Production of chemicals from renewable sources

Abstract

The present disclosure provides biosynthetic polypeptides, methods, and non-naturally occurring microorganisms, etc. for making a variety of compounds such as 1,5-pentanediol, adipic acid, 1,6-hexanediol, 6-hydroxyhexanoic acid, and 2-ketocarboxylic acids.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 838,793, filed April 25, 2019, and U.S. Patent Application No. 62 / 868,824, filed June 28, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to compositions and methods for preparing industrially usable chemicals. Background Technology

[0004] Adipic acid (AA) is a widely used chemical, with an estimated demand of 2.3 million metric tons in 2012 (IHS Chemical, Process Economics Program Report: Bio-Based Adipic Acid (Dec. 2012)). It is used in conjunction with hexamethylenediamine (HMDA) in the production of nylon 6,6, polyester resins, plasticizers, food products, and other materials. Therefore, there is a strong need for methods to produce adipic acid in high yields using renewable resources.

[0005] 1,5-Pentanediol is a major component of polyurethanes and polyesters (PDLs). 1,6-Hexanediol (HDO) is a linear diol with terminal hydroxyl groups. It is used in polyesters for industrial coatings applications and in two-component polyurethane coatings for automotive applications. It is also used in the production of macromolecular diols, such as adipates and polycarbonate diols for elastomers, and polyurethane dispersions for parquet flooring and leather coatings.

[0006] 6-Hydroxyhexanoic acid (6HH) can be cyclized to form ε-caprolactone, which can then be amination to form ε-caprolactam. ε-caprolactam is used to produce nylon 6, a polymer widely used in many different industries. ε-caprolactone can be polymerized to produce polycaprolactone (PCL), a biodegradable polyester that can be used to produce specialized polyurethanes.

[0007] 2-Ketocarboxylic acids are available intermediates for the preparation of many industrially relevant chemicals and pharmaceuticals. They are precursors for the production of amino acids and industrially available α-hydroxycarboxylic acids. Summary of the Invention

[0008] This disclosure covers the recognition that certain biosynthetic peptides, such as various enzymes, can be used in many embodiments to efficiently prepare a variety of compounds from substrates that are structurally different from their natural and / or characterized substrates. In some embodiments, this disclosure provides techniques for preparing a variety of compounds (e.g., enzymes, nucleic acids, organisms, cultures, etc.) using one or more such enzymes.

[0009] For example, in some embodiments, the present disclosure provides aldol dehydration product biosynthesis polypeptides, such as a plurality of hydratase- aldolase enzymes, that can be effectively used to make a plurality of compounds from aliphatic aldehydes rather than their typical aromatic aldehyde substrates. In some embodiments, the present disclosure provides methods comprising:

[0010] contacting a pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthesis polypeptide to produce an aldol dehydration product, wherein:

[0011] the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aryl group; and

[0012] the aldol dehydration product is a compound comprising an aldehyde group or a ketone group and a double bond conjugated to the aldehyde group or the ketone group.

[0013] In some embodiments, the aldehyde, such as the aliphatic aldehyde, has the structure of Formula A-1:

[0014] R a -L 2 -L 1 -C(O)H,

[0015] A-1

[0016] or a salt thereof, wherein:

[0017] R a is R” or -OR”, and

[0018] L 1 and L 2 are each independently a covalent bond, or a bivalent, optionally substituted, straight-chain or branched C 1-20 aliphatic or C 1-20 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0019] -C≡C-, -C(R”)2-, -Cy-, -O-, -S-, -S-S-, -N(R”)-, -C(O)-, -C(S)-, -C(NR”)-, -C(O)N(R”)-, -N(R”)C(O)N(R”)-, -N(R”)C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R”)-, -C(O)S-, or -C(O)O-;

[0020] -Cy- is a bivalent, optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms;

[0021] each R" is independently -R', -C(O)R', -CO2R', or -SO2R';

[0022] R' is hydrogen, or an optionally substituted group selected from C 1-10 aliphatic, C 1-10 heteroaliphatic, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring having 1 to 5 heteroatoms, and 3- to 10-membered heterocyclic ring having 1 to 5 heteroatoms, or:

[0023] two or more R' groups, together with their intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0 to 5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms.

[0024] In some embodiments, L 1 is an optionally substituted -CH2-. In some embodiments, L 1 is an optionally monosubstituted -CH2-. In some embodiments, L 1 is -CH2-.

[0025] In some embodiments, the aldol dehydration product has the structure of Formula P-2:

[0026] R a -L 2 -L 1 -CH=CH-C(O)-C(O)OH,

[0027] P-2 or a salt thereof, wherein each variable is independently as described herein.

[0028] As described herein, the aldol dehydration product, e.g., the compound of Formula P-2 or a salt thereof, in some embodiments, can be further processed to provide a variety of products, e.g., 1,5-pentanediol, HDO, 6HH, adipic acid, etc., by one or more biosynthetic processes, e.g., as described in Figures 2 to 5 ) and a variety of products made therefrom, including a variety of polymeric products made therefrom.

[0029] In some embodiments, as shown herein, the aldol dehydration product, e.g., the compound of Formula P-2 or a salt thereof, can also be made from the aldol product, e.g., the compound of Formula P-1:

[0030] R a -L 2 -L 1 -CH(OH)-CH2-C(O)-C(O)OH,

[0031] P-1

[0032] or salts thereof, wherein each variable is independently as described herein.

[0033] In some embodiments, the aldol dehydration product is made by contacting an aldol product with an aldol dehydration product biosynthetic polypeptide.

[0034] In some embodiments, the aldol product is made by contacting a suitable substrate with an aldol product biosynthetic polypeptide.

[0035] In some embodiments, the present disclosure demonstrates that a variety of olefin reduction product biosynthetic polypeptides are useful for making a variety of compounds from their natural or unnatural substrates. In some embodiments, the present disclosure provides methods comprising:

[0036] contacting an olefin with an olefin reduction product biosynthetic polypeptide to produce an olefin reduction product, wherein:

[0037] the olefin comprises a double bond conjugated to a carbonyl group; and

[0038] reducing the double bond conjugated to the carbonyl group in the olefin to a single bond to provide the olefin reduction product.

[0039] In some embodiments, the olefin is an aldol dehydration product, such as one of formula P-2 or a salt thereof. In some embodiments, the olefin reduction product has the structure of formula P-3:

[0040] R a -L 2 -L 1 -CH2-CH2-C(O)-C(O)OH,

[0041] P-3

[0042] or salts thereof, wherein each variable is independently as described herein.

[0043] wherein enzymes, methods, and recombinant microorganisms for making 2-keto carboxylic acids, 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxyhexanoic acid using renewable resources are disclosed.

[0044] In one aspect, provided herein is a method of producing a 2-keto carboxylic acid of the following formula:

[0045]

[0046] wherein R is H, CH3, or CH2OH;

[0047] the method comprising or consisting essentially of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase in a culture or organism comprising one or more non-naturally occurring microorganisms to produce a 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microorganisms.

[0048] In another aspect, provided herein is a method of producing a 2-keto carboxylic acid of the formula:

[0049]

[0050] wherein R is H, CH3, or CH2OH;

[0051] The method comprises or consists essentially of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase in a culture or organism comprising two or more non-naturally occurring microorganisms to produce a 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microorganisms.

[0052] In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising or consisting essentially of:

[0053] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0054]

[0055] wherein R is CH2OH;

[0056] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxy-pentanal; and

[0057] contacting the 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce 1,5-pentanediol, wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0058] In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising or consisting essentially of:

[0059] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0060]

[0061] wherein R is CH2OH;

[0062] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxy-pentanal; and

[0063] contacting 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce 1,5- pentanediol, wherein the method is performed in a culture comprising two or more non- naturally occurring microbial organisms.

[0064] In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising: contacting pyruvate and 3-hydroxy-propanal with a hydratase- aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0065]

[0066] wherein R is CH2OH;

[0067] contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2- reductase to produce 2,6-dihydroxy-hexanoic acid;

[0068] contacting the 2,6-dihydroxy-hexanoic acid with a 2,6-dihydroxy-hexanoate CoA- transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0069] contacting the 2,6-dihydroxy-hexanoyl-CoA with a 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0070] contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA;

[0071] contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoic acid;

[0072] contacting the 6-hydroxyhexanoic acid with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxy-hexanal; and

[0073] contacting the 6-hydroxyhexanal with a 6-hydroxyhexanal 1-reductase to produce 1,6- hexanediol, wherein the method is performed in a culture comprising one or more non- naturally occurring microbial organisms.

[0074] In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising: contacting pyruvate and 3-hydroxy-propanal with a hydratase- aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0075]

[0076] wherein R is CH2OH;

[0077] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0078] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0079] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0080] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with 2,3-dehydro-hexanoyl-CoA 2,3- reductase to produce 6-hydroxy-hexanoyl-CoA;

[0081] contacting 6-hydroxy-hexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0082] contacting 6-hydroxyhexanoate with 6-hydroxyhexanoate 1 -reductase to produce 6- hydroxy-hexanal; and

[0083] contacting 6-hydroxyhexanal with 6-hydroxyhexanal 1 -reductase to produce 1,6- hexanediol, wherein the method is performed in a culture comprising two or more non- naturally occurring microbial organisms.

[0084] In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising:

[0085] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce 2-ketocarboxylic acid of the formula:

[0086]

[0087] wherein R is CH2OH;

[0088] contacting 2-ketocarboxylic acid with 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxy-hexanoate;

[0089] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0090] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0091] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 2,3-dihydro-hexanoyl-CoA 2,3- reductase to produce 6-hydroxy-hexanoyl-CoA; and

[0092] contacting 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0093] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0094] In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising:

[0095] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0096]

[0097] wherein R is CH2OH;

[0098] contacting 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxy-hexanoate;

[0099] contacting 2,6-dihydroxy-hexanoate with a 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0100] contacting 2,6-dihydroxy-hexanoyl-CoA with a 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0101] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 2,3-dihydro-hexanoyl-CoA 2,3- reductase to produce 6-hydroxy-hexanoyl-CoA; and

[0102] contacting 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0103] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0104] In another aspect, provided herein is a method for producing adipic acid, the method comprising: contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0105]

[0106] wherein R is CH2OH;

[0107] contacting 2-ketocarboxylic acid with 6-hydroxy-2-oxohexanoate 2-reductase to produce 2,6-dihydroxy-hexanoic acid;

[0108] contacting 2,6-dihydroxy-hexanoic acid with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0109] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0110] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with 2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA; and

[0111] contacting 6-hydroxy-hexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoic acid;

[0112] contacting 6-hydroxy-hexanoic acid with 6-hydroxyhexanoate dehydrogenase to produce 6-oxo-hexanoic acid; and

[0113] contacting 6-oxo-hexanoic acid with 6-oxo-hexanoate oxidase to produce adipic acid,

[0114] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0115] In another aspect, provided herein are methods for producing adipic acid, the method comprising:

[0116] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce 2-ketocarboxylic acid of the formula:

[0117]

[0118] wherein R is CH2OH;

[0119] contacting 2-ketocarboxylic acid with 6-hydroxy-2-oxohexanoate 2-reductase to produce 2,6-dihydroxy-hexanoic acid;

[0120] contacting 2,6-dihydroxy-hexanoic acid with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0121] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0122] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0123] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0124] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0125] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate,

[0126] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0127] In some embodiments, the hydratase-aldolase is an enzyme of EC number 4.1.2.45, EC number 4.1.2.34, or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID No.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.

[0128] In some embodiments, the hydratase-aldolase is an enzyme of EC number 4.1.2.45, EC number 4.1.2.34, or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID No.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0129] In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by the following GenBank, RefSeq, or Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1, or a portion thereof (e.g., a domain, a set of amino acid residues (which can be contiguous or separated), etc.) that facilitates the formation of an aldol dehydration product.In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0130] In some embodiments, the hydratase-aldolase is selected from the enzymes of Tables 1 and 5-8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from Tables 1 and 5-8.

[0131] In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the sequence of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0132] In some embodiments, the hydratase-aldolase and the quinone oxidoreductase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by one or more exogenous genes expressed by one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by one or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microbial organisms.

[0133] In some embodiments, the hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by one or more exogenous genes expressed by two or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by two or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microbial organisms.

[0134] In some embodiments, one or more of the hydratase-aldolase and quinone oxidoreductase further comprise one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of polyhistidine tag, GST tag (glutathione-S-transferase tag), HA tag (hemagglutinin tag), FLAG tag, Myc tag, maltose binding protein tag, chitin binding protein tag, and fluorescent tag.

[0135] In some embodiments, the method for producing a 2-keto carboxylic acid further comprises or consists essentially of isolating the 2-keto carboxylic acid from one or more non-naturally occurring microbial organisms or cultures comprising one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of isolating the 2-keto carboxylic acid from two or more non-naturally occurring microbial organisms or cultures comprising two or more non-naturally occurring microbial organisms.

[0136] In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by EC number 4.1.1.1; EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number 4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11; EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number 4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number 4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number 4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number 4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77. In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by Uniprot ID No. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto acid decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0137] In some embodiments, the primary alcohol dehydrogenase is an enzyme with EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase is an enzyme comprising the sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

[0138] In some embodiments, the hydratase-aldolase is the enzyme identified with Uniprot ID No. A0A286PH18; the quinone oxidoreductase is the enzyme identified with Uniprot ID No. P28304; the 2-keto acid decarboxylase is the enzyme identified with Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase is the enzyme identified with the following Uniprot or GenBank ID Nos.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694. In some embodiments, the hydratase-aldolase is the enzyme comprising the sequence of SEQ ID NO: 8; the quinone oxidoreductase is the enzyme comprising the sequence of SEQ ID NO: 45; the 2-keto acid decarboxylase is the enzyme comprising the sequence of SEQ ID NO: 83; and the primary alcohol dehydrogenase is the enzyme comprising the sequence of SEQ ID NO: 70.

[0139] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are heterologously expressed by one or more non-naturally occurring microbial organisms.

[0140] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are heterologously expressed by two or more non-naturally occurring microbial organisms.

[0141] In some embodiments, one or more of the hydratase-aldolase, the quinone oxidoreductase, the 2-keto acid decarboxylase, and the primary alcohol dehydrogenase further comprise one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0142] In some embodiments, the method for producing 1,5-pentanediol further comprises or consists essentially of isolating the 1,5-pentanediol from the one or more non-naturally occurring microbial organisms or cultures comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of isolating the 1,5-pentanediol from the two or more non-naturally occurring microbial organisms or cultures comprising the two or more non-naturally occurring microbial organisms.

[0143] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy- hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1- reductase, and 6-hydroxyhexanal 1-reductase are expressed by one or more non- naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase are exogenously expressed by one or more non-naturally occurring microbial organisms.

[0144] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy- hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1- reductase, and 6-hydroxyhexanal 1-reductase are expressed by two or more non- naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase are exogenously expressed by two or more non-naturally occurring microbial organisms.

[0145] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme selected from the group of enzymes identified by EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate 1-reductase is an enzyme of EC number 1.2.99.6; and the 6-hydroxyhexanal 1-reductase is an enzyme of EC number 1.1.1.

[0146] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme selected from the group of enzymes identified with Uniprot ID No. T4VW93; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme selected from the group of enzymes identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93; the 6-hydroxyhexanoate 1-reductase is an enzyme identified with Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified with Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0147] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, or the sequence of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 6-hydroxyhexanoate 1-reductase is an enzyme comprising the sequence of SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68; and the 6-hydroxyhexanal 1-reductase is an enzyme comprising the sequence of SEQ ID NO: 70.

[0148] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63 or the sequence of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58;6-hydroxyhexanoate 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68; and 6-hydroxyhexanal 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 70.

[0149] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified with the following Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0150] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No.The enzyme identified by T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9 has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity; the 6-hydroxyhexanoate 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by Uniprot or GenBank ID No: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by Uniprot or GenBank ID No: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1.

[0151] In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2- reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase further comprise one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0152] In some embodiments, the method of producing 1,6-hexanediol further comprises or consists essentially of isolating the 1,6-hexanediol from one or more non-naturally occurring microbial organisms or cultures comprising one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of isolating the 1,6-hexanediol from two or more non-naturally occurring microbial organisms or cultures comprising two or more non-naturally occurring microbial organisms.

[0153] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by one or more non-naturally occurring microbial organisms.

[0154] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by two or more non-naturally occurring microbial organisms.

[0155] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

[0156] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0157] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0158] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0159] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 5, SEQ ID NO: 54, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0160] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase further comprise one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0161] In some embodiments, the method for producing 6-hydroxy-hexanoate further comprises or consists essentially of isolating the 6-hydroxy-hexanoate from one or more non-naturally occurring microbial organisms or cultures comprising one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of isolating the 6-hydroxy-hexanoate from two or more non-naturally occurring microbial organisms or cultures comprising two or more non-naturally occurring microbial organisms.

[0162] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6- hydroxyhexanoate dehydrogenase, and 6-oxo-hexanoate oxidase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2- oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl- CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxo-hexanoate oxidase are exogenously expressed by one or more non-naturally occurring microbial organisms.

[0163] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6- hydroxyhexanoate dehydrogenase, and 6-oxo-hexanoate oxidase are expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2- oxohexanoate-2-reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6- hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxo- hexanoate oxidase are exogenously expressed by two or more non-naturally occurring microbial organisms.

[0164] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA- transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; the 6- hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate dehydrogenase is an enzyme of EC number 1.1.1.258; and the 6-oxo-hexanoate oxidase is an enzyme of EC number 1.2.1.63.

[0165] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925 and Q5U923; or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified with Uniprot ID No. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase is an enzyme identified with Uniprot ID No. Q9R2F4.

[0166] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No.The enzymes identified by Q9R2F4 have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity.

[0167] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified comprising the sequence of SEQ ID NO: 71 or SEQ ID NO: 72; and the 6-oxo-hexanoate oxidase is an enzyme comprising the sequence of SEQ ID NO: 75.

[0168] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 71 and SEQ ID NO: 72 as identified.and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 75.

[0169] In some embodiments, wherein one or more of the 6-hydroxy-2-oxohexanoate-2- reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxo-hexanoate oxidase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0170] In some embodiments, the method of producing adipic acid further comprises or consists essentially of isolating the adipic acid from the one or more non-naturally occurring microbial organisms or cultures comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of isolating the adipic acid from the two or more non-naturally occurring microbial organisms or cultures comprising the two or more non-naturally occurring microorganisms.

[0171] In some embodiments, the pyruvate is produced from a carbon source selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof. In some embodiments, is 3-hydroxy-propanal. In some embodiments, the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase exogenously expressed by the one or more non-naturally occurring microbial organisms.

[0172] In another aspect, provided herein is a recombinant microbial organism comprising a first exogenous nucleic acid encoding an aldolase hydratase, wherein the recombinant microbial organism is further modified to express an increased amount of a quinone oxidoreductase as compared to a wild-type or unmodified same microbial organism, and optionally wherein the microbial organism is Corynebacterium glutamicum, Clostridium sp., or E. coli. In some embodiments, the organism comprises a second exogenous nucleic acid encoding a quinone oxidoreductase. In some embodiments, the first and / or second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. Alternatively, the first and second nucleic acids are under the control of the same promoter regulatory element. In some embodiments, the regulatory element is selected from a promoter or an enhancer. In some embodiments, the aldolase hydratase has EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the aldolase hydratase is an enzyme selected from the group of enzymes identified with the following Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.In some embodiments, the aldolase hydratase is an enzyme selected from the group of enzymes identified by the following Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the aldolase hydratase is an enzyme comprising a sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0173] In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in a vector, e.g., a plasmid or a viral vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in their own separate vector. In some embodiments, the vector is a plasmid. In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0 or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. In some embodiments, the recombinant microbial organism is capable of producing a 2-ketocarboxylic acid of the formula:

[0174]

[0175] wherein R is H, CH3, or CH2OH. In some embodiments, the recombinant microbial organism is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid. In some embodiments, the recombinant microbial organism is genetically modified to increase production of pyruvate from a carbon source. In some embodiments, the carbon source is selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof.

[0176] In another aspect, provided herein is a culture comprising a recombinant microbial organism disclosed herein.

[0177] In another aspect, provided herein is a population of recombinant microbial organisms as disclosed herein. In some embodiments, the population is substantially homogeneous.

[0178] In another aspect, provided herein is a culture comprising a population disclosed herein.

[0179] In another aspect, provided herein is a method for producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid comprising culturing a population or microbial organism as disclosed herein under conditions that promote expression of an exogenous nucleic acid as disclosed herein. In one aspect, the exogenous nucleic acid is overexpressed as compared to a wild type or unmodified counterpart microbial organism. In some embodiments, the method further comprises isolating 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid from the culture or microbial organism. BRIEF DESCRIPTION OF DRAWINGS

[0180] Figure 1 A two-enzyme biosynthetic pathway for producing 2-ketocarboxylic acids from pyruvate and an aldehyde is shown as an example. The aldol dehydration product (e.g., an aldol condensation product described herein) can be produced by a single enzyme (e.g., an aldol dehydration product biosynthetic polypeptide, such as a hydratase-aldolase (in some embodiments, referred to as Ads-Hyd)) by steps 1 and 2 as depicted, without intending to be limited by theory. As will be appreciated by one of skill in the art, the double bond in the aldol condensation product shown can exist as E or Z. In many embodiments, step 3 as shown can be catalyzed by an oxidoreductase, such as one belonging to EC 1.6.5 (e.g., EC 1.6.5.5), which utilizes NADH and / or NADPH to reduce quinones. As described herein, a variety of aldehydes can be utilized. For example, in some embodiments the aldehyde shown, R is H, CH3, CH2CH3, OH, CH2OH, or CH2CH2OH.

[0181] Figure 2 A biosynthetic pathway for producing 1,5-pentanediol via a 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate is shown. As used herein, 3HPA refers to 3-hydroxy- propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexanoate; and 5HPeA refers to 5-hydroxy- pentanal. NADH is depicted as a cofactor for many of the reduction steps in this pathway for illustrative purposes. NADPH or NADH can both be cofactors.

[0182] Figure 3A biosynthetic pathway to 1,6-hexanediol via a 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate is shown. As used herein, 3HPA refers to 3-hydroxy- propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexanoate; 6H2HH refers to 2,6-dihydroxy- hexanoate; 6HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6- hydroxyhexanoate; 6H2HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA; and 6HHA refers to 6-hydroxyhexanal. Either NADPH or NADH can be the cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase. For illustrative purposes, 6HH-CoA is depicted as the donor for the step 5 reaction and 6H2HH is depicted as the acceptor. Other CoA esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

[0183] Figure 4 A biosynthetic pathway to 6-hydroxyhexanoate via a 6-hydroxy-2-keto- hexanoate (6H2KH) intermediate is shown. As used herein, 3HPA refers to 3- hydroxy-propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexanoate; 6H2HH refers to 2,6- dihydroxy-hexanoate; 6HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6-hydroxyhexanoate; 6H2HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA. Either NADPH or NADH can be the cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase. For illustrative purposes, 6HH-CoA is depicted as the donor for the step 5 reaction and 6H2HH is depicted as the acceptor. Other CoA esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

[0184] Figure 5 A biosynthetic pathway to adipic acid via a 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate is shown. As used herein, 3HPA refers to 3-hydroxy- propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexanoate; 6H2HH refers to 2,6-dihydroxy- hexanoate; 6HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6- hydroxyhexanoate; 6H2HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA; 6KHA refers to 6-oxo-hexanoate. Either NADPH or NADH can be the cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase. For illustrative purposes, 6HH-CoA is depicted as the donor for the step 5 reaction and 6H2HH is depicted as the acceptor. Other CoA esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

[0185] Figure 6 Activity of quinone oxidoreductase-1 (Qor-1) to reduce 6-hydroxy-3,4-dehydro-2-keto-hexanoic acid to 6-hydroxy-2-keto-hexanoic acid is shown in the presence of the co-factors NADH and NADPH. DETAILED DESCRIPTION

[0186] DEFINITIONS

[0187] As used herein, certain terms can have the following defined meanings. As used herein, the singular forms "a", "an" and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0188] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" shall have the meaning ascribed in U.S. Patent law. "Consisting of" shall mean excluding more than trace elements of other ingredients. Aspects defined by each of these transition terms are within the scope of the present disclosure. Thus, it is intended that the methods and compositions can include additional steps and components (comprising), or alternatively, exclude steps and components not specifically listed (consisting essentially of) or further exclude steps and components not specifically listed (consisting of).

[0189] As used herein, the term "aldol dehydration product biosynthetic polypeptide" refers to a polypeptide involved in the synthesis of an aldol dehydration product as described herein. In some embodiments, an aldol dehydration product biosynthetic polypeptide can be or comprise an aldolase polypeptide, a hydratase, a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol dehydration product biosynthetic polypeptide can be or comprise a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol dehydration product biosynthetic polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference aldol dehydration biosynthetic polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol dehydration biosynthetic polypeptide shares characteristic sequence elements and / or overall percent identity with a suitable reference aldol dehydration biosynthetic polypeptide (e.g., as found in nature and / or as presented herein (e.g., in one or more relevant tables (e.g., Tables 1 and 5-8)) or a portion thereof (e.g., a portion of amino acid residues (which can be contiguous or isolated) that facilitate a relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or group).

[0190] As used herein, “aldol dehydration product” refers to a compound comprising an aldehyde group or a ketone group and a double bond conjugated to the aldehyde group or ketone group. In some embodiments, the aldol dehydration product is a compound of Formula P-2 or a salt thereof.

[0191] As used herein, the term “aldol product” refers to a compound comprising an aldehyde group or a ketone group and a hydroxyl group attached to the beta-carbon of the carbonyl group of the aldehyde or ketone. In some embodiments, the aldol product is the product of an aldol reaction. In some embodiments, the aldol product has the structural formula P-1 or a salt thereof.

[0192] As used herein, the term “aldol product biosynthetic polypeptide” refers to a polypeptide involved in the synthesis of an aldol product as described herein. In some embodiments, the aldol product biosynthetic polypeptide can be or comprise an aldolase polypeptide, a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, the aldol product biosynthetic polypeptide is or comprises an aldolase polypeptide as described herein. In some embodiments, the aldol product biosynthetic polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference aldol biosynthetic polypeptide found in nature). Alternatively or additionally, in some embodiments, the aldol biosynthetic polypeptide shares characteristic sequence elements and / or overall percent identity with a suitable reference aldol biosynthetic polypeptide (e.g., as found in nature and / or as presented herein (e.g., in one or more relevant tables)) or a portion thereof (e.g., a portion of amino acid residues (which can be contiguous or isolated) that facilitate a relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or a group).

[0193] As used herein, the term “olefin reduction product biosynthetic polypeptide” refers to a polypeptide involved in the conversion of a double bond to a single bond (and formation of an olefin reduction product) as described herein. In some embodiments, the olefin reduction product biosynthetic polypeptide can be or comprise a quinone oxidoreductase as described herein. In some embodiments, the olefin reduction product biosynthetic polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference olefin reduction biosynthetic polypeptide found in nature). Alternatively or additionally, in some embodiments, the aldol biosynthetic polypeptide shares characteristic sequence elements and / or overall percent identity with a suitable reference aldol biosynthetic polypeptide (e.g., as found in nature and / or as presented herein (e.g., in one or more relevant tables)) or a portion thereof (e.g., a portion of amino acid residues (which can be contiguous or isolated) that facilitate a relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or a group).

[0194] As used herein, the term "aliphatic" refers to a straight chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain which is completely saturated or which contains one or more units of unsaturation; or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring (which is completely saturated or which contains one or more units of unsaturation (but not aromatic), or a combination thereof. In some embodiments, an aliphatic group contains 1 to 50 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 5 aliphatic carbon atoms, while in yet other embodiments, an aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl groups.

[0195] As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl group has 1 to 100 carbon atoms. In certain embodiments, straight-chain or branched alkyl groups have about 1 to 20 carbon atoms in their backbone (e.g., C1-C20for straight-chain, and C3-C20for branched-chain alkyl groups). In certain embodiments, straight-chain or branched alkyl groups have about 1 to 10 carbon atoms in their backbone. In some embodiments, a cycloalkyl group has from about 3 to 10 carbon atoms in their ring structure, with such rings being single, double, or triple ring structures, and either 20 ; for branched-chain, C2-C 20 ), or about 1 to 10. In some embodiments, an alkyl group can be lower alkyl, wherein lower alkyl groups contain 1 to 4 carbon atoms (e.g., C1-C4for straight-chain lower alkyl groups).

[0196] As used herein, the term "aryl," used alone or as part of a larger moiety, e.g., in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, the aryl group is a biaryl group. The term "aryl" can be used interchangeably with the term "aromatic ring." In certain embodiments of the disclosure, "aryl" refers to an aromatic ring system, including but not limited to phenyl, biphenyl, naphthyl, binaphthyl, anthryl, and the like, which can bear one or more substituent groups. Groups wherein an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthylidene, phenanthridinyl, or tetrahydronaphthyl, and the like, are also within the scope of the term "aryl."

[0197] As used herein, the terms "alicyclic," "carbocyclic," "carbocyclyl," "carbocyclic group," and "carbocycle" are used interchangeably and refer to a saturated or partially unsaturated but non-aromatic monocyclic, bicyclic, or polycyclic ring system having, unless otherwise specified, 3 to 30 ring members, as described herein. A cycloaliphatic group includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic group has 3 to 6 carbons. In some embodiments, the cycloaliphatic group is saturated and is a cycloalkyl group. The term "cycloaliphatic" can also include aliphatic rings that are fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, the cycloaliphatic group is bicyclic. In some embodiments, the cycloaliphatic group is tricyclic. In some embodiments, the cycloaliphatic group is polycyclic. In some embodiments, "cycloaliphatic" refers to a C3-C6monocyclic hydrocarbon, or a C8-C 10 bicyclic or polycyclic hydrocarbon that is fully saturated or that contains one or more units of unsaturation, except it is not aromatic, which has a single point of attachment to the rest of the molecule, or a C9-C 16 polycyclic hydrocarbon that is fully saturated or that contains one or more units of unsaturation, except it is not aromatic, which has a single point of attachment to the rest of the molecule.

[0198] As used herein, the term "heteroaliphatic" is given its ordinary meaning in the art and refers to aliphatic groups as described herein, wherein one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH2, and CH3 are independently replaced with one or more heteroatoms, including oxidized and / or substituted forms thereof. In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.

[0199] As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to alkyl groups as described herein, wherein one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.

[0200] As used herein, the terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety, e.g., "heteroaralkyl," or "heteroaralkoxy," refer to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one of the aromatic ring atoms is a heteroatom. In some embodiments, the heteroaryl group is a group having 5 to 10 ring atoms (i.e., a monocyclic, bicyclic, or polycyclic ring), in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, the heteroaryl group has 6, 10, or 14 p-electrons shared in a cyclic array; and in addition to carbon atoms, one to five heteroatoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, triazolyl, triazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, the heteroaryl group is a heterobiaryl, such as bipyridyl and the like. As used herein, the terms "heteroaryl" and "heteroar- " also include groups in which a heteroaromatic ring is fused to one or more aryl, aliphatic or heterocyclyl rings, where the radical or point of attachment is at a carbon of the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl, phenazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyridino[2,3-b]-l,4-oxazinyl. oxazinyl, Azine-3(4H)-one. The heteroaryl group can be monocyclic, bicyclic, or polycyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl," or "heteroarylene," any of which includes the optionally substituted ring. The term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl portions are optionally substituted independently.

[0201] As used herein, the term "heteroatom" refers to an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen, phosphorus, sulfur, or oxygen (e.g., quaternized forms, such as those in imine groups)). In some embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0202] As used herein, the terms “heterocyclic,” “heterocyclic group,” “heterocyclic group,” and “heterocyclic” are used interchangeably to refer to a monocyclic, bicyclic, or polycyclic ring portion (e.g., 3 to 30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, the heterocyclic group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic portion that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When used to refer to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, nitrogen can be N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or + NR (e.g., in N-substituted pyrrolidinyl groups). The heterocycle can be attached to its side group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, etc. azolealkyl, piperazine, di Alkyl, dioxacyclopentyl, diaza Basic, oxygen and nitrogen Basic, sulfur-nitrogen Heterocyclic, morpholino, and quinine cyclic groups. The terms “heterocyclic,” “heterocyclic group,” “heterocyclic ring,” “heterocyclic moiety,” and “heterocyclic group” are used interchangeably herein and also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl, or alicyclic rings, such as dihydroindolyl, 3H-indolyl, chromyl, phenanthrenediyl, or tetrahydroquinolinyl. Heterocyclic groups can be monocyclic, bicyclic, or polycyclic. The term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl and heterocyclic moiety are optionally substituted independently.

[0203] Optionally substituted: As described herein, the chemical entities of this disclosure, such as various compounds, may comprise optionally substituted and / or substituted portions. Generally, the term “substituted” means that one or more hydrogens of the specified portion are substituted by a suitable substituent. Unless otherwise stated, an “optionally substituted” group may have a suitable substituent at each substituted position of the group, and the substituent may be the same or different at each position when more than one position in any given structure can be substituted by more than one substituent selected from a particular group. In some embodiments, the optionally substituted group is substituted. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated in this disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term “stable” means a compound that remains substantially unchanged when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more purposes disclosed herein. Some substituents are described below.

[0204] A suitable monovalent substituent on a substituted atom, such as a suitable carbon atom, is independently a halogen; -(CH2) 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0-4 Ph, which can be R o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be R o Substitution; -CH=CHPh, which can be replaced by R o Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl, which can be R o replace;

[0205] -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(Ro )C(O)NR o 2; -N(R o )C(S)NR o 2; -(CH2) 0-4 N(R o )C(O)OR o ; -N(R o )N(R o )C(O)R o ; -N(R o )N(R o )C(O)NR o 2; -N(R o )N(R o )C(O)OR o ; -(CH2) 0-4 C(O)R o ; -C(S)R o ; -(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 C(O)SR o ; -(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ; -OC(O)(CH2) 0-4 SR o , -SC(S)SR o ; -(CH2) 0-4 SC(O)R o ; -(CH2) 0-4 C(O)NR o 2; -C(S)NR o 2; -C(S)SR o ; -(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR o )R o ; -C(O)C(O)R o ; -C(O)CH2C(O)R o ; -C(NOR o )R o ; -(CH2) 0-4 SSR o ; -(CH2) 0-4 S(O)2R o ; -(CH2) 0-4 S(O)2OR o ; -(CH2) 0-4 OS(O)2Ro -S(O)2NR o 2; -(CH2) 0-4 S(O)R o -N(R o )S(O)2NR o 2; -N(R o )S(O)2R o -N(OR o )R o -C(NH)NR o 2; -Si(R o )3; -OSi(R o )3; -B(R o )2; -OB(R o )2; -OB(OR o )2; -P(R o )2; -P(OR o )2; -P(R o )(OR o ); -OP(R o )2; -OP(OR o )2; -OP(R o )(OR o ); -P(O)(R o )2; -P(O)(OR o )2; -OP(O)(R o )2; -OP(O)(OR o )2; -OP(O)(OR o )(SR o ); -SP(O)(R o )2; -SP(O)(OR o )2; -N(R o )P(O)(R o )2; -N(R o )P(O)(OR o )2; -P(R o )2[B(R o )3]; -P(OR o )2[B(R o )3]; -OP(R o )2[B(R o )3]; -OP(OR o )2[B(R o )3];

[0206] -(C 1-4 straight-chained or branched alkylene)O-N(R o )2; or -(C 1-4 straight-chained or branched alkylene)S(O)2NRstraight or branched alkylene)C(O)O-N(R o )2, wherein each R o may be substituted as defined herein and is independently hydrogen, C 1-20 aliphatic, Ci-20heteroaliphatic having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, -CH2-(C6-i4 aryl), -O(CH2) 0-1 (C6-i4 aryl), -CH2-(5- to 14-membered heteroaryl ring), a 5- to 20-membered monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, or, notwithstanding the definition above, two independent occurrences of R o , together with their intervening atoms, form a 5- to 20-membered monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which can be substituted as defined below.

[0207] R o (or the ring formed by taking two independent occurrences of R o , together with their intervening atoms) is a suitable monovalent substituent on the ring and is independently halogen,

[0208] -(CH2) 0-2 R · , - (haloR · ), -(CH2) 0-2 OH, -(CH2) 0-2 OR · , -(CH2) 0-2 CH(OR · )2; -O(haloR · ), -CN, -N3, -(CH2) 0-2 C(O)R · , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR · , -(CH2) 0-2 SR · , -(CH2) 0- 2SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR · , -(CH2) 0-2 NR · 2, -NO2, -SiR · 3, -OSiR · 3, -C(O)SR · , -(C 1-4 straight or branched alkylene)C(O)OR· , or SSR · , or SSR · each is unsubstituted or, if preceded by the prefix "halo," substituted only with one or more halogen, and is independently selected from the group consisting of C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, and a 5-6 membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. R o Suitable divalent substituents on a saturated carbon atom of R

[0209] Suitable divalent substituents, for example on a suitable carbon atom, are independently =0, =s, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2- 3O-, or -S(C(R * 2)) 2-3 S-, wherein each independently occurring R* is selected from the group consisting of hydrogen; C 1-6 aliphatic, which can be substituted as defined below; and an unsubstituted 5-6 membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents bound to an ortho- substituted carbon of an "optionally substituted" group include: -O(CR*2) 2-3 O-, wherein each independently occurring R* is selected from the group consisting of hydrogen; C1-6 aliphatic, which can be substituted as defined below; and an unsubstituted 5-6 membered saturated, partially unsaturated, and aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0210] Suitable substituents on the aliphatic group of R* are independently halogen, -R · , -(haloR · ), -OH, -OR · , -O(haloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2, or -NO2 wherein each R · is unsubstituted or, if preceded by the prefix "halo," substituted only with one or more halogen, and is independently selected from the group consisting of C 1-4aliphatic, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0211] In some embodiments, suitable substituents on the replaceable nitrogen are independently wherein each is independently hydrogen; C 1-6 aliphatic, which can be substituted as defined below; unsubstituted -OPh; or unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of together with their intervening atoms form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0212] suitable substituents on the aliphatic group of -C(O)OR · , -(haloR · ), -OH, -OR · , -O(haloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2, or -NO2, wherein each R · is unsubstituted or, when preceded by the designation “halo,” substituted only with one or more halogens, and is independently selected from C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0213] As used herein, the term “partially unsaturated” means a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to include rings that have multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as these terms are defined herein.

[0214] “Wild type” defines a cell, composition, tissue, or other biological material that occurs in nature.

[0215] In some embodiments, the 3-hydroxy-propanal and pyruvic acid are prepared from one or more of glycerol, C5 sugars, C6 sugars, phosphoglycerate, other carbon sources, intermediates of the glycolysis pathway, and combinations thereof. In some embodiments, the C5 sugars comprise, or alternatively consist essentially of, or further consist of one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose, and the C6 sugars comprise, or alternatively consist of, or further consist of, allose, altrose, glucose, mannose, gulose, idose, talose, fructose, psicose, sorbose, and tagatose. In some embodiments, the other carbon sources are feedstocks suitable as carbon sources for microorganisms, wherein the feedstocks comprise, or alternatively consist essentially of, or further consist of one or more of amino acids, lipids, corn stover, miscanthus, municipal waste, energy cane, sugar cane, bagasse, starch streams, dextrose streams, formic acid, methanol, and combinations thereof.

[0216] As used herein, the term "C5 sugar" refers to a sugar molecule containing 5 carbons.

[0217] As used herein, the term "C6 sugar" refers to a sugar molecule containing 6 carbons.

[0218] In some embodiments, the term "aldol addition" refers to a chemical reaction in which an aldehyde functional group of a pyruvic acid molecule reacts with an aldehyde functional group of a 3-hydroxy-propanal to form a C N 4-hydroxy-2-keto-carboxylic acid intermediate. In some embodiments, the C N+3 4-hydroxy-2-keto-carboxylic acid intermediate is 4,6-dihydroxy-2-keto-hexanoic acid. N aldehyde is 3-hydroxy-propanal, the C N+3 4-hydroxy-2-keto-carboxylic acid intermediate is 4,6-dihydroxy-2-keto-hexanoic acid.

[0219] In some embodiments, the term "aldol condensation" refers to a chemical reaction in which an aldehyde functional group of a pyruvic acid molecule reacts with an aldehyde functional group of a 3-hydroxy-propanal to form a C N 4-hydroxy-2-keto-carboxylic acid. In some embodiments, the C N+3 4-hydroxy-2-keto-carboxylic acid. In some embodiments, the C N aldehyde is 3-hydroxy-propanal, the C N+3 4-hydroxy-2-keto-carboxylic acid is 6-hydroxy-3,4-dehydro-2-keto-hexanoic acid.

[0220] As used herein, the term "solution" refers to a liquid composition comprising a solvent and a solute, such as the starting materials used in the methods described herein. In some embodiments, the solvent is water. In some embodiments, the solvent is an organic solvent.

[0221] As used herein, the term "enzymatic step" or "enzymatic reaction" refers to a molecular reaction catalyzed by an enzyme selected to facilitate a desired enzymatic reaction. Enzymes are biological macromolecules and highly selective catalysts. Most enzymes are proteins, but a few catalytic RNA molecules have been identified.

[0222] Throughout the application, enzymatic steps can be denoted as "Step 1", "Step 2", etc., and enzymes that specifically catalyze these steps are denoted as "1", "2", etc., respectively. Such enzymes are also referred to as "reaction-specific enzymes".

[0223] As used herein, the term "CoA" or "CoA" is intended to mean an organic cofactor or prosthetic group (non-protein part of an enzyme) whose presence is required for the activity of many enzymes to form an active enzyme system.

[0224] As used herein, the term "substantially anaerobic" when used in reference to culture or growth conditions is intended to mean an amount of oxygen that is less than about 10% of the saturation of dissolved oxygen in the liquid culture medium. The term is also intended to include a sealed chamber of liquid or solid media maintained in an atmosphere of less than about 1% oxygen.

[0225] As used herein, the term "non-naturally occurring" or "non-native" when used in reference to a microbial organism or microorganism of the disclosure is intended to mean that the microbial organism has at least one genetic alteration that is not normally found in a natural strain of the reference species, including a wild-type strain of the reference species. Genetic alterations include, for example, but are not limited to, modifications that introduce expressible nucleic acids encoding polypeptides, other nucleic acid additions, nucleic acid deletions, and / or other functional disruptions of the genetic material of the microbial organism. Such modifications include, for example, but are not limited to, coding regions and functional fragments thereof, heterologous, homologous, or heterologous and homologous polypeptides to the reference species. Additional modifications include, for example, but are not limited to, non-coding regulatory regions, modifications in which alter the expression of a gene or operon.

[0226] As used herein, "exogenous" is intended to mean the introduction of the referenced molecule or the activity into a host microbial organism. For example, a molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material, for example by integration into the host chromosome, or as non-chromosomal genetic material, for example a plasmid. Thus, when used in reference to the expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid into the microbial organism in a form that is expressible. When used in reference to an enzyme activity, the term refers to the activity introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the reference activity upon introduction into the host microbial organism. Thus, the term "endogenous" refers to a reference molecule or activity that is originally or naturally present in the wild-type host. Similarly, when used in reference to the expression of an encoding nucleic acid, the term refers to the expression of the encoding nucleic acid comprised in the wild-type microorganism.

[0227] The term "heterologous" refers to a molecule or activity that is derived from a source other than the reference species, while "homologous" as used herein refers to a molecule or activity that is derived from the host microbial organism. Thus, exogenous expression of an encoding nucleic acid can utilize one or both of a heterologous or homologous encoding nucleic acid.

[0228] It should be understood that when more than one exogenous nucleic acid is comprised in a microbial organism, more than one exogenous nucleic acid refers to the referenced encoding nucleic acid or enzyme activity, as discussed above. It should also be understood that more than one exogenous nucleic acid can be introduced into a host microbial organism for separate nucleic acid molecules, multi-cistronic nucleic acid molecules, or combinations thereof, and still be considered more than one exogenous nucleic acid, as disclosed herein. For example, as disclosed herein, a microbial organism can be engineered to express two or more exogenous nucleic acids that encode enzymes or proteins of a desired pathway. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it should be understood that the two exogenous nucleic acids can be introduced as a single nucleic acid (e.g., 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 two exogenous nucleic acids. Similarly, it should be understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination (e.g., 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 two or more exogenous nucleic acids, e.g., three exogenous nucleic acids. Thus, the amount of referenced exogenous nucleic acid or enzyme activity refers to the amount of encoding nucleic acid or the amount of enzyme activity, not the amount of individual nucleic acids introduced into the host organism.

[0229] In some embodiments, exogenous expression of the encoding nucleic acids is used. Exogenous expression confers the ability to the host and the application to tailor expression and / or regulatory elements to achieve desired expression levels controlled by the user. However, endogenous expression can also be used in other embodiments, for example by removing negative regulatory effectors or induction of a gene promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene with a naturally occurring inducible promoter can be upregulated by providing a suitable inducer, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, allowing for the regulation of increased expression of the endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

[0230] Those skilled in the art will appreciate that genetic alterations are described with reference to a suitable host organism, such as E. coli, and its corresponding metabolic reactions or suitable source organisms for the desired genetic material, such as genes for a desired biosynthetic pathway. However, given the high level of technology in the field of genomics and the complete genome sequencing of a variety of organisms, those skilled in the art will be able to readily apply the teachings and guidance provided herein to substantially all other organisms. For example, the E. coli metabolic alterations exemplified herein can be readily applied to other species by incorporating the same or analogous encoding nucleic acids from species other than the reference species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and particularly, orthologs, paralogs, or non-orthologous gene displacement.

[0231] Nucleic acid sources encoding enzymes of the pathway can include, for example, any species in which the encoded gene product is capable of catalyzing a reference reaction. Such species include both prokaryotic and eukaryotic organisms, including but not limited to bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plants, insects, animals, and mammals, including humans. Exemplary species of such sources include, for example, Escherichia coli, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas fluorescens, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces 2065, Pseudomonas aeruginosa, Ralstonia eutropha, Clostridium acetobutylicum, Euglena gracilis, Treponema denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter ADP1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium thermoaceticum (thermoaceticum), Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, and the like.Mobilis), Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp. M62 / 1, Fusobacterium nucleatum, and other exemplary species disclosed herein or available as source organisms for the corresponding genes (see Examples). However, as there are now over 400 microbial genomes and complete genome sequences for a variety of yeast, fungi, plants, and mammalian genomes, it is routine and well known in the art to identify genes encoding essential pathway enzymes, including, for example, homologs, orthologs, paralogs, and non-orthologous gene displacement of known genes, for one or more genes in related or distant species, as well as the exchange of genetic changes between organisms.

[0232] Orthologs refer to genes in different species that have evolved from a common ancestral gene through speciation. Typically, orthologs retain the same function over the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

[0233] Paralogs refer to genes that are related through duplication within a genome. While orthologs typically retain the same function over the course of evolution, paralogs can evolve new functions, even if these functions are related to the original function.

[0234] Paralog replacement is a paralog gene from one species that can replace the function of a reference gene in a different species. Replacement includes, for example, the ability to perform substantially the same or similar function in the source species as compared to the reference function in the different species. While generally, a paralog replacement will be identified as structurally related to the known gene encoding the reference function, genes and their corresponding gene products that are less structurally related but functionally similar will still fall within the meaning of the term as used herein. For example, functional similarity requires that the active site or binding region of the paralog gene product has at least some structural similarity as compared to the gene encoding the function sought to be replaced. Thus, paralogs include, for example, orthologs or unrelated genes.

[0235] As used herein, the terms "microorganism" or "microbial organism" or "microbe" are used interchangeably to refer to living organisms and isolated prokaryotic or eukaryotic cells that can be transformed or transfected by insertion of exogenous or recombinant nucleic acids (e.g., DNA or RNA). Any suitable prokaryotic or eukaryotic microorganism can be used in the present disclosure so long as it remains viable after transformation with a nucleic acid sequence. Suitable microorganisms of the present disclosure are microorganisms that are capable of expressing one or more nucleic acid constructs that encode one or more recombinant proteins that can catalyze at least one step in a method. Microorganisms can be selected from bacteria, yeast, fungi, molds, and archaea. These are commercially available.

[0236] As used herein, "fungi" refers to any eukaryotic organism classified within the kingdom Fungi. Phyla within the kingdom Fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. As used herein, "yeast" refers to fungi that grow in single cell form (e.g., by budding), while "mold" refers to fungi that grow in filaments made of multicellular hyphae or mycelia (McGinnis, M. R. and Tyring, S. K. "Introduction to Mycology." Medical Microbiology. 4th ed. Galveston: Univ. of TX Medical Branch at Galveston, 1996). th ed. Galveston: Univ. of TX Medical Branch at Galveston, 1996).

[0237] In some embodiments, the microorganism is a yeast cell. In some embodiments, the yeast cell is from a Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species.

[0238] In some embodiments, the microorganism is a mold cell. In some embodiments, the mold host cell is from a Neurospora, Trichoderma, Aspergillus, Fusarium, or Chrysosporium species.

[0239] In some embodiments, the microorganism is an archaeon. In some embodiments, the suitable archaeon is from an Archaeoglobus, Aeropyrum, Halobacterium, Pyrobaculum, Pyrococcus, Sulfolobus, Methanococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, Methanocaldococcus, or Methanosarcina species.

[0240] The term "bacteria" refers to any microorganism within the domain or kingdom of prokaryotic organisms. Phyla within the bacterial domain or kingdom include Acidobacteria, Actinobacteria, Actinobacillus, Agrobacterium, Anaerobiospirrulum, Aquificae, Armatimonadetes, Bacteroidetes, Burkholderia, Caldiserica, Chlamydiae, Chlorobi, Chlorella, Chloroflexi, Chrysiogenetes, Citrobacter, Clostridium, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Enterobacter, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Geobacillus, Gemmatimonadetes, Gluconobacter, Halanaerobium, Klebsiella, Kluyvera, Lactobacillus, Lentisphaerae, Methylobacterium, Nitrospira, Pasteurellaceae, Paenibacillus, Planctomycetes, Propionibacterium, Pseudomonas, Proteobacteria, Ralstonia, Schizochytrium, Spirochaetes, Spirochaetes, Synergistetes, Tenericutes,Thermoanaerobacterium, Thermodesulfobacteria, Thermotogae, Verrucomicrobia, Zobellella, and Zymomonas. In some embodiments, the bacterial microorganism is an Escherichia coli cell. In some embodiments, the bacterial microorganism is a Bacillus cell. Examples of Bacillus species include, but are not limited to, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus licheniformis.

[0241] The carboxylic acid compounds produced by the methods of the present disclosure can form salts with counterions, including but not limited to metal ions, such as alkali metal ions, for example sodium, potassium, alkaline earth metal ions, for example calcium, magnesium, or aluminum ions; or coordinate with organic bases such as tetraalkylammonium, ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, and the like. The acid can form a salt with a counterion or organic base present in the reaction conditions, or can be converted to a salt by reaction with an inorganic or organic base.

[0242] Any carboxylic acid-containing compound herein is referred to as an acid or salt, which are used interchangeably throughout to refer to the compound in any of its neutral or ionized form, including any salt form thereof. The skilled artisan appreciates that the particular form will depend on the pH.

[0243] Solvates of a compound are solid forms of the compound that contain less than one, one, or more than one solvent molecule in the crystal lattice when crystallized. Some examples of solvents that can be used to produce solvates (e.g., pharmaceutically acceptable solvates) include, but are not limited to, water, the common C1-C6 alcohols (e.g., methanol, ethanol, isopropanol, butanol, and can optionally be substituted), tetrahydrofuran, acetone, ethylene glycol, propylene glycol, acetic acid, formic acid, and mixtures thereof. Other such biocompatible solvents that can be helpful in making pharmaceutically acceptable solvates are well known in the art. In addition, a wide variety of organic and inorganic acids and bases can be added to produce the desired solvate. Such acids and bases are known in the art. When the solvent is water, the solvate can be referred to as a hydrate. In some embodiments, one molecule of a compound can form a solvate with 0.1 to 5 molecules of solvent, such as with 0.5 molecules of solvent (a hemi-solvate, such as a hemi-hydrate), one molecule of solvent (a mono-solvate, such as a monohydrate), and two molecules of solvent (a di-solvate, such as a dihydrate).

[0244] When referring to a compound for which multiple isomers exist (e.g., cis and trans isomers, as well as R and S isomers, or combinations thereof), the compound in principle includes all possible enantiomeric, diastereomeric, and cis / trans isomers of the compound that are useful in the methods of the disclosure.

[0245] For each species, any cell belonging to that species is considered a suitable microorganism of the disclosure. A host cell of any species can exist as it is isolated from nature, or it can contain any number of genetic modifications (e.g., a string of mutations, deletions, or recombined polynucleotides).

[0246] As used herein, the term “recombinant nucleic acid” or “recombinant polynucleotide” refers to a polymer of nucleic acids in which at least one of the following is true: (a) the nucleic acid sequence is foreign to (i.e., does not naturally occur in) a given microorganism; (b) the sequence can naturally occur in the given microorganism, but is present in an amount that is not natural (e.g., greater than expected); (c) the nucleic acid sequence comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, for case (c), the recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to produce a new functional nucleic acid.

[0247] In some embodiments, the recombinant polypeptides or proteins or enzymes of the disclosure can be encoded by genetic material that is part of one or more expression vectors. An expression vector comprises one or more polypeptide-encoding nucleic acids, and it can further comprise any desired elements that control expression of the nucleic acids, as well as any elements that enable the expression vector to replicate and be maintained within a given host cell. All of the recombinant nucleic acids can be present on a single expression vector, or they can be encoded by multiple expression vectors.

[0248] One or more expression vectors can be constructed to include one or more pathway- encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors suitable for the provided microbial host organisms include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into the host chromosome. In addition, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes can also be included, for example, to provide resistance to antibiotics or toxins, to complement auxotrophic deficiencies, or to provide a key nutrient not in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more exogenous coding nucleic acids are co-expressed, the two nucleic acids can be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the coding nucleic acids can be operably linked to one common expression control sequence or to different expression control sequences, for example, an inducible promoter and a constitutive promoter. Such vectors, which contain both a promoter and cloning sites to which a polynucleotide can be operably linked, are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). To optimize expression and / or in vitro transcription, it can be necessary to remove, add to, or alter the 5' and / or 3' untranslated portions of the cloned sequence to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that can interfere with expression at the transcriptional or translational levels. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the start codon to enhance expression.

[0249] Exogenous nucleic acid sequences involved in the synthetic pathways of desired compounds described herein can be stably or transiently introduced into host cells using techniques known in the art, including but not limited to conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasonic transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals, such as N-terminal mitochondrial or other targeting signals, which can be removed, if desired, prior to transformation into the prokaryotic host cell. For example, removal of the mitochondrial leader sequence results in increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, the genes can be expressed in the cytosol without addition of a leader sequence, or can be targeted to the mitochondria or other organelle by addition of appropriate targeting sequences (e.g., mitochondrial targeting or secretion signals appropriate for the host cell), or targeted for secretion. It will be appreciated that appropriate modifications to the nucleic acid sequences for removal or inclusion of targeting sequences can be incorporated into the exogenous nucleic acid sequences to impart desired properties. Furthermore, genes can be codon-optimized for optimal expression of the protein using techniques known in the art.

[0250] All numerical designations, including numerical designations of ranges, for example, pH, temperature, time, concentrations, and molecular weight, are approximations. Variations (plus or minus) of less than 0.1 are included in the approximation. It should be understood that, although not always explicitly stated, the term "about" is always meant to be included in the description of all numerical designations. As used herein, "about" will mean up to plus or minus 10% of the indicated value. It is also to be understood that, although not always explicitly stated, reagents described herein are merely exemplary and equivalents thereof are known in the art.

[0251] "Operably linked" refers to a juxtaposition wherein the elements involved are in a relationship permitting them to function as intended.

[0252] The term "culturing" refers to the in vitro propagation of cells or organisms on or in a variety of media (cultures). It will be appreciated that progeny of a cell grown in culture can not be completely identical to the parent cell (i.e., in morphology, genetics, or phenotype) both for biological reasons and because of somatic mutations which can arise in culture.

[0253] A "gene" refers to a polynucleotide comprising at least one open reading frame (ORF) that, upon transcription and translation, is capable of encoding a particular polypeptide or protein. Any of the polynucleotide sequences described herein can be used to identify larger fragments or full-length coding sequences of the gene with which it is associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0254] The term "expression" refers to the production of a gene product. The term overexpression refers to the production of more mRNA transcribed from a gene or protein product encoded by the gene than in a normal or control cell, e.g., 0.5-fold, 1.0-fold, 1.5-fold or, alternatively, 2-fold, or, alternatively, at least 2.5-fold, or, alternatively, at least 3.0-fold, or, alternatively, at least 3.5-fold, or, alternatively, at least 4.0-fold, or, alternatively, at least 5-fold, or, alternatively, 10-fold, the level of expression detected in a control sample or wild-type cell.

[0255] As used herein, "homology" refers to sequence similarity between a reference sequence and at least one segment of a second sequence. Homologs can be identified by any method known in the art, preferably by comparing a reference sequence to a single second sequence or to a segment of a sequence or to a database of sequences using the BLAST tool. BLAST will compare sequences based on percent identity and similarity as described below.

[0256] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" means that two or more sequences are the same. Two sequences are "substantially identical" when they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity over a specified region, or, if not specified over the entire sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region) when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0257] Methods for sequence alignment for comparison are well known in the art. For example, a mathematical algorithm can be used to determine the percent sequence identity between any two sequences. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS 4: 117 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math. 2: 482 (1981); the search-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 2448 (1988); the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 90: 5873 5877 (1993).

[0258] For sequence comparison, typically one sequence acts as the reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the program parameters. When comparing two sequences, the sequences are not necessarily contiguous, but any gaps between sequences are taken into account in calculating the maximum identity match. For blastn, the default parameters are gap creation penalty = 5 and gap extension penalty = 2. For blastp, the default parameters are gap creation penalty = 11 and gap extension penalty = 1.

[0259] As used herein, "comparison window" includes reference to a segment of any one of the number of contiguous positions deemed to be meaningful for comparison purposes. Exemplary, non-limiting, comparison windows are between twenty and 600, commonly about 50 to about 200, more commonly about 100 to about 150 contiguous positions, where the terms "position" refers to a position in the sequence. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol 48(3):443-453 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci USA 85(8):2444-2448 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection [see, e.g., Brent et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringsbou Ed)].

[0260] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997) and Altschul et al., J. Mol Biol 215(3):403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words within the query sequence that either match or exceed the specified positive- valued threshold or cutoff score T when aligned with a word of the same length within a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc Natl Acad Sci USA 89(22): 10915-10919 (1992)) of 50, expectation value (E) of 10, M=5, N=-4, and a comparison of both strands.

[0261] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc Natl Acad Sci USA 90(12):5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0262] In addition to the percentage sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with an antibody raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical if two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that identical primers can be used to amplify the sequences.

[0263] The phrase "functionally equivalent protein" refers to a protein that hybridizes to an exemplary polynucleotide under stringent conditions and exhibits similar or enhanced biological activity in vivo compared to standard or control biological activity, e.g., more than 120%, or alternatively more than 110%, or alternatively more than 100%, or alternatively more than 90%, or alternatively more than 85%, or alternatively more than 80%. Other embodiments within the scope of the disclosure are identified by having more than 80%, or alternatively more than 85%, or alternatively more than 90%, or alternatively more than 95%, or alternatively more than 97%, or alternatively more than 98% or 99% sequence homology. Percent homology can be determined by a sequence comparison program run under appropriate conditions, e.g., BLAST. In some embodiments, the program is run under default parameters. In some embodiments, reference to an enzyme or protein includes functionally equivalent enzymes or proteins thereof.

[0264] A cell population is intended to be a collection of more than one cell that is either phenotypically and / or genotypically identical (clonal) or different. A substantially homogenous cell population is one that is at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical in phenotype, as measured by preselected markers.

[0265] When enzymes are referred to by reference to Enzyme Class (EC), the Enzyme Class is the classification of the enzyme according to, or in which the enzyme can be classified, according to the Enzyme Nomenclature provided by the International Union of Biochemistry and Molecular Biology. Also included are other suitable enzymes that have not yet been assigned to a particular class but can be so classified.

[0266] Non-naturally occurring microorganisms

[0267] The non-naturally occurring microorganisms provided herein are constructed using methods well known in the art, as exemplified herein, to exogenously express at least one nucleic acid encoding an enzyme or protein used in a biosynthetic pathway described herein in an amount sufficient to produce a compound, e.g., 2-ketovalerate, 2-ketohexanoate, 6-hydroxy-2-keto-hexanoate, 1,5-pentanediol, adipic acid, 1,6-hexanediol, or 6-hydroxyhexanoate.

[0268] Successful engineering of a microbial host capable of producing the desired products described herein involves the identification of a suitable set of enzymes with sufficient activity and specificity to catalyze each step in the pathway, such as those described in the examples and literature herein. Individual enzyme or protein activity from an exogenous DNA sequence can also be assayed using methods well known in the art. Furthermore, these enzymes can be engineered using modern protein engineering methods (Protein Engineering Handbook; Lutz S., & Bornscheuer U. T. Wiley-VCH Verlag GmbH & Co. KGaA: 2008; Vol. 1 & 2) such as directed evolution, rational mutagenesis, computational design (Zanghelini, A et al, 2008), or combinations thereof, for achieving desired substrate specificity, controlling stereoselectivity for synthesis of enantiomerically pure or racemic products, stabilizing enzymes for withstanding harsh industrial process conditions by improving half-life, thermal stability, inhibitor / product tolerance, and improving expression and solubility of enzymes in the desired selected microbial production host. Once the desired enzymes capable of catalyzing each step of the pathway are characterized, the genes encoding these enzymes will be cloned into the selected microorganism, fermentation conditions will be optimized and product formation will be monitored after fermentation. Once the enzymes are identified, the genes corresponding to one or more of the enzymes are cloned into the microbial host. In some embodiments, the genes encoding each enzyme of a particular pathway described herein are cloned into the microbial host.

[0269] Methods of introducing recombinant / exogenous nucleic acids / proteins into microorganisms and vectors suitable for this purpose are well known in the art. For example, a variety of techniques are described in Current Protocols in Molecular Biology, edited by Ausubel et al. (Wiley & Sons, New York, 1988, updated quarterly). Methods for transferring expression vectors into microbial host cells are well known in the art. The particular method and vector can vary depending on the type of microbial host desired. For example, bacterial host cells can be transformed by heat shock, calcium chloride treatment, electroporation, liposomes, or phage infection. Yeast host cells can be transformed by lithium acetate treatment (which can further include vector DNA and PEG treatment) or electroporation. The inclusion of these methods is for illustrative purposes only and is in no way limiting or comprehensive. Routine experimentation by one of ordinary skill in the art, in light of known methods, can be used to determine whether a particular expression vector or transformation method is suitable for a given microbial host. In addition, reagents and vectors suitable for a number of different microbial hosts are commercially available and well known in the art.

[0270] Methods of constructing, expressing or overexpressing enzymes in non-naturally occurring microbial hosts and testing expression levels in non-naturally occurring microbial hosts are well known in the art (Protein Expression Technologies: Current Status and Future Trends, Baneyx F., ed. Horizon Bioscience, 2004, Norfolk, UK; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)).

[0271] Methods for carrying out microbial fermentations are well known in the art. For example, a variety of techniques are described in Clark et al., eds., Biochemistry Engineering (CRC Press, 2nded. 1997). The specific method of fermentation can vary depending on the type of microbial host desired. Typically, the microorganism is grown in a suitable medium with a carbon source in batch or continuous fermentation mode. The use of agents known to modulate catabolite repression or enzyme activity can be used to enhance the production of adipic acid or glutaric acid. Suitable pH values for fermentation are between 3 and 10. Fermentation can be carried out under aerobic, anaerobic, or anoxic conditions, based on the needs of the microorganism. Fermentation can be carried out in batch, fed-batch, or continuous fashion. If desired, fermentation can also be carried out in two stages. For example, the first stage can be aerobic to achieve high growth and high productivity, followed by an anaerobic stage for high lactone production.

[0272] The carbon source can include, for example, any source of carbohydrates that can provide a carbon source for the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch. Other sources of carbohydrates include, for example, renewable feedstocks and biomass. Exemplary biomass types that can be used as feedstocks in the methods of the present disclosure include cellulosic biomass, hemicellulosic biomass, and lignin feedstocks, or portions of the feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates such as glucose, xylose, arabinose, galactose, mannose, fructose, and starch that can be used as carbon sources. Those skilled in the art will appreciate, in light of the teachings and guidance provided herein, that renewable feedstocks and biomass other than those exemplified above can also be used to cultivate the microbial organisms of the present disclosure to produce the desired compounds.

[0273] The culture medium can be analyzed to monitor the reactions described herein and to identify starting materials, products, or intermediates in the fermentation medium by using high pressure liquid chromatography (HPLC) analysis, GC-MS (gas chromatography-mass spectrometry), and LC-MS (liquid chromatography-mass spectrometry), or other suitable analytical methods using routine procedures well known in the art.

[0274] Any of the non-naturally occurring microbial organisms described herein can be cultivated to produce and / or secrete the products of the present disclosure.

[0275] Compounds prepared by the methods described herein can be isolated by methods well known in the art for isolating organic compounds prepared by biosynthesis or fermentation. For example, compounds can be isolated from solution by crystallization, salt formation, pervaporation, reactive extraction, extraction (liquid-liquid and two-phase), adsorption, ion exchange, dialysis, distillation, stripping, and membrane-based separations (Roffler et al., Trends Biotechnolgy. 2: 129-136 (1984)). Distillation, extraction (liquid-liquid and two-phase), pervaporation, and membrane-based separations (Roffler et al., Trends Biotechnolgy. 2: 129-136 (1984)) can be used to isolate 1,5-pentanediol from solution.

[0276] As described herein, one exemplary growth condition for effecting biosynthesis of a desired product includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microorganism of the present disclosure can be maintained, cultured, or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment without oxygen. Substantially anaerobic conditions include, for example, culturing, batch fermentation, or continuous fermentation such that the dissolved oxygen concentration in the culture medium is maintained between 0 and 10% of saturation. Substantially anaerobic conditions also include growing or resting cells in liquid media or on solid agar within a sealed chamber that maintains an atmosphere of less than 1% oxygen. The percentage of oxygen can be maintained, for example, by sparging the culture with a N2 / CO2 mixture or other suitable non-oxygen gas.

[0277] The culture conditions described herein can be scaled up and grown continuously to produce the product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these methods are well known in the art. Fermentation procedures are particularly useful for biosynthetic production in commercial quantities.

[0278] The term "pathway enzyme expressed in sufficient amounts" means that the enzyme is expressed in an amount sufficient to allow detection of the desired pathway product.

[0279] In another aspect, provided herein is a recombinant microbial organism comprising a first exogenous nucleic acid encoding an aldehyde:ferredoxin hydratase, wherein the recombinant microbial organism is further modified to express an increased amount of a quinone oxidoreductase as compared to a wild-type or unmodified same microbial organism, and optionally wherein the microorganism is Corynebacterium glutamicum, Clostridium sp., or Escherichia coli.

[0280] In some embodiments, the organism comprises a second exogenous nucleic acid encoding a quinone oxidoreductase. In some embodiments, the first exogenous nucleic acid and / or the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid further comprise a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid or the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the regulatory element is selected from a promoter or an enhancer. In some embodiments, the regulatory element is a promoter. In some embodiments, the regulatory element is an enhancer.

[0281] In some embodiments, the aldolase hydratase has EC number 4.1.2.45, EC number 4.1.2.34, or EC number 4.1.1.4. In some embodiments, the aldolase hydratase is an enzyme selected from the group of enzymes identified by Uniprot ID No. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified by GenBank, RefSeq, or Uniprot ID No. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.In some embodiments, the hydratase-aldolase is an enzyme comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0282] In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by the following GenBank, RefSeq, or Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1, or a portion thereof (e.g., a domain, a set of amino acid residues (which can be contiguous or separated), etc.) that facilitates formation of an aldehyde alcohol dehydration product.In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0283] In some embodiments, the hydratase-aldolase is selected from the enzymes of Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from Tables 1, 5, 6, 7, and 8.

[0284] In some embodiments, the hydratase-aldolase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0285] In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in a vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each comprised in their own separate vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector.

[0286] In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0 or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0287] In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0288] In some embodiments, the quinone oxidoreductase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0289] In some embodiments, the recombinant microorganism is capable of producing a 2-keto carboxylic acid of the formula:

[0290]

[0291] wherein R is H, CH3, or CH2OH.

[0292] In some embodiments, the recombinant microbial organism is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid.

[0293] In some embodiments, the recombinant microorganism is genetically modified to increase production of pyruvate from a carbon source. In some embodiments, the carbon source is selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof.

[0294] In another aspect, provided herein is a population of the recombinant microbial organisms disclosed herein. In some embodiments, the population is substantially homogeneous. In some embodiments, substantially homogeneous means at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homogeneous.

[0295] In another aspect, provided herein is a method of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid, comprising culturing the population disclosed herein under suitable conditions. In some embodiments, the method further comprises isolating 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoic acid from the culture or microbial organism.

[0296] Detailed description of certain embodiments

[0297] The present disclosure includes, among other things, the recognition that certain polypeptides, e.g., a variety of aldol dehydration product biosynthetic polypeptides that are or comprise a hydratase-aldolase polypeptide, can be used to efficiently produce a variety of compounds. In some embodiments, the present disclosure demonstrates that a variety of aldehydes that are structurally different from natural and / or known aldehyde substrates of such polypeptides, e.g., a variety of aliphatic aldehydes described herein, can be used to efficiently manufacture a number of products using the aldol dehydration product biosynthetic polypeptides described herein. Among other things, the present disclosure demonstrates that production of a variety of aldol dehydration products can be catalyzed by a single aldol dehydration product biosynthetic polypeptide, e.g., a variety of hydratase-aldolase polypeptides as described herein.

[0298] In some embodiments, the present disclosure provides a method comprising:

[0299] contacting pyruvate and an aldehyde with an aldol dehydration product biosynthetic polypeptide, thereby producing an aldol dehydration product, wherein:

[0300] The aldol dehydration product is a compound comprising an aldehyde group or a ketone group and a double bond conjugated to the aldehyde group or the ketone group.

[0301] In some embodiments, the aldehyde is an aliphatic aldehyde. In some embodiments, the -CHO group of the aldehyde is not conjugated to, e.g., a double bond, a triple bond, or an aromatic group.

[0302] In some embodiments, the present disclosure provides methods comprising:

[0303] contacting pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthesis polypeptide, thereby producing an aldol dehydration product, wherein:

[0304] the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aryl group; and

[0305] the aldol dehydration product is a compound comprising an aldehyde group or a ketone group and a double bond conjugated to the aldehyde group or the ketone group.

[0306] In some embodiments, the aldol dehydration product biosynthesis polypeptide is or comprises a hydratase-aldolase polypeptide, e.g., those exemplified herein. In some embodiments, the provided methods comprise contacting pyruvate and an aliphatic aldehyde with a hydratase-aldolase to produce an aldol dehydration product.

[0307] In some embodiments, the aldol dehydration product biosynthesis polypeptide comprises an aldolase polypeptide. In some embodiments, the aldol dehydration product biosynthesis polypeptide comprises a hydratase polypeptide. In some embodiments, the aldol dehydration product biosynthesis polypeptide comprises a hydratase-aldolase polypeptide. In some embodiments, the aldol dehydration product biosynthesis polypeptide is a hydratase-aldolase polypeptide. In some embodiments, the hydratase-aldolase polypeptide is or comprises a hydratase-aldolase as described herein, e.g., having EC number 4.1.2.45 or EC number 4.1.2.34 or EC 4.1.1.4, or an enzyme selected from Tables 1 and 5-8.

[0308] In some embodiments, the aldol dehydration product biosynthesis polypeptide is within an organism, e.g., a microorganism. In some embodiments, the organism expresses an engineered aldol dehydration product biosynthesis polypeptide. In some embodiments, the organism expresses an increased level and / or activity of an aldol dehydration product biosynthesis polypeptide. In some embodiments, the organism provides an increased rate and / or yield for production of an aldol dehydration product. In some embodiments, the organism provides an increased substrate utilization for production of an aldol dehydration product.

[0309] In some embodiments, the conversion of pyruvate and an aliphatic aldehyde to an aldol dehydration product is catalyzed by an aldol dehydration product biosynthesis polypeptide.

[0310] In some embodiments, the aldol dehydration product can be provided by an alternative pathway. In some embodiments, the aldol dehydration product is produced from an aldol product.

[0311] In some embodiments, the present disclosure provides methods comprising:

[0312] contacting pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthesis polypeptide, thereby producing an aldol dehydration product, wherein:

[0313] The aldol dehydration product is a compound comprising an aldehyde group or a ketone group and a double bond conjugated to the aldehyde group or the ketone group.

[0314] In some embodiments, the aldehyde is an aliphatic aldehyde. In some embodiments, the -CHO group of the aldehyde is not conjugated to a double bond, a triple bond, or an aromatic group.

[0315] In some embodiments, the present disclosure provides methods comprising:

[0316] contacting pyruvate and an aliphatic aldehyde with an aldol product biosynthesis polypeptide to produce an aldol product, wherein:

[0317] the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aryl group; and

[0318] The aldol product is a compound comprising an aldehyde group or a ketone group and a hydroxyl group attached to the beta-carbon of the aldehyde or ketone carbonyl.

[0319] The methods of the present disclosure include the use of biosynthesis polypeptides. In some embodiments, a biosynthesis polypeptide, when used with a particular product, e.g., an aldol product biosynthesis polypeptide, a reduction product biosynthesis polypeptide, etc., refers to a polypeptide that participates in the synthesis of the particular product. In some embodiments, a biosynthesis polypeptide, when used with a particular product, is or comprises an enzyme that catalyzes the formation of the particular product. In some embodiments, a biosynthesis polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference biosynthesis polypeptide for the particular product found in nature). Alternatively or additionally, in some embodiments, a biosynthesis polypeptide shares characteristic sequence elements and / or overall percent identity with a suitable reference biosynthesis polypeptide (e.g., as found in nature and / or as presented herein (e.g., in one or more relevant tables) or a portion thereof (e.g., a portion of amino acid residues (which can be contiguous or isolated) that facilitate a relevant reaction (such as a relevant catalytic domain (e.g., a relevant catalytic domain) and / or group).

[0320] In some embodiments, the aldol product biosynthesis polypeptide is or comprises an aldolase polypeptide. It is understood by one of skill in the art reading the present disclosure that a variety of aldolase polypeptides can be utilized in accordance with the present disclosure. In some embodiments, the aldolase polypeptide is or comprises an aldolase described in US20170044551, which is incorporated by reference herein.

[0321] In some embodiments, the aldol product biosynthesis polypeptide is or comprises an aldolase-hydratase as described herein.

[0322] In some embodiments, the aldol product biosynthesis polypeptide is present in an organism, e.g., a microorganism. In some embodiments, the organism is engineered to express an engineered or exogenous aldol product biosynthesis polypeptide, typically at higher protein levels and / or activity levels. In some embodiments, the conversion of pyruvate and an aliphatic aldehyde to an aldol product is catalyzed by the aldol product biosynthesis polypeptide. In some embodiments, the method is performed in a culture, e.g., a bacterial culture. As with other biosynthesis polypeptides, the aldol product biosynthesis polypeptide can be in an organism, e.g., a bacterium, can be engineered, and / or can be expressed in an enhanced manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0323] In some embodiments, the aldol product is converted to an aldol dehydration product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the conversion comprises contacting the aldol product with a dehydration product biosynthesis polypeptide, thereby producing the aldol dehydration product. In some embodiments, the dehydration product biosynthesis polypeptide is or comprises a hydratase. In some embodiments, the dehydration product biosynthesis polypeptide is or comprises a dehydratase. In some embodiments, the hydratase or dehydratase is described in US20170044551, which is incorporated herein by reference. As with other biosynthesis polypeptides, the dehydration product biosynthesis polypeptide can be in an organism, e.g., a bacterium, can be engineered, and / or can be expressed in an enhanced manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0324] As will be appreciated by those skilled in the art, the aldol dehydration product can be used to make a variety of products, e.g., 1,5-pentanediol, 1,6-hexanediol, 6HH, adipic acid, etc., which can be used to make a wide range of products, e.g., polymers, resins, coating products, etc. In some embodiments, utilization of the aldol dehydration product includes one or more chemical transformations, each of which can independently be catalyzed by a polypeptide (e.g., an enzyme) optionally in an organism, or by traditional chemical processes without the use of enzymes. As will be appreciated by those skilled in the art, one or more or all of the steps can be performed in one or more organisms, each of which can use substrates produced within itself or from outside the organism, and / or one or more cultures independently comprising one or more types of organisms (each of which can use substrates produced by itself or in the culture (e.g., a feed compound, a compound produced by another) to independently perform one or more reactions), to independently perform one or more reactions. In some embodiments, one or more or all of the biosynthetic polypeptides are independently present in an organism, e.g., an optionally engineered bacterium. In some embodiments, one or more of a set of biosynthetic polypeptides used to produce a product are expressed in one organism, e.g., an optionally engineered bacterium, and one or more other biosynthetic polypeptides in the set are expressed in one or more other organisms, e.g., optionally engineered bacteria. In some embodiments, an organism, e.g., a bacterium, is engineered to comprise one or more exogenous nucleic acids encoding one or more or all of the biosynthetic polypeptides. In some embodiments, production of a product includes multiple reaction steps performed in a single culture comprising one or more bacteria, each of which independently comprises one or more or all of the required biosynthetic polypeptides, and collectively comprises all of the required biosynthetic polypeptides. In some embodiments, production of a product includes multiple reaction steps performed in two or more cultures, each of which independently comprises one or more bacteria, each of which independently comprises one or more or all of the required biosynthetic polypeptides, and collectively comprises all of the required biosynthetic polypeptides.

[0325] For example, in some embodiments, a double bond in the aldol dehydration product is converted to a single bond.

[0326] In some embodiments, the disclosure provides a method comprising:

[0327] contacting an olefin with an olefin reduction product biosynthetic polypeptide to produce an olefin reduction product, wherein:

[0328] the olefin comprises a double bond conjugated to a carbonyl group; and

[0329] the double bond conjugated to the carbonyl group in the olefin is reduced to a single bond to provide the olefin reduction product.

[0330] In some embodiments, the olefin is an aldol dehydration product.

[0331] In some embodiments, the olefin reduction product biosynthesis polypeptide is or comprises an enzyme that catalyzes the reduction of an aldol dehydration product (e.g., a 2-oxo-3-enoate), as described herein. In some embodiments, such an enzyme is a quinone oxidoreductase described herein. In some embodiments, such an enzyme belongs to EC 1.6.5. In some embodiments, such an enzyme belongs to EC 1.6.5.5. In some embodiments, such an enzyme is selected from Table 9.

[0332] In some embodiments, the olefin reduction product biosynthesis polypeptide is within an organism, e.g., a microorganism. In some embodiments, the organism expresses an engineered olefin reduction product biosynthesis polypeptide. In some embodiments, the organism expresses an elevated level and / or activity of an olefin reduction product biosynthesis polypeptide. In some embodiments, the organism provides an increased rate and / or yield for producing an olefin reduction product. In some embodiments, the organism provides an increased utilization of a substrate for producing an olefin reduction product.

[0333] In some embodiments, the olefin reduction product biosynthesis polypeptide is or comprises an enzyme that is endogenously encoded and / or expressed by an organism, without engineering.

[0334] It is understood by one of skill in the art reading the present disclosure that a variety of aldehydes can be used in accordance with the present disclosure. In some embodiments, the aldehyde is a natural or known substrate of a biosynthesis polypeptide, e.g., an aldol dehydration product biosynthesis polypeptide, which is or comprises a hydratase-aldolase. In some embodiments, the aldehyde is not a natural or known substrate. For example, the present disclosure demonstrates, among other things, that aliphatic aldehydes can be used in product manufacturing using a hydratase-aldolase, the natural or known substrate of which is an aromatic or conjugated aldehyde.

[0335] In some embodiments, the aldehyde is an aliphatic aldehyde. In some embodiments, the aldehyde has one or two a-hydrogens. In some embodiments, the aldehyde has the structure of Formula A-1:

[0336] R a -L 2 -L 1 -C(O)H,

[0337] A - 1

[0338] or a salt thereof, wherein:

[0339] Ra is R” or -OR”, R” is H or C1-C6alkyl, and

[0340] L 1 and L 2each independently is a covalent bond, or a divalent, optionally substituted, straight chain or branched C 1-20 aliphatic or C 1-20 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0341] -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -S-S-, -N(R")-, -C(O)-, -C(S)-, -C(NR")-, -C(O)N(R")-, -N(R")C(O)N(R")-, -N(R")C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R")-, -C(O)S-, or -C(O)O-;

[0342] -Cy- is a divalent, optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms;

[0343] each R" is independently -R', -C(O)R', -CO2R', or -SO2R';

[0344] R' is hydrogen, or an optionally substituted group selected from C 1-10 aliphatic, C 1-10 heteroaliphatic, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring having 1 to 5 heteroatoms, and 3- to 10-membered heterocyclic ring having 1 to 5 heteroatoms, or:

[0345] two or more R' groups, together with their intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0 to 5 heteroatoms in addition to the intervening atoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms.

[0346] In some embodiments, the aldol product has the structure of Formula P-1:

[0347] R a -L 2 -L 1 -CH(OH)-CH2-C(O)-C(O)OH,

[0348] P-1

[0349] or a salt thereof, wherein:

[0350] R a is R" or -OR",

[0351] L 1 and L 2 each independently is a covalent bond, or a bivalent, optionally substituted, straight-chain or branched C 1-20 aliphatic or C 1-20 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0352] -CºC-, -C(R")2-, -Cy-, -O-, -S-, -S-S-, -N(R")-, -C(O)-, -C(S)- -C(NR")-, -C(O)N(R")-, -N(R")C(O)N(R")-, -N(R")C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R")-, -C(O)S-, or -C(O)O-;

[0353] -Cy- is a bivalent, optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms;

[0354] each R" is independently -R', -C(O)R', -CO2R', or -SO2R';

[0355] R' is hydrogen, or an optionally substituted group selected from C 1-10 aliphatic, C 1-10 heteroaliphatic, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring having 1 to 5 heteroatoms, and 3- to 10-membered heterocyclic ring having 1 to 5 heteroatoms, or:

[0356] two or more R' groups, together with their intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0 to 5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms.

[0357] In some embodiments, the aldol dehydration product has the structure of Formula P-2:

[0358] R a -L 2 -L 1 -CH=CH-C(O)-C(O)OH,

[0359] P-2

[0360] or a salt thereof, wherein:

[0361] R a is R" or -OR",

[0362] L 1 and L 2 each independently is a covalent bond, or a bivalent, optionally substituted, straight-chain or branched C 1-20 aliphatic or C 1-20 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0363] -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -S-S-, -N(R")-, -C(O)-, -C(S)-, -C(NR")-, -C(O)N(R")-, -N(R")C(O)N(R")-, -N(R")C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R")-, -C(O)S-, or -C(O)O-;

[0364] -Cy- is a bivalent, optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms;

[0365] each R" is independently -R', -C(O)R', -CO2R', or -SO2R';

[0366] R' is hydrogen, or an optionally substituted group selected from C 1-10 aliphatic, C 1-10 heteroaliphatic, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring having 1 to 5 heteroatoms, and 3- to 10-membered heterocyclic ring having 1 to 5 heteroatoms, or:

[0367] two or more R' groups, together with their intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0 to 5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms.

[0368] In some embodiments, the -CH=CH- in formula P-2 is in the E configuration. In some embodiments, the -CH=CH- in formula P-2 is in the Z configuration.

[0369] In some embodiments, the olefin reduction product has the structure of formula P-3:

[0370] R a -L2 -L 1 -CH2-CH2-C(O)-C(O)OH,

[0371] P-3

[0372] or a salt thereof, wherein:

[0373] R a is R" or -OR",

[0374] L 1 and L 2 each independently is a covalent bond, or a bivalent, optionally substituted, straight-chain or branched C 1-20 aliphatic or C 1-20 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0375] -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -S-S-, -N(R")-, -C(O)-, -C(S)- -C(NR")-, -C(O)N(R")-, -N(R")C(O)N(R")-, -N(R")C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R")-, -C(O)S-, or -C(O)O-;

[0376] -Cy- is a bivalent, optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms;

[0377] each R" is independently -R', -C(O)R', -CO2R', or -SO2R';

[0378] R' is hydrogen, or an optionally substituted group selected from C 1-10 aliphatic, C 1-10 heteroaliphatic, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring having 1 to 5 heteroatoms, and 3- to 10-membered heterocyclic ring having 1 to 5 heteroatoms, or:

[0379] two or more R' groups, together with their intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0 to 5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0 to 5 heteroatoms.

[0380] In some embodiments, R aR". In some embodiments, R a is -OR".

[0381] In some embodiments, R" is R'. In some embodiments, R" is -C(O)R'. In some embodiments, R" is -CO2R'. In some embodiments, R" is -SO2R'.

[0382] In some embodiments, R' is hydrogen. In some embodiments, R' is not hydrogen.

[0383] In some embodiments, R a is R'. In some embodiments, R a is -OR'. In some embodiments, R a is -H. In some embodiments, R a is -OH.

[0384] In some embodiments, L 1 is a covalent bond. In some embodiments, L 1 is not a covalent bond.

[0385] In some embodiments, L 1 is optionally substituted C 1-6 alkylene. In some embodiments, L 1 is optionally substituted straight chain C 1-6 alkylene. In some embodiments, L 1 is optionally substituted -CH2-. In some embodiments, L 1 is optionally substituted -CH2CH2-. In some embodiments, L 1 is optionally substituted -CH2CH2CH2-. In some embodiments, L 1 is optionally substituted -CH2CH2CH2CH2-. In some embodiments, L 1 is optionally substituted -CH2CH2CH2CH2CH2-. In some embodiments, L 1 is optionally substituted -CH2CH2CH2CH2CH2CH2-. In some embodiments, the -CH2- bonded to -C(O)H is unsubstituted. In some embodiments, the -CH2- bonded to -C(O)H is monosubstituted. In some embodiments, L 1 is substituted. In some embodiments, L 1 is unsubstituted. In some embodiments, L 1 is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L

[0386] is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L 2 is a covalent bond. In some embodiments, L 2 is not a covalent bond.

[0387] is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L 2 is an optionally substituted C 1-6 alkylene. In some embodiments, L 2 is an optionally substituted straight chain C 1-6 alkylene. In some embodiments, L 2 is -CH2-. In some embodiments, L 2 is -CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2CH2CH2-. In some embodiments, the -CH2- bonded to -C(O)H is unsubstituted. In some embodiments, the -CH2- bonded to -C(O)H is monosubstituted. In some embodiments, L 2 is substituted. In some embodiments, L 2 is unsubstituted. In some embodiments, L 2 is -CH2-. In some embodiments, L 2 is -CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2CH2-. In some embodiments, L 2 is -CH2CH2CH2CH2CH2CH2-.

[0388] is -CH2CH2CH2CH2CH2CH2-. In some embodiments, L1 and L 2 At least one of them is not a covalent bond.

[0389] In some embodiments, the aldehyde is CH3CHO. In some embodiments, the aldehyde is CH3CH2CHO. In some embodiments, the aldehyde is CH3CH2CH2CHO. In some embodiments, the aldehyde is CH2OHCHO. In some embodiments, the aldehyde is CH2OHCH2CHO. In some embodiments, the aldehyde is CH2OHCH2CH2CHO.

[0390] In some embodiments, the aldol product is CH3CH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH3CH2CH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH3CH2CH2CH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH2OHCH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH2OHCH2CH2CH(OH)CH2C(O)COOH.

[0391] In some embodiments, the dehydration product of the aldehyde is CH3CH=CHC(O)COOH. In some embodiments, the dehydration product of the aldehyde is CH3CH2CH=CHC(O)COOH. In some embodiments, the dehydration product of the aldehyde is CH3CH2CH2CH=CHC(O)COOH. In some embodiments, the dehydration product of the aldehyde is CH2OHCH=CHC(O)COOH. In some embodiments, the dehydration product of the aldehyde is CH2OHCH2CH2CH=CHC(O)COOH.

[0392] In some embodiments, the olefin reduction product is CH3CH2CH2C(O)COOH. In some embodiments, the olefin reduction product is CH3CH2CH2CH2C(O)COOH. In some embodiments, the olefin reduction product is CH3CH2CH2CH2CH2C(O)COOH. In some embodiments, the olefin reduction product is CH2OHCH2CH2CH2C(O)COOH. In some embodiments, the olefin reduction product is CH2OHCH2CH2CH2CH2C(O)COOH.

[0393] In some embodiments, the alkene reduction product is converted to a carbonyl reduction product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the alkene reduction product comprises a carbonyl group, and the carbonyl group is converted to -CH(OH)-. In some embodiments, the method comprises contacting the alkene reduction product with a carbonyl reduction product biosynthesis polypeptide to produce the carbonyl reduction product, wherein:

[0394] the alkene reduction product comprises a carbonyl group; and

[0395] the carbonyl group of the alkene reduction product is converted to -CH(OH)-.

[0396] In some embodiments, the carbonyl reduction product biosynthesis polypeptide is or comprises a reductase. In some embodiments, the carbonyl reduction product biosynthesis polypeptide is or comprises a keto reductase as described herein. In some embodiments, the carbonyl reduction product biosynthesis polypeptide is or comprises a 2-keto acid-2-reductase as described herein. In some embodiments, such an enzyme is a 6-hydroxy-2-oxohexanoate-2-reductase as described herein. In some embodiments, such an enzyme is described in US20170044551, which is incorporated herein by reference.

[0397] In some embodiments, the conversion of the alkene reduction product to the carbonyl reduction product is catalyzed by the carbonyl reduction product biosynthesis polypeptide.

[0398] For many other biosynthesis polypeptides, the carbonyl reduction product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0399] In some embodiments, the carbonyl reduction product has the structure of Formula P-4:

[0400] R a -L 2 -L 1 -CH2-CH2-CH(OH)-C(O)OH,

[0401] P-4

[0402] or a salt thereof, wherein each variable is independently as described herein.

[0403] In some embodiments, the carbonyl reduction product is CH3CH2CH2CH(OH)COOH. In some embodiments, the carbonyl reduction product is CH3CH2CH2CH2CH(OH)COOH. In some embodiments, the carbonyl reduction product is CH3CH2CH2CH2CH2CH(OH)COOH. In some embodiments, the carbonyl reduction product is CH2OHCH2CH2CH(OH)COOH. In some embodiments, the carbonyl reduction product is CH2OHCH2CH2CH2CH(OH)COOH. In some embodiments, the carbonyl reduction product is CH2OHCH2CH2CH2CH2CH(OH)COOH.

[0404] In some embodiments, the carbonyl reduction product is converted to a CoA transfer product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the CoA transfer product is a compound of formula P-5:

[0405] R a -L 2 -L 1 -CH2-CH2-CH(OH)-C(O)-S-CoA,

[0406] P-5

[0407] or salts thereof, wherein each variable is independently as described herein.

[0408] In some embodiments, such conversion is catalyzed by a CoA (CoA = coenzyme A) transfer product biosynthesis polypeptide. In some embodiments, the CoA transfer product biosynthesis polypeptide is or comprises a CoA transferase as described herein, e.g., a 2,6-dihydroxy-hexanoate CoA-transferase. In some embodiments, the CoA transferase is one described in US20170044551, which is incorporated herein by reference. In some embodiments, such conversion is catalyzed by a CoA transfer product biosynthesis polypeptide.

[0409] For many other biosynthesis polypeptides, CoA transfer product biosynthesis polypeptides can be engineered in organisms, e.g., bacteria, and / or can be expressed in an improved manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0410] In some embodiments, the CoA transfer product is CH3CH2CH2CH(OH)C(O)S-CoA. In some embodiments, the CoA transfer product is CH3CH2CH2CH2CH(OH)C(O)S-COA. In some embodiments, the CoA transfer product is CH3CH2CH2CH2CH2CH(OH)C(O)S-COA. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH(OH)C(O)S-COA. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH(OH)C(O)S-COA. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2CH(OH)C(O)S-COA.

[0411] In some embodiments, the CoA transfer product is converted to a dehydration product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the dehydration product is a compound of formula P-6:

[0412] R a -L 2 -L 1 -CH2-CH=CH-C(O)-S-CoA,

[0413] P-6

[0414] or salts thereof, wherein each variable is independently as described herein.

[0415] In some embodiments, such conversion is catalyzed by a dehydration product biosynthesis polypeptide. In some embodiments, the dehydration product biosynthesis polypeptide is or comprises a dehydration enzyme as described herein. In some embodiments, the dehydration enzyme is or comprises a 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase as described herein. In some embodiments, the dehydration enzyme is described in US20170044551, which is incorporated by reference.

[0416] In some embodiments, such conversion is catalyzed by a dehydration product biosynthesis polypeptide.

[0417] For many other biosynthesis polypeptides, the dehydration product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0418] In some embodiments, the dehydration product is CH3CH2CH=CHC(0)S-CoA. In some embodiments, the dehydration product is CH3CH2CH2CH=CHC(0)S-COA. In some embodiments, the dehydration product is CH3CH2CH2CH2CH=CHC(0)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH=CHC(0)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH2CH=CHC(0)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH2CH2CH=CHC(0)S-COA.

[0419] In some embodiments, the dehydration product, e.g., a compound of formula P-6 or a salt thereof, is converted to a reduction product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the reduction product is a compound of formula P-7:

[0420] R a -L 2 -L 1 -CH2-CH2-CH2-C(0)-S-CoA,

[0421] P-7

[0422] or a salt thereof, wherein each variable is independently as described herein.

[0423] In some embodiments, such conversion is catalyzed by a reduction product biosynthesis polypeptide. In some embodiments, the reduction product biosynthesis polypeptide is or comprises a 2,3-enoyl-CoA reductase, a 2,3-dehydro-carboxyl CoA 2’3-reductase, e.g., a 2,3-dehydro-hexanoyl-CoA 2,3-reductase as described herein. In some embodiments, suitable reductases are described in US20170044551, which is incorporated by reference herein. In some embodiments, such conversion is catalyzed by a reduction product biosynthesis polypeptide.

[0424] For many other biosynthesis polypeptides, the reduction product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0425] In some embodiments, the reduction product is CH3CH2CH2CH2C(O)S-CoA. In some embodiments, the reduction product is CH3CH2CH2CH2CH2C(O)S-COA. In some embodiments, the reduction product is CH3CH2CH2CH2CH2CH2C(O)S-COA. In some embodiments, the reduction product is CH2OHCH2CH2CH2C(O)S-COA. In some embodiments, the reduction product is CH2OHCH2CH2CH2CH2C(O)S-COA. In some embodiments, the reduction product is CH2OHCH2CH2CH2CH2CH2C(O)S-COA.

[0426] In some embodiments, the reduction product, e.g., a compound of Formula P-7, or a salt thereof, is converted to a CoA transfer product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the CoA transfer product is a compound of Formula P-8:

[0427] R a -L 2 -L 1 -CH2-CH2-CH2-C(O)-OH,

[0428] P-8

[0429] or a salt thereof, wherein each variable is independently as described herein.

[0430] In some embodiments, such conversion is catalyzed by a CoA transfer product biosynthesis polypeptide. In some embodiments, the CoA transfer product biosynthesis polypeptide is or comprises a CoA transferase as described herein, e.g., a 6-hydroxyhexanoyl-CoA transferase as described herein. In some embodiments, the CoA transferase is described in US20170044551, which is incorporated herein by reference. In some embodiments, such conversion is catalyzed by a CoA transfer product biosynthesis polypeptide.

[0431] For many other biosynthesis polypeptides, CoA transfer product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0432] In some embodiments, the CoA transfer product is CH3CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH3CH2CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH3CH2CH2CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2CH2C(O)OH.

[0433] In some embodiments, the CoA transfer product, e.g., a compound of Formula P-8 or a salt thereof, wherein R a is -OH, is converted to an oxidation product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the oxidation product is a compound of Formula P-9:

[0434] H-C(O)-L 2’ -L 1 -CH2-CH2-CH2-C(O)-OH,

[0435] P-9

[0436] or a salt thereof, wherein L 2’ is a covalent bond, or a bivalent, optionally substituted, straight-chain or branched, C 1-19 aliphatic or C 1-19 heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by:

[0437] -CºC-, -C(R")2-, -Cy-, -O-, -S-, -S-S-, -N(R")-, -C(O)-, -C(S)-, -C(NR")-, -C(O)N(R")-, -N(R")C(O)N(R")-, -N(R")C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R")-, -C(O)S-, or -C(O)O-, and each other variable is independently as described herein.

[0438] In some embodiments, L 2’ is a covalent bond. In some embodiments, L 2’ is not a covalent bond. In some embodiments, at least one of L 1 and L 2’ is not a covalent bond.

[0439] In some embodiments, L 2’is optionally substituted C 1-6 alkylene. In some embodiments, L 2’ is optionally substituted straight chain C 1-6 alkylene. In some embodiments, L 2’ is optionally substituted -CH2-. In some embodiments, L 2’ is optionally substituted -CH2CH2-. In some embodiments, L 2’ is optionally substituted -CH2CH2CH2-. In some embodiments, L 2’ is optionally substituted -CH2CH2CH2CH2-. In some embodiments, L 2’ is optionally substituted -CH2CH2CH2CH2CH2-. In some embodiments, L 2’ is optionally substituted -CH2CH2CH2CH2CH2CH2-. In some embodiments, the -CH2- bonded to -C(O)H is unsubstituted. In some embodiments, the -CH2- bonded to -C(O)H is monosubstituted. In some embodiments, L 2’ is substituted. In some embodiments, L 2’ is unsubstituted. In some embodiments, L 2’ is -CH2-. In some embodiments, L 2’ is -CH2CH2-. In some embodiments, L 2’ is -CH2CH2CH2-. In some embodiments, L 2’ is -CH2CH2CH2CH2-. In some embodiments, L 2’ is -CH2CH2CH2CH2CH2-. In some embodiments, L 2’ is -CH2CH2CH2CH2CH2CH2-.

[0440] In some embodiments, such conversion is catalyzed by an oxidation product biosynthesis polypeptide. In some embodiments, the oxidation product biosynthesis polypeptide is or comprises an alcohol dehydrogenase, such as a primary alcohol dehydrogenase, such as a 6-hydroxyhexanoate dehydrogenase as described herein. In some embodiments, the alcohol dehydrogenase is described in US20170044551, which is incorporated by reference herein. In some embodiments, such conversion is catalyzed by an oxidation product biosynthesis polypeptide.

[0441] For many other biosynthesis polypeptides, the oxidation product biosynthesis polypeptides can be engineered in an organism, such as a bacterium, and / or can be expressed in an improved manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0442] In some embodiments, the oxidation product is HC(O)CH2CH2CH2C(O)OH. In some embodiments, the oxidation product is HC(O)CH2CH2CH2CH2C(O)OH. In some embodiments, the oxidation product is HC(O)CH2CH2CH2CH2CH2C(O)OH.

[0443] In some embodiments, the oxidation product, e.g., a compound of Formula P-9, or a salt thereof, is converted to an aldehyde oxidation product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the oxidation product is a compound of Formula P-10:

[0444] HO-C(O)-L 2’ -L 1 -CH2-CH2-CH2-C(O)-OH,

[0445] P-10

[0446] or a salt thereof, wherein each other variable is independently as described herein.

[0447] In some embodiments, such conversion is catalyzed by an aldehyde oxidation product biosynthesis polypeptide. In some embodiments, the aldehyde oxidation product biosynthesis polypeptide is or comprises an aldehyde dehydrogenase, e.g., a 6-hydroxyhexanoate dehydrogenase as described herein. In some embodiments, the aldehyde dehydrogenase is described in US20170044551, which is incorporated by reference herein. In some embodiments, such conversion is catalyzed by an aldehyde oxidation product biosynthesis polypeptide.

[0448] For many other biosynthesis polypeptides, the aldehyde oxidation product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0449] In some embodiments, the aldehyde oxidation product is HOC(O)CH2CH2CH2C(O)OH. In some embodiments, the oxidation product is HOC(O)CH2CH2CH2CH2C(O)OH. In some embodiments, the oxidation product is HOC(O)CH2CH2CH2CH2CH2C(O)OH.

[0450] In some embodiments, the CoA transfer product, e.g., a compound of Formula P-8, or a salt thereof, is converted to a carboxyl reduction product by enzyme catalysis, by biosynthesis, or by traditional organic synthesis without enzyme catalysis. In some embodiments, the carboxyl reduction product is a compound of Formula P-9’:

[0451] R a -L 2 -L 1 -CH2-CH2-CH2-C(O)-H,

[0452] P-9’

[0453] or salts thereof, wherein each variable is independently as described herein.

[0454] In some embodiments, such conversion is catalyzed by a carboxyl reduction product biosynthesis polypeptide. In some embodiments, the carboxyl reduction product biosynthesis polypeptide is or comprises a carboxylate reductase or an aldehyde dehydrogenase as described herein. In some embodiments, the carboxyl reduction product biosynthesis polypeptide is or comprises a 6-hydroxyhexanoate 1 -reductase. In some embodiments, the carboxyl reduction product biosynthesis polypeptide is or comprises a carboxylate reductase or an aldehyde dehydrogenase described in US20170044551, which is incorporated by reference herein. In some embodiments, such conversion is catalyzed by a carboxyl reduction product biosynthesis polypeptide.

[0455] For many other biosynthesis polypeptides, the carboxyl reduction product biosynthesis polypeptides can be engineered in organisms, e.g., bacteria, and / or can be expressed in an improved manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0456] In some embodiments, the carboxyl reduction product is CH3CH2CH2CH2C(O)H. In some embodiments, the carboxyl reduction product is CH3CH2CH2CH2CH2C(O)H. In some embodiments, the carboxyl reduction product is CH3CH2CH2CH2CH2CH2C(O)H. In some embodiments, the carboxyl reduction product is CH2OHCH2CH2CH2C(O)H. In some embodiments, the carboxyl reduction product is CH2OHCH2CH2CH2CH2C(O)H. In some embodiments, the carboxyl reduction product is CH2OHCH2CH2CH2CH2CH2C(O)H.

[0457] In some embodiments, a carboxyl reduction product, e.g., a compound of formula P-9’ or a salt thereof, is converted to an aldehyde reduction product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the aldehyde reduction product is a compound of formula P-10’:

[0458] R a -L 2 -L 1 -CH2-CH2-CH2-CH2-OH,

[0459] P-10’

[0460] or salts thereof, wherein each variable is independently as described herein.

[0461] In some embodiments, such conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide. In some embodiments, the aldehyde reduction product biosynthesis polypeptide is or comprises an aldehyde reductase or an alcohol (e.g., primary alcohol) dehydrogenase as described herein. In some embodiments, the aldehyde reductase or alcohol (e.g., primary alcohol) dehydrogenase is described in US20170044551, which reductase and dehydrogenase are incorporated by reference herein. In some embodiments, such conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide.

[0462] For many other biosynthesis polypeptides, the aldehyde reduction product biosynthesis polypeptides can be engineered, and / or can be expressed in an improved manner at increased protein and / or activity levels in an organism, e.g., a bacterium, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0463] In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2CH2OH.

[0464] In some embodiments, an alkene reduction product, e.g., a compound of formula P-3 or a salt thereof, is converted to a decarboxylation product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the decarboxylation product is a compound of formula P-4’:

[0465] R a -L 2 -L 1 -CH2-CH2-C(O)-H,

[0466] P-4’

[0467] or salts thereof, wherein each variable is independently as described herein.

[0468] In some embodiments, such conversion is catalyzed by a decarboxylation product biosynthesis polypeptide. In some embodiments, the decarboxylation product biosynthesis polypeptide is or comprises a decarboxylase as described herein. In some embodiments, the decarboxylase is a 2-keto acid decarboxylase as described herein. In some embodiments, the decarboxylase is described in US20170044551, which is incorporated herein by reference. In some embodiments, such conversion is catalyzed by a decarboxylation product biosynthesis polypeptide.

[0469] For many other biosynthesis polypeptides, the decarboxylation product biosynthesis polypeptides can be engineered in organisms, e.g., bacteria, and / or can be expressed in an improved manner at increased protein and / or activity levels, and their products can be produced at increased rates and / or yields and / or substrate utilization.

[0470] In some embodiments, the decarboxylation product is CH3CH2CH2CHO. In some embodiments, the decarboxylation product is CH3CH2CH2CH2CHO. In some embodiments, the decarboxylation product is CH3CH2CH2CH2CH2CHO. In some embodiments, the decarboxylation product is CH2OHCH2CH2CHO. In some embodiments, the decarboxylation product is CH2OHCH2CH2CH2CHO. In some embodiments, the decarboxylation product is CH2OHCH2CH2CH2CH2CHO.

[0471] In some embodiments, the decarboxylation product, e.g., a compound of Formula P-4’ or a salt thereof, is converted to an aldehyde reduction product by enzymatic catalysis, by biosynthesis, or by traditional organic synthesis without enzymatic catalysis. In some embodiments, the aldehyde reduction product is a compound of Formula P-5’:

[0472] R a -L 2 -L 1 -CH2-CH2-CH2-OH,

[0473] P-5’

[0474] or a salt thereof, wherein each variable is independently as described herein.

[0475] In some embodiments, such conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide. In some embodiments, the aldehyde reduction product biosynthesis polypeptide is or comprises a primary alcohol dehydrogenase as described herein. In some embodiments, the primary alcohol dehydrogenase is described in US20170044551, which is incorporated herein by reference. In some embodiments, such conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide.

[0476] For many other biosynthetic polypeptides, the aldehyde reduction product biosynthetic polypeptides can be engineered, and / or can be expressed in an organism, e.g., a bacterium, in an elevated manner, e.g., at elevated protein and / or activity levels, and their products can be produced at elevated rates and / or yields and / or substrate utilization.

[0477] In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2OH.

[0478] In some embodiments, the present disclosure provides nucleic acids encoding one or more biosynthetic polypeptides. In some embodiments, such nucleic acids comprise non-native sequences. In some embodiments, such nucleic acids are optimized for expression in a producing organism, e.g., a bacterium.

[0479] As indicated herein, a variety of techniques can be used to assess the activity of a biosynthetic activity of a polypeptide. For example, a variety of techniques for assessing the activity of an aldol dehydration product biosynthetic polypeptide (e.g., a hydratase-aldolase) or an olefin reduction product biosynthetic polypeptide (e.g., an enzyme for reducing an aldol dehydration product) are described in the Examples.

[0480] In some embodiments, a variety of biosynthetic polypeptides, e.g., aldol dehydration product biosynthetic polypeptides, are expressed in an organism, in many embodiments, a microorganism, e.g., a bacterium, a fungus, etc. In some embodiments, they are expressed from one or more recombinant nucleic acids. In some embodiments, the transformations are carried out biosynthetically, e.g., in an organism, e.g., a bacterium. In some embodiments, the organism (e.g., a microorganism, e.g., a bacterium) is engineered to comprise an exogenous nucleic acid encoding a biosynthetic polypeptide (e.g., an aldol dehydration product biosynthetic polypeptide, e.g., a hydratase-aldolase).

[0481] In some embodiments, an organism, e.g., those designed for the production of aldol dehydration products, express an aldol dehydration product biosynthetic polypeptide, e.g., a hydratase-aldolase polypeptide, at modulated levels, typically elevated levels and / or activities.

[0482] In some embodiments, the organism comprises an engineered nucleic acid and / or expresses an engineered biosynthetic polypeptide, e.g., an aldol dehydration product biosynthetic polypeptide (e.g., a plurality of hydratase-aldolase). In some embodiments, the engineered nucleic acid comprises one or more sequence differences compared to a reference nucleic acid. In some embodiments, the reference nucleic acid is the corresponding nucleic acid in the organism into which the engineered nucleic acid is introduced. In some embodiments, the reference nucleic acid is a native nucleic acid. In some embodiments, the engineered nucleic acid encodes the same polypeptide or characteristic element thereof as the reference nucleic acid, e.g., a native nucleic acid. In some embodiments, the engineered nucleic acid encodes a different polypeptide or characteristic element thereof than the polypeptide or characteristic element thereof encoded by the reference nucleic acid. In some embodiments, the engineered polypeptide comprises one or more differences compared to a reference polypeptide (e.g., encoded by a reference nucleic acid, found in nature, etc.). In some embodiments, the engineered polypeptide comprises one or more amino acid residues that are different from the reference polypeptide. In some embodiments, the engineered polypeptide is a polypeptide that is not present in the organism into which it is introduced. In some embodiments, the engineered polypeptide is homologous to a reference polypeptide, e.g., shares 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99%, or more homology to a reference polypeptide or characteristic element thereof. In some embodiments, a characteristic element is a domain that catalyzes a relevant reaction. In some embodiments, a characteristic element is a set of amino acid residues. In some embodiments, a characteristic element is a set of amino acid residues that form contacts with substrates, products, cofactors, etc., and / or facilitate a relevant reaction. As will be understood by one of skill in the art, residues in a set of amino acid residues can be adjacent to one another in sequence, or can be separated. In some embodiments, two or more amino acid residues in a set can be in close spatial proximity to one another, e.g., in a catalytic pocket.

[0483] In some embodiments, for biosynthetic production, the organism can express high levels and / or activities of one or more biosynthetic polypeptides. In some embodiments, the organism provides an improved rate and / or yield to produce a desired product.

[0484] As described herein, in some embodiments, the present disclosure provides high product yields. In some embodiments, the yield, e.g., yield of one or more steps of a process involving one or more biosynthetic polypeptides, is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg / L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g / L. In some embodiments, the provided technology provides high utilization of a substrate, e.g., pyruvate, for a desired product. In some embodiments, the percent utilization for a desired product is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0485] One skilled in the art understands that various compounds of the disclosure, such as compounds of Formula P-1, P-2, P-3, P-4, P-4', P-5, P-5', P-6, P-7, P-8, P-9, P-9', P-10, or P-10', or salts thereof, can be used as feedstocks to produce various compounds, materials, and products. For example, adipic acid can be used to produce nylon 6,6, polyester polyols, polyester resins, plasticizers, foodstuffs, and other materials. 1,5-pentanediol can be used to make various polyurethanes, polyester polyols, and polyesters. 1,6-hexanediol (HDO) can be used to make various polyesters, some of which can be used in industrial coating applications. HDO can also be used to produce polyurethanes, which can be used, inter alia, as coatings for automotive applications. In some embodiments, HDO is used to produce macroglycols, such as adipate and polycarbonate glycols, for use in, e.g., elastomers and polyurethane dispersions (e.g., for parquet flooring and leather coatings). 6-hydroxyhexanoic acid can be cyclized to produce epsilon-caprolactone, which can then be aminated to produce epsilon-caprolactam, by traditional chemical or biosynthetic processes, or combinations thereof. 6-hydroxyhexanoic acid can be aminated to produce 6-aminohexanoic acid, which can then be cyclized to produce epsilon-caprolactam, by traditional chemical or biosynthetic processes, or combinations thereof. Epsilon-caprolactam can be used, inter alia, to produce nylon 6, a polymer that is widely used in many different industries. Epsilon-caprolactone can be polymerized to produce polycaprolactone (PCL), a biodegradable polyester with various applications, including the production of specialized polyurethanes. Various 2-ketocarboxylic acids can be used to produce various industrially relevant chemicals and pharmaceuticals. In some embodiments, such chemicals and pharmaceuticals, or intermediates thereof, are amino acids or alpha-hydroxy carboxylic acids. In some embodiments, compounds of the disclosure are used to make polyesters, polyester polyols, polyurethanes, nylons (e.g., from adipic acid), polycarbonate glycols (e.g., from HDO or 1,5-pentanediol, etc.), diacrylates (e.g., from HDO or 1,5-pentanediol, etc.), diglycidyl ethers (e.g., from HDO or 1,5-pentanediol, etc.), and the like.

[0486] In some embodiments, the disclosure provides the production of the provided methods, such as the production of compounds of Formula P-1, P-2, P-3, P-4, P-4', P-5, P-5', P-6, P-7, P-8, P-9, P-9', P-10, or P-10', or salts thereof, and the production of various compounds, materials, products, and the like, produced from such compounds.

[0487] The provided technology provides a number of advantages. Among others, the provided methods utilize one or more biosynthetic polypeptides and / or materials from renewable resources, which can increase efficiency and / or reduce pollution. In some embodiments, formulations of the present disclosure (e.g., compounds of Formula P-1, P-2, P-3, P-4, P-4', P-5, P-5', P-6, P-7, P-8, P-9, P-9', P-10, or P-10', or salts thereof, as well as a variety of compounds, materials, products, etc. prepared from such compounds) comprise enriched levels of one or more isotopes (e.g., 14 C) compared to those prepared from fossil carbon sources. In some embodiments, formulations using fossil carbon sources have 0 or nearly 0 14 C levels. Techniques for assessing the isotopic ratios and / or levels of a variety of atoms in compounds, compositions, prepared products, etc. are well known to those of skill in the art and can be used in accordance with the present disclosure. For example, in some embodiments, isotopic enrichment can be readily assessed by mass spectrometry using techniques such as accelerated mass spectrometry (AMS) and / or Stable Isotope Ratio Mass Spectrometry (SIRMS) and / or by Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR).

[0488] As will be appreciated by those of skill in the art, the provided methods can be performed in vitro in a system comprising one or more biosynthetic polypeptides. In many embodiments, the provided technology is performed using an organism (e.g., a microorganism, such as a bacterium) that expresses one or more biosynthetic polypeptides. In some embodiments, the present disclosure provides an organism, such as a bacterium, that expresses one or more biosynthetic polypeptides as described herein. In some embodiments, such organisms are engineered. In some embodiments, such organisms are engineered and / or cultured to express increased levels of one or more biosynthetic polypeptides and / or increased activity. In some embodiments, such organisms are engineered and / or cultured to utilize carbon sources to more efficiently produce desired products.

[0489] In some embodiments, the present disclosure provides an organism that produces an aldol product of an aliphatic aldehyde, the microorganism comprising increased expression or activity of an aldol product biosynthetic polypeptide. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium.

[0490] In some embodiments, the present disclosure provides organisms that produce an aldol dehydration product of an aldehyde, the microorganism comprising increased expression or activity of a hydroxyaldehyde product biosynthesis polypeptide, an aldol dehydration product biosynthesis polypeptide, a dehydration product biosynthesis polypeptide, and combinations thereof. In some embodiments, the present disclosure provides organisms that produce an aldol dehydration product of an aldehyde, the microorganism comprising increased expression or activity of a hydroxyaldehyde dehydration product biosynthesis polypeptide. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium. In some embodiments, the aldehyde is an aliphatic aldehyde.

[0491] In some embodiments, the present disclosure provides organisms that produce an alkene reduction product, the microorganism comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide. In some embodiments, the present disclosure provides organisms that produce an alkene reduction product from pyruvate and an aldehyde, the microorganism comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium.

[0492] In some embodiments, the present disclosure provides a culture of an organism as described herein. In some embodiments, the present disclosure provides a culture of a bacterium. In some embodiments, the culture comprises one or more products of one or more biosynthesis polypeptides, for example one or more compounds of Formula P-1, P-2, P-3, P-4, P-4', P-5, P-5', P-6, P-7, P-8, P-9, P-9', P-10, or P-10', or a salt thereof.

[0493] As understood by one of skill in the art, pyruvate can be provided as pyruvate or a salt thereof.

[0494] In one aspect, provided herein is a method for making a compound of Formula I, or a salt thereof, or a solvate of the compound or salt:

[0495]

[0496] wherein R is CH2OH, CH3, or H, wherein the method comprises an enzymatic step.

[0497] In some embodiments, the method comprises, or alternatively consists essentially of, or yet further consists of, combining or incubating in solution a C N an aldehyde (wherein R is CH2OH, CH3, or H) and pyruvate, such that C is produced by an aldol condensation catalyzed by a hydratase-aldolase enzyme (referred to herein as Ads-Hyd) of EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4.N aldehyde and pyruvate to C N+3 3,4-dehydro-2-keto-carboxylic acid intermediate; then (b) using an oxidoreductase enzyme of EC number 1.6.5. (e.g., EC number 1.6.5.5.) to convert C N+3 3,4-dehydro-2-keto-carboxylic acid to C N+3 2-keto carboxylic acid (i.e., a compound of Formula I), or a salt thereof, or a solvate of the compound or salt.

[0498] In some embodiments, the method comprises, or alternatively consists essentially of, or yet further consists of, combining or incubating in solution C aldehyde and pyruvate under conditions such that A) C N aldehyde (wherein R is CH2OH, CH3, or H) and pyruvate, such that A) C N aldehyde and pyruvate to C N+3 4-hydroxy-2-keto carboxylic acid intermediate; then (b) using a hydratase- aldolase to convert the 4-hydroxy-2-keto carboxylic acid to C N+3 3,4-dehydro-2-keto-carboxylic acid; then (c) using an oxidoreductase enzyme of EC number 1.6.5. (e.g., EC number 1.6.5.5.) to convert C N+3 3,4-dehydro-2-keto-carboxylic acid to C N+3 2-keto carboxylic acid (i.e., a compound of Formula I), or a salt thereof, or a solvate of the compound or salt.

[0499] In another aspect, provided herein is a method for making a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxyhexanoic acid, the method comprising, or alternatively consisting essentially of, or yet further consisting of, a) using a combination of a hydratase-aldolase of EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4 and an oxidoreductase enzyme of EC number 1.6.5 (e.g., EC number 1.6.5.5) to convert 3-hydroxy-propanal and pyruvate to a 6-hydroxy-2-keto carboxylic acid intermediate; and b) converting the 6-hydroxy-2-keto carboxylic acid intermediate to the compound by an enzymatic step.

[0500] In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidene pyruvate hydratase-aldolase with EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-2'-carboxybenzylidene pyruvate hydratase-aldolase with EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is an acetoacetate decarboxylase with EC number 4.1.1.4.

[0501] In some embodiments, the microorganism is used as a host for making a compound of Formula I or a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxyhexanoic acid, or a salt thereof, or a solvate of the compound or salt. As used herein, "host" refers to a cell or microorganism that can produce one or more enzymes capable of catalyzing a reaction inside the cell or microorganism (by, for example, taking up a starting material and optionally secreting a product) or outside the cell or microorganism (by, for example, secreting an enzyme).

[0502] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of isolating a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxyhexanoic acid, or a salt thereof, or a solvate of the compound or salt, from the solution, culture, and / or host cell.

[0503] In some embodiments, the conditions of the methods disclosed herein comprise, or alternatively consist essentially of, or yet further consist of, incubating or contacting the components at a temperature of about 10 to about 200 °C, or alternatively at least (all temperatures provided are in degrees Celsius) 10, 15, 20, 25, 28, 29, 30, 31, 32, 33, 34, 35, 37, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 °C, or no more than 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, or 25 °C (lower temperature limit is 10 °C). In some embodiments, the conditions comprise, or alternatively consist essentially of, or yet further consist of, incubating the solution at a pH of about 2 to about 12. In some embodiments, the pH is at least 2, or 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 9, up to about 12. In some embodiments, the pH is no more than 12, 11, 10, 9, 8, 7.5, 7, 6.5, 6, 5.5, or 4, with a lower pH limit of no less than 2.

[0504] In some embodiments, the conditions include, or alternatively consist essentially of, or further consist of, pyruvate and C N The molar concentration of the aldehyde is present at a concentration of about 0.1 mM to about 5 M. In some embodiments, the concentration is at least about 0.1, 0.5, 1, 10, 100, 500 mM, or 1 M. In some embodiments, the concentration is no more than about 4 M, 3 M, 2 M, 1 M, 500 mM, 200 mM, 100 mM, or 10 mM. The concentration of pyruvate and C N The concentrations of pyruvate and C

[0505] In some embodiments, the conditions include the presence of a non-natural microorganism that produces one or more enzymes selected from the group consisting of Class I / II pyruvate-dependent aldolases, hydratase-aldolases, dehydratases, quinone oxidoreductases, enoyl-CoA reductases, primary alcohol dehydrogenases, keto acid decarboxylases, coenzyme A transferases, and carboxylate reductases. Each of these enzymes is a reaction-specific enzyme.

[0506] In some embodiments, the microorganism or host is genetically engineered to overexpress an enzyme or to express an enzyme in greater amounts than a wild-type counterpart. Methods of determining expression levels of enzymes or expression products are known in the art, for example by PCR.

[0507] In some embodiments, C N The aldehyde is 3-hydroxy-propanal.

[0508] In some embodiments, the method further includes, or alternatively consists essentially of, or yet further consists of, preparing 3-hydroxy-propanal and pyruvate from glycerol, C5 sugars, C6 sugars, phosphoglycerides, other carbon sources, glycolysis pathway intermediates, propionic acid metabolism intermediates, or combinations thereof.

[0509] In some embodiments, 3-hydroxy-propanal is obtained by dehydration of glycerol.

[0510] In some embodiments, the C5 sugars include, or alternatively consist essentially of, or yet further consist of, one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose.

[0511] In some embodiments, the C6 sugars include, or alternatively consist essentially of, or yet further consist of, one or more of allose, altrose, glucose, mannose, gulose, idose, talose, galactose, fructose, psicose, sorbose, and tagatose.

[0512] In some embodiments, the other carbon source is a feedstock suitable as a carbon source for a microorganism, wherein the feedstock comprises, or alternatively consists essentially of, or further consists of, an amino acid, a lipid, corn stover, miscanthus, municipal waste, energy cane, sugar cane, sugar cane bagasse, a starch stream, a dextrose stream, methanol, formic acid, or combinations thereof.

[0513] In some embodiments, the microorganism is used as a host for producing 1,5-pentanediol, adipic acid, 1,6-hexanediol, or 6-hydroxyhexanoic acid.

[0514] In some embodiments, the microorganism has the ability to convert a C5 sugar, a C6 sugar, glycerol, another carbon source, or combinations thereof to pyruvate.

[0515] In some embodiments, the microorganism is engineered for enhanced sugar uptake, such as C5 sugar uptake, simultaneous C6 / C5 sugar uptake, simultaneous C6 sugar / glycerol uptake, simultaneous C5 sugar / glycerol uptake, or combinations thereof.

[0516] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0517]

[0518] wherein R is H, CH3, or CH2OH;

[0519] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms.

[0520] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0521]

[0522] wherein R is H, CH3, or CH2OH;

[0523] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms.

[0524] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0525]

[0526] wherein R is H, CH3, or CH2OH;

[0527] The method comprises, consists essentially of, or consists of contacting pyruvate and with a trans-o-hydroxybenzylidene pyruvate hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the trans-o-hydroxybenzylidene pyruvate hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms.

[0528] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0529]

[0530] wherein R is H, CH3, or CH2OH;

[0531] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms; and wherein the pyruvate and performing an aldol condensation catalyzed only by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid performs a reaction catalyzed only by the quinone oxidoreductase to produce the 2-ketocarboxylic acid.

[0532] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0533]

[0534] wherein R is H, CH3, or CH2OH;

[0535] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms; and wherein the pyruvate and performing an aldol condensation catalyzed only by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and performing a reaction catalyzed only by the quinone oxidoreductase on the 2-oxo-3-enoic acid to produce the 2-keto carboxylic acid.

[0536] In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of the formula:

[0537]

[0538] wherein R is H, CH3, or CH2OH;

[0539] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms.

[0540] In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of the formula:

[0541]

[0542] wherein R is H, CH3, or CH2OH;

[0543] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms, and the method is performed in the presence of the two or more non-naturally occurring microbial organisms.

[0544] In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of the formula:

[0545]

[0546] wherein R is H, CH3, or CH2OH;

[0547] The method comprises, consists essentially of, or consists of contacting pyruvate and with a trans-o-hydroxybenzylidene pyruvate hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the trans-o-hydroxybenzylidene pyruvate hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms, and the method is performed in the presence of the two or more non-naturally occurring microbial organisms.

[0548] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0549]

[0550] wherein R is H, CH3, or CH2OH;

[0551] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms; and wherein the pyruvate and performing an aldol condensation catalyzed only by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid performs a reaction catalyzed only by the quinone oxidoreductase to produce the 2-ketocarboxylic acid.

[0552] In another aspect, provided herein is a method for producing a 2-ketocarboxylic acid of the formula:

[0553]

[0554] wherein R is H, CH3, or CH2OH;

[0555] The method comprises, consists essentially of, or consists of contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by two or more non-naturally occurring microbial organisms; and wherein the pyruvate and performing an aldol condensation catalyzed only by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid performs a reaction catalyzed only by the quinone oxidoreductase to produce the 2-ketocarboxylic acid.

[0556] In some embodiments, is 3-hydroxy-propanal. In some embodiments, 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase exogenously expressed by one or more non-naturally occurring microbial organisms.

[0557] In some embodiments, the method for producing 2-keto carboxylic acid further comprises isolating the 2-keto carboxylic acid from the one or more non-naturally occurring microbial organisms or the culture comprising the one or more non-naturally occurring microbial organisms.

[0558] In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising

[0559] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0560]

[0561] wherein R is CH2OH;

[0562] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5- hydroxy-pentanal; and

[0563] contacting the 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce 1,5- pentanediol,

[0564] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0565] In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising

[0566] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0567]

[0568] wherein R is CH2OH;

[0569] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5- hydroxy-pentanal; and

[0570] contacting the 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce 1,5- pentanediol,

[0571] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0572] In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising

[0573] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0574]

[0575] wherein R is CH2OH;

[0576] contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2- reductase to produce 2,6-dihydroxy-hexanoic acid;

[0577] contacting the 2,6-dihydroxy-hexanoic acid with a 2,6-dihydroxy-hexanoate CoA- transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0578] contacting the 2,6-dihydroxy-hexanoyl-CoA with a 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0579] contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA;

[0580] contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoic acid;

[0581] contacting the 6-hydroxyhexanoic acid with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxy-hexanal; and

[0582] contacting the 6-hydroxyhexanal with a 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol,

[0583] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0584] In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0585]

[0586] wherein R is CH2OH;

[0587] contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2- reductase to produce 2,6-dihydroxy-hexanoic acid;

[0588] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0589] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0590] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with 2,3-dehydro-hexanoyl-CoA 2,3- reductase to produce 6-hydroxy-hexanoyl-CoA;

[0591] contacting 6-hydroxy-hexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0592] contacting 6-hydroxy-hexanoate with 6-hydroxyhexanoate 1-reductase to produce 6- hydroxy-hexanal; and

[0593] contacting 6-hydroxy-hexanal with 6-hydroxyhexanal 1-reductase to produce 1,6- hexanediol,

[0594] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0595] In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising

[0596] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0597]

[0598] wherein R is CH2OH;

[0599] contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxy-hexanoate;

[0600] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0601] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0602] contacting 6-hydroxy-2-oxohexanoate-2-reductase with 2-ketocarboxylic acid to produce 2,6-dihydroxy-hexanoate;

[0603] contacting 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0604] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0605] In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0606]

[0607] wherein R is CH2OH;

[0608] contacting 6-hydroxy-2-oxohexanoate-2-reductase with 2-ketocarboxylic acid to produce 2,6-dihydroxy-hexanoate;

[0609] contacting 2,6-dihydroxy-hexanoate with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0610] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0611] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with 2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA; and

[0612] contacting 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0613] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0614] In another aspect, provided herein is a method for producing adipic acid (AA), the method comprising contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0615]

[0616] wherein R is CH2OH;

[0617] contacting 2-ketocarboxylic acid with 6-hydroxy-2-oxo-hexanoate 2-reductase to produce 2,6-dihydroxy-hexanoic acid;

[0618] contacting 2,6-dihydroxy-hexanoic acid with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0619] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0620] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with 2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA; and

[0621] contacting 6-hydroxy-hexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoic acid;

[0622] contacting 6-hydroxy-hexanoic acid with 6-hydroxyhexanoate dehydrogenase to produce 6-oxo-hexanoic acid; and

[0623] contacting 6-oxo-hexanoic acid with 6-oxo-hexanoate oxidase to produce adipic acid,

[0624] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0625] In another aspect, provided herein are methods for producing adipic acid (AA), the method comprising

[0626] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce 2-ketocarboxylic acid of the formula:

[0627]

[0628] wherein R is CH2OH;

[0629] contacting 2-ketocarboxylic acid with 6-hydroxy-2-oxo-hexanoate 2-reductase to produce 2,6-dihydroxy-hexanoic acid;

[0630] contacting 2,6-dihydroxy-hexanoic acid with 2,6-dihydroxy-hexanoate CoA-transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0631] contacting 2,6-dihydroxy-hexanoyl-CoA with 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0632] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0633] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0634] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0635] contacting 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate,

[0636] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0637] In some embodiments, the hydratase-aldolase is an enzyme with EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme with EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidene pyruvate hydratase-aldolase with EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is an enzyme with EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is an enzyme with EC number 4.1.1.4.

[0638] In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified by the following GenBank or RefSeq or Uniprot ID Nos: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified by the following GenBank, RefSeq or Uniprot ID Nos: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0639] In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by the following Genbank or RefSeq or Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising a sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0640] In some embodiments, the hydratase-aldolase is selected from the enzymes of Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from Tables 1, 5, 6, 7, and 8.

[0641] In some embodiments, the hydratase-aldolase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0642] In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme of EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID No.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0 or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0643] In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by the following GenBank, RefSeq, or Uniprot ID No.: P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising a sequence of: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0644] In some embodiments, the quinone oxidoreductase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0645] In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is heterologously expressed by one or more non-naturally occurring microbial organism. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is heterologously expressed by two or more non-naturally occurring microbial organisms.

[0646] In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by one or more exogenous genes expressed by one or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by one or more exogenous genes expressed by two or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by two or more exogenous genes expressed by one or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed by two or more exogenous genes expressed by two or more non-naturally occurring microorganisms. The one or more exogenous genes comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more exogenous genes. The two or more exogenous genes comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more exogenous genes.

[0647] In some embodiments, the hydratase-aldolase is expressed by one or more non- naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is expressed by two or more non-naturally occurring microbial organisms.

[0648] In some embodiments, the quinone oxidoreductase is expressed by one or more non- naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microbial organisms.

[0649] In some embodiments, the hydratase-aldolase is expressed by one or more non- naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is expressed by two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microbial organisms.

[0650] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by one or more non-naturally occurring microbial organisms.

[0651] In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by EC number 4.1.1.1; EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number 4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11; EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number 4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number 4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number 4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number 4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77. In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by Uniprot ID No. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto acid decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified by Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0652] In some embodiments, the 2-keto acid decarboxylase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0653] In some embodiments, the primary alcohol dehydrogenase is an enzyme with EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase is an enzyme comprising the sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

[0654] In some embodiments, the primary alcohol dehydrogenase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0655] In some embodiments, the hydratase-aldolase is the enzyme identified with Uniprot ID No. A0A286PH18; the quinone oxidoreductase is the enzyme identified with Uniprot ID No. P28304; the 2-keto acid decarboxylase is the enzyme identified with Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase is the enzyme identified with the following Uniprot or GenBank ID Nos.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by Uniprot ID No. A0A286PH18; the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by Uniprot ID No. P28304; the 2-keto acid decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified by the following Uniprot or GenBank ID No.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0656] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6- hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase are expressed by one or more non-naturally occurring microbial organisms.

[0657] In some embodiments, wherein 6-hydroxy-2-oxohexanoate-2-reductase, 2,6- dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, 2,3- dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6- hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase are expressed by one or more non-naturally occurring microbial organisms exogenously.

[0658] In some embodiments, 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; 6-hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; 6-hydroxyhexanoate 1-reductase is an enzyme of EC number 1.2.99.6; and 6-hydroxyhexanal 1-reductase is an enzyme of EC number 1.1.1.

[0659] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925 and Q5U923; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93; the 6-hydroxyhexanoate 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1 or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0660] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or 0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923, or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified with the following Uniprot or GenBank ID No. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0661] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or 0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923, or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No.The identified enzyme has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with the following Uniprot or GenBank ID No.: D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0662] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 6-hydroxyhexanoate 1-reductase is an enzyme comprising the sequence of SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68; and the 6-hydroxyhexanal 1-reductase is an enzyme comprising the sequence of SEQ ID NO: 70.

[0663] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58;6-hydroxyhexanoate 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68; and 6-hydroxyhexanal 1 -reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 70.

[0664] In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2- reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1 -reductase, and 6-hydroxyhexanal 1 -reductase further comprises one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0665] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

[0666] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923, or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified with Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0667] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot or GenBank ID No. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923 or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0668] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0669] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 53, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0670] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group of enzymes identified by EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme of EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme of EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme of EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate dehydrogenase is an enzyme of EC number 1.1.1.258; and the 6-oxo-hexanoate oxidase is an enzyme of EC number 1.2.1.63.

[0671] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is the enzyme identified by Uniprot ID No. Q5FTU6; the 2,6-dihydroxy-hexanoate CoA-transferase is the enzyme identified by Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is the enzyme identified by Uniprot ID No. Q5U924, Q5U925, and Q5U923 or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is the enzyme identified by Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is the enzyme identified by Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase is the enzyme identified by Uniprot ID No. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase is the enzyme identified by Uniprot ID No. Q9R2F4.

[0672] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5FTU6; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q5U924, Q5U925, and Q5U923 or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to the enzyme identified with Uniprot ID No. Q9R2F4.

[0673] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO: 53; the 2,6-dihydroxy-hexanoate CoA- transferase is an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63 or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 6-hydroxyhexanoate dehydrogenase is an identified enzyme comprising the sequence of SEQ ID NO: 71 or SEQ ID NO: 72; and the 6-oxo-hexanoate oxidase is an enzyme comprising the sequence of SEQ ID NO: 75.

[0674] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 53; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63; or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an identified enzyme comprising the sequence of SEQ ID NO: 71 and SEQ ID NO: 72; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identity to an enzyme comprising the sequence of SEQ ID NO: 75.

[0675] In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2- reductase, 2,6-dihydroxy-hexanoate CoA-transferase, 2,6-dihydroxy-hexanoyl-CoA 2- dehydratase, 2,3-dehydro-hexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxo-hexanoate oxidase further comprise one or more protein tags. In some embodiments, the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag

[0676] In some embodiments, the pyruvate is produced from a carbon source selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

[0677] In some embodiments, the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase exogenously expressed by one or more non-naturally occurring microbial organisms.

[0678] The one or more non-naturally occurring microbial organisms comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more non-naturally occurring microbial organisms. The two or more non-naturally occurring microbial organisms comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more non-naturally occurring microbial organisms. In some embodiments, the methods disclosed herein are performed in the presence of one non-naturally occurring microbial organism. In some embodiments, the methods disclosed herein are performed in the presence of two non-naturally occurring microbial organisms. In some embodiments, the methods disclosed herein are performed in the presence of three non-naturally occurring microbial organisms. In some embodiments, the methods disclosed herein are performed in the presence of four non-naturally occurring microbial organisms. In some embodiments, the methods disclosed herein are performed in the presence of five non-naturally occurring microbial organisms.

[0679] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0680] In some embodiments, the present application provides the following embodiments as examples:

[0681] 1. A method for producing a 2-ketocarboxylic acid of the formula:

[0682]

[0683] wherein R is H, CH3, or CH2OH;

[0684] the method comprising contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce the 2-ketocarboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms.

[0685] 2. The method of embodiment 1, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0686] 3. The method of embodiment 1, wherein the hydratase-aldolase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0687] 4. The method of embodiment 1, wherein the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0688] 5. The method of embodiment 1, wherein the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms.

[0689] 6. The method of embodiment 1, wherein the hydratase-aldolase is expressed exogenously by the one or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms.

[0690] 7. The method of any one of embodiments 1-6, wherein is 3-hydroxy-propanal.

[0691] 8. The method of embodiment 7, wherein the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0692] 9. The method of any one of embodiments 1-8, further comprising isolating the 2-ketocarboxylic acid from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms.

[0693] 10. A method for producing a 2-ketocarboxylic acid of the formula:

[0694]

[0695] wherein R is H, CH3, or CH2OH;

[0696] The method comprises contacting pyruvate and with a hydratase-aldolase and a quinone oxidoreductase to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms.

[0697] 11. The method of embodiment 10, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0698] 12. The method of embodiment 10, wherein the hydratase-aldolase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0699] 13. The method of embodiment 10, wherein the quinone oxidoreductase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0700] 14. The method of embodiment 10, wherein the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

[0701] 15. The method of embodiment 10, wherein the hydratase-aldolase is expressed exogenously by the two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

[0702] 16. The method of any one of embodiments 10-15, wherein is 3-hydroxy-propanal.

[0703] 17. The method of embodiment 16, wherein the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0704] 18. The method of any one of embodiments 10-17, further comprising isolating the 2-keto carboxylic acid from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

[0705] 19. The method of any one of embodiments 1-18, wherein the hydratase-aldolase is an enzyme with EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4.

[0706] 20. The method of any one of embodiments 1-18, wherein the hydratase-aldolase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID Nos.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.

[0707] 21. The method of any one of embodiments 1-18, wherein the hydratase-aldolase is an enzyme comprising the sequence of:

[0708] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0709] 22. The method of any one of embodiments 1-18, wherein the hydratase-aldolase has at least 50% identity to an enzyme comprising a sequence of:

[0710] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0711] 23. The method of any one of embodiments 1-18, wherein the hydratase-aldolase has at least 70% identity to an enzyme comprising a sequence of:

[0712] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0713] 24. The method of any one of embodiments 1-18, wherein the hydratase-aldolase has at least 90% identity to an enzyme comprising a sequence of:

[0714] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0715] 25. The method of any one of embodiments 1-18, wherein the hydratase-aldolase is an enzyme selected from Tables 1, 5-8.

[0716] 26. The method of any one of embodiments 1-25, wherein the quinone oxidoreductase is an enzyme of EC number 1.6.5 (e.g., EC 1.6.5.5).

[0717] 27. The method of any one of embodiments 1-25, wherein the quinone oxidoreductase is an enzyme comprising a sequence of:

[0718] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0719] 28. The method of any one of embodiments 1-25, wherein the quinone oxidoreductase has at least 50% identity to an enzyme comprising a sequence of:

[0720] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0721] 29. The method of any one of embodiments 1-25, wherein the quinone oxidoreductase enzyme has at least 70% identity to an enzyme comprising a sequence of:

[0722] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0723] 30. The method of any one of embodiments 1-25, wherein the quinone oxidoreductase enzyme has at least 90% identity to an enzyme comprising a sequence of:

[0724] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0725] 31. The method of any one of embodiments 1-30, wherein one or more of the hydratase- aldolase and quinone oxidoreductase further comprises one or more protein tags.

[0726] 32. The method of embodiment 31, wherein the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0727] 33. The method of any one of embodiments 1-32, wherein the pyruvate is produced from a carbon source selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

[0728] 34. The method of any one of embodiments 1-11, wherein R is CH2OH.

[0729] 35. A method for producing 1,5-pentanediol, the method comprising:

[0730] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0731]

[0732] wherein R is CH2OH;

[0733] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5- hydroxy-pentanal; and

[0734] contacting the 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce 1,5- pentanediol,

[0735] wherein the method is performed in a culture comprising one or more non-naturally occurring microbial organisms.

[0736] 36. The method of embodiment 35, wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms.

[0737] 37. The method of embodiment 35, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0738] 38. The method of embodiment 35, wherein the hydratase-aldolase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0739] 39. The method of embodiment 35, wherein the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0740] 40. The method of embodiment 35, wherein the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms.

[0741] 41. The method of any one of embodiments 35-40, wherein the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by the one or more non-naturally occurring microorganisms.

[0742] 42. The method of any one of embodiments 35-40, wherein the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0743] 43. The method of any one of embodiments 35-40, wherein one or more of the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are overexpressed by the one or more non-naturally occurring microbial organisms.

[0744] 44. The method of any one of embodiments 35-43, further comprising isolating the 1,5- pentanediol from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms.

[0745] 45. A method for producing 1,5-pentanediol, the method comprising:

[0746] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-keto carboxylic acid of the formula:

[0747]

[0748] wherein R is CH2OH;

[0749] contacting the 2-keto carboxylic acid with a 2-keto acid decarboxylase to produce 5- hydroxy-pentanal; and

[0750] contacting the 5-hydroxy-pentanal with a primary alcohol dehydrogenase to produce the 1,5- pentanediol,

[0751] wherein the method is performed in a culture comprising two or more non-naturally occurring microbial organisms.

[0752] 46. The method of embodiment 45, wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms.

[0753] 47. The method of embodiment 45, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0754] 48. The method of embodiment 45, wherein the hydratase-aldolase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0755] 49. The method of embodiment 45, wherein the quinone oxidoreductase is expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0756] 50. The method of embodiment 45, wherein the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

[0757] 51. The method of any one of embodiments 45-50, wherein the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by the two or more non-naturally occurring microbial organisms.

[0758] 52. The method of any one of embodiments 45-50, wherein the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed exogenously by the two or more non-naturally occurring microbial organisms.

[0759] 53. The method of any one of embodiments 45-50, wherein one or more of the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are overexpressed by the two or more non-naturally occurring microbial organisms.

[0760] 54. The method of any one of embodiments 45-53, further comprising isolating the 1,5-pentanediol from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

[0761] 55. The method of any one of embodiments 35-54, wherein the hydratase-aldolase is an enzyme with EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4.

[0762] 56. The method of any one of embodiments 35-54, wherein the hydratase-aldolase is an enzyme selected from the group of enzymes identified with the following GenBank, RefSeq, or Uniprot ID No.: D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.

[0763] 57. The method of any one of embodiments 35-54, wherein the hydratase-aldolase is an enzyme comprising a sequence of:

[0764] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0765] 58. The method of any one of embodiments 35-54, wherein the hydratase-aldolase has at least 50% identity to an enzyme comprising a sequence of:

[0766] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0767] 59. The method of any one of embodiments 35-54, wherein the hydratase-aldolase has at least 70% identity to an enzyme comprising a sequence of:

[0768] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0769] 60. The method of any one of embodiments 35-54, wherein the hydratase-aldolase has at least 90% identity to an enzyme comprising a sequence of:

[0770] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0771] 61. The method of any one of embodiments 35-54, wherein the hydratase-aldolase is an enzyme selected from Tables 1, 5-8.

[0772] 62. The method of any one of embodiments 35-61, wherein the quinone oxidoreductase is an enzyme with EC number 1.6.5 (e.g., EC 1.6.5.5).

[0773] 63. The method of any one of embodiments 35-61, wherein the quinone oxidoreductase is an enzyme comprising a sequence of:

[0774] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0775] 64. The method of any one of embodiments 35-61, wherein the quinone oxidoreductase has at least 50% identity to an enzyme comprising a sequence of:

[0776] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0777] 65. The method of any one of embodiments 35-61, wherein the quinone oxidoreductase has at least 70% identity to an enzyme comprising a sequence of:

[0778] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0779] 66. The method of any one of embodiments 35-61, wherein the quinone oxidoreductase enzyme has at least 90% identity to an enzyme comprising a sequence of:

[0780] SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO: 97.

[0781] 67. The method of any one of embodiments 35-66, wherein the 2-keto acid decarboxylase enzyme is an enzyme having an EC number of: EC number 4.1.1.1; EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number 4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11; EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number 4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number 4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number 4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number 4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77.

[0782] 68. The method of any one of embodiments 35-66, wherein the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified with Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0783] 69. The method of any one of embodiments 35-66, wherein the 2-keto acid decarboxylase has at least 50% identity to an enzyme selected from the group of enzymes identified with Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0784] 70. The method of any one of embodiments 35-66, wherein the 2-keto acid decarboxylase has at least 70% identity to an enzyme selected from the group of enzymes identified with Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0785] 71. The method of any one of embodiments 35-66, wherein the 2-keto acid decarboxylase has at least 90% identity to an enzyme selected from the group of enzymes identified with Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

[0786] 72. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase is an enzyme with EC number 1.1.1.61.

[0787] 73. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified with the following Uniprot or GenBank ID No.: NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1.

[0788] 74. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase is an enzyme comprising the sequence of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0789] 75. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase has at least 50% identity to an enzyme comprising the sequence of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0790] 76. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase has at least 70% identity to an enzyme comprising the sequence of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0791] 77. The method of any one of embodiments 35-71, wherein the primary alcohol dehydrogenase has at least 90% identity to an enzyme comprising the sequence of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0792] 78. The method of any one of embodiments 35-54, wherein

[0793] the hydratase-aldolase is an enzyme comprising the sequence of SEQ ID NO: 8;

[0794] the quinone oxidoreductase is an enzyme comprising the sequence of SEQ ID NO: 45;

[0795] the 2-keto acid decarboxylase is an enzyme comprising the sequence of SEQ ID NO: 83; and

[0796] the primary alcohol dehydrogenase is an enzyme comprising the sequence of SEQ ID NO: 70.

[0797] 79. The method of any one of embodiments 35-78, wherein one or more of the hydratase-aldolase, quinone oxidoreductase, 2-keto acid decarboxylase, and primary alcohol dehydrogenase further comprises one or more protein tags.

[0798] 80. The method of embodiment 79, wherein the protein tag is selected from the group consisting of a polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

[0799] 81. The method of any one of embodiments 35-80, wherein the pyruvate is produced from a carbon source selected from the group consisting of glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

[0800] 82. The method of any one of embodiments 35-81, wherein the 3-hydroxy- propanal is produced by dehydration of glycerol by a glycerol dehydratase exogenously expressed by the one or more non-naturally occurring microbial organisms.

[0801] 83. A method for producing 1,6-hexanediol, the method comprising

[0802] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0803]

[0804] wherein R is CH2OH;

[0805] contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2- reductase to produce 2,6-dihydroxy-hexanoate;

[0806] contacting the 2,6-dihydroxy-hexanoate with a 2,6-dihydroxy-hexanoate CoA transferase to produce 2,6-dihydroxy-hexanoyl-CoA;

[0807] contacting the 2,6-dihydroxy-hexanoyl-CoA with a 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydro-hexanoyl-CoA;

[0808] contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a 2,3-dehydro- hexanoyl-CoA 2,3-reductase to produce 6-hydroxy-hexanoyl-CoA;

[0809] contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxy-hexanoate;

[0810] contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxy-hexanal; and

[0811] contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol,

[0812] wherein the method is performed in a culture comprising one or more non- naturally occurring microbial organisms.

[0813] 84. The method of embodiment 83, wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms.

[0814] 85. The method of embodiment 83, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0815] 86. The method of embodiment 83, wherein the hydratase-aldolase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0816] 87. The method of embodiment 83, wherein the quinone oxidoreductase is expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0817] 88. The method of embodiment 83, wherein the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms.

[0818] 89. The method of any one of embodiments 83-88, wherein the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are expressed by the one or more non-naturally occurring microbial organisms.

[0819] 90. The method of any one of embodiments 83-88, wherein the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are expressed exogenously by the one or more non-naturally occurring microbial organisms.

[0820] 91. The method of any one of embodiments 83-88, wherein one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are overexpressed by the one or more non-naturally occurring microbial organisms.

[0821] 92. The method of any one of embodiments 83-91, further comprising isolating the 1,6-hexanediol from the one or more non-naturally occurring microbial organisms or the culture comprising the one or more non-naturally occurring microbial organisms.

[0822] 93. A method for producing 1,6-hexanediol, the method comprising

[0823] contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula:

[0824]

[0825] wherein R is CH2OH;

[0826] contacting the 2-ketocarboxyli...

Claims

1. A method, comprising: Pyruvate and 3-hydroxypropanal (HO-CH2-CH2-CHO) are contacted with a biosynthetic polypeptide of hydroxyl dehydration product with the amino acid sequence SEQ ID NO:27 to produce 6-hydroxy-2-oxo-3-hexenoic acid (HO-CH2-CH2-CH=CH-C(O)-COOH) or its salt.

2. The method of claim 1, wherein the dehydration product of the hydroxyl group is a biosynthesized polypeptide in a microorganism.

3. The method of claim 2, wherein the microorganism is engineered to contain exogenous nucleic acid encoding the biosynthetic polypeptide of the aldol dehydration product.

4. The method of claim 2, wherein the microorganism expresses the hydroxyl dehydration product biosynthetic polypeptide at a regulated level.

5. The method of claim 2, wherein the microorganism is Escherichia coli.

6. The method of claim 3, wherein the microorganism is Escherichia coli.

7. The method of claim 4, wherein the microorganism is Escherichia coli.

8. The method of claim 1, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate and HO-CH2-CH2-CHO.

9. The method of claim 2, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate and HO-CH2-CH2-CHO.

10. The method of claim 3, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate, and HO-CH2-CH2-CHO.

11. The method of claim 4, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate and HO-CH2-CH2-CHO.

12. The method of claim 5, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate, and HO-CH2-CH2-CHO.

13. The method of claim 6, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate, and HO-CH2-CH2-CHO.

14. The method of claim 7, wherein the contact is carried out in a culture medium containing microorganisms, pyruvate, and HO-CH2-CH2-CHO.

15. The method of any one of claims 1-14, wherein the hydroxyl dehydration product biosynthetic polypeptide is expressed by plasmid.

16. The method of any one of claims 1-14, wherein the aldol dehydration product biosynthetic polypeptide is expressed via plasmid and the DNA encoding the aldol dehydration product biosynthetic polypeptide is codon-optimized for expression in Escherichia coli.

17. The method of claim 16, wherein the DNA encoding the hydroxyl dehydration product biosynthetic polypeptide is cloned into the pB11 backbone plasmid downstream of the T7 RNA polymerase promoter and upstream of the T7 terminator sequence.

18. The method of any one of claims 1-14, wherein the pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch or combinations thereof.

19. The method of claim 15, wherein the pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch or combinations thereof.

20. The method of claim 16, wherein the pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch or combinations thereof.

21. The method of claim 17, wherein the pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch or combinations thereof.

Images