABLE 4C. Accordingly, in some embodiments, the recombinant nucleic acid encodes an engineered ADH having: A, S, or T at a residue corresponding to position 215 in SEQ ID NO: 15; H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0149] In some embodiments, the recombinant nucleic acid encodes an engineered ADH having: A or S at a residue corresponding to position 215 in SEQ ID NO: 15; H or Q at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0150] In some embodiments, the recombinant nucleic acid encodes an engineered ADH having one or more amino acid alterations comprising: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15.

[0151] In some embodiments, the recombinant nucleic acid encodes an engineered ADH having one or more amino acid alterations comprising: D215A, D215S, L216H, or L216Q, more than one of the foregoing; or a combination thereof, in reference to SEQ ID NO: 15.

[0152] In some embodiments, the recombinant nucleic acid encodes an engineered ADH having one or more amino acid alterations comprising: D215A, L216H, more than one of the foregoing; or a combination thereof, in reference to SEQ ID NO: 15.

[0153] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered ADH described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered ADH that is a variant of a wild-type ADH (e.g., SEQ ID NO: 15), such as an engineered ADH having one or more alterations at a position described in TABLE 4C. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered ADH having one or more alterations at a position described in TABLE 4C. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered ADH having a combination of alterations described in TABLE 4C

[0154] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C, wherein the portion, other than the one or more alterations described in TABLE 4C, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 65% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 70% sequence identity to an amino acid sequencereferenced as SEQ ID NO: 15 In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 75% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 80% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 85% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 90% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15 In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 95% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 96% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 97% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15 In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 98% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH has at least 99% sequence identity to an amino acid sequence referenced as SEQ ID NO: 15. In other embodiments, arecombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C, other than the alteration described in TABLE 4C, of the engineered ADH is identical to an amino acid sequence referenced as SEQ ID NO: 15

[0155] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered ADH described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered ADH that is a variant of a wild-type or parent ADH (e.g., SEQ ID NO: 15), such as an engineered ADH having one or more alterations at a position described in TABLE 4C, and / or, in some embodiments, a one or more, or a combination of alterations described in TABLE 3B. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered ADH having one or more alterations at a position described in TABLE 4C and / or TABLE 3B.

[0156] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 65% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterationsdescribed in TABLE 3B, of the engineered ADH has at least 70% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 75% identical to SEQ ID NO: 15 In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 80% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 85% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 90% identical to SEQ ID NO: 15 In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 95% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 98% identical to SEQ ID NO: 15. In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has anamino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH has at least 99% identical to SEQ ID NO: 15 In some embodiments, a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more, or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more, or a combination of alterations described in TABLE 3B, of the engineered ADH is identical to SEQ ID NO: 15.

[0157] In some embodiments, provided herein is a recombinant nucleic acid that includes a nucleotide sequence encoding an engineered ADH described herein that is operatively linked to a promoter. Such a promoter can express the engineered ADH in a microbial organism as described herein.

[0158] In some embodiments, provided herein is a vector containing a recombinant nucleic acid described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises double stranded DNA.

[0159] A recombinant nucleic acid encoding an engineered CAR and / or an engineered ADH described herein also includes a nucleic acid that hybridizes to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a recombinant nucleic acid that can be used in the compositions and methods described herein can be described as having a certain percent sequence identity to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein. For example, the nucleic acid can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleotide described herein.

[0160] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridizedpolynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleotide sequences that form stable hybridized polynucleotides in 0.018MNaCl at 65°C, for example, if a hybrid is not stable in 0.018MNaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0. IX SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleotide sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and 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).

[0161] A recombinant nucleic acid encoding an engineered CAR and / or an engineered ADH described herein can have at least a certain sequence identity to a nucleotide sequence disclosed herein. Accordingly, in some aspects described herein, a recombinant nucleic acid encoding an engineered CAR and / or an engineered ADH has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed herein.

[0162] It is understood that a recombinant nucleic acid described herein or an engineered CAR described herein excludes a wild-type parental sequence, for example a parental sequence, such as SEQ ID NO: 1 It is understood that a recombinant nucleic acid described herein or an engineered ADH described herein excludes a wild-type parental sequence, for example a parental sequence, such as SEQ ID NO: 15. One skilled in the art will readily understand the meaning of a parental wild-type sequence based on what is well known in the art. It is further understood that such a recombinant nucleic acid described herein can exclude a nucleotide sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, an engineered CAR and / or an engineered ADH described herein can exclude an amino acid sequence as found in nature. Thus, in some embodiments, the recombinant nucleic acid or engineered CAR and / or an engineered ADH described herein is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild-type parental sequence or a naturally occurring amino acid sequence and / or that the nucleotide sequence is not a wild-type or naturally occurring nucleotide sequence. A naturally occurring amino acid or nucleotide sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleotide or amino acid sequence that is not found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a recombinant nucleotide and / or amino acid sequence described herein. For example, a nucleotide or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including variants as described herein, are included within the meaning of a nucleotide or amino acid sequence described herein that is not naturally occurring. A recombinant nucleic acid described herein excludes a naturally occurring chromosome that contains the nucleotide sequence, and can further exclude other molecules, as found in a naturally occurring cell, such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

[0163] Thus, a recombinant nucleic acid described here has physical and chemical differences compared to a naturally occurring nucleic acid. A recombinant or non-naturally occurring nucleic acid described herein does not contain or does not necessarily have some or all of the chemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid as found in nature. A recombinant nucleic acid described herein thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid as found in a chromosome. A different chemical structure can occur, forexample, by cleavage of phosphodiester bonds that release a recombinant nucleic acid from a naturally occurring chromosome. A recombinant nucleic acid described herein can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non-naturally occurring nucleic acid described herein does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such as polymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non-naturally occurring nucleic acid described herein also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid described herein is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non-naturally occurring nucleic acids described herein are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and / or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be a recombinant or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that recombinant or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.

[0164] A recombinant nucleic acid encoding an engineered CAR and / or an engineered ADH provided herein, and optionally exogenous nucleic acid sequences involved in a pathway for production of Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof, can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. col or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or othertargeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in / ■ / coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.Microbial Organisms

[0165] In some embodiments, provided herein is a microbial organism, in particular a non- naturally occurring microbial organism, that expresses a CAR and / or ah ADH described herein, that is, an engineered CAR and / or an engineered ADH described herein. Thus, provided herein is a non-naturally occurring microbial organism having a recombinant nucleic acid encoding an engineered CAR and / or an engineered ADH described herein. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that is a variant of a wild-type or parent CAR (e g , SEQ ID NO: 1), such as an engineered CAR having one or more alterations at a position described in TABLE 3B, or a combination of alterations described in TABLE 3B and / or TABLE 4C.

[0166] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, and the engineered CAR has one or more alterations at a position described in TABLE 3B relative to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered CAR that includes one or more amino acid alterations at a residue corresponding to position: 48, 98, 134, 179, 238, 299, 354, 379, 389, 937, more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered CAR that includes one or more amino acid alterations at a residue correspondingto position 379, 354, 937, more than one of the foregoing, or a combination thereof, in SEQ ID NO: 1

[0167] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR having one or more alterations described in TABLE 3B. Accordingly, in some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; W at a residue corresponding to position 299 in SEQ ID NO: 1; G at a residue corresponding to position 238 in SEQ ID NO: 1; E or R at a residue corresponding to position 134 in SEQ ID NO: 1; R at a residue corresponding to position 134 in SEQ ID NO: 1; S at a residue corresponding to position 389 in SEQ ID NO: 1; V at a residue corresponding to position 48 in SEQ ID NO: 1; D at a residue corresponding to position 179 in SEQ ID NO: 1; E at a residue corresponding to position 98 in SEQ ID NO: 1; more than one of the foregoing; or a combination thereof.

[0168] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR having: R at a residue corresponding to position 379 in SEQ ID NO: 1; H at a residue corresponding to position 354 in SEQ ID NO: 1; T at a residue corresponding to position 937 in SEQ ID NO: 1; more than one of the foregoing; or a combination thereof.

[0169] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR having one or more amino acid alterations comprising: Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, more than one of the foregoing, or any combination thereof, in reference to SEQ ID NO: 1.

[0170] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR having one or more amino acid alterations comprising: Y379R, R354H, and / or Q937T, more than one of the foregoing, or any combination thereof, in reference to SEQ ID NO: 1

[0171] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR provided hereinhas an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 3B, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 65% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 70% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 75% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 80% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 85% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 90% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 95% identical to SEQ ID NO: 1. In someembodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 98% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR has at least 99% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and the portion, other than the alteration described in TABLE 3B, of the engineered CAR is identical to SEQ ID NO: 1.

[0172] ] In some embodiments, provided herein is a microbial organism, in particular a non- naturally occurring microbial organism, that expresses an engineered CAR described herein, that is, an engineered CAR described herein. Thus, provided herein is a non-naturally occurring microbial organism having a recombinant nucleic acid encoding an engineered CAR described herein. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that is a variant of a wild-type or parent CAR (SEQ ID NO: 1), such as an engineered CAR having one or more alterations at a position described in TABLE 3B, and / or one or more, or a combination of alterations described in TABLE 4C.

[0173] ] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that is a variant of SEQ ID NO: 1 that includes one or more alterations at a position described in TABLE 3B, and wherein the engineered CAR further includes one or more or a combination of alterations described in TABLE 4C.

[0174] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR an engineered CAR provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C, wherein the portion, other than the one ormore alterations described in TABLE 3B and / or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 65% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 70% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 75% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 80% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 85% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodesan engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 90% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 95% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 98% identical to SEQ ID NO: 1 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR has at least 99% identical to SEQ ID NO: 1. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered CAR that has an amino acid sequence that includes one or more alterations as described in TABLE 3B and / or one or more or a combination of alterations described in TABLE 4C and the portion, other than the alteration described in TABLE 3B or the one or more or combination of alterations described in TABLE 4C, of the engineered CAR is identical to SEQ ID NO: 1

[0175] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that is a variant of a wild-type or parent ADH (e.g., SEQ ID NO: 15), such as an engineered ADH having oneor more alterations at a position described in TABLE 4C, or a combination of alterations described in TABLE 4C and / or TABLE 3B.

[0176] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 15, and the engineered ADH has one or more alterations at a position described in TABLE 4C relative to SEQ ID NO: 15. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered ADH that includes one or more amino acid alterations at a residue corresponding to position: 215, 216 or both, in SEQ ID NO: 15.

[0177] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH having one or more alterations described in TABLE 4C. Accordingly, in some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered ADH having: A, S, or T at a residue corresponding to position 215 in SEQ ID NO: 15; H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0178] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH having A or S at a residue corresponding to position 215 in SEQ ID NO: 15; H or Q at a residue corresponding to position 216 in SEQ ID NO: 15; more than one of the foregoing; or a combination thereof.

[0179] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH having one or more amino acid alterations comprising: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15

[0180] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH having one or more amino acid alterations comprising: D215A, D215S, L216H, or L216Q more than one of the foregoing, or a combination thereof, in reference to SEQ ID NO: 15.

[0181] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH having one or more amino acid alterations comprising: D215A, L216H, more than one of the foregoing, or any combination thereof., in reference to SEQ ID NO: 15

[0182] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C, wherein the portion, other than the one or more alterations described in TABLE 4C, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 65% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 70% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 75% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 80% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 85% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinantnucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 90% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 95% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 98% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH has at least 99% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and the portion, other than the alteration described in TABLE 4C, of the engineered ADH is identical to SEQ ID NO: 15.

[0183] In some embodiments, provided herein is a microbial organism, in particular a non- naturally occurring microbial organism, that expresses an engineered ADH described herein, that is, an engineered ADH described herein. Thus, provided herein is a non-naturally occurring microbial organism having a recombinant nucleic acid encoding an engineered ADH described herein. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that is a variant of a wild-type or parent ADH (SEQ ID NO: 15), such as an engineered ADH having one or more alterations at a position described in TABLE 4C, and / or one or more, or a combination of alterations described in TABLE 3B. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that is a variant of SEQ ID NO: 15 that includes one or more alterations at a position described in TABLE 4C, and wherein the engineered ADH further includes one or more or a combination of alterations described in TABLE 3B.

[0184] In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH an engineered ADH provided herein has an amino acid sequence that is a variant of SEQ ID NO: 15 that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 15. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 65% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 70% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 75% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 80% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodesan engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 85% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 90% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 95% identical to SEQ ID NO: 15 In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 98% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE 4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH has at least 99% identical to SEQ ID NO: 15. In some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encodes an engineered ADH that has an amino acid sequence that includes one or more alterations as described in TABLE 4C and / or one or more or a combination of alterations described in TABLE 3B, wherein the portion, other than the one or more alterations described in TABLE4C or the one or more or combination of alterations described in TABLE 3B, of the engineered ADH is identical to SEQ ID NO: 15.

[0185] In one embodiment, the cell comprises a pathway that produces Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof. In one embodiment, the cell is capable of fermentation. In one embodiment, the cell further includes at least one substrate for the engineered CAR described herein present or produced in the cell. In some embodiments, the substrate is HHA. In some embodiments, the cell has higher activity for HHA over propionate, acetate, adipate, succinate, or 4- hydroxybutyrate (4-HB), or any combination thereof. In one embodiment, the cell further includes at least one substrate for the engineered CAR described herein present or produced in the cell. In some embodiments, the substrate is 6-ACA. In some embodiments, the cell has higher activity for 6-ACA over propionate, acetate, adipate, succinate, or 4-hydroxybutyrate (4-HB), or any combination thereof. In one embodiment, the cell further includes at least one cofactor for the engineered ADH described herein present or produced in the cell. In some embodiments, the substrate is NAD(P). In some embodiments, the cell has higher activity for NAD(P) over NAD.

[0186] Also provided herein is a culture medium comprising a cell, or a population of cells as described herein.

[0187] The engineered CAR described herein can be utilized in a pathway that converts carboxylic acid to an aldehyde. Exemplary pathways for Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, that comprise a CAR have been described, for example, in US 2019 / 0300919, US 2007 / 0254341, US 2011 / 0091944, US 2011 / 0171699, WO 2009 / 151728, WO 2010 / 129936, and WO 2021 / 216952, all of which are incorporated herein by reference.Pathways, By-Products, Products

[0188] Exemplary 6-HCA, 6-HH, and / or HDO pathways are shown in FIG. 1. Such an HHA, 6-HH, and / or HDO pathway as described herein can include one or more of: (1A) P- ketoadipyl-CoA thiolase that converts AcCoA and succinyl-CoA (SucCoA) to 3-oxoadipoyl- CoA; (IB) 3-hydroxyadipyl-CoA dehydrogenase that converts 3-oxoadipoyl-CoA to 3- hydroxyadipoyl CoA, in some embodiments, to (3S)- hydroxyadipoyl-CoA; (1C) 2,3- dehydroadipyl-CoA hydratase that converts 3 -hydroxy adipoyl CoA, in some embodiments,(3S)- hydroxyadipoyl-CoA, to trans-2,3-dehydroadipyl-CoA; (ID) TER that converts trans- 2,3-dehydroadipyl-CoA to adipoyl-CoA; (IE) aldehyde dehydrogenase (ALDH) or acyl-CoA reductase / dehydrogenase that converts adipoyl-CoA to adipate semialdehyde and / or that converts HHA-CoA to 6-HH; (1G) ACL or acyl-CoA transferase that converts ADA to adipoyl-CoA; (II) ADH that converts 6HH to HDO; (1 J) FAR that converts adipyl-CoA to 6- HCA; (IK) CAR, such as an engineered CAR described herein, or CAR and PPTase that converts adipic acid to Ad-SAld; (IL) acyl-CoA ligase (ACL) or acyl-CoA transferase that converts 6-HCA to HHA-CoA; (1H) aldehyde dehydrogenase (ALDH) or acyl-CoA reductase / dehydrogenase that converts HHA-CoA to 6-HH; (IM) carboxylic acid reductase (CAR) or CAR and phosphopantetheinyl transferase (PPTase) that converts 6-HCA to 6-HH;(IN) alcohol-forming fatty acyl-CoA reductase (FAR) that converts HHA-CoA to HDO; and(IO) ADHthat converts Ad-SAld to 6-HCA.

[0189] An exemplary HCA pathway from Ac-CoA and SucCoA, as described herein as a pathway for a CAR substrate (e.g, HCA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 1 A; IB; 1C; ID; IE; and 10, such as an engineered ADH described herein.

[0190] Another exemplary HCA pathway from Ac-CoA and SucCoA, as described herein as a pathway for a CAR substrate (e.g, HCA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 1 A; IB; 1C; ID; and 1J.

[0191] An exemplary HCA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g, HCA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 1G; IE; and 10, such as an engineered ADH described herein.

[0192] Another exemplary HCA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g, HCA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 1G and 1 J.

[0193] Another exemplary HCA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g, HCA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: IK, such as an engineered CAR described herein; and 10, such as an engineered ADH described herein.

[0194] An exemplary 6-HH pathway from Ac-CoA and SucCoA that comprises an engineered CAR as described herein can include: 1A; IB; 1C; ID; IE; 10, such as an engineered ADH described herein; and IM, such as an engineered CAR described herein.

[0195] Another exemplary 6-HH pathway from Ac-CoA and SucCoA that comprises an engineered CAR as described herein can include: 1A; IB; 1C; ID; 1J; and IM, such as an engineered CAR described herein.

[0196] An exemplary 6-HH pathway from ADA that comprises an engineered CAR as described herein can include: 1G; IE; 10, such as an engineered ADH described herein; and IM, such as an engineered CAR described herein.

[0197] Another exemplary 6-HH pathway from ADA that comprises an engineered CAR as described herein can include: 1G; 1J; and IM, such as an engineered CAR described herein.

[0198] Another exemplary 6-HH pathway from ADA that comprises an engineered CAR as described herein can include: IK, such as an engineered CAR described herein; 10, such as an engineered ADH described herein; and IM, such as an engineered CAR described herein.

[0199] An exemplary HDO pathway from Ac-CoA and SucCoA that comprises an engineered CAR as described herein can include: 1A; IB; 1C; ID; IE; 10, such as an engineered ADH described herein; IM, such as an engineered CAR described herein; and II, such as an engineered ADH described herein.

[0200] Another exemplary HDO pathway from Ac-CoA and SucCoA that comprises an engineered CAR as described herein can include: 1A; IB; 1C; ID; 1J; IM, such as an engineered CAR described herein, and II, such as an engineered ADH described herein.

[0201] An exemplary HDO pathway from ADA that comprises an engineered CAR as described herein can include: 1G; IE; 10, such as an engineered ADH described herein; IM, such as an engineered CAR described herein, and II, such as an engineered ADH described herein.

[0202] Another exemplary HDO pathway from ADA that comprises an engineered CAR as described herein can include: 1G; 1J; IM, such as an engineered CAR described herein; and II, such as an engineered ADH described herein.

[0203] Another exemplary HDO pathway from ADA that comprises an engineered CAR as described herein can include: IK, such as an engineered CAR described herein; 10, such as an engineered ADH described herein; IM, such as an engineered CAR described herein; and II, such as an engineered ADH described herein.

[0204] Another exemplary HDO pathway from ADA that comprises an engineered CAR as described herein can include: IK, such as an engineered CAR described herein; 10, such as an engineered ADH described herein; IL; 1H; and II, such as an engineered ADH described herein.

[0205] Another exemplary HDO pathway from ADA that comprises an engineered CAR as described herein can include: IK, such as an engineered CAR described herein; 10, such as an engineered ADH described herein; IL; and IN.

[0206] Exemplary HHA, 6-HH, and / or HDO pathways are shown in FIG. 2. Such an HHA, 6- HH, and / or HDO pathway as described herein can include one or more of: (2A) a THL (alternatively referred to as “3-oxoadipyl-CoA thiolase” or “Beta-ketoadipyl-CoA thiolase”) that converts SucCoA and AcCoA into 3-oxoadipyl-CoA; (2B) a HBD (alternatively referred to as “3-oxoadipyl-CoA dehydrogenase”) that converts 3-oxoadipyl-CoA to 3- hydroxyadipyl- CoA; (2C) a CRT (alternatively referred to as “3-oxoadipyl-CoA dehydratase”) that converts 3- hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; (2D) a TER (alternatively referred to as “5-carboxy-2-pentenoyl-CoA reductase”) that converts 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; (2E) a 6ACA-ALD that converts adipyl CoA to Ad-SAld; (2F) an alcohol dehydrogenase which can convert adipate semialdehyde to 6-HH; an (2G) an ACL or acyl- CoA transferase that catalyzes the conversion of HHA to HHA-CoA; (2H) acetylating aldehyde dehydrogenase (ALDH) or acyl-CoA reductase that catalyzes the conversion of HHA-CoA to 6-HH; (21) an ADH that catalyzes the conversion of 6-HH to HDO; and (2K) a CAR, such as an engineered CAR described herein, which can catalyze the conversion of HHA to 6-HH, or a CAR and a PPTase.

[0207] An exemplary HHA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, HHA) and / or a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 2A; 2B; 2C; 2D; 2E; and 2F.

[0208] An exemplary 6-HH pathway from SucCoA and AcCoA that comprises an engineered CAR as described herein can include: 2A; 2B; 2C; 2D; 2E; 2F; and 2K, such as an engineered CAR as described herein.

[0209] An exemplary HDO pathway from SucCoA and AcCoA that comprises an engineered CAR as described herein can include: 2A; 2B; 2C; 2D; 2E; 2F; and 2K, such an engineered CAR as described herein; and 21, such as an engineered ADH described herein.

[0210] Exemplary 6ACA, 6ACA-SAld, CPL and HMD pathways are shown in FIG. 3. Such a 6ACA, 6ACA-SAld, CPL and / or HMD pathway as described herein can include one or more of: (3A) 3-oxoadipyl-CoA thiolase, succinyl-CoA:acetyl-CoA transferase or a thiolase that converts succinyl-CoA and acetyl -CoA to 3-oxoadipyl-CoA; (3B) 3-oxoadipyl-CoA reductase or 3-hydroxyacyl-CoA dehydrogenase that converts 3-oxoadipyl-CoA to 3-hydroxyadipyl- CoA; (3C) 3-hydroxyadipyl-CoA dehydratase which converts that converts 3-hydroxyadipyl- CoA to 5-carboxy-2-pentenoyl-CoA; (3D) adipate semialdehyde reductase or 5-carboxy-2- pentenoyl-CoA reductase which converts 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; (3E) 3- oxoadipyl-CoA / acyl-CoA transferase, (3F) 3-oxoadipyl-CoA synthase, or (3G) 3-oxoadipyl- CoA hydrolase which converts 3-oxoadipyl-CoA to 3 -oxoadipate; (3H) 3 -oxoadipate reductase which converts 3-oxoadipate to 3 -hydroxy adipate; (31) 3 -hydroxy adipate dehydratase which converts 3 -hydroxy adipate to 5-carboxy-2-pentenoate; (3J) 5-carboxy-2- pentenoate reductase which converts 5-carboxy-2-pentenoate to adipate; (3K) adipyl- CoA / acyl-CoA transferase, (3L) adipyl-CoA synthase or (3M) adipyl-CoA hydrolase which can reversibly convert adipyl-CoA and adipate; (3N) adipyl-CoA reductase (aldehyde forming) or 6-ACA-aldehyde dehydrogenase (ALD) which converts adipyl-CoA to adipate semialdehyde; (30) 6-aminocaproate transaminase or (3P) 6-aminocaproate dehydrogenase which converts adipate semialdehyde to 6ACA; (3Q) 6-aminocaproyl-CoA / acyl-CoA transferase or (3R) 6-aminocaproyl-CoA synthase or acyl-CoA synthetase (ACS) which converts 6 AC A to 6ACA-C0A; (3S) amidohydrolase which converts 6 AC A to CPL; (3T) spontaneous and / or chemical cyclization of 6ACA-CoAto caprolactam; (3U) 6-aminocaproyl- CoA reductase (aldehyde forming) or ALDH which converts 6ACA-C0A to 6ACA-SAld; (3 V) HMD transaminase or (3W) HMD dehydrogenase which converts 6ACA-SAM to HMD; (3X) adipate reductase or CAR which converts adipate to adipate semialdehyde; (3 Y) adipate kinase which converts adipate to adipylphosphate; (3Z) adipylphosphate reductase whichconverts adipylphosphate to adipate semialdehyde; and (3 AB) CAR which converts 6ACA to 6ACA-SAld.

[0211] An exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAld, CPL and HMD, can include: 3A; 3B; 3C; 3D; 3N; and 30 or 3P.

[0212] Another exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAld, CPL and HMD, can include: 3A; 3B; 3C; 3D; 3K, 3L, or 3M; 3X; and 30 or 3P.

[0213] Another exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAld, CPL and HMD, can include: 3A; 3B; 3C; 3D; 3K, 3L, or 3M; 3Y; 3Z; and 30 or 3P.

[0214] Another exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAM, CPL and HMD, can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3K, 3L, or 3M; 3N; and 30 or 3P.

[0215] Another exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAld, CPL and HMD, can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3X; and 30 or 3P.

[0216] Another exemplary 6ACA pathway from SucCoA and AcCoA, as described herein as a pathway for a CAR substrate (e.g, 6ACA) and / or as a precursor pathway for the downstream production of 6ACA-SAld, CPL and HMD, can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3Y; 3Z; and 30 or 3P.

[0217] An exemplary CPL pathway from SucCoA and AcCoA that comprises a CAR as described herein, can include: 3A; 3B; 3C; 3D; 3K, 3L or 3M; 3X, such as an engineered CAR described herein; 30 or 3P; and 3S.

[0218] Another exemplary CPL pathway from SucCoA and AcCoA that comprises a CAR as described herein, can include: 3A; 3B; 3C; 3D; 3K, 3L or 3M; 3X, such as an engineered CAR described herein; 30 or 3P; 3Q or 3R; and 3T.

[0219] Another exemplary CPL pathway from SucCoA and AcCoA that comprises a CAR as described herein, can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3X, such as an engineered CAR described herein; 30 or 3P; and 3S.

[0220] Another exemplary CPL pathway from SucCoA and AcCoA that comprises a CAR as described herein, can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3X, such as an engineered CAR described herein; 30 or 3P; 3Q or 3R; and 3T.

[0221] An exemplary 6ACA-SAld pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3 A; 3B; 3C; 3D; 3N; 30 or 3P; and 3AB, such as an engineered CAR described herein.

[0222] Another exemplary 6ACA-SAld from SucCoA and AcCoA pathway that comprises a CAR as described herein can include: 3 A; 3B; 3C; 3D; 3K, 3L, or 3M; 3X; 30 or 3P; and 3AB, such as an engineered CAR described herein.

[0223] Another exemplary 6ACA-SAld from SucCoA and AcCoA pathway that comprises a CAR as described herein can include: 3A; 3B; 3C; 3D; 3K, 3L, or 3M; 3Y; 3Z; 30 or 3P; and 3 AB, such as an engineered CAR described herein.

[0224] Another exemplary 6ACA-SAld from SucCoA and AcCoA pathway that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3K, 3L, or 3M; 3N; 30 or 3P; and 3AB, such as an engineered CAR described herein.

[0225] Another exemplary 6ACA-SAld pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3X; 30 or 3P; and 3AB, such as an engineered CAR described herein.

[0226] Another 6ACA-SAld pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3Y; 3Z; 30 or 3P; and 3AB, such as an engineered CAR described herein.

[0227] An exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3B; 3C; 3D; 3N; 30 or 3P; 3AB, such as an engineered CAR described herein; and 3 V or 3W.

[0228] Another exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3B; 3C; 3D; 3K, 3L, or 3M; 3X; 30 or 3P; 3AB, such as an engineered CAR described herein; and 3 V or 3W.

[0229] Another exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3B; 3C; 3D; 3K, 3L, or 3M; 3Y; 3Z; 30 or 3P; 3AB, such as an engineered CAR described herein; and 3 V or 3W.

[0230] Another exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3K, 3L, or 3M; 3N; 30 or 3P; 3 AB, such as an engineered CAR described herein; and 3 V or 3W.

[0231] Another exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3X; 30 or 3P; 3AB, such as an engineered CAR described herein; and 3 V or 3W.

[0232] Another exemplary HMD pathway from SucCoA and AcCoA that comprises a CAR as described herein can include: 3A; 3E, 3F, or 3G; 3H; 31; 3J; 3Y; 3Z; 30 or 3P; 3AB, such as an engineered CAR described herein; and 3 V or 3W.

[0233] Exemplary 6ACA-SAld and HMD pathways are also shown in FIG. 4. Such a 6ACA- SAld, 6-acetamidohexanal, and / or HMD pathway as described herein can include one or more of: (4 A) 6-aminocaproate kinase that converts 6- AC A to 6-AHOP; (4B) 6-AHOP oxidoreductase that converts (6-aminohexanoyl)oxy]phosphonate to 6ACA-SAld; (4C) 6- aminocaproic semialdehyde aminotransferase and / or 6-aminocaproic semialdehyde oxidoreductase (aminating) which converts 6ACA-SAld to HMD; (4D) 6-aminocaproate N- acetyltransferase which converts 6ACA to 6-acetamidohexanoate; (4E) 6-acetamidohexanoate kinase which converts 6-acetamidohexanoate to 6-AAHOP; (4F) 6-AAHOP oxidoreductase which converts 6-AAHOP to 6-acetamidohexanal; (4G) 6-acetamidohexanal aminotransferase and / or 6-acetamidohexanal oxidoreductase (aminating) which convert 6-acetamidohexanal to 6-acetamidohexanamine; (4H) 6-acetamidohexanamine N-acetyltransferase and / or 6- acetamidohexanamine hydrolase (amide) which convert 6-acetamidohexanamine to HMD; (41)6-acetamidohexanoate CoA transferase and / or 6-acetamidohexanoate CoA ligase which convert 6-acetamidohexanoate to 6-acetamidohexanoyl-CoA; (4J) 6-acetamidohexanoyl-CoA oxidoreductase which converts 6-acetamidohexanoyl-CoA to 6-acetamidohexanal, (4K) 6- AAHOP acyltransferase which converts 6-AAHOP to 6-acetamidohexanoyl-CoA; (4L) 6- AHOP acyltransferase which converts 6-AHOP to 6ACA-C0A; (4M) 6-aminocaproate CoA transferase, 6-aminocaproate CoA ligase and / or ACL which converts 6ACA to 6ACA-C0A; (4N) 6-aminocaproyl-CoA oxidoreductase and / or ALDH which converts 6ACA-C0A to 6ACA-SAld; (40) CAR which converts 6ACA to 6ACA-SAld; and (4P) CAR which converts 6-acetamidohexanoate to 6-acetamidohexanal.

[0234] An exemplary 6ACA-SAld pathway from 6ACA that comprises a CAR as described herein, can include: 40, such as an engineered CAR as described herein.

[0235] An exemplary 6-acetamidohexanal pathway from 6ACA that comprises a CAR as described herein, can include: 4D; and 4P, such as an engineered CAR as described herein.

[0236] An exemplary 6-acetamidohexanal pathway from 6-acetamidohexanoate that comprises a CAR as described herein, can include: 4P, such as an engineered CAR as described herein.

[0237] An exemplary HMD pathway from 6ACA that comprises a CAR as described herein, can include: 40, such as an engineered CAR as described herein; and 4C.

[0238] Another exemplary HMD pathway from 6ACA that comprises a CAR as described herein, can include: 4D; 4P, such as an engineered CAR as described herein; 4G; and 4H.

[0239] An exemplary HMD pathway from 6-acetamidohexanoate that comprises a CAR as described herein, can include: 4P, such as an engineered CAR as described herein; 4G; and 4H.

[0240] Exemplary HHA, 6ACA-SAld, 6-aminohexanol, 6-HH, and / or HDO pathways are shown in FIG. 5. Such a 6ACA-C0A, 6ACA-SAld, 6-aminohexanol, 6-HH, and / or HDO pathway as described herein can include one or more of: (5 A) 6-aminocaproyl-CoA transferase or synthetase, or an ACL which converts 6ACA to 6ACA-C0A; (5B) 6-aminocaproyl-CoA reductase or an ALDH which converts 6ACA-C0A to 6ACA-SAld; (5C) 6ACA-SAld reductase which converts 6ACA-SAld to 6-aminohexanol; (5D) 6-aminocaproate reductase or CAR which converts 6ACA to 6 ACA-S Aid; (5E) adipyl-CoA reductase which converts adipyl-CoA to Ad-SAld; (5F) adipate semialdehyde reductase which converts Ad-SAld to 6- hydroxyhexanoate; (5G) 6-hydroxyhexanoyl-CoA transferase or synthetase or an ACL which converts 6-hydroxyhexanoate to 6-hydroxyhexanoyl-CoA; (5H) 6-hydroxyhexanoyl-CoA reductase or an ALDH which converts 6-hydroxyhexanoyl-CoA to 6-HH; (51) 6-HH reductase which converts 6-HH to HDO; (5J) 6-aminohexanol aminotransferase or oxidoreductases which converts 6-aminohexanol to 6-HH; (5K) 6-hydroxyhexanoate reductase or CAR, such as an engineered CAR described herein, which converts 6-hydroxyhexanoate to 6-HH; (5L) adipate reductase or CAR, such as an engineered CAR described herein, which converts ADA to adipate semialdehyde; (5M) adipyl-CoA transferase which reversibly converts adipyl-CoA to ADA; (5N) adipyl-CoA hydrolase which converts adipyl-CoA to ADA; and (50) adipyl- CoA synthase which converts ADA to adipyl-CoA.

[0241] An exemplary HHA pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5E; and 5F.

[0242] Another exemplary HHA pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5M; 5L such as an engineered CAR described herein; and 5F.

[0243] Another exemplary HHA pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5N; 5L such as an engineered CAR described herein; and 5F.

[0244] Another exemplary HHA pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5N; 50; 5E; and 5F. Another exemplary HHA pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5M; 50; 5E; and 5F.

[0245] An exemplary HHA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5M; 5E; and 5F.

[0246] Another exemplary HHA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 50; 5E; and 5F.

[0247] Another exemplary HHA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 50; 5N; 5L; and 5F.

[0248] Another exemplary HHA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 50; 5M; 5L; and 5F.

[0249] Another exemplary HHA pathway from ADA, as described herein as a pathway for a CAR substrate (e.g., HHA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., 6-HH, and / or HDO), can include: 5L, such as an engineered CAR described herein; and 5F.

[0250] An exemplary 6ACA-SAld pathway from 6ACA, that comprises a CAR as described herein, can include: 5D, such as an engineered CAR described herein.

[0251] An exemplary 6-HH pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5E; 5F; and 5K, such as an engineered CAR described herein.

[0252] Another exemplary 6-HH pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5M; 5L such as an engineered CAR described herein; 5F; and 5K, such as an engineered CAR described herein.

[0253] Another exemplary 6-HH pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5N; 5L such as an engineered CAR described herein; 5F; and 5K, such as an engineered CAR described herein.

[0254] Another 6-HH pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5N; 50; 5E; 5F; and 5K, such as an engineered CAR described herein.

[0255] Another 6-HH pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5M; 50; 5E; 5F; and 5K, such as an engineered CAR described herein.

[0256] An exemplary 6-HH pathway from ADA, that comprises a CAR as described herein, can include: 5M; 5E; 5F; and 5K, such as an engineered CAR described herein.

[0257] Another exemplary 6-HH pathway from ADA, that comprises a CAR as described herein, can include: 50; 5E; 5F; and 5K, such as an engineered CAR described herein.

[0258] Another exemplary 6-HH pathway from ADA, that comprises a CAR as described herein, can include: 50; 5N; 5L; 5F; and 5K, such as an engineered CAR described herein.

[0259] Another exemplary 6-HH pathway from ADA, that comprises a CAR as described herein, can include: 50; 5M; 5L; 5F; and 5K, such as an engineered CAR described herein.

[0260] Another exemplary 6-HH pathway from ADA, that comprises a CAR as described herein, can include: 5L, such as an engineered CAR described herein; 5F; and 5K, such as an engineered CAR described herein.

[0261] Another exemplary 6-HH pathway from 6ACA, that comprises a CAR as described herein, can include: 5D, such as an engineered CAR described herein; 5C; and 5J.

[0262] An exemplary HDO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5E; 5F; 5K, such as an engineered CAR described herein; and 51.

[0263] Another exemplary HDO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5M; 5L such as an engineered CAR described herein; 5F; 5K, such as an engineered CAR described herein; and 51.

[0264] Another exemplary HDO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5N; 5L such as an engineered CAR described herein; 5F; 5K, such as an engineered CAR described herein; and 51.

[0265] Another exemplary HDO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5N; 50; 5E; 5F; 5K, such as an engineered CAR described herein; and 51.

[0266] Another exemplary HDO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 5M; 50; 5E; 5F; 5K, such as an engineered CAR described herein; and 51.

[0267] An exemplary HDO pathway from ADA, that comprises a CAR as described herein, can include: 5M; 5E; 5F; 5K, such as an engineered CAR described herein; and 51.

[0268] Another exemplary HDO pathway from ADA, that comprises a CAR as described herein, can include: 50; 5E; 5F; 5K, such as an engineered CAR described herein; and 51.

[0269] Another exemplary HDO pathway from ADA, that comprises a CAR as described herein, can include: 50; 5N; 5L; 5F; 5K, such as an engineered CAR described herein; and 51.

[0270] Another exemplary HDO pathway from ADA, that comprises a CAR as described herein, can include: 50; 5M; 5L; 5F; 5K, such as an engineered CAR described herein; and 51.

[0271] Another exemplary HDO pathway from ADA, that comprises a CAR as described herein, can include: 5L, such as an engineered CAR described herein; 5F; 5K, such as an engineered CAR described herein; and 51.

[0272] Another exemplary HDO pathway from 6ACA, that comprises a CAR as described herein, can include: 5D, such as an engineered CAR described herein; 5C; 5J; and 51.

[0273] Exemplary Ad-SAld, HHA, and / or CPO pathways are shown in FIG. 6. Such an Ad- SAld, HHA, and / or CPL pathway as described herein can include one or more of: (6A) adipyl- CoA reductase that converts adipyl-CoA to Ad-SAld; (6B) adipate semialdehyde reductase that converts Ad-SAld to HHA; (6C) 6-hydroxyhexanoyl-CoA transferase or synthetase that converts HHA to 6-hydroxyhexanoyl-CoA, (6D) 6-hydroxyhexanoyl-CoA cyclase or spontaneous cyclization which converts 6-hydroxyhexanoyl-CoA to CPO; (6E) adipate reductase or CAR that converts adipate to Ad-SAld; (6F) adipyl-CoA transferase, synthetase or hydrolase which can reversibly converts adipate and adipyl-CoA; (6G) 6-hydroxyhexanoate cyclase which converts HHA to CPO; (6H) 6-hydroxyhexanoate kinase which converts HHA to 6-hydroxyhexanoyl phosphate; (61) 6-hydroxyhexanoyl phosphate cyclase or spontaneous cyclization which converts 6-hydroxyhexanoyl phosphate to CPO; (6J) phosphotrans-6- hydroxyhexanoylase which converts 6-hydroxyhexanoyl-CoA to 6-hydroxyhexanoyl phosphate.

[0274] An exemplary adipate pathway from adipyl-CoA, as described herein as a pathway for a CAR substrate (e.g., adipate) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPO), can include: 6F.

[0275] An exemplary Ad-SAld pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; and 6E, such as an engineered CAR described herein.

[0276] An exemplary Ad-SAld pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein.

[0277] An exemplary HHA pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; 6E, such as an engineered CAR described herein; and 6B.

[0278] A exemplary HHA pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein; and 6B.

[0279] An exemplary CPO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; 6E, such as an engineered CAR described herein; 6B; 6C; and 6D.

[0280] Another exemplary CPO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; 6E, such as an engineered CAR described herein; 6B; and 6G.

[0281] Another exemplary CPO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; 6E, such as an engineered CAR described herein; 6B; 6H; and 61.

[0282] Another exemplary CPO pathway from adipyl-CoA, that comprises a CAR as described herein, can include: 6F; 6E, such as an engineered CAR described herein; 6B; 6C; 6J; and 61.

[0283] An exemplary CPO pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein; 6B; 6C; and 6D.

[0284] Another exemplary CPO pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein; 6B; and 6G.

[0285] Another exemplary CPO pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein; 6B; 6H; and 61.

[0286] Another exemplary CPO pathway from adipate, that comprises a CAR as described herein, can include: 6E, such as an engineered CAR described herein; 6B; 6C; 6J; and 61.

[0287] Exemplary 6ACA, 6ACA-SAM, and HMD pathways are shown in FIG. 7. Such a 6ACA, 6ACA-SAM, and HMD pathway as described herein can include one or more of: (7 A) thiolase that converts acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA; (7B) HBD thatconverts 3-oxoadipyl-CoAto 3-hydroxyadipyl-CoA; (7C) CRT that converts 3-hydroxyadipyl- CoA to 2,3-dehydroadipyl-CoA; (7D) TER that converts 2,3-dehydroadipyl-CoA to adipyl- CoA; (7E) 6ACA-aldehyde dehydrogenase (ALD) that converts adipyl-CoA to Ad-SAld; (7F) 6ACA-transaminase (TA) that converts Ad-SAld to 6 AC A; (7G) Co A transferase / CoA ligase that converts 6ACA to 6ACA-C0A; (7H) HMD-aldehyde dehydrogenase (ALD) that converts 6ACA-C0A to 6-ACA-SADH; (71) CAR or CAR and PPTase that converts 6ACA to 6ACA- SAld; and (7J) HMD-transaminase (TA2) that converts 6ACA-SAld to HMD.

[0288] An exemplary 6ACA pathway from AcCoA and SucCoA, as described herein as a pathway for a CAR substrate (e.g., 6 AC A) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., HMD), can include: 7A; 7B; 7C; 7D; 7E; and 7F.

[0289] An exemplary 6ACA-SAld pathway from AcCoA and SucCoA, that comprises a CAR as described herein, can include: 7A; 7B; 7C; 7D; 7E; 7F; and 71, such an engineered CAR as described herein.

[0290] An exemplary HMD pathway from AcCoA and SucCoA, that comprises a CAR as described herein, can include: 7A; 7B; 7C; 7D; 7E; 7F; 71, such an engineered CAR as described herein; and 7J.

[0291] Exemplary 6ACA pathways are shown in FIG. 8. Such a 6ACA pathway as described herein can include one or more of: (8 A) HODH aldolase which converts pyruvate (PYR) and succinic semialdehyde to HODH; (8B) OHED hydratase which converts HODH to OHED; (8C) OHED reductase which converts OHED to 2-OHED; (8D) 2-OHD decarboxylase which converts 2-OHD to Ad-SAld; (8E) Ad-SAld aminotransferase and / or Ad-SAld oxidoreductase (aminating) which converts Ad-SAld to 6ACA; (8F) OHED decarboxylase which converts OHED to 6-OHE; (8G) 6-OHE reductase which converts 6-OHE to Ad-SAld; (8H) 2-OHD aminotransferase and / or 2-OHD oxidoreductase (aminating) which converts 2-OHD to 2- AHD; (81) 2-AHD decarboxylase which converts 2-AHD to 6ACA; (8J) OHED aminotransferase and / or OHED oxidoreductase (aminating) which converts OHED to 2-AHE; (8K) 2-AHE reductase 2-AHE to 2-ADH; (8L) HODH formate-lyase and / or HODH dehydrogenase which converts HODH to 3-hydroxyadipyl-CoA; (8M) 3-hydroxyadipyl-CoA dehydratase which converts 3-hydroxyadipyl-CoA to 2, 3-dehydroadipyl-CoA; (8N) 2,3- dehydroadipyl-CoA reductase which converts 2, 3-dehydroadipyl-CoA to adipyl-CoA; (80)adipyl-CoA dehydrogenase which converts adipyl-CoA to Ad-SAld; (8P) OHED formatelyase and / or OHED dehydrogenase which converts OHED to 2, 3-dehydroadipyl-CoA; and (8Q) 2-OHD formate-lyase and / or 2-OHD dehydrogenase which converts 2-OHD to adipyl- CoA.

[0292] An exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8C; 8D; and 8E.

[0293] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8L; 8M; 8N; 80; and 8E.

[0294] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8P; 8N; 80; and 8E.

[0295] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8 A; 8B; 8F; 8G; and 8E.

[0296] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8C; 8Q; 80; and 8E.

[0297] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8C; 8H; and 81.

[0298] Another exemplary 6ACA pathway from PYR and succinic semialdehyde, as described herein as a pathway for a CAR substrate (e.g., 6ACA) and / or as a precursor pathway for the downstream production of a desired downstream product (e.g., CPL, HMD, and HDO, such as the CPL, HMD, and HDO pathways described in FIGS. 3, 4, and 5), can include: 8A; 8B; 8J; 8K; and 81.

[0299] An engineered CAR and / or engineered ADH described herein can be used in any of the biosynthetic pathways described herein so long as the substrate and products of the referenced CAR or ADH enzymatic conversion or conversions are intermediates within the described pathway. For example, as disclosed herein, an engineered CAR described herein can function in a pathway to convert HHA to 6-HH. In the pathway described above that includes a CAR that converts HHA to 6-HH, the pathway converts HHA to 6-HH. An engineered CAR described herein can also be used in other product pathways (e.g., CPO, HMD, and / or HDO pathways) that comprise HHA as a substrate / product in the pathway. One skilled in the art can readily utilize an engineered CAR described herein to convert HHA to 6-HH in any desired pathway that comprises such a reaction.

[0300] In another example, as disclosed herein, an engineered CAR described herein can function in a pathway to convert 6ACA to 6ACA-SAld. In the pathway described above that includes a CAR that converts 6ACA to 6ACA-SAld, the pathway converts 6ACA to 6ACA- SAld. An engineered CAR described herein can also be used in other product pathways (e.g., CPL, HMD, and / or HDO) pathways that comprise 6ACA as a substrate / product in the pathway. One skilled in the art can readily utilize an engineered CAR described herein to convert 6ACA to 6ACA-SAM in any desired pathway that comprises such a reaction.

[0301] In another example, as disclosed herein, an engineered CAR described herein can function in a pathway to convert ADA to Ad-SAld. In the pathway described above that includes a CAR that converts ADA to Ad-SAld, the pathway converts ADA to Ad-SAld. An engineered CAR described herein can also be used in other product pathways (e.g., CPL, CPO,HMD, and / or HDO) pathways that comprise ADA as a substrate / product in the pathway. One skilled in the art can readily utilize an engineered CAR described herein to convert ADA to Ad-SAld in any desired pathway that comprises such a reaction.

[0302] In another example, as disclosed herein, an engineered ADH described herein can function in a pathway to convert Ad-SAld to HHA. In the pathway described above that includes a ADH that converts Ad-SAld to HHA, the pathway converts ADA to Ad-SAld. An engineered ADH described herein can also be used in other product pathways (e.g., CPL, CPO, HMD, and / or HDO) pathways that comprise Ad-SAld as a substrate / product in the pathway. One skilled in the art can readily utilize an engineered ADH described herein to convert Ad- SAld to HHA in any desired pathway that comprises such a reaction.

[0303] In another example, as disclosed herein, an engineered ADH described herein can function in a pathway to convert 6ACA-SAld to 6-AHL. In the pathway described above that includes a ADH that converts 6ACA-SAld to 6-AHL, the pathway converts 6ACA-SAld to 6- AHL. An engineered ADH described herein can also be used in other product pathways (e.g., CPL, CPO, HMD, and / or HDO) pathways that comprise 6ACA-SAld as a substrate / product in the pathway. One skilled in the art can readily utilize an engineered ADH described herein to convert 6ACA-SAld to 6-AHL in any desired pathway that comprises such a reaction.

[0304] Enzyme types required to convert common central metabolic intermediates into HDO, HMD or intermediates are indicated above with representative Enzyme Commission (EC) numbers (see also WO 2010 / 127319, WO 2013 / 036764, WO 2008 / 115840, WO 2010 / 030711, WO 2010 / 141920, WO 2011 / 047101, WO 2013 / 184602, WO 2014 / 176514, US Patent No. 9,017,983, US Patent No. 8,067,214, US Patent No. 7,858,350, US Patent No. 8,129,169, US Patent No. 8,377,666, US 2013 / 0066035, US 2013 / 0029381, US 2014 / 0030779, US 2015 / 0148513, US 2014 / 0371417, US 2019 / 0300919, US 2007 / 0254341, US 2011 / 0091944, US 2011 / 0171699, WO 2009 / 151728, WO 2010 / 129936, and WO 2021 / 216952). The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity. Exemplary enzymes include: 1.1.1.a, Oxidoreductase (ketone to hydroxyl or aldehyde to alcohol); 1.1.1.c, Oxidoreductase (2 step, acyl-CoA to alcohol); 1.2.1.b, Oxidoreductase (acyl-CoA to aldehyde); 1.2.1.C, Oxidoreductase (2-oxo acid to acyl-CoA, decarboxylation); 1.2. l.d, Oxidoreductase (phosphorylating / dephosphorylating); 1.3. La, Oxidoreductase operating on CH-CH donors; 1.4. La, Oxidoreductase operating on amino acids (deaminating); 2.3. La, Acyltransferase(transferring phosphate group); 2.6.1. a, Aminotransferase; 2.7.2. a, Phosphotransferase, carboxyl group acceptor; 2.8.3. a, Coenzyme-A transferase; 3.1.2. a, Thiolester hydrolase (CoA specific); 3.5.1. a, hydrolase; 3.5.2.-, in cyclic amides; 4.1.1. a, Carboxy-lyase; 4.2.1. a, Hydrolyase; 4.3.1. a, Ammonia-lyase; 5.3.3. a, Isomerase; 5.4.3. a, Aminomutase; and 6.2.1. a, Acid- thiol ligase.

[0305] The bio-derived form of Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD can be utilized for production of downstream products in which a nonpetroleum based product is desired, for example, by substituting petroleum-derived HDO with the bio-derived form of HDO as described herein. Accordingly, in some embodiments, provided herein is a bioderived HDO, or downstream products related thereto, such as an ester or amide thereof, or the biosynthesis of nylon and intermediates thereof.

[0306] An engineered CAR described herein can be utilized in a cell or in vitro to convert a carboxylic acid to into its corresponding aldehyde. As disclosed herein, the engineered CARs described herein have beneficial and useful properties, including but not limited to increased specificity for HHA or 6ACA, or both over: propanoic acid, hexanoic acid, adipic acid, acetic acid, succinic acid, benzoate, 4HBAld, either isomer of 3HbAld, butyraldehyde, or a combination thereof; increased activity; decreased by-product production, and the like.

[0307] An engineered ADH described herein can be utilized in a cell or in vitro to reversibly convert an aldehyde to into its corresponding alcohol. As disclosed herein, the engineered ADHs described herein have beneficial and useful properties, including but not limited to increased specificity for NAD(P)H; increased activity; decreased by-product production, and the like.Construction and Culture

[0308] While generally described herein as a cell that contains a Ad-SAld, 6ACA, 6ACA- SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway comprising an engineered CAR provided herein, it is understood that provided herein is a cell comprising at least one recombinant nucleic acid encoding an engineered CAR provided herein. The CAR can be expressed in a sufficient amount to produce a desired product, such a product of a Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, or a downstream product related thereto such as an ester or amide thereof. Likewise, generally described herein as a cell that contains a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL,HHA, 6-HH, HDO, and / or HMD pathway comprising an engineered ADH provided herein, it is understood that provided herein is a cell comprising at least one recombinant nucleic acid encoding an engineered ADH provided herein. The ADH can be expressed in a sufficient amount to produce a desired product, such a product of a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, or a downstream product related thereto such as an ester or amide thereof. Exemplary Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathways are described herein.

[0309] It is understood that any of the pathways disclosed herein, as described in the Examples, including the pathways described herein, can be utilized to generate a cell that produces any pathway intermediate or product, as desired, in particular a pathway that utilizes an engineered CAR and / or an engineered ADH as provided herein. As disclosed herein, such a cell that produces an intermediate can be used in combination with another cell expressing one or more upstream or downstream pathway enzymes to produce a desired product. However, it is understood that a cell that produces a Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate can be utilized to produce the intermediate as a desired product.

[0310] The subject matter described herein includes general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well- known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

[0311] The cells provided herein can be produced by introducing an expressible nucleic acid encoding an engineered CAR and / or an engineered ADH provided herein, and optionally expressible nucleic acids encoding one or more of the enzymes or proteins participating in oneor more Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathways, and further optionally a nucleic acid encoding an enzyme that produces a downstream product related to Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD such as an ester or amide thereof. Depending on the host cell chosen, nucleic acids for some or all of a particular Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway, or downstream product related thereto, such as an ester or amide thereof, can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is included for the deficient enzyme(s) or protein(s) to achieve Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6- AHL, HHA, 6-HH, HDO, and / or HMD biosynthesis, or exogenous expression of endogenously expressed genes can be provided to increase expression of pathway enzymes, if desired. Thus, a cell provided herein can be produced by introducing an engineered CAR provided herein, and optionally exogenous enzyme or protein activities to obtain a desired biosynthetic pathway, or by introducing one or more exogenous enzyme or protein activities, including an engineered CAR and / or an engineered ADH provided herein that, together with one or more endogenous enzymes or proteins, produces a desired product such as Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof.

[0312] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and / or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene’s promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non- naturally occurring cell.

[0313] Sources of encoding nucleic acids for a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6- AHL, HHA, 6-HH, HDO, and / or HMD pathway enzyme or protein, a derivative thereof, or a downstream product related thereto, such as an ester or amide thereof, can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Clostridium khiyveri. Clostridium acelohulylicum. Clostridium heijerinckii. Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate-producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei,Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Aquifex aeolicus, Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus niger, Aspergillus terreus, Bacillus subtilis, Bos Taurus, Candida albicans, Candida tropicalis, Chlamydomonas reinhardtii, Chlorobium tepidum, Citrobacter koseri, Citrus junos, Clostridium acetobutylicum, Clostridium kluyveri, Clostridium saccharoperbutylacetonicum, Cyanobium PCC7001, Desulfatibacillum alkenivorans, Dictyostelium discoideum, Fusobacterium nucleatum, Haloarcula marismortui, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumoniae, Kluyveromyces lactis, Lactobacillus brevis, Leuconostoc mesenteroides, Metallosphaera sedula, Methanothermobacter thermautotrophicus, Mus musculus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium smegmatis, Nicotiana tabacum, Nocardia iowensis, Oryctolagus cuniculus, Penicillium chrysogenum, Pichia pastoris, Porphyromonas gingivalis, Porphyromonas gingivalis, Pseudomonas aeruginos, Pseudomonas putida, Pyrobaculum aerophilum, Ralstonia eutropha, Rattus norvegicus, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Salmonella enteric, Salmonella typhimurium, Schizosaccharomyces pombe, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoanaerobacter tengcongensis, Thermus thermophilus, Trypanosoma brucei, Tsukamurella paurometabola, Yarrowia lipolytica, Zoogloea ramigera and Zymomonas mobilis, Clostridum species, including but no limited to Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium botulinum, Clostridium methylpentosum, Clostridium sticklandii, Clostridium phytofermentans, Clostridium saccharolyticum, Clostridium asparagiforme, Clostridium celatum, Clostridium carboxidivorans, Clostridium clostridioforme, Clostridium bolteae, Caldalkalibacillus thermarum, Clostridium botulinum, Pelosinus fermentans, Thermoanaerobacterium thermosaccharolyticum, Desulfosporosinus speices, Thermoanaerobacterium species, including but not limited to Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium xylanolyticum,Acetonema longum, Geobacillus species, including but not limited to Geobacillus thermoglucosidans, Bacillus azotoformans, Thermincola potens, Fusobacterium species, including but not limited to Fusobacterium nucleatum, Fusobacterium ulcerans, Fusobacterium varium, Ruminococcus species, including but not limited to Ruminococcus gnavus, Ruminococcus obeum, Lachnospiraceae bacterium, Flavonifractor plautii, Roseburia inulinivorans, Acetobacterium woodii, Eubacterium species, including but not limited to Eubacterium plexicaudatum, Eubacterium hallii, Eubacterium limosum, Eubacterium yurii, Eubacteriaceae bacterium, Thermosediminibacter oceani, Ilyobacter polytropus, Shuttleworthia satelles, Halanaerobium saccharolyticum, Thermoanaerobacter ethanolicus, Rhodospirillum rubrum, Vibrio, Propionibacterium propionicum as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the fatty aldehydes, fatty alcohols, fatty alcohol acetates, saturated and / or monounsaturated compounds thereof biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof, and / or a downstream product related thereto, such as an ester or amide thereof, including expression of a CAR and / or ADH provided herein, described herein with reference to a particular organism such as E. coli can be readily applied to other cells such as microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[0314] In some instances, such as when an alternative Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway exists in an unrelated species, Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods provided herein can be applied to all cells using the cognate metabolic alterations to those exemplified herein to construct a cell in a species of interest that will synthesize Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or an ester or amide thereof, and / or a downstream product related thereto, such as an ester or amide thereof, if desired, including introducing a CAR and / or ADH provided herein.

[0315] Host cells can be selected from, and the non-naturally cells expressing an engineered car provided herein generated in, for example, bacteria, yeast, fungus or any of a variety of microorganisms applicable or suitable to fermentation processes. In some embodiments, the microbial organism is a species of bacteria, yeast or fungus. In some embodiments, the microbial organism is a species of bacteria. In some embodiments, the microbial organism is a species of yeast. In some embodiments, the microbial organism is a species of fungus. Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae , including the genera Escherichia and Klebsiella, the order Aeromonadales, family Succinivibrionaceae , including the genus Anaerobiospirillunr, the order Pasteurellales, family Pasteurellaceae, including the genera Actinobacillus and Mannheimia, the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobiunr, the order Bacillales, family Bacillaceae, including the genus Bacillus,' the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Ghiconobacter, the order Sphingomonadales, family Sphingomonadaceae , including the genus Zymomonas,' the order Lactobacillales, families Lactobacillaceae and Streptococcaceae , including the genus Lactobacillus and the genus Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium,' and the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas. Non-limiting species of host bacteria include 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. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.

[0316] Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycelales, family Saccaromycelaceae, including the genera Saccharomyces, Kluyveromyces and Pichler, the order Saccharomycelales. family Dipodascaceae. including the genus Yarrowia the order Schizosaccharomycelales, family Schizosaccaromycelaceae, including the genus Schizosaccharomyces: the order Eurotiales, family Trichocomaceae, including the genus Aspergillus,' and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolylica, and the like. A particularly useful host organism that is a yeast includes Saccharomyces cerevisiae.

[0317] Although generally described herein as utilizing a cell that is a microbial organism as a host cell, particularly for producing Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof, it is understood that a host cell can be a cell line of a higher eukaryote, such as a mammalian cell line or insect cell line. Thus, it is understood that reference herein to a host cell that is a microbial organism can alternatively utilize a higher eukaryotic cell line to produce a desired product. Exemplary higher eukaryotic cell lines include, but are not limited to, Chinese hamster ovary (CHO), human (Hela, Human Embryonic Kidney (HEK) 293, Jurkat), mouse (3T3), primate (Vero), insect (Sf9), and the like. Such cell lines are commercially available (see, for example, the American Type Culture Collection (ATCC; Manassas VA); Life Technologies, Carlsbad CA). It is understood that any suitable host cell can be used to introduce an engineered CAR provided herein, and optionally metabolic and / or genetic modifications to produce a desired product.

[0318] Depending on the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway constituents of a selected host cell, the non-naturally occurring cells provided herein will include at least one exogenously Ad-SAld, 6ACA, 6ACA- SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathways, or a downstream product related thereto such as an ester or amide thereof, including an engineered CAR provided herein. For example, Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / orHMD biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid, including an engineered CAR provided herein. In a host deficient in all enzymes or proteins of a Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, or a downstream product related thereto such as an ester or amide thereof, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD pathway, or a downstream product related thereto such as an ester or amide thereof, can be included, including an engineered CAR and / or an engineered ADH provided herein.

[0319] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway deficiencies of the selected host cell if a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway is to be included in the cell. Therefore, a non-naturally occurring cell provided herein can have one, two, three, four, five, six, seven, eight, and so forth, depending on the particular pathway, up to all nucleic acids encoding the enzymes or proteins constituting a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6- AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring cells also can include other genetic modifications that facilitate or optimize Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthesis or that confer other useful functions onto the host cell. One such other functionality can include, for example, augmentation of the synthesis of one or more of the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway precursors such acetyl-CoA or succinyl-CoA.

[0320] Generally, a host cell is selected such that it can express an engineered CAR provided herein, and optionally produces the precursor of a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, in a cell containing such a pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced bythe host cell. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a cell that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a Ad- SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, or a downstream product related thereto such as an ester or amide thereof, if desired.

[0321] In some embodiments, a non-naturally occurring cell provided herein is generated from a host that contains the enzymatic capability to synthesize Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof. In this specific embodiment it can be useful to increase the synthesis or accumulation of a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD pathway product to, for example, drive Ad-SAld, 6ACA, 6AC A-S Aid, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway reactions toward Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD production, or a downstream product related thereto such as an ester or amide thereof. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway enzymes or proteins, including an engineered CAR provided herein. Overexpression of the enzyme or enzymes and / or protein or proteins of the Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes, including exogenous expression of an engineered CAR provided herein. Therefore, naturally occurring organisms can be readily converted to non-naturally occurring cells provided herein, for example, producing Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD or a downstream product related thereto such as an ester or amide thereof, through overexpression of one, two, three, four, five, six, seven, eight, or more, depending on the Ad- SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway, that is, up to all nucleic acids encoding Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway enzymes or proteins, or enzymes that produce a downstream product related thereto such as an ester or amide thereof. In addition, a non- naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO,6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway, or a downstream product related thereto such as an ester or amide thereof.

[0322] It is understood that, in methods described herein, any of the one or more recombinant and / or exogenous nucleic acids can be introduced into a cell to produce a non-naturally occurring cell provided herein. The nucleic acids can be introduced so as to confer, for example, a Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof, biosynthetic pathway onto the cell, including introducing a nucleic acid encoding an engineered CAR provided herein. Alternatively, encoding nucleic acids can be introduced to produce a cell having the biosynthetic capability to catalyze some of the required reactions to confer Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic capability to produce an intermediate. For example, a non-naturally occurring cell having a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, including an engineered CAR provided herein. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell provided herein, including an engineered CAR provided herein. Likewise, a non-naturally occurring cell having an Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, including an engineered ADH provided herein. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell provided herein, including an engineered ADH provided herein. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell provided herein, as desired, so long as the combination of enzymes and / or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of five, six, seven, eight, nine, ten, eleven, twelve or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring cell provided herein, as desired, so long as the combination of enzymes and / or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.I l l

[0323] In addition to the biosynthesis of Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof, as described herein, the non-naturally occurring cells and methods provided herein also can be utilized in various combinations with each other and / or with other cells and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce Ad-SAld, 6 AC A, 6 AC A- S Aid, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD other than use of the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD producers is through addition of another cell capable of converting a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate to Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD. One such procedure can include the fermentation of a cell that produces a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate. The Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6- HH, HDO, and / or HMD pathway intermediate can then be used as a substrate for a second cell that converts the Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate to Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD. The Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate can be added directly to another culture of the second organism or the original culture of the Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD pathway intermediate producers can be depleted of these cells by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. A cell that produces a downstream product related to Ad-SAld, 6ACA, 6ACA-SAM, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD such as an ester or amide thereof, can optionally be included to produce such a downstream product.

[0324] Alternatively, such enzymatic conversions can be carried out in vitro, with a combination of enzymes or sequential exposure of substrates to enzymes that result in conversion of a substrate to a desired product. As another alternative, a combination of cellbased conversions and in vitro enzymatic conversions can be used, if desired.

[0325] In other embodiments, the non-naturally occurring cells and methods provided herein can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD or adownstream product related thereto such as an ester or amide thereof. In these embodiments, biosynthetic pathways for a desired product provided herein can be segregated into different cells, and the different cells can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one cell is the substrate for a second cell until the final product is synthesized. For example, the biosynthesis of Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof, can be accomplished by constructing a cell that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD also can be biosynthetically produced from cells through coculture or co-fermentation using two different cells in the same vessel, where the first cell produces a Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD intermediate and the second cell converts the intermediate to Ad-SAld, 6ACA, 6ACA- SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HMD, or a downstream product related thereto such as an ester or amide thereof.

[0326] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring cells and methods provided herein together with other cells, with the co-culture of other non-naturally occurring cells having subpathways and with combinations of other chemical and / or biochemical procedures well known in the art to produce Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, 6-AHL, HHA, 6-HH, HDO, and / or HM...

Claims

CLAIMSWhat is claimed is:

1. An engineered carboxylic acid reductase (CAR) comprising a variant of amino acid sequence SEQ ID NO: 1 or a functional fragment thereof, wherein the engineered CAR comprises one or more alterations at a position described in TABLE 3B.

2. The engineered CAR of claim 1, wherein the engineered CAR is capable of: a) catalyzing the conversion of 6-hydroxycaproic acid (6-HCA) to 6- hydroxyhexanal (6-HH); b) catalyzing the conversion of 6-HCA to 6-HH at a greater rate compared to wild-type CAR; c) having a higher specificity for the 6-HCA substrate as compared to the wild type CAR; or d) catalyzing the conversion of 6-aminocaproic acid (6ACA) to 6-aminocaproate semialdehyde (6ACA-SAld); e) catalyzing the conversion of 6ACA to 6ACA-SAld at a greater rate compared to wild-type CAR; f) having a higher specificity for the 6ACA substrate as compared to the wild type CAR; or g) any combination thereof.

3. The engineered CAR of claim 1 or claim 2, wherein the engineered CAR has higher specificity for conversion of 6-HCA to 6-HH or 6ACA to 6ACA-SAM, or both, over conversion of: a) propanoic acid to propanoate semialdehyde; b) hexanoic acid to hexanoate semialdehyde; c) adipic acid to adipate semialdehyde; d) acetic acid to acetaldehyde; e) succinic acid to succinate semialdehyde; f) benzoate to benzaldehyde; g) 4HB to 4HBAld;h) 3HB to the (R) or (S) isomer of 3HBAld; i) butyric acid to butyraldehyde; or j) any combination thereof.

4. The engineered CAR of any one of claims 1 to 3, wherein the engineered CAR has higher specificity for conversion of 6-HCA to 6-HH over conversion of 6ACA to 6ACA-SAld.

5. The engineered CAR of any one of claims 1 to 4, wherein the engineered CAR comprises an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a CAR consisting of the amino acid sequence of SEQ ID NO: 1.

6. The engineered CAR of any one of claims 1 to 5, wherein the one or more amino acid alterations comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110 or more alterations.

7. The engineered CAR of any one of claims 1 to 6, wherein the one or more amino acid alterations are conservative or non-conservative amino acid substitutions, deletions, or insertions, or any combination thereof.

8. The engineered CAR of any one of claims 1 to 7, wherein the CAR comprises one or more amino acid alterations at a position corresponding to position 48, 98, 134, 179, 238, 299, 354, 379, 389, 937, or a combination thereof, in SEQ ID NO: 1.

9. The engineered CAR of any one of claims 1 to 8, wherein the CAR comprises one or more amino acid alterations at a position corresponding to position 379, 354, 937, or a combination thereof, in SEQ ID NO:

110. The engineered CAR of any one of claims 1 to 9, wherein the one or more amino acid alterations result in an engineered CAR comprising: a) R at a residue corresponding to position 379 in SEQ ID NO: 1; b) H at a residue corresponding to position 354 in SEQ ID NO: 1; c) T at a residue corresponding to position 937 in SEQ ID NO: 1; d) W at a residue corresponding to position 299 in SEQ ID NO: 1;e) G at a residue corresponding to position 238 in SEQ ID NO: 1; f) E or R at a residue corresponding to position 134 in SEQ ID NO: 1; g) R at a residue corresponding to position 134 in SEQ ID NO: 1; h) S at a residue corresponding to position 389 in SEQ ID NO: 1; i) V at a residue corresponding to position 48 in SEQ ID NO: 1; j) D at a residue corresponding to position 179 in SEQ ID NO: 1; k) E at a residue corresponding to position 98 in SEQ ID NO: 1; or a combination thereof.

11. The engineered CAR of claim 10, wherein the one or more amino acid alterations result in an engineered CAR comprising: a) R at a residue corresponding to position 379 in SEQ ID NO: 1; b) H at a residue corresponding to position 354 in SEQ ID NO: 1; c) T at a residue corresponding to position 937 in SEQ ID NO: 1; or a combination thereof.

12. The engineered CAR of any one of claims 1 to 7, wherein the engineered CAR comprises one or more amino acid alterations selected from the group consisting of: Y379R, R354H, S299W, A238G, A134E, A134R, M389S, I48V, G179D, S98E, Q937T, and any combination thereof.

13. The engineered CAR claim 12, wherein the engineered CAR comprises one or more amino acid alterations selected from the group consisting of: Y379R, R354H, and / or Q937T, and any combination thereof.

14. The engineered CAR of any one of claims 1 to 13, wherein the engineered CAR comprises a single alteration or a combination of alterations described in TABLE 3B or TABLE 4A15. The engineered CAR of any one of claims 1 to 14, wherein the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence referenced in SEQ ID NO:

116. An engineered alcohol dehydrogenase (ADH) comprising a variant of amino acid sequence SEQ ID NO: 15 or a functional fragment thereof, wherein the engineered ADH comprises one or more alterations at a position described in TABLE 4C.

17. The engineered ADH of claim 16, wherein the engineered ADH is capable of: a) catalyzing the conversion of Ad-SAld to HHA; b) catalyzing the conversion of 6-HH to HDO; c) catalyzing the conversion of 6ACA-SAld to 6-AHL; d) converting NAD(P) to NAD(P)H; e) converting NAD to NADH; f) having a higher specificity for the NAD(P) substrate as compared to the wild type ADH; or g) any combination thereof.

18. The engineered ADH of claim 16 or claim 17, wherein the engineered ADH has higher specificity for NAD(P), over NAD.

19. The engineered ADH of any one of claims 16-18, wherein the engineered ADH comprises an activity that 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%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a ADH consisting of the amino acid sequence of SEQ ID NO: 15.

20. The engineered ADH of any one of claims 16-19, wherein the one or more amino acid alterations comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110 or more alterations.

21. The engineered ADH of any one of claims 16-20, wherein the one or more amino acid alterations are conservative or non-conservative amino acid substitutions, deletions, or insertions, or any combination thereof.

22. The engineered ADH of any one of claims 16-21, wherein the ADH comprises one or more amino acid alterations at a position corresponding to position 215, 216, or a combination thereof, in SEQ ID NO: 15.

23. The engineered ADH of claim 22, wherein the one or more amino acid alterations result in an engineered ADH comprising: a) A, S, or T at a residue corresponding to position 215 in SEQ ID NO: 15; b) H, Q, S, or T at a residue corresponding to position 216 in SEQ ID NO: 15; or a combination thereof.

24. The engineered ADH of any one of claim 23, wherein the one or more amino acid alterations result in an engineered ADH comprising: a) A or S at a residue corresponding to position 215 in SEQ ID NO: 15; b) H or Q at a residue corresponding to position 216 in SEQ ID NO: 15; c) or a combination thereof.

25. The engineered ADH of any one of claims 16 to 23, wherein the engineered ADH comprises one or more amino acid alterations selected from the group consisting of: D215A, D215S, L216H, L216Q, D215T, L216S, D215T, L216T, and any combination thereof.

26. The engineered ADH of claim 25, wherein the engineered ADH comprises one or more amino acid alterations selected from the group consisting of: D215A, D215S, L216H, or L216Q, and any combination thereof.

27. The engineered ADH of claim 25 or claim 26, wherein the engineered ADH comprises one or more amino acid alterations selected from the group consisting of: D215A, L216H, and any combination thereof.

28. The engineered ADH of any one of claims 16 to 27., wherein the engineered ADH comprises a single alteration or a combination of alterations described in TABLE 4C or TABLE 3B29. The engineered ADH of any one of claims 16 to 28, wherein the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence referenced in SEQ ID NO: 1530. A recombinant nucleic acid encoding: a) the engineered CAR of any one of claims 1 to 15; b) the engineered ADH of any one of claims 16 to 29; or c) both.

31. The recombinant nucleic acid of claim 30, wherein the nucleic acid comprises a nucleotide sequence encoding the engineered CAR and / or the engineered ADH operatively linked to a promoter.

32. A vector comprising the recombinant nucleic acid of claims 30 or 31.

33. A non-naturally occurring microbial organism comprising: a) a recombinant nucleic acid encoding an engineered CAR selected from any one of claims 1 to 15; b) a recombinant nucleic acid encoding an engineered ADH selected from any one of claims 16 to 29; or c) a recombinant nucleic acid encoding both the engineered CAR and the engineered ADH.

34. The non-naturally occurring microbial organism of claim 33, wherein the non- naturally occurring microbial organism further comprises a pathway that produces adipate semialdehyde (Ad-SAld), 6-aminocaproic acid (6ACA), 6-hydroxycaproic acid (HHA), 6-hydroxyhexanal (6-HH), 6-aminocaproate semialdehyde (6ACA- SAld), caprolactam (CPL), caprolactone (CPO), 6-aminohexanol (6-AHL), hexamethylenediamine (HMD), and / or 1,6 hexanediol (HDO), or an ester or amide thereof.

35. The non-naturally occurring microbial organism of claim 34, wherein the non- naturally occurring microbial organism is capable of producing at least 10% more Ad- SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof compared to a control microbial organism that does not comprise any one of the nucleic acids of claims 30 or 31.

36. The non-naturally occurring microbial organism of any one of claims 33 to 35, wherein the microbial organism produces a decreased amount of one or more byproduct as compared to a control microbial organism that does not comprise the recombinant nucleic acid of any one of claims 30 to 31, respectively.

37. The non-naturally occurring microbial organism of claim 36, wherein the one or more by-product comprises propanoate semialdehyde, hexanoate semialdehyde, acetaldehyde, succinate semialdehyde, 4HBAld, either isomer of 3HBAld, butyraldehyde, 4HB, BDO, or combinations thereof.

38. The non-naturally occurring microbial organism of any one of claims 33 to 37, wherein the microbial organism is capable of producing at least 10% less by-product compared to a control microbial organism that does not comprise the recombinant nucleic acid of any one of claims 30 to 31.

39. The non-naturally occurring microbial organism of any one of claims 33 to 38, wherein the one or more enzymes of the pathway are encoded by an exogenous nucleic acid.

40. The non-naturally occurring microbial organism of claim 39, wherein the exogenous nucleic acid is heterologous or homologous.

41. The non-naturally occurring microbial organism of any one of claims 33 to 40, wherein the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

42. The non-naturally occurring microbial organism of any one of claims 33 to 41, wherein the microbial organism is a species of bacteria, yeast, or fungus.

43. A method for producing Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6- HH, HDO, and / or HMD, or an ester or amide thereof, comprising culturing the non- naturally occurring microbial organism of any one of claims 33 to 42 under conditions and for a sufficient period of time to produce the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof.

44. The method of claim 43, wherein the method further comprises separating the Ad- SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof from other components in the culture.

45. The method of claim 44, wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

46. A culture medium comprising the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof produced by the method of any one of claims 43 to 45, wherein the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source.

47. An Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof produced according to the method of any one of claims 43 to 45.

48. The Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof of claim 46 or 47, wherein the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

49. A composition comprising the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6- AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof of claim 47 or 48 and a compound other than the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof.

50. The composition of claim 49, wherein the compound other than the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a pathway that produces Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6-AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof.

51. A composition comprising the Ad-SAld, 6ACA, 6ACA-SAld, CPL, CPO, HHA, 6- AHL, 6-HH, HDO, and / or HMD, or an ester or amide thereof of claim 47 or 48 or a cell lysate or culture supernatant thereof.

52. Use of the engineered CAR of any one of claims 1 to 15 as a biocatalyst.

53. A composition comprising the engineered CAR of any one of claims 1 to 15 and at least one substrate for the engineered CAR.

54. The composition of claim 53, wherein the engineered CAR can react with the substrate under in vitro conditions.

55. The composition of claim 53 or 54, wherein the substrate is adipate or adipic acid.

56. The composition of claim 53 or 54, wherein the substrate is 6-hydroxycaproic acid.

57. The composition of claim 53 or 54, wherein the substrate is 6-hydroxyhexanoate.

58. The composition of claim 53 or 54, wherein the substrate is 6-aminocaproate.

59. Use of the engineered ADH of any one of claims 16 to 29 as a biocatalyst.

60. A composition comprising the engineered ADH of any one of claims 16 to 29 and at least one substrate for the engineered ADH.

61. The composition of claim 60, wherein the engineered ADH can react with the substrate under in vitro conditions.

62. The composition of claim 60 or 61 wherein the substrate is Ad-SAld.

63. The composition of claim 60 or 61 wherein the substrate is 6-HH.

64. The composition of claim 60 or 61 wherein the substrate is 6ACA-SAM.

65. The composition of any one of claims 60 to 64 further comprising NAD(P) orNAD(P)H.

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