Production of chemicals from renewable resources

Abstract

Among other things, the disclosure provides biosynthetic polypeptides, methods, and non-naturally occurring microorganisms for preparing various compounds such as 1,5-pentanediol, adipic acid, 1,6-hexanediol, 6-hydroxyhexanoic acid, and 2-ketocarboxylic acids.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 62 / 838,793, filed April 25, 2019, and 62 / 868,824, filed June 28, 2019, each of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to compositions and methods of preparing industrially useful chemicals. [Background technology]

[0003] 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, December 2012). AA, along with hexamethylenediamine (HMDA), is used in the production of nylon 6,6, polyester resins, plasticizers, food products, and other materials. Therefore, a method for preparing adipic acid in high yields using renewable resources is highly desirable.

[0004] 1,5-Pentanediol (PDL) is the main component of polyurethanes and polyesters. 1,6-Hexanediol (HDO) is a linear diol with terminal hydroxyl groups. HDO is used in polyesters for industrial coatings and in two-component polyurethane coatings for automotive applications. HDO is also used to produce macrodiols, such as adipates, polycarbonate diols used in elastomers, and polyurethane dispersions for parquet flooring and leather coatings.

[0005] 6-Hydroxyhexanoic acid (6HH) can be cyclized to produce ε-caprolactone, which can then be aminated to produce 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 with applications in producing specialty polyurethanes.

[0006] 2-Ketocarboxylic acids are useful intermediates in the preparation of numerous industrially relevant chemicals and pharmaceuticals. 2-Ketocarboxylic acids are precursors for the production of amino acids and industrially useful α-hydroxycarboxylic acids. Summary of the Invention [Means for solving the problem]

[0007] Among other things, the present disclosure includes the recognition that certain biosynthetic peptides, e.g., enzymes, can be used to efficiently prepare compounds, in many embodiments, from substrates that are structurally distinct from the enzyme's natural and / or characterized substrate. In some embodiments, the present disclosure provides techniques (e.g., enzymes, nucleic acids, organisms, culture media, etc.) for preparing compounds using one or more of these enzymes.

[0008] For example, in some embodiments, the present disclosure provides that various hydratase-aldolase-like aldol dehydration product biosynthetic polypeptides can be effectively used to prepare numerous compounds from aliphatic aldehydes, as opposed to their typical substrates, aromatic aldehydes. 1. A method comprising contacting pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthetic polypeptide to produce an aldol dehydration product, the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aromatic group; the aldol dehydration product is a compound containing an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group; A method is provided.

[0009] In some embodiments, the aldehyde, e.g., the aliphatic aldehyde, has the structure of formula A-1: R a -L 2 -L 1 -C(O)H A-1 or a salt thereof, wherein R a is R" or -OR" L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 heteroaliphatic groups, one or more of the methylene units of which are optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-; -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; each R" is independently -R', -C(O)R', -CO2R', or -SO2R'; R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Two or more R' groups, together with the intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0-5 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 0-5 heteroatoms.

[0010] In some embodiments, L 1 is an optionally substituted —CH—. In some embodiments, L 1 is an optionally monosubstituted —CH—. In some embodiments, L 1 is -CH2-.

[0011] In some embodiments, the aldol dehydration product has the structure of formula P-2: R a -L 2 -L 1 -CH=CH-C(O)-C(O)OH P-2 or a salt thereof, wherein each variable is independently as described herein.

[0012] As described herein, the aldol dehydration product, e.g., a compound of formula P-2 or a salt thereof, can, in some embodiments, be further processed through one or more biosynthetic processes to provide various products such as 1,5-pentanediol, HDO, 6HH, adipic acid (see, e.g., Figures 2-5), and the like, and various products made therefrom, including various polymeric products made therefrom.

[0013] In some embodiments, as shown herein, the aldol dehydration product, e.g., a compound of formula P-2 or a salt thereof, can be converted to an aldol product, e.g., a compound of formula P-1 R a -L 2 -L 1 -CH(OH)-CH2-C(O)-C(O)OH P-1 or a salt thereof, wherein each variable is independently as described herein.

[0014] In some embodiments, the aldol dehydration product is produced by contacting an aldol product with a dehydration product biosynthetic polypeptide.

[0015] In some embodiments, the aldol product is produced by contacting an appropriate substrate with an aldol product biosynthetic polypeptide.

[0016] In some embodiments, the present disclosure demonstrates that various alkene reduction product biosynthetic polypeptides can be used to produce a variety of compounds from their natural or unnatural substrates. contacting an alkene with an alkene reduction product biosynthetic polypeptide to produce an alkene reduction product; the alkene contains a double bond conjugated to a carbonyl group, reducing the double bond in the alkene that is conjugated to the carbonyl group to a single bond to provide an alkene reduction product; A method is provided.

[0017] In some embodiments, the alkene is an aldol dehydration product, such as one of formula P-2 or a salt thereof. In some embodiments, the alkene reduction product has the structure of formula P-3 below: R a -L 2 -L 1 -CH2-CH2-C(O)-C(O)OH P-3 or a salt thereof, wherein each variable is independently as described herein.

[0018] Among other things, the present invention discloses enzymes, methods and recombinant microorganisms for the preparation of 2-ketocarboxylic acids, 1,5-pentanediol, adipic acid, 1,6-hexanediol and 6-hydroxyhexanoic acid using renewable resources.

[0019] In one aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: producing pyruvate and thiamin in a culture or organism comprising one or more naturally occurring microorganisms; [ka] 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 one or more non-naturally occurring microorganisms.

[0020] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises the step of: detecting pyruvate and ATP in a culture or organism comprising two or more naturally occurring microorganisms; [ka] 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 microorganisms.

[0021] In another aspect, the present invention provides a method for producing 1,5-pentanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxypentanal; contacting the 5-hydroxypentanal with a primary alcohol dehydrogenase to produce 1,5-pentanediol; comprising or consisting essentially of The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0022] In another aspect, the present invention provides a method for producing 1,5-pentanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxypentanal; contacting the 5-hydroxypentanal with a primary alcohol dehydrogenase to produce 1,5-pentanediol; comprising or consisting essentially of This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0023] In another aspect, the present invention provides a method for producing 1,6-hexanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH, contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxyhexanal; and contacting the 6-hydroxyhexanal with 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol; Including, The method is carried out in a culture medium containing one or more non-naturally occurring microorganisms.

[0024] In another aspect, the present invention provides a method for producing 1,6-hexanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxyhexanal; and contacting the 6-hydroxyhexanal with 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0025] In another aspect, the present invention provides a method for producing 6-hydroxyhexanoate, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0026] In another aspect, the present invention provides a method for producing 6-hydroxyhexanoate, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH, contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0027] In another aspect, the present invention provides a method for producing adipic acid, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH, contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with 6-hydroxyhexanoate dehydrogenase to produce 6-oxohexanoate; contacting the 6-oxohexanoate with 6-oxohexanoate oxidase to produce adipic acid; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0028] In another aspect, the present invention provides a method for producing adipic acid, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with 6-hydroxyhexanoate dehydrogenase to produce 6-oxohexanoate; contacting the 6-oxohexanoate with 6-oxohexanoate oxidase to produce adipic acid; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0029] In some embodiments, the hydratase-aldolase is an enzyme having 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 having GenBank, RefSeq, or Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_11547803 3, 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.

[0030] In some embodiments, the hydratase-aldolase is an enzyme that is an EC No. 4.1.2.45, EC No. 4.1.2.34, or EC No. 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme that is an EC No. 4.1.2.45, EC No. 4.1.2.34, or EC No. 4.1.1.4, or is an enzyme that is an ... 0A370X7D8, 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.

[0031] In some embodiments, the hydratase-aldolase is selected from the group consisting of GenBank, RefSeq, or Uniprot ID numbers 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_1078 18191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1 or KZL92449.1, or a portion thereof that promotes formation of the aldol dehydration product (e.g., a domain, a series of amino acid residues, which can be contiguous or spaced, etc.). 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 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.

[0032] In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1 and 5-8. In some embodiments, the hydratase-aldolase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme selected from Tables 1 and 5-8.

[0033] In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified by GenBank, RefSeq, or Uniprot ID numbers 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 selected from the group consisting of GenBank, RefSeq, and Uniprot ID numbers P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, and Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A21 2, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1, 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 in WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising 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.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 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.

[0034] In some embodiments, the hydratase-aldolase and quinone oxidoreductase are expressed by one or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and quinone oxidoreductase enzymes 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 microorganisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by one or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by one or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microorganisms.

[0035] In some embodiments, the hydratase-aldolase and quinone oxidoreductase are expressed by two or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and quinone oxidoreductase enzymes 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 microorganisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by two or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microorganisms.

[0036] In some embodiments, one or more of the hydratase-aldolase and quinone oxidoreductase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0037] 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 microorganisms or a culture medium comprising one or more non-naturally occurring microorganisms. In some embodiments, the method further comprises, or consists essentially of, isolating the 2-keto carboxylic acid from two or more non-naturally occurring microorganisms or a culture medium comprising two or more non-naturally occurring microorganisms.

[0038] In some embodiments, the 2-keto acid decarboxylase is selected from the group consisting of EC No. 4.1.1.1, EC No. 4.1.1.2, EC No. 4.1.1.3, EC No. 4.1.1.4, EC No. 4.1.1.5, EC No. 4.1.1.6, EC No. 4.1.1.7, EC No. 4.1.1.11, EC No. 4.1.1.12, EC No. 4.1.1.15, EC No. 4.1.1.16, EC No. 4.1.1.17, EC No. 4.1.1.18, E In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by Uniprot ID Nos. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto acid decarboxylase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more identical to an enzyme selected from the group of enzymes identified by Uniprot ID numbers Q6QBS4, A7M7D6 or P20906.

[0039] In some embodiments, the primary alcohol dehydrogenase is an enzyme having the EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers 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 selected from the group consisting of those identified by Uniprot or GenBank ID numbers 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 ... 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 as BX39192.1, 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.

[0040] In some embodiments, the hydratase-aldolase is an enzyme identified by Uniprot ID number A0A286PH18, the quinone oxidoreductase is an enzyme identified by Uniprot ID number P28304, and the 2-keto acid decarboxylase is an enzyme identified by Uniprot ID number P28304. The enzyme is identified by ID number Q6QBS4, and primary alcohol dehydrogenases are identified by Uniprot or GenBank ID numbers 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, and YP_001703694. In some embodiments, the hydratase-aldolase is an enzyme comprising the sequence of SEQ ID NO:8, the quinone oxidoreductase is an enzyme comprising the sequence of SEQ ID NO:45, the 2-keto acid decarboxylase is an enzyme comprising the sequence of SEQ ID NO:83, and the primary alcohol dehydrogenase is an enzyme comprising the sequence of SEQ ID NO:70.

[0041] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by one or more non-naturally occurring microorganisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by one or more non-naturally occurring microorganisms.

[0042] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by two or more non-naturally occurring microorganisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by two or more non-naturally occurring microorganisms.

[0043] In some embodiments, one or more of the hydratase-aldolase, quinone oxidoreductase, 2-keto acid decarboxylase, and primary alcohol dehydrogenase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0044] In some embodiments, the method for producing 1,5-pentanediol further comprises, or consists essentially of, isolating 1,5-pentanediol from one or more non-naturally occurring microorganisms or a broth comprising one or more non-naturally occurring microorganisms. In some embodiments, the method further comprises, or consists essentially of, isolating 1,5-pentanediol from two or more non-naturally occurring microorganisms or a broth comprising two or more non-naturally occurring microorganisms.

[0045] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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.

[0046] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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.

[0047] 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, and the 2,6-dihydroxyhexanoate 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, and produces 2,6-dihydroxyhexanoyl CoA. A2-dehydratase is an enzyme having the EC number 4.2.1.167, 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44, 6-hydroxyhexanoyl-CoA transferase is an enzyme having the 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 having the EC number 1.2.99.6, and 6-hydroxyhexanal 1-reductase is an enzyme having the EC number 1.1.1.

[0048] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme selected from the group of enzymes identified by Uniprot ID number T4VW93; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1. The enzyme is selected from the group of enzymes identified by ID numbers Q5U924, Q5U925 and Q5U923, the 2,3-dehydrohexanoyl CoA 2,3-reductase is the enzyme identified by Uniprot ID number Q73Q47, and the 6-hydroxyhexanoyl CoA transferase is the enzyme identified by Uniprot ID number Q73Q47. The enzyme identified by ID number T4VW93, 6-hydroxyhexanoate 1-reductase, is listed in Uniprot or GenBank ID numbers 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 6-hydroxyhexanal 1-reductase is an enzyme identified by Uniprot or GenBank ID numbers 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.

[0049] 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-dihydroxyhexanoate 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; and the 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63. Or it is an enzyme having the sequence of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, the 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the sequence of SEQ ID NO: 65, the 6-hydroxyhexanoyl-CoA transferase is an enzyme having 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 having 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 having the sequence of SEQ ID NO: 70.

[0050] 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, and the 2,6-dihydroxyhexanoate 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 identity is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, and the 2,6-dihydroxyhexanoyl-CoA2-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, and the 2,3-dehydrohexanoyl-CoA2,The 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; 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 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 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.

[0051] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1. The enzymes are identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9. 2,3-Dehydrohexanoyl CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47. 6-Hydroxyhexanoyl CoA transferase is an enzyme identified by Uniprot ID number Q73Q47. The enzymes identified by ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or A0A2X3BTQ9 are 6-hydroxyhexanoate 1-reductases, which have Uniprot or GenBank ID numbers 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 6-hydroxyhexanal 1-reductase is identified by Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.The enzymes identified are ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, and YP_001703694.

[0052] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group consisting of Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.2,6-dihydroxyhexanoate CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified in 1, Uniprot 2,6-dihydroxyhexanoyl CoA2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified by ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or A0A2X3BTQ9. 2,3-dehydrohexanoyl-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% identity to the enzymes identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9, and The 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q73Q47, and the 6-hydroxyhexanoate 1-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or A0A2X3BTQ9. Ze has Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.6-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% identity to the enzymes identified in Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7. 407.1, 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, or YP_001703694, and have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the enzymes identified in

[0053] In some embodiments, one or more of 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0054] In some embodiments, the method for producing 1,6-hexanediol further comprises, or consists essentially of, separating 1,6-hexanediol from one or more non-naturally occurring microorganisms or a broth comprising one or more non-naturally occurring microorganisms. In some embodiments, the method further comprises, or consists essentially of separating 1,6-hexanediol from two or more non-naturally occurring microorganisms or a broth comprising two or more non-naturally occurring microorganisms.

[0055] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate-CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are expressed by one or more non-naturally occurring microorganisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate-CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by one or more non-naturally occurring microorganisms.

[0056] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate-CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are expressed by two or more non-naturally occurring microorganisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate-CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by two or more non-naturally occurring microorganisms.

[0057] 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, and the 2,6-dihydroxyhexanoate CoA transferase is selected from the group of enzymes identified by EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme having the EC number 4.2.1.167; 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44; and 6-hydroxyhexanoyl-CoA transferase is an enzyme having the EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

[0058] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1. The 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47, and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified by Uniprot ID number T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0059] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1, and the 2,6-dihydroxyhexanoate CoA transfer ... 2,6-dihydroxyhexanoyl CoA2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified by ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16 or A0A175L1W4. 2,3-dehydrohexanoyl-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% identity to the enzymes identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9, and The enzyme is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q73Q47, and the 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16 or A0A175L1W4.

[0060] 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-dihydroxyhexanoate 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-dihydroxyhexanoyl-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-dehydrohexanoyl-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.

[0061] 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% 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. 5% or more, and the 2,6-dihydroxyhexanoate 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, and the 2,6-dihydroxyhexanoyl-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 an enzyme comprising the sequence of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64 has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity, and the 2,3-dehydrohexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35% identity with an enzyme comprising the sequence of SEQ ID NO: 65. , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, 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.

[0062] In some embodiments, one or more of 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate-CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, and 6-hydroxyhexanoyl-CoA transferase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0063] In some embodiments, the method for producing 6-hydroxyhexanoate further comprises, or consists essentially of, isolating 6-hydroxyhexanoate from one or more non-naturally occurring microorganisms or a culture medium comprising one or more non-naturally occurring microorganisms. In some embodiments, the method further comprises, or consists essentially of, isolating 6-hydroxyhexanoate from two or more non-naturally occurring microorganisms or a culture medium comprising two or more non-naturally occurring microorganisms.

[0064] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase are expressed by one or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase are exogenously expressed by one or more non-naturally occurring microbial organisms.

[0065] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase are expressed by two or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase are exogenously expressed by two or more non-naturally occurring microbial organisms.

[0066] 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, and the 2,6-dihydroxyhexanoate CoA transferase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12, and ...yl CoA 2-dehydratase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12. 2,3-Dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44; 6-hydroxyhexanoyl-CoA transferase is an enzyme having the EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; 6-hydroxyhexanoate dehydrogenase is an enzyme having the EC number 1.1.1.258; and 6-oxohexanoate oxidase is an enzyme having the EC number 1.2.1.63.

[0067] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93 or A0A2X3BTQ9; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409. 2,3-Dehydrohexanoyl-CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47; 6-hydroxyhexanoyl-CoA transferase is an enzyme identified by Uniprot ID number T4VW93 or A0A2X3BTQ9; 6-hydroxyhexanoate dehydrogenase is an enzyme identified by Uniprot ID number Q7WVD0 or Q84H78; and 6-oxohexanoate oxidase is an enzyme identified by Uniprot ID number Q9R2F4.

[0068] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1, and the 2,6-dihydroxyhexanoate CoA transfer ... 2,6-dihydroxyhexanoyl CoA2-dehydratase that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID numbers T4VW93 or A0A2X3BTQ9. 2,3-dehydrohexanoyl CoA2, ...The 3-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number T4VW93 or A0A2X3BTQ9; and the 6-hydroxyhexanoate dehydrogenase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number T4VW93 or A0A2X3BTQ9. The 6-oxohexanoate oxidase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q7WVD0 or Q84H78, and the 6-oxohexanoate oxidase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number Q9R2F4.

[0069] 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-dihydroxyhexanoate CoA transferase is an enzyme comprising the sequence of SEQ ID NO:55 or SEQ ID NO:58; and the 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO:59, SEQ ID NO:61, and and SEQ ID NO:63, or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme comprising the sequence of SEQ ID NO:65; 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising the sequence of SEQ ID NO:55 or SEQ ID NO:58; 6-hydroxyhexanoate dehydrogenase is an enzyme comprising the sequence of SEQ ID NO:71 or SEQ ID NO:72; and 6-oxohexanoate oxidase is an enzyme comprising the sequence of SEQ ID NO:75.

[0070] 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, and the 2,6-dihydroxyhexanoate 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. , at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, and the 2,6-dihydroxyhexanoyl-CoA2-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, and 2,3-dehydrohexanoyl-CoA2,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% 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% identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58 ... The 6-oxohexanoate 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, and the 6-oxohexanoate 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.

[0071] In some embodiments, one or more of 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0072] In some embodiments, the method for producing adipic acid further comprises, or consists essentially of, separating adipic acid from one or more non-naturally occurring microorganisms or a broth comprising one or more non-naturally occurring microorganisms. In some embodiments, the method further comprises, or consists essentially of, separating adipic acid from two or more non-naturally occurring microorganisms or a broth comprising two or more non-naturally occurring microorganisms.

[0073] In some embodiments, pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof. [ka] is 3-hydroxypropanal. In some embodiments, the 3-hydroxypropanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by one or more non-naturally occurring microorganisms.

[0074] In another aspect, the present invention provides a recombinant microorganism comprising a first exogenous nucleic acid encoding an aldolase hydratase enzyme, and further modified to express a quinone oxidoreductase in an amount greater than that of a wild-type or unmodified version of the same microorganism, optionally 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 driving 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 enzyme is designated EC No. 4.1.2.45, EC No. 4.1.2.34, or EC No. 4.1.1.4. In some embodiments, the aldolase hydratase enzyme is designated Uniprot ID number D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F 2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_01365480 7, 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 enzyme is selected from the group consisting of Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J 6L6, 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 enzyme 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.

[0075] In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a vector, such as a plasmid vector or a viral vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a unique, separate vector. In some embodiments, the vector is a plasmid. In some embodiments, the quinone oxidoreductase is an enzyme having EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme having EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified by GenBank, RefSeq, or Uniprot ID numbers 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 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. In some embodiments, a recombinant microorganism of the invention is capable of producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH3, or CH2OH. In some embodiments, the recombinant microorganisms of the invention are capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate. In some embodiments, the recombinant microorganisms of the invention are genetically modified to improve pyruvate production from a carbon source. In some embodiments, the carbon source is selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

[0076] In another aspect, the present invention provides a culture medium comprising a recombinant microorganism disclosed herein.

[0077] In another aspect, the invention provides a population of recombinant microorganisms as disclosed herein. In some embodiments, the population is substantially homogeneous.

[0078] In another aspect, the present invention provides a culture medium comprising the populations disclosed herein.

[0079] In another aspect, the present invention provides a method for producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate, comprising culturing a population or recombinant microorganism as disclosed herein under suitable conditions that promote expression of an exogenous nucleic acid as disclosed herein. In one aspect, the exogenous nucleic acid is overexpressed relative to a wild-type or equivalent unmodified microorganism. In some embodiments, the method further comprises isolating 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate from the culture medium or microorganism. In particular embodiments, for example, the following items are provided: (Item 1) 1. A method comprising contacting pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthetic polypeptide to produce an aldol dehydration product, the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aromatic group; The method, wherein the aldol dehydration product is a compound containing an aldehyde group or a ketone group and a double bond conjugated with the aldehyde group or the ketone group. (Item 2) 2. The method of claim 1, wherein the aldol dehydration product biosynthetic polypeptide is or comprises a hydratase-aldolase. (Item 3) 2. The method according to Item 1, wherein the aldol dehydration product biosynthetic polypeptide is or comprises an enzyme having EC number 4.1.2.45, EC number 4.1.2.34, or EC 4.1.1.4, or an enzyme selected from Tables 1 and 5 to 8. (Item 4) 4. The method of claim 3, wherein the aldol dehydration product biosynthetic polypeptide is in a microorganism. (Item 5) contacting an alkene with an alkene reduction product biosynthetic polypeptide to produce an alkene reduction product; the alkene contains a double bond conjugated to a carbonyl group; The process wherein the double bond in the alkene conjugated to the carbonyl group is reduced to a single bond to provide an alkene reduction product. (Item 6) Item 6. The method according to Item 5, wherein the alkene is the aldol dehydration product according to Item 1. (Item 7) 7. The method of claim 6, wherein the alkene reduction product biosynthetic polypeptide is or comprises an enzyme belonging to EC 1.6.5 or an enzyme selected from Table 9. (Item 8) The aliphatic aldehyde has the structure of the following formula A-1: R a -L 2 -L 1 -C(O)H A-1 or a salt thereof, wherein R a is R" or -OR"; L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 A heteroaliphatic group, one or more of whose methylene units are optionally and independently -C≡C-, -C(R") 2 -, -Cy-, -O-, -S-, -SS-, -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) 2 is replaced by -N(R")-, -C(O)S- or -C(O)O-, -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; Each R" is independently -R', -C(O)R', -CO 2 R' or -SO 2 R', R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or 5. The method of claim 4, wherein two or more R' groups together with intervening atoms form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms in addition to the intervening atoms, and each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3-20 membered ring having 0-5 heteroatoms. (Item 9) The aldol dehydration product has the structure of formula P-2 below: R a -L 2 -L 1 -CH=CH-C(O)-C(O)OH P-2 or a salt thereof, wherein R a is R" or -OR"; L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 A heteroaliphatic group, one or more of whose methylene units are optionally and independently -C≡C-, -C(R") 2 -, -Cy-, -O-, -S-, -SS-, -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) 2 is replaced by -N(R")-, -C(O)S- or -C(O)O-, -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; Each R" is independently -R', -C(O)R', -CO 2 R' or -SO 2 R', R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Item 9. The method of item 8, wherein two or more R' groups together with intervening atoms form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms in addition to the intervening atoms, and each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3-20 membered ring having 0-5 heteroatoms. (Item 10) Item 10. The method according to Item 9, wherein in the formula, -CH=CH- is an E-configuration. (Item 11) Item 10. The method according to Item 9, wherein -CH=CH- is a Z-form. (Item 12) The alkene reduction product has the structure of formula P-3 below: R a -L 2 -L 1 -CH 2 -CH 2 -C(O)-C(O)OH P-3 or a salt thereof, wherein R a is R" or -OR"; L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 A heteroaliphatic group, one or more of whose methylene units are optionally and independently -C≡C-, -C(R") 2 -, -Cy-, -O-, -S-, -SS-, -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) 2 is replaced by -N(R")-, -C(O)S- or -C(O)O-, -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; Each R" is independently -R', -C(O)R', -CO 2 R' or -SO 2 R', R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or 12. The method according to any one of items 5 to 11, wherein two or more R' groups together with intervening atoms form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0 to 5 heteroatoms in addition to the intervening atoms, and each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0 to 5 heteroatoms. (Item 13) The alkene reduction product is treated to form a compound of the following formula P-10: HO-C(O)-L 2’ -L 1 -CH 2 -CH 2 -CH 2 -C(O)-OH P-10 or a salt thereof. (Item 14) The alkene reduction product is treated to form a compound of the following formula P-10' R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -CH 2 -OH P-10’ or a salt thereof. (Item 15) The method comprises reacting the compound of formula P-3 or a salt thereof with a compound of formula P-4: R a -L 2 -L 1 -CH 2 -CH 2 -CH(OH)-C(O)OH P-4 or a salt thereof, wherein R a is R" or -OR"; L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 A heteroaliphatic group, one or more of whose methylene units are optionally and independently -C≡C-, -C(R") 2 -, -Cy-, -O-, -S-, -SS-, -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) 2 is replaced by -N(R")-, -C(O)S- or -C(O)O-, -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; Each R" is independently -R', -C(O)R', -CO 2 R' or -SO 2 R', R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Item 13. The method of item 12, wherein two or more R' groups together with intervening atoms form an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms in addition to said intervening atoms, and each monocyclic ring is independently an optionally substituted, saturated, partially saturated, or aromatic 3-20 membered ring having 0-5 heteroatoms. (Item 16) 16. The method of claim 15, wherein the converting comprises contacting the compound of formula P-3 or a salt thereof with a carbonyl reduction product biosynthetic polypeptide. (Item 17) The compound of formula P-4 or a salt thereof is reacted with a compound of formula P-5 R a -L 2 -L 1 -CH 2 -CH 2 -CH(OH)-C(O)-S-CoA P-5 or a salt thereof. (Item 18) 18. The method of claim 17, wherein the converting comprises contacting the compound of formula P-4 or a salt thereof with a CoA transfer product biosynthetic polypeptide. (Item 19) The compound of formula P-5 or a salt thereof is reacted with a compound of formula P-6 below. R a -L 2 -L 1 -CH 2 -CH=CH-C(O)-S-CoA P-6 or a salt thereof. (Item 20) 20. The method of claim 19, wherein the converting comprises contacting the compound of formula P-5 or a salt thereof with a dehydration product biosynthetic polypeptide. (Item 21) The compound of formula P-6 or a salt thereof is reacted with a compound of formula P-7 below. R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -C(O)-S-CoA P-7 or a salt thereof. (Item 22) 22. The method of claim 21, wherein the converting comprises contacting the compound of formula P-6 or a salt thereof with a reduction product biosynthetic polypeptide that is or includes a 2,3-enoyl-CoA reductase. (Item 23) The compound of formula P-7 or a salt thereof is reacted with a compound of formula P-8 below. R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -C(O)-OH P-8 22. The method according to item 21, comprising converting the compound of formula (I) to a salt thereof. (Item 24) 24. The method of claim 23, wherein the converting comprises contacting the compound of formula P-7 or a salt thereof with a CoA transfer product biosynthetic polypeptide. (Item 25) The compound of formula P-8, wherein L 2 Ga-CH 2 -L 2’ or a salt thereof, HC(O)-L 2’ -L 1 -CH 2 -CH 2 -CH 2 -C(O)-OH P-9 or a salt thereof, wherein L 2’ is a covalent bond or a divalent optionally substituted straight or branched chain C 1-19 Aliphatic group or C 1-19 A heteroaliphatic group, one or more of whose methylene units are optionally and independently -C≡C-, -C(R") 2 -, -Cy-, -O-, -S-, -SS-, -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) 2 24. The method of claim 23, wherein the substituted aryl group is N(R")-, -C(O)S-, or -C(O)O-. (Item 26) 26. The method of claim 25, wherein the converting comprises contacting the compound of formula P-8 or a salt thereof with an oxidation product biosynthetic polypeptide that is or includes an alcohol dehydrogenase. (Item 27) The compound of formula P-9 or a salt thereof is reacted with a compound of formula P-10 HO-C(O)-L 2’ -L 1 -CH 2 -CH 2 -CH 2 -C(O)-OH P-10 26. The method according to item 25, comprising converting the compound of formula (I) to a salt thereof. (Item 28) 28. The method of claim 27, wherein the converting comprises contacting the compound of formula P-9 or a salt thereof with an aldehyde oxidation product biosynthetic polypeptide. (Item 29) The compound of the formula P-8 or a salt thereof is reacted with a compound of the following formula P-9' R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -C(O)-H P-9’ or a salt thereof. (Item 30) 30. The method of claim 29, comprising contacting the compound of formula P-8 or a salt thereof with a carboxyl reduction product biosynthetic polypeptide. (Item 31) The compound of formula P-9' or a salt thereof is reacted with a compound of formula P-10' R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -CH 2 -OH P-10’ 30. The method according to item 29, comprising converting the compound of formula (I) to a salt thereof. (Item 32) 32. The method of claim 31, comprising contacting the compound of formula P-9' or a salt thereof with an aldehyde reduction product biosynthetic polypeptide that is an aldehyde reductase or a primary alcohol dehydrogenase, or that includes an aldehyde reductase or a primary alcohol dehydrogenase. (Item 33) The compound of formula P-3 or a salt thereof is reacted with a compound of formula P-4' below. R a -L 2 -L 1 -CH 2 -CH 2 -C(O)-H P-4’ or a salt thereof. (Item 34) 34. The method according to item 33, comprising contacting the compound of formula P-3 or a salt thereof with a decarboxylation product biosynthetic polypeptide. (Item 35) The compound of formula P-4' or a salt thereof is reacted with a compound of formula P-5' R a -L 2 -L 1 -CH 2 -CH 2 -CH 2 -OH P-5’ or a salt thereof. (Item 36) 36. The method of claim 35, comprising contacting the compound of formula P-4' or a salt thereof with an aldehyde reduction product biosynthetic polypeptide. (Item 37) In the above formula, R a 37. The method according to any one of items 1 to 36, wherein is —H. (Item 38) In the above formula, R a 37. The method according to any one of items 1 to 36, wherein is —OH. (Item 39) In the formula, L 1 is arbitrarily substituted C 1-6 39. The method according to any one of items 1 to 38, wherein the alkylene is alkylene. (Item 40) In the formula, L 1 But unsubstituted C 1-6 39. The method according to any one of items 1 to 38, wherein the alkylene is alkylene. (Item 41) The alkylene is —CH 2 - 41. The method according to any one of items 39 to 40, wherein (Item 42) The alkylene is —CH 2 CH 2 - 41. The method according to any one of items 39 to 40, wherein (Item 43) The alkylene is —CH 2 CH 2 CH 2 - 41. The method according to any one of items 39 to 40, wherein (Item 44) In the formula, L 1 is a covalent bond. (Item 45) In the formula, L 2 45. The method according to any one of items 1 to 44, wherein is a covalent bond. (Item 46) In the formula, L 2 is arbitrarily substituted C 1-6 45. The method according to any one of items 1 to 44, wherein the alkylene is alkylene. (Item 47) In the formula, L 2 But unsubstituted C 1-6 45. The method according to any one of items 1 to 44, wherein the alkylene is alkylene. (Item 48) The alkylene is —CH 2 48. The method according to any one of items 46 to 47, wherein (Item 49) The alkylene is —CH 2 CH 2 48. The method according to any one of items 46 to 47, wherein (Item 50) The alkylene is —CH 2 CH 2 CH 2 48. The method according to any one of items 46 to 47, wherein (Item 51) The aliphatic aldehyde is HO-CH 2 -CH 2 - The method according to item 8, wherein the IgG4-related gene is CHO. (Item 52) The aldol dehydration product is HO-CH 2 -CH 2 Item 10. The method according to item 9, wherein the compound is —CH═CH—C(O)—COOH or a salt thereof. (Item 53) The alkene reduction product is HO-CH 2 -CH 2 -CH 2 -CH 2 Item 13. The method according to item 12, wherein the compound is —C(O)—COOH or a salt thereof. (Item 54) The carbonyl reduction product is HO-CH 2 -CH 2 -CH 2 -CH 2 Item 16. The method according to item 15, wherein the compound is —CH(OH)—COOH or a salt thereof. (Item 55) The compound of formula P-5 or a salt thereof is HO-CH 2 -CH 2 -CH 2 -CH 2 Item 17. The method according to item 16, wherein the compound is —CH(OH)—CO—S-CoA or a salt thereof. (Item 56) The compound of formula P-6 or a salt thereof is HO-CH 2 -CH 2 -CH 2 Item 19. The method according to item 18, wherein the compound is —CH═CH—CO—S-CoA or a salt thereof. (Item 57) The compound of formula P-7 or a salt thereof is HO-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 21. The method according to item 20, wherein the hydroxylase is -CO-S-CoA or a salt thereof. (Item 58) The compound of formula P-8 or a salt thereof is HO-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 Item 24. The method according to item 23, wherein the compound is —CO—OH or a salt thereof. (Item 59) The compound of formula P-9 or a salt thereof is HC(O)-CH 2 -CH 2 -CH 2 -CH 2 Item 26. The method according to item 25, wherein the compound is —CO—OH or a salt thereof. (Item 60) The compound of formula P-10 or a salt thereof is HO-CO-CH 2 -CH 2 -CH 2 -CH 2 Item 28. The method according to item 27, wherein the compound is —CO—OH or a salt thereof. (Item 61) The compound of formula P-9′ or a salt thereof is HO—CH 2 -CH 2 -CH 2 -CH 2 -CH 2 26. The method according to item 25, wherein the compound is —C(O)—H or a salt thereof. (Item 62) The compound of formula P-10′ or a salt thereof is 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 Item 28. The method according to item 27, wherein the compound is —OH or a salt thereof. (Item 63) The compound of formula P-4′ or a salt thereof is HO—CH 2 -CH 2 -CH 2 -CH 2 Item 33. The method of item 32, wherein the compound is —C(O)—H or a salt thereof. (Item 64) The compound of formula P-5′ or a salt thereof is 2 -CH 2 -CH 2 -CH 2 -CH 2 Item 33. The method according to item 32, wherein the compound is —OH or a salt thereof. (Item 65) A preparation prepared by the method of any one of the preceding items. (Item 66) 1. A preparation of a compound 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, or a preparation prepared by a method according to any one of the preceding paragraphs, wherein said preparation contains isotopes 14 The preparation wherein C is enriched relative to the isotope observed in a reference preparation of the compound, the reference preparation being prepared using a fossil carbon source. (Item 67) 10. A polyester, polyester polyol, polyurethane, nylon 6, nylon 6,6, polycarbonate diol, diacrylate ester or diglycidyl ether preparation produced using a preparation prepared by the method of any one of the preceding items. (Item 68) In the preparation, isotopes 14 68. The preparation of item 67, wherein C is enriched relative to the isotope observed in a reference preparation of the compound, the reference preparation being prepared using a fossil carbon source. (Item 69) A nucleic acid encoding a biosynthetic polypeptide according to any one of the preceding items. (Item 70) 1. An engineered microorganism that produces an aldol dehydration product of an aliphatic aldehyde, wherein the microorganism has enhanced expression or activity of an aldol product biosynthetic polypeptide, an aldol dehydration product biosynthetic polypeptide, a dehydration product biosynthetic polypeptide, or any combination thereof; the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aromatic group; The engineered microorganism, wherein the aldol dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated to the aldehyde or ketone group. (Item 71) 1. An engineered microorganism that produces an alkene reduction product, wherein the microorganism has enhanced expression or activity of an alkene reduction product biosynthetic polypeptide; the alkene contains a double bond conjugated to a carbonyl group; The engineered microorganism reduces the double bond conjugated to the carbonyl group in the alkene to a single bond to provide the alkene reduction product. (Item 72) 72. A culture medium comprising the microorganism according to any one of items 70 to 71, wherein the culture medium independently comprises one or more of the compounds of formulae P-1 to P-10, P-9', P-10', P-4', or P-5', or salts thereof. (Item 73) The method, preparation, nucleic acid, microorganism or culture medium according to any one of embodiments 1 to 386. [Brief explanation of the drawings]

[0080] [Figure 1] As an example, a bienzymatic biosynthetic pathway for producing a 2-ketocarboxylic acid from pyruvate and an aldehyde is shown. Without intending to be limited by theory, aldol dehydration products (e.g., aldol condensation products described herein) can be produced through steps 1 and 2 as shown in the figure, from a process catalyzed 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)). As will be apparent to one of skill in the art, the double bond in the illustrated aldol condensation product can exist in either the E or Z configuration. In many embodiments, step 3 as shown can be catalyzed by an oxidoreductase, e.g., an oxidoreductase belonging to EC 1.6.5 (e.g., EC 1.6.5.5) that uses NADH and / or NADPH for the reduction of a quinone. As described herein, a variety of aldehydes may be used. For example, in the aldehydes exemplified in some embodiments, R is H, CH3, CH2CH3, OH, CH2OH, or CH2CH2OH. [Figure 2] This figure shows a biosynthetic pathway for producing 1,5-pentanediol via the intermediate 6-hydroxy-2-ketohexanoate (6H2KH). As used herein, 3HPA refers to 3-hydroxypropanal, 6H4H2KH refers to 4,6-dihydroxy-2-ketohexanoate, 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-ketohexenoate, and 5HPeA refers to 5-hydroxypentanal. For illustrative purposes, NADH is shown as a cofactor in many of the reduction steps of this pathway. Either NADPH or NADH can be the cofactor. [Figure 3]Figure 1 shows a biosynthetic pathway for producing 1,6-hexanediol via the intermediate 6-hydroxy-2-ketohexanoate (6H2KH). As used herein, 3HPA refers to 3-hydroxypropanal, 6H4H2KH refers to 4,6-dihydroxy-2-ketohexanoate, 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-ketohexenoate, 6H2HH refers to 2,6-dihydroxyhexanoate, 6HH-CoA refers to 6-hydroxyhexanoyl-CoA, 6HH refers to 6-hydroxyhexanoate, 6H2HH-CoA refers to 2,6-dihydroxyhexanoyl-CoA, and 6HHA refers to 6-hydroxyhexanal. Either NADPH or NADH can be a cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase enzyme. For illustrative purposes, 6HH-CoA is shown as the donor and 6H2HH as the acceptor for the reaction in step 5. In vivo, other CoA esters or carboxylic acids can also function as donors and acceptors for this enzyme. [Figure 4] Figure 1 shows a biosynthetic pathway for producing 6-hydroxyhexanoate via the intermediate 6-hydroxy-2-ketohexanoate (6H2KH). As used herein, 3HPA refers to 3-hydroxypropanal, 6H4H2KH refers to 4,6-dihydroxy-2-ketohexanoate, 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-ketohexenoate, 6H2HH refers to 2,6-dihydroxyhexanoate, 6HH-CoA refers to 6-hydroxyhexanoyl-CoA, 6HH refers to 6-hydroxyhexanoate, and 6H2HH-CoA refers to 2,6-dihydroxyhexanoyl-CoA. Either NADPH or NADH can be the cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase enzyme. For illustrative purposes, 6HH-CoA is shown as the donor and 6H2HH as the acceptor in the reaction of step 5. In vivo, other CoA esters or carboxylic acids can also function as donors and acceptors for this enzyme. [Figure 5] Figure 1 shows a biosynthetic pathway for producing adipic acid via the intermediate 6-hydroxy-2-ketohexanoate (6H2KH). As used herein, 3HPA refers to 3-hydroxypropanal, 6H4H2KH refers to 4,6-dihydroxy-2-ketohexanoate, 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-ketohexenoate, 6H2HH refers to 2,6-dihydroxyhexanoate, 6HH-CoA refers to 6-hydroxyhexanoyl-CoA, 6HH refers to 6-hydroxyhexanoate, 6H2HH-CoA refers to 2,6-dihydroxyhexanoyl-CoA, and 6KHA refers to 6-oxohexanoate. Either NADPH or NADH can be a cofactor. Steps 5 and 8 are catalyzed by a single CoA transferase enzyme. For illustrative purposes, 6HH-CoA is shown as the donor and 6H2HH as the acceptor in the reaction of step 5. In vivo, other CoA esters or carboxylic acids can also function as donors and acceptors for this enzyme. [Figure 6] Figure 1 shows the activity of quinone oxidoreductase-1 (Qor-1) in reducing 6-hydroxy-3,4-dehydro-2-ketohexenoate to 6-hydroxy-2-ketohexenoate using the cofactors NADH and NADPH. DETAILED DESCRIPTION OF THE INVENTION

[0081] definition As used herein, certain terms may have the following defined meanings: As used herein, the singular forms "a," "an," and "the" include singular as well as plural referents unless the context clearly indicates otherwise.

[0082] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean that the composition or method excludes other elements of some essential significance. "Consisting of" is intended to mean excluding other ingredients beyond trace elements for the claimed composition and more than substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure. That is, the methods and compositions may include additional steps and components (comprising), or may include insignificant steps and compositions (consisting essentially of), or may contemplate only the recited method steps or compositions (consisting of).

[0083] As used herein, the term "aldol dehydration product biosynthesis polypeptide" refers to a polypeptide that is involved in the synthesis of an aldol dehydration product, as described herein. In some embodiments, an aldol dehydration product biosynthesis polypeptide may be or include an aldolase polypeptide, a hydratase, or a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol dehydration product biosynthesis polypeptide may be or include a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol dehydration product biosynthesis polypeptide of the invention has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference aldol dehydration biosynthesis 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 an appropriate reference aldol dehydration biosynthetic polypeptide (e.g., as found in nature and / or as shown herein (e.g., in one or more of the relevant tables, e.g., Tables 1 and 5-8)), or a portion thereof (e.g., a portion that facilitates the relevant reaction, e.g., a domain (e.g., a relevant catalytic domain) and / or a series of amino acid residues, which can be contiguous or spaced).

[0084] As used herein, "aldol dehydration product" refers to a compound containing an aldehyde or ketone group and a double bond conjugated to the aldehyde or ketone group. In some embodiments, the aldol dehydration product is a compound of formula P-2, or a salt thereof:

[0085] As used herein, the term "aldol product" refers to a compound containing an aldehyde or ketone group and a hydroxyl group attached to the β-carbon of the aldehyde or ketone carbonyl group. In some embodiments, the aldol product is the product of an aldol reaction. In some embodiments, the aldol product has a structure of formula P-1 or a salt thereof:

[0086] As used herein, the term "aldol product biosynthesis polypeptide" refers to a polypeptide involved in the synthesis of an aldol product as described herein. In some embodiments, an aldol product biosynthesis polypeptide may be or include an aldolase polypeptide, a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol product biosynthesis polypeptide is or includes an aldolase polypeptide as described herein. In some embodiments, an aldol product biosynthesis polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference aldol biosynthesis polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol biosynthesis polypeptide shares characteristic sequence elements and / or overall percent identity with an appropriate reference aldol biosynthesis polypeptide (e.g., as found in nature and / or as set forth herein (e.g., in one or more of the relevant Tables)), or a portion thereof (e.g., a portion that facilitates the relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or a series of amino acid residues, which can be contiguous or spaced).

[0087] As used herein, the term "alkene reduction product biosynthesis polypeptide" refers to a polypeptide involved in the conversion of a double bond to a single bond (and the formation of an alkene reduction product), as described herein. In some embodiments, an alkene reduction product biosynthesis polypeptide may be or include a quinone oxidoreductase, as described herein. In some embodiments, an alkene reduction product biosynthesis polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference alkene reduction biosynthesis polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol biosynthesis polypeptide shares characteristic sequence elements and / or overall percent identity with an appropriate reference aldol biosynthesis polypeptide (e.g., as found in nature and / or as set forth herein (e.g., in one or more of the relevant Tables)), or a portion thereof (e.g., a portion that facilitates the relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or a series of amino acid residues, which can be contiguous or spaced).

[0088] As used herein, the term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring (but is not aromatic) that is fully saturated or contains one or more units of unsaturation, or combinations thereof. In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0089] As used herein, the term "alkyl" is given its ordinary meaning in the art, and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl contains 1 to 100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1 to 20 carbon atoms in its backbone (e.g., C1-C6 for a straight chain). 20 , C2-C for branched chains 20), or about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure (which rings may be monocyclic, bicyclic or polycyclic), or alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group can be a lower alkyl group, which lower alkyl group contains 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0090] As used herein, the term "aryl," whether used alone or as part of a larger moiety, as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having 5 to 30 total ring members, in which at least one ring of the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic, or polycyclic ring system having 5 to 14 total ring members, in which at least one ring of the system is aromatic, and in which each ring of the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system (including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, etc.), which may have one or more substituents. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl.

[0091] As used herein, the terms "alicyclic," "carbocycle," "carbocyclyl," "carbocyclic radical," and "carbocyclic ring" are used interchangeably and, unless otherwise specified, refer to a saturated or partially unsaturated non-aromatic cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein, having 3 to 30 ring members. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, alicyclic groups have 3 to 6 carbons. In some embodiments, alicyclic groups are saturated and cycloalkyl. The term "alicyclic" may also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, or a C8-C 10 Bicyclic or polycyclic hydrocarbons that are not aromatic and have one point of attachment to the rest of the molecule, or C9-C that are fully saturated or contain one or more unsaturated units 16 Refers to a polycyclic hydrocarbon that is not aromatic and has one point of attachment to the rest of the molecule.

[0092] As used herein, the term "heteroaliphatic" is given its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which 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, CH, and CH are independently replaced with one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0093] As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an alkyl group, as described herein, in which one or more carbon atoms are independently replaced by 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, etc.

[0094] As used herein, the terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to a monocyclic, bicyclic, or polycyclic ring system having 5 to 30 total ring members, in which at least one ring of the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), and in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 shared pi electrons in a cyclic arrangement and, in addition to the carbon atoms, has 1 to 5 heteroatoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on 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, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups may be monocyclic, bicyclic, or polycyclic.The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0095] As used herein, the term "heteroatom" refers to an atom that is neither carbon nor 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 (including, for example, quaternized forms, forms such as in iminium groups), phosphorus, sulfur, oxygen, etc.). In some embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0096] As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocycle" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +It may be NR (as in N-substituted pyrrolidinyl). The heterocycle can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings (such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl). Heterocyclyl groups can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0097] Optionally substituted: As described herein, chemical entities, e.g., various compounds of the present disclosure, may contain optionally substituted and / or substituted moieties. Generally, the term "substituted" means that one or more of the hydrogens of the designated moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a given group, the substituents may be the same or different at all positions. In some embodiments, an optionally substituted group is substituted. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to a compound that remains substantially unchanged when subjected to conditions that allow for the production, detection, and, in certain embodiments, recovery, purification, and use of the compound for one or more of the purposes disclosed herein. Specific substituents are described below.

[0098] Suitable monovalent substituents on substitutable atoms, e.g., suitable carbon atoms, are independently halogen, —(CH) 0-4 R°, -(CH2) 0-4 OR°, -O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR°, -(CH2) 0-4 CH(OR°)2, -(CH2) 0-4 Ph (optionally substituted with R°), —(CH2) 0-4 O(CH2) 0-1 Ph (optionally substituted with R°), -CH=CHPh (optionally substituted with R°), -(CH2) 0-4 O(CH2) 0-1 -pyridyl (optionally substituted by R°), -NO2, -CN, -N3, -(CH2) 0-4 N(R°)2, -(CH2) 0-4N(R°)C(O)R°、-N(R°)C(S)R°、-(CH2) 0-4 N(R°)C(O)NR°2、-N(R°)C(S)NR°2、-(CH2) 0-4 N(R°)C(O)OR°、-N(R°)N(R°)C(O)R°、-N(R°)N(R°)C(O)NR°2、-N(R°)N(R°)C(O)OR°、-(CH2) 0-4 C(O)R°、-C(S)R°、-(CH2) 0-4 C(O)OR°、-(CH2) 0-4 C(O)SR°、-(CH2) 0-4 C(O)OSiR°3、-(CH2) 0-4 OC(O)R°、-OC(O)(CH2) 0-4 SR°、-SC(S)SR°、-(CH2) 0-4 SC(O)R°、-(CH2) 0-4 C(O)NR°2、-C(S)NR°2、-C(S)SR°、-(CH2) 0-4 OC(O)NR°2、-C(O)N(OR°)R°、-C(O)C(O)R°、-C(O)CH2C(O)R°、-C(NOR°)R°、-(CH2) 0-4 SSR°、-(CH2) 0-4 S(O)2R°、-(CH2) 0-4 S(O)2OR°、-(CH2) 0-4 OS(O)2R°、-S(O)2NR°2、-(CH2) 0-4 S(O)R°、-N(R°)S(O)2NR°2、-N(R°)S(O)2R°、-N(OR°)R°、-C(NH)NR°2、-Si(R°)3、-OSi(R°)3、-B(R°)2、-OB(R°)2、-OB(OR°)2、-P(R°)2、-P(OR°)2、-P(R°)(OR°)、-OP(R°)2、-OP(OR°)2、-OP(R°)(OR°)、-P(O)(R°)2、-P(O)(OR°)2、-OP(O)(R°)2、-OP(O)(OR°)2、-OP(O)(OR°)(SR°)、-SP(O)(R°)2、-SP(O)(OR°)2、-N(R°)P(O)(R°)2、-N(R°)P(O)(OR°)2、-P(R°)2[B(R°)3]、-P(OR°)2[B(R°)3]、-OP(R°)2[B(R°)3]、-OP(OR°)2[B(R°)3]、-(C1-4 linear or branched alkylene)ON(R°)2, or -(C 1-4 straight or branched chain alkylene)C(O)ON(R°), where each R° may be optionally substituted as defined herein and independently represents hydrogen, C 1-20 Aliphatic, C having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus 1-20 Heteroaliphatic group, -CH2-(C 6-14 aryl), -O(CH2) 0-1 (C 6-14 aryl), -CH2- (a 5-14 membered heteroaryl ring), a 5-20 membered mono-, bi- or polycyclic saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus; or, notwithstanding the above definitions, two independent occurrences of R° together with their intervening atom(s) form a 5-20 membered mono-, bi- or polycyclic saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which ring may be substituted as defined below.

[0099] Suitable monovalent substituents on R° (or the ring formed by two independent R° together with their intervening atoms) are independently halogen, -(CH2), 0-2 R ● ,-(Halo R ● ), -(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) 0-2 SH, -(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 chain alkylene)C(O)OR ● , or -SSR ● where each R ● is unsubstituted or, if preceded by "halo", is substituted with only one or more halogens, and independently represents C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 and Ph, and a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0100] For example, suitable divalent substituents on suitable carbon atoms are independently: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-, wherein each independently occurring R * is hydrogen, C 1-6 "Optionally substituted" groups are selected from aliphatic groups (which may be substituted as defined below) and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents attached to adjacent substitutable carbon atoms of "optionally substituted" groups include -O(CR * 2) 2-3 O—, wherein each independently occurring R * is hydrogen, C1-6 Selected from aliphatic groups (which may be substituted as defined below), and unsubstituted 5-6 membered saturated, partially unsaturated, and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0101] R * Suitable substituents on the aliphatic group are independently halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, where each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0102] In some embodiments, suitable substituents on a substitutable nitrogen are independently —R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2 or -N(R † )S(O)2R † where each R † are independently hydrogen, C 1-6an aliphatic group (which may be substituted as defined below), an unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or two R independently present, regardless of the above definitions. † together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0103] R † Suitable substituents on the aliphatic group are independently halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, where each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0104] As used herein, the term "partially unsaturated" refers to a ring moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to include rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0105] "Wild-type" defines a cell, composition, tissue or other biological material as it exists in nature.

[0106] In some embodiments, 3-hydroxypropanal and pyruvate are prepared from one or more of glycerol, C5 sugars, C6 sugars, phosphoglycerate, other carbon sources, glycolytic pathway intermediates, and combinations thereof. In some embodiments, the C5 sugars include, consist essentially of, or even consist of one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose, and the C6 sugars include, consist essentially of, or even consist of allose, altrose, glucose, mannose, gulose, idose, talose, fructose, psicose, sorbose, and tagatose. In some embodiments, the other carbon source is a feedstock suitable as a carbon source for the microorganism, the feedstock comprising, consisting essentially of, or even consisting of one or more of amino acids, lipids, corn stover, miscanthus, municipal solid waste, energy grade sugarcane, sugar grade sugarcane, bagasse, starch stream, dextrose stream, formate, methanol, and combinations thereof.

[0107] As used herein, the term "C5 sugar" refers to a sugar molecule containing five carbons.

[0108] As used herein, the term "C6 sugar" refers to a sugar molecule containing six carbons.

[0109] In some embodiments, the term "aldol addition" refers to the addition of a pyruvate molecule to a C N reacting with the aldehyde functional group of the aldehyde to form the corresponding enol ion or enolate ion, or a Schiff base or enamine to give intermediate C N+3 refers to a chemical reaction that produces a 4-hydroxy-2-ketocarboxylic acid. N The aldehyde is 3-hydroxypropanal, intermediate C N+34-Hydroxy-2-ketocarboxylic acid is 4,6-dihydroxy-2-ketohexanoic acid.

[0110] In some embodiments, the term "aldol condensation" refers to a process in which a pyruvate molecule undergoes a C N Reacts with the aldehyde functional group of the aldehyde to form the corresponding enol or enolate ion, or a Schiff base or enamine, to give C N+3 refers to a chemical reaction that produces 3,4-dehydro-2-ketocarboxylic acid. In some embodiments, C N The aldehyde is 3-hydroxypropanal, C N+3 3,4-Dehydro-2-ketocarboxylic acid is 6-hydroxy-3,4-dehydro-2-ketohexanoic acid.

[0111] As used herein, the term "solution" refers to a liquid composition comprising a solvent and a solute, such as the starting material used in the methods described herein. In some embodiments, the solvent is water. In some embodiments, the solvent is an organic solvent.

[0112] As used herein, the term "enzymatic process" or "enzymatic reaction" refers to a molecular reaction catalyzed by an enzyme that has been selected to facilitate a desired enzymatic reaction. Enzymes are large biological molecules that are highly selective catalysts. Most enzymes are proteins, although some catalytic RNA molecules have been identified.

[0113] Throughout this application, enzymatic steps may be designated as "step 1," "step 2," etc., and the enzymes that specifically catalyze these steps are designated as "1," "2," etc. Such enzymes are also referred to as "reaction-specific enzymes."

[0114] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (the non-protein portion of an enzyme), the presence of which is required for the activity of many enzymes to form an active enzyme system.

[0115] As used herein, the term "substantially anaerobic," when used in reference to culture or growth conditions, is intended to mean that the amount of oxygen is less than about 10% saturation of dissolved oxygen in the liquid medium. The term is also intended to include maintaining a sealed chamber containing a liquid or solid medium in an atmosphere with less than about 1% oxygen.

[0116] As used herein, the terms "non-naturally occurring" or "non-naturally occurring," when used with respect to a microbial organism or microorganism of the present disclosure, are intended to mean that the microorganism possesses at least one genetic alteration that is not normally found in naturally occurring strains of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, but are not limited to, modifications that introduce expressible nucleic acids encoding polypeptides, additions of other nucleic acids, deletions of nucleic acids, and / or other functional disruptions of the genetic material of the microorganism. Such modifications include, but are not limited to, coding regions and functional fragments thereof for heterologous polypeptides, homologous polypeptides, or both heterologous and homologous polypeptides relative to the referenced species. Additional modifications include, but are not limited to, non-coding regulatory regions, where the modification alters expression of a gene or operon.

[0117] As used herein, "exogenous" is intended to mean that the referenced molecule or referenced activity is one that has been introduced into the host microbial organism. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome or as non-chromosomal genetic material (such as a plasmid). That is, when used in reference to expression of an encoding nucleic acid, the term refers to the encoding nucleic acid being introduced into the microbial organism in an expressible form. When used in reference to an enzymatic activity, the term refers to the activity being introduced into the host organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity after introduction into the host microbial organism. Thus, the term "endogenous" refers to the referenced molecule or activity being natively or naturally present in the wild-type host. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid contained in a wild-type microbial organism.

[0118] The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species, whereas "homologous," when used in a similar context, refers to a molecule or activity derived from the host microbial organism. Thus, exogenous expression of an encoding nucleic acid can utilize either heterologous or homologous encoding nucleic acid, or both.

[0119] When two or more exogenous nucleic acids are contained within a microorganism, it is understood that the two or more exogenous nucleic acids refer to the encoding nucleic acids or enzymatic activities referenced, as discussed above. Furthermore, as disclosed herein, it is understood that two or more exogenous nucleic acids can be introduced into a host microorganism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or combinations thereof, and still be considered two or more exogenous nucleic acids. For example, as disclosed herein, a microorganism can be engineered to express two or more exogenous nucleic acids encoding enzymes or proteins of a desired pathway. When two exogenous nucleic acids encoding desired activities are introduced into a host microorganism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, e.g., on a single plasmid, on separate plasmids, integrated into the host chromosome at a single site or multiple sites, and still be considered two or more exogenous nucleic acids. Similarly, it is understood that three or more exogenous nucleic acids can be introduced into a host organism in any desired combination, e.g., on a single plasmid, on separate plasmids, integrated into the host chromosome at a single site or multiple sites, and still be considered three or more exogenous nucleic acids, e.g., three exogenous nucleic acids. Thus, the number of exogenous nucleic acids or enzymatic activities referenced refers to the number of encoding nucleic acids or enzymatic activities, and not the number of separate nucleic acids introduced into the host organism.

[0120] In some embodiments, exogenous expression of the encoding nucleic acid is used. Exogenous expression allows for customization of expression and / or regulatory elements for the host and application, achieving a desired expression level controlled by the user. However, in other embodiments, endogenous expression can be used, such as by removing negative regulatory effectors or inducing the gene's promoter if linked to an inducible promoter or other regulatory element. That is, endogenous genes with native inducible promoters can be upregulated by providing an appropriate inducer, or the regulatory region of the endogenous gene can be engineered to incorporate inducible regulatory elements, allowing for tunable increased expression of the endogenous gene at the desired time. Similarly, inducible promoters can be included as regulatory elements for exogenous genes introduced into non-naturally occurring microorganisms.

[0121] Those skilled in the art will understand that genetic alterations are described with reference to a suitable host organism, such as E. coli, and its corresponding metabolic reaction or a suitable source organism for desired genetic material, such as genes in a desired biosynthetic pathway. However, those skilled in the art will readily apply the teachings and guidance provided herein to essentially any other organism, given the advanced knowledge in the field of genomics and whole genome sequencing of a wide variety of organisms. For example, the metabolic alterations exemplified herein in E. coli can be readily applied to other species by incorporating the same or similar encoding nucleic acid from a species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, broadly, and specifically, orthologs, paralogs, or non-orthologous gene replacements.

[0122] Sources of nucleic acids encoding pathway enzymes can include, for example, any species in which the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotes and eukaryotes, 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 sp. 2065, Pseudomonas aeruginosa, Ralstonia eutropha, Clostridium acetobutylicum, Euglena gracilis, Treponema denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp.ADP1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium thermoaceticum(Moorella thermoaceticum), Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium Examples of suitable species include: A. 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 corresponding genes (see Examples). However, with the current availability of complete genome sequences for over 400 microbial genomes, as well as a variety of yeast, fungal, plant, and mammalian genomes, the identification of genes encoding required pathway enzymes (including, for example, homologs, orthologs, paralogs, and nonorthologous gene displacements of known genes) for one or more genes in closely or distantly related species, and the shuffling of genetic changes between organisms, are routine and well known in the art.

[0123] Orthologs are genes that have evolved from a common ancestor through speciation in different species. Orthologs typically retain the same function throughout evolution. Identifying orthologs is essential for reliable prediction of gene function in newly sequenced genomes.

[0124] Paralogs are genes related by duplication within a genome. While orthologs generally retain the same function during evolution, paralogs can acquire new functions that are related to the original.

[0125] A non-orthologous gene displacement is a non-orthologous gene that can substitute for a target gene function in a different species from a non-orthologous gene in one species. Substitution includes, for example, being able to perform substantially the same or similar function in the original species compared to the target function in the different species. Generally, a non-orthologous gene displacement will be identified as being structurally related to a known gene encoding the target function; however, as used herein, less structurally related but functionally similar genes and their corresponding gene products also fall within the meaning of this term. Functional similarity requires at least some structural similarity, for example, in the active site or binding region of the non-orthologous gene product, compared to the gene encoding the function being substituted. Thus, a non-orthologous gene includes, for example, a paralog or an unrelated gene.

[0126] As used herein, the terms "microorganism," "microbial organism," or "microbe" are used interchangeably and refer to an isolated, living prokaryotic or eukaryotic cell of a living organism that can be transformed or transfected by the insertion of exogenous or recombinant nucleic acids, such as DNA or RNA. Any suitable prokaryotic or eukaryotic microorganism may be used in the present disclosure, as long as it remains viable after transformation with a nucleic acid sequence. Suitable microorganisms of the present disclosure are those that are capable of expressing one or more nucleic acid constructs encoding one or more recombinant proteins capable of catalyzing at least one of the steps in the methods of the present invention. The microorganism can be selected from the group of bacteria, yeast, fungi, molds, and archaea. They are commercially available.

[0127] As used herein, "fungi" refers to any eukaryotic organism classified in the kingdom Fungi. Phylums within the kingdom Fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. As used herein, "yeast" refers to fungi that grow in a unicellular form (e.g., by budding), whereas "mold" refers to fungi that grow in filaments consisting of multicellular hyphae or mycelium (McGinnis, MR and Tyring, SK "Introduction to Mycology." Medical Microbiology. 4 th ed.Galveston:Univ.of TX Medical Branch at Galveston,1996).

[0128] In some embodiments, the microorganism is a yeast cell. In some embodiments, the yeast cell is a cell of a species of Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia.

[0129] In some embodiments, the microorganism is a fungal cell. In some embodiments, the fungal host cell is a cell of a species of Neurospora, Trichoderma, Aspergillus, Fusarium, or Chrysosporium.

[0130] In some embodiments, the microorganism is an archaea. In some embodiments, suitable archaea are bacteria of the species Archaeoglobus, Aeropyrum, Halobacterium, Pyrobaculum, Pyrococcus, Sulfolobus, Methanococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, Methanocaldococcus, or Methanosarcina.

[0131] The term "bacteria" refers to any microorganism of the domain Prokaryote or kingdom Prokaryote. Phylums within the domain or kingdom Bacteria include Acidobacteria, Actinobacteria, Actinobacillus, Agrobacterium, Anaerobiospirrulum, Aquificae, Armatimonadetes, Bacteroidetes, Burkholderia, Caldiserica, Chlamydiae, Chlorobi, Chlorella, Chloroflexi, Chrysiogenetes, Citrobacter, Clostridium, Cyanobacteria, Deferribacteres, Deinococcus thermomus, Dictyoglomi, Enterobacter, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Geobacillus, Gemma, and others. In some embodiments, the bacterial microorganism is an E. coli cell. In some embodiments, the bacterial microorganism is a Bacillus sp. cell.Examples of Bacillus species include, but are not limited to, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus licheniformis.

[0132] The carboxylic acid compounds prepared by the methods of the present disclosure can form salts with counterions (metal ions, including but not limited to alkali metal ions such as sodium and potassium, alkaline earth ions such as calcium and magnesium, or aluminum ions) or can coordinate with organic bases such as tetraalkylammonium, ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, etc. The acids can form salts with counterions or organic bases present in the reaction conditions, or can be converted to salts by reaction with inorganic or organic bases.

[0133] Any carboxylic acid containing compound of the present invention may be referred to as either the acid or the salt, which are used interchangeably throughout this specification to refer to the compound in either its neutral or ionized form, including any salt forms thereof, as will be understood by those skilled in the art, the particular form being determined by pH.

[0134] A solvate of a compound is a solid form of the compound that crystallizes with less than one, one, or more than two solvent molecules inside the crystal lattice. Some examples of solvents that can be used to prepare solvates, such as pharmaceutically acceptable solvates, include, but are not limited to, water, typically C1-C6 alcohols (methanol, ethanol, isopropanol, butanol, etc., optionally substituted), tetrahydrofuran, acetone, ethylene glycol, propylene glycol, acetic acid, formic acid, and mixtures of these solvents. Other biocompatible solvents that can help prepare pharmaceutically acceptable solvates, such as those mentioned above, are well known in the art. In addition, various organic and inorganic acids and organic and inorganic bases can be added to prepare the desired solvates. Such acids and bases are well known in the art. When the solvent is water, the solvate can be referred to as a hydrate. In some embodiments, one molecule of compound can form a solvate with 0.1 to 5 molecules of solvent, including 0.5 molecules of solvent (hemisolvate, such as a hemihydrate), 1 molecule of solvent (monosolvate, such as a monohydrate), and 2 molecules of solvent (disolvate, such as a dihydrate).

[0135] When referring to a compound that exists in several isomeric forms (e.g., cis and trans isomers, and R and S isomers, or combinations thereof), the compound essentially includes all possible enantiomers, diastereomers, and cis / trans isomers of that compound that may be used in the methods of the present disclosure.

[0136] For each species, any cell belonging to that species is considered a suitable microorganism of the present disclosure. Host cells of any species may exist in a state isolated from the natural state or may contain any number of genetic modifications (e.g., gene mutations, deletions, or recombinant polynucleotides).

[0137] The term "recombinant nucleic acid" or "recombinant polynucleotide," as used herein, refers to a nucleic acid polymer in which at least one of the following conditions is met: (a) the nucleic acid sequence is foreign to a given microorganism (i.e., not found in nature); (b) the sequence may be found in a given microorganism in its natural state, but in an amount not found in nature (e.g., greater than expected); or (c) the nucleic acid sequence includes two or more subsequences that are not found in the same relationship to each other in nature. For example, with respect to item (c), a recombinant nucleic acid sequence would include two or more sequences from unrelated genes arranged for the purpose of creating a new functional nucleic acid.

[0138] In some embodiments, recombinant polypeptides or proteins or recombinant enzymes of the present disclosure may be encoded by genetic material as part of one or more expression vectors. An expression vector contains one or more polypeptide-encoding nucleic acids and may further contain any desired elements that control expression of the nucleic acid(s) and any elements that allow for replication and maintenance of the expression vector in a given host cell. All recombinant nucleic acids may be present on a single expression vector or may be encoded by multiple expression vectors.

[0139] An expression vector or vectors can be constructed containing one or more pathway-encoding nucleic acids, such as those exemplified herein, operably linked to expression control sequences functional in the host organism. Applicable expression vectors for the indicated host microorganisms include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes containing a vector and a selection sequence or selectable marker operable for stable integration into a host chromosome. In addition, the expression vector can contain one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes that confer resistance to antibiotics or toxins, complement auxotrophic defects, or provide critical nutrients not found in the culture medium can also be included. Expression control sequences can include constitutive promoters, inducible promoters, transcriptional enhancers, transcriptional terminators, and the like, as are well known in the art. When two or more exogenous encoding nucleic acids are to be coexpressed, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. For expression in a single vector, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Vectors containing both promoters 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 may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted directly 5' of the initiation codon to enhance expression.

[0140] Exogenous nucleic acid sequences involved in the pathway for synthesizing the desired compounds described herein can be stably or transiently introduced into host cells using techniques well known in the art, including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and sonic transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the gene or cDNA of the eukaryotic nucleic acid can encode a targeting signal, such as an N-terminal mitochondrial signal or other targeting signal, and if desired, this signal can be removed before transformation into the prokaryotic host cell. For example, in E. coli, removal of the mitochondrial leader sequence increased expression (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of a leader sequence, or can be targeted to mitochondria or other organelles or secreted by the addition of an appropriate targeting sequence, such as a mitochondrial targeting signal or secretion signal, appropriate for the host cell. It is understood that appropriate modifications to the nucleic acid sequence to remove or include targeting sequences can be incorporated into the exogenous nucleic acid sequence to confer desired properties. Furthermore, genes can be codon-optimized using techniques well known in the art to achieve optimized protein expression.

[0141] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values ​​that vary in increments of 0.1 (±). It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." As used herein, "about" means up to ±10%. It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and equivalents thereof are known in the art.

[0142] "Operably linked" refers to a juxtaposition wherein the elements are in an arrangement permitting them to function.

[0143] The term "culture" refers to the propagation of cells or organisms in vitro on or in various types of media (culture medium). It is understood that the progeny of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0144] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that, after transcription and translation, is capable of encoding a particular polypeptide or protein. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of genes to which the polynucleotide sequences relate. Methods for isolating larger fragment sequences are known to those of skill in the art.

[0145] The term "express" refers to the production of a gene product. The term overexpression refers to the production of mRNA transcribed from the gene or the protein product encoded by the gene in greater amounts than normal or control cells, e.g., 0.5-fold, 1.0-fold, 1.5-fold, or 2-fold, or at least 2.5-fold, or at least 3.0-fold, or at least 3.5-fold, or at least 4.0-fold, or at least 5-fold, or 10-fold greater than the expression level detected in a control sample or wild-type cells.

[0146] As used herein, "homology" refers to the sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologs can be identified by comparing a reference sequence with a single second sequence or a fragment of a sequence, or a database of sequences, by any method known in the art, preferably by using the BLAST tool. As explained below, BLAST will compare sequences based on percent identity and similarity.

[0147] The term "identical" or percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if the two sequences have a specified percentage of the same amino acid residues or nucleotides (i.e., 29% identity, or, optionally, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity in 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, as measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. Optionally, the identity is observed over a region that is at least about 50 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100-500 nucleotides in length, or 1000 or more nucleotides in length (or 20, 50, 200 or more amino acids in length).

[0148] The method of aligning sequences for comparison is well known in the art.For example, the determination of the sequence identity percentage between any two sequences can be carried out using mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS4:11 17 (1988), the local homology algorithm of Smith et al., Adv. Appl. Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 453 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci.85:2444 2448 (1988), and the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993).

[0149] In sequence comparison, typically, one sequence serves as a 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 of the test sequence relative to the reference sequence based on the program parameters. When comparing two sequences for identity, the sequences do not have to be contiguous, but any gaps will be penalized, reducing the overall percent identity. For blastn, the default parameters are a gap opening penalty of 5 and a gap extension penalty of 2. For blastp, the default parameters are a gap opening penalty of 11 and a gap extension penalty of 1.

[0150] As used herein, a "comparison window" refers to any segment of a number of contiguous positions, including but not limited to, 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence in that segment can be compared to a reference sequence having the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. For example, sequences can be optimally aligned for comparison using the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch, J Mol Biol 48(3):443-453 (1970), the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci USA 85(8):2444-2448 (1988), computer 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. (Ringbou Ed.)).

[0151] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms; BLAST is described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997), and BLAST 2.0 is described in Altschul et al., J. Mol Biol 215(3)-403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of the same length in a query sequence that, when aligned with words of length W in a database sequence, match or meet some positive threshold score T, where 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 those words. 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 greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is terminated when the cumulative alignment score drops by an amount X from its maximum achieved value, when the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero, or when 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 the following defaults: word length (W) = 11, expectation (E) = 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 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc Natl Acad Sci USA 89(22):10915-10919 (1992)) of alignment (B) of 50, expectation (E) of 10, M of 5, N of -4, and a comparison of both strands.

[0152] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc Natl Acad Sci USA 90(12):5873-5877(1993)). One of the measures of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. For example, a nucleic acid is considered to be similar to a reference sequence when the minimum sum probability in the comparison between the test nucleic acid and 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.

[0153] In addition to the above-mentioned sequence identity percentage, another indicator 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 the antibody produced against the polypeptide encoded by the second nucleic acid. That is, for example, when two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions. Yet another indicator that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0154] The phrase "functionally equivalent protein" refers to a protein or polynucleotide that hybridizes to an exemplified polynucleotide under stringent conditions and has similar or, for example, enhanced in vivo biological activity compared to the biological activity of a standard or control, e.g., greater than 120%, alternatively greater than 110%, alternatively greater than 100%, alternatively greater than 90%, alternatively greater than 85%, alternatively greater than 80%. Additional embodiments within the scope of the present disclosure are identified by sequence homology of greater than 80%, alternatively greater than 85%, alternatively greater than 90%, alternatively greater than 95%, alternatively greater than 97%, alternatively greater than 98 or 99%. Percent homology can be determined using a sequence comparison program such as BLAST run under appropriate conditions. In some embodiments, the program is run under default parameters. In some embodiments, a reference to a specific enzyme or protein includes its functionally equivalent enzyme or protein.

[0155] A cell population is intended to be a collection of two or more cells that may or may not be phenotypically and / or genotypically identical (clonal). A substantially homogeneous cell population is one that has a phenotype that is at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 98% identical as measured by preselected markers.

[0156] When an enzyme is referred to by its enzyme class (EC), the enzyme class is the class into which the enzyme is or could be classified based on enzyme nomenclature established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Included are other suitable enzymes not yet classified in a given class, but which may be classified in that class.

[0157] Non-naturally occurring microorganisms As exemplified herein, using methods well known in the art, the non-naturally occurring microbial organisms provided herein are constructed to exogenously express at least one nucleic acid encoding an enzyme or protein used in a biosynthetic pathway described herein in sufficient amounts to produce a compound such as 2-ketopentanoic acid, 2-ketohexanoic acid, 6-hydroxy-2-ketohexanoic acid, 1,5-pentanediol, adipic acid, 1,6-hexanediol, or 6-hydroxyhexanoic acid.

[0158] Successfully engineering a microbial host capable of producing the desired products described herein requires identifying an appropriate set of enzymes with sufficient activity and specificity to catalyze the various steps in the pathway, such as those described in the Examples and literature herein. The activity of individual enzymes or proteins derived from the exogenous DNA sequence can also be assayed using methods well known in the art. Additionally, these enzymes can be engineered using modern protein engineering approaches (Protein Engineering Handbook; Lutz S., & Bornscheuer UTWiley-VCH Verlag GmbH & Co. KGaA: 2008; Vol. 1 & 2), such as directed evolution, rational mutagenesis, computational design (Zanghellini, A et al., 2008), or a combination thereof, to achieve desired substrate specificity, control stereoselectivity, synthesize enantiomerically pure or racemic products, and stabilize enzymes by improving half-life, thermostability, inhibitor / product tolerance, and improving enzyme expression and solubility in the desired production microbial host of choice, enabling them to withstand harsh industrial process conditions. Once the desired enzymes capable of catalyzing each step of the pathway are characterized, the genes encoding these enzymes are cloned in a selected microorganism, fermentation conditions are optimized, and product formation is monitored after fermentation. After the enzymes are identified, the genes corresponding to one or more of the enzymes are cloned into a microbial host. In some embodiments, the genes encoding each enzyme in a particular pathway described herein are cloned into a microbial host.

[0159] Methods for introducing recombinant nucleic acids / exogenous nucleic acids / recombinant proteins / exogenous proteins into microorganisms, and vectors suitable for this purpose, are well known in the art. For example, various teachings are exemplified in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988 and quarterly updates). Methods for transferring expression vectors into microbial host cells are well known in the art. Specific methods and vectors may vary depending on the desired microbial host species. For example, bacterial host cells may be transformed by heat shock, calcium chloride treatment, electroporation, liposome, or phage infection. Yeast host cells may be transformed by lithium acetate treatment (which may further include carrier DNA and PEG treatment) or electroporation. These methods are included for illustrative purposes and are not intended to be limiting or comprehensive in any way. Routine experimentation using means well known in the art may be used to determine whether a particular expression vector or transformation method is suitable for a given microbial host. Moreover, suitable reagents and vectors for many different microbial hosts are commercially available and well known in the art.

[0160] Methods for constructing, expressing, or overexpressing enzymes in non-naturally occurring microbial hosts and testing expression levels are well known in the art (Protein Expression Technologies: Current Status and Future Trends, Baneyx F. eds. Horizon Bioscience, 2004, Norfolk, UK, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)).

[0161] Methods for fermenting microorganisms are well known in the art. For example, various techniques are described in Biochemical Engineering, Clark et al., eds. (CRC press, 1997, 2 nd Examples are provided in the "Synthetic Methods for Fermentation of Microorganisms" (edition 1). Specific fermentation methods may vary depending on the desired microbial host species. Typically, microorganisms are grown in a suitable medium with a carbon source in batch or continuous fermentation mode. Adipic acid or glutaric acid production can be enhanced using agents known to modulate catabolite repression or enzyme activity. A suitable pH for fermentation is between 3 and 10. Fermentation can be carried out under aerobic, anaerobic, or anoxic conditions, depending on the requirements of the microorganism. Fermentation can be carried out in batch, fed-batch, or continuous mode. Fermentation can be carried out in two phases, if desired. For example, the first phase can be aerobic to achieve high growth, i.e., high productivity, followed by an anaerobic phase for high caprolactone yield.

[0162] Carbon sources can include, for example, any carbohydrate source capable of providing a carbon source to a non-naturally occurring microorganism. Such carbohydrate sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch. Other carbohydrate sources include, for example, renewable feedstocks and biomass. Exemplary types of biomass that can be used as feedstock in the methods of the present disclosure include cellulosic biomass, hemicellulosic biomass, and lignin feedstocks, or portions of feedstocks. Such biomass feedstocks include carbohydrate substrates useful as carbon sources, such as, for example, glucose, xylose, arabinose, galactose, mannose, fructose, and starch. Given the teachings and guidance provided herein, one of skill in the art will understand that renewable feedstocks and biomass other than those exemplified above can also be used to culture the microorganisms of the present disclosure to produce desired compounds.

[0163] The reactions described herein can be monitored and the starting materials, products or intermediates in the fermentation medium can be identified by analyzing the medium using high performance 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.

[0164] Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and / or secrete the products of the disclosure.

[0165] The compounds prepared by the methods described herein can be isolated by methods commonly known in the art for isolating organic compounds prepared by biosynthesis or fermentation. For example, the compounds can be isolated from solution by crystallization, salt formation, pervaporation, reactive extraction, extraction (liquid-liquid extraction and two-phase extraction), adsorption, ion exchange, dialysis, distillation, gas stripping, and membrane-based separation (Roffler et al., Trends Biotechnolgy. 2:129-136 (1984)). 1,5-pentanediol can be isolated from solution using distillation, extraction (liquid-liquid extraction and two-phase extraction), pervaporation, and membrane-based separation (Roffler et al., Trends Biotechnolgy. 2:129-136 (1984)).

[0166] As described herein, exemplary growth conditions for biosynthesis of a desired product include anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microorganisms of the present disclosure can be maintained, cultured, or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment lacking oxygen. Substantially anaerobic conditions include, for example, culturing, batch fermentation, or continuous fermentation such that the dissolved oxygen concentration in the medium is maintained at 0-10% saturation. Substantially anaerobic conditions also include growing or placing cells in a liquid medium or on solid agar in a sealed chamber maintained in an atmosphere containing less than 1% oxygen. The oxygen percentage can be maintained, for example, by sparging the culture medium with a N2 / CO2 mixture or other suitable gas(es) other than oxygen.

[0167] For product production, the culture conditions described herein can be scaled up to allow continuous growth. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation, fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for commercial-scale biosynthetic production.

[0168] The term "pathway enzyme expressed in sufficient amounts" indicates that the enzyme is expressed in sufficient amounts to allow detection of the desired pathway product.

[0169] In another aspect, the invention provides a recombinant microorganism that includes a first exogenous nucleic acid encoding an aldolase hydratase enzyme, and that has been further modified to express a quinone oxidoreductase in an amount that is greater than that of a wild-type or unmodified version of the same microorganism, and optionally the recombinant microorganism is Corynebacterium glutamicum, Clostridium species, or E. coli.

[0170] 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 comprise a regulatory element driving 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 driving expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid or the second exogenous nucleic acid further comprise a regulatory element driving expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid further comprises a regulatory element driving expression of the second exogenous nucleic acid. In some embodiments, the second exogenous nucleic acid further comprises a regulatory element driving 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.

[0171] In some embodiments, the aldolase hydratase enzyme is EC No. 4.1.2.45, EC No. 4.1.2.34, or EC No. 4.1.1.4. In some embodiments, the aldolase hydratase enzyme is selected from the group consisting of 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_01365480 7, 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 selected from the group consisting of GenBank, RefSeq, or Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A 0A370X7D8, 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.

[0172] In some embodiments, the hydratase-aldolase is selected from the group consisting of GenBank, RefSeq, or Uniprot ID numbers 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_1078 18191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1 or KZL92449.1, or a portion thereof that promotes formation of the aldol dehydration product (e.g., a domain, a series of amino acid residues, which can be contiguous or spaced, etc.). 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 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.

[0173] In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme selected from Tables 1, 5, 6, 7, and 8.

[0174] In some embodiments, the hydratase-aldolase further comprises one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0175] In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a unique, separate vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector.

[0176] In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified by GenBank, RefSeq, or Uniprot ID numbers 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 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.

[0177] In some embodiments, the quinone oxidoreductase is selected from the group consisting of GenBank, RefSeq, and Uniprot ID numbers P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, and Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A21 2, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1 or WP_136898000.1, or 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 in 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 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.

[0178] In some embodiments, the quinone oxidoreductase further comprises one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0179] In some embodiments, a recombinant microorganism of the invention is capable of producing a 2-ketocarboxylic acid of the following formula: [ka] wherein R is H, CH or CHOH.

[0180] In some embodiments, a recombinant microorganism of the invention is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate.

[0181] In some embodiments, the recombinant microorganisms of the invention are genetically modified to enhance pyruvate production from a carbon source, in some embodiments, selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof.

[0182] In another aspect, the present invention provides a population of recombinant microorganisms disclosed herein. In some embodiments, the population is substantially homogeneous. In some embodiments, substantially homogeneous refers to being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homogeneous.

[0183] In another aspect, the present invention provides a method for producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate, the method comprising culturing under suitable conditions a population disclosed herein. In some embodiments, the method further comprises isolating 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxyhexanoate from the culture medium or the microorganism.

[0184] Detailed Description of Specific Embodiments Among other things, the present disclosure includes the recognition that various aldol dehydration product biosynthesis polypeptides, e.g., hydratase-aldolase polypeptides or including hydratase-aldolase polypeptides, can be used to effectively produce a variety of compounds. In some embodiments, the present disclosure demonstrates that various aldehydes, e.g., various aliphatic aldehydes described herein that are structurally distinct from natural and / or known aldehyde substrates of such polypeptides, can be utilized to efficiently produce numerous products using the aldol dehydration product biosynthesis polypeptides described herein. Among other things, the present disclosure demonstrates that a single aldol dehydration product biosynthesis polypeptide (e.g., various hydratase-aldolase polypeptides as described herein) can catalyze the production of a variety of aldol dehydration products.

[0185] In some embodiments, the present disclosure provides: 1. A method comprising contacting pyruvate and an aldehyde with an aldol dehydration product biosynthetic polypeptide to produce an aldol dehydration product, The method provides a method in which the aldol dehydration product is a compound containing an aldehyde or ketone group and a double bond conjugated to the aldehyde or ketone group.

[0186] In some embodiments, the aldehyde is an aliphatic aldehyde. In some embodiments, the -CHO group of the aldehyde is not conjugated to, for example, a double bond, a triple bond, or an aromatic group.

[0187] In some embodiments, the present disclosure provides: 1. A method comprising contacting pyruvate and an aliphatic aldehyde with an aldol dehydration product biosynthetic polypeptide to produce an aldol dehydration product, the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aromatic group; The method provides a method in which the aldol dehydration product is a compound containing an aldehyde or ketone group and a double bond conjugated to the aldehyde or ketone group.

[0188] In some embodiments, the aldol dehydration product biosynthetic polypeptide is or includes a hydratase-aldolase polypeptide, e.g., a hydratase-aldolase polypeptide exemplified herein. In some embodiments, provided methods include contacting pyruvate and a fatty aldehyde with a hydratase-aldolase to produce an aldol dehydration product.

[0189] In some embodiments, the aldol dehydration product biosynthetic polypeptide comprises an aldolase polypeptide. In some embodiments, the aldol dehydration product biosynthetic polypeptide comprises a hydratase polypeptide. In some embodiments, the aldol dehydration product biosynthetic polypeptide comprises a hydratase-aldolase polypeptide. In some embodiments, the aldol dehydration product biosynthetic polypeptide is a hydratase-aldolase polypeptide. In some embodiments, the hydratase-aldolase polypeptide is or comprises a hydratase-aldolase as described herein, e.g., an enzyme having EC No. 4.1.2.45 or EC No. 4.1.2.34 or EC 4.1.1.4, or an enzyme selected from Tables 1 and 5-8.

[0190] In some embodiments, the aldol dehydration product biosynthetic polypeptide is in an organism, e.g., a microorganism. In some embodiments, the organism expresses an engineered aldol dehydration product biosynthetic polypeptide. In some embodiments, the organism expresses an improved level and / or activity of the aldol dehydration product biosynthetic polypeptide. In some embodiments, the organism improves the rate and / or yield of aldol dehydration product production. In some embodiments, the organism improves substrate utilization in producing the aldol dehydration product.

[0191] In some embodiments, the conversion of pyruvate and aliphatic aldehydes to aldol dehydration products is catalyzed by aldol dehydration product biosynthetic polypeptides.

[0192] In some embodiments, the aldol dehydration product can be provided through an alternative pathway: In some embodiments, the aldol dehydration product is produced from the aldol product.

[0193] In some embodiments, the present disclosure provides: contacting pyruvate and an aldehyde with an aldol product biosynthetic polypeptide to produce an aldol product, The method provides a method in which the aldol dehydration product is a compound containing an aldehyde or ketone group and a double bond conjugated to the aldehyde or ketone group.

[0194] 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.

[0195] In some embodiments, the present disclosure provides: 1. A method comprising contacting pyruvate and an aliphatic aldehyde with an aldol product biosynthetic polypeptide to produce an aldol product, the carbonyl group of the aliphatic aldehyde is not conjugated to an alkenyl group, an alkynyl group, or an aromatic group; The method provides a method in which the aldol product is a compound containing an aldehyde or ketone group and a hydroxyl group attached to the β-carbon of the aldehyde or ketone carbonyl group.

[0196] Various methods of the disclosure involve using biosynthetic polypeptides. In some embodiments, when used with a particular product, a biosynthetic polypeptide, e.g., an aldol product biosynthetic polypeptide, a reduction product biosynthetic polypeptide, etc., refers to a polypeptide that is involved in the synthesis of that particular product. In some embodiments, a biosynthetic polypeptide, when used with a particular product, is or includes an enzyme that catalyzes the formation of that particular product. In some embodiments, a biosynthetic polypeptide has an amino acid sequence found in nature, e.g., in a microorganism (e.g., in a reference biosynthetic polypeptide for a particular product found in nature). Alternatively or additionally, in some embodiments, a biosynthetic polypeptide shares characteristic sequence elements and / or overall percent identity with an appropriate reference biosynthetic polypeptide (e.g., as found in nature and / or as set forth herein (e.g., in one or more of the relevant tables)), or a portion thereof (e.g., a portion that facilitates a relevant reaction (e.g., a domain (e.g., a relevant catalytic domain) and / or a series of amino acid residues, which can be contiguous or spaced).

[0197] In some embodiments, the aldol product biosynthetic polypeptide is or includes an aldolase polypeptide. It will be apparent to one of skill in the art upon reading this disclosure that a variety of aldolase polypeptides can be used in accordance with this disclosure. In some embodiments, the aldolase polypeptide is or includes an aldolase described in US20170044551, which aldolases are incorporated herein by reference.

[0198] In some embodiments, the aldol product biosynthetic polypeptide is or comprises an aldolase-hydratase as described herein.

[0199] In some embodiments, the aldol product biosynthetic polypeptides are in an organism, such as a microorganism. In some embodiments, the organism has been engineered to express engineered or exogenous aldol product biosynthetic polypeptides, often at elevated protein and / or activity levels. In some embodiments, the conversion of pyruvate and aliphatic aldehydes to aldol products is catalyzed by the aldol product biosynthetic polypeptides. In some embodiments, the method is carried out in a culture, e.g., a bacterial culture. In other biosynthetic polypeptides, the aldol product biosynthetic polypeptides may be in an organism, such as a bacterium, engineered, and / or expressed at elevated protein and / or activity levels, and the products may be produced at improved rates and / or yields and / or substrate utilization.

[0200] In some embodiments, the aldol product is converted to an aldol dehydration product, either enzymatically, biosynthetically, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the conversion comprises contacting the aldol product with a dehydration product biosynthetic polypeptide to produce the aldol dehydration product. In some embodiments, the dehydration product biosynthetic polypeptide is or comprises a hydratase. In some embodiments, the dehydration product biosynthetic polypeptide is or comprises a dehydratase. In some embodiments, the hydratase or dehydratase is described in US20170044551, which hydratases and dehydratases are incorporated herein by reference. As with other biosynthetic polypeptides, the dehydration product biosynthetic polypeptide may be in an organism such as a bacterium, may be engineered, and / or may be expressed at elevated protein and / or activity levels, and the product may be produced at an improved rate and / or yield and / or substrate utilization.

[0201] As will be apparent to those skilled in the art, the aldol dehydration products can be used to produce a variety of products, such as 1,5-pentanediol, 1,6-hexanediol, 6HH, adipic acid, and the like, which can be used to produce a wide range of products, such as polymers, resins, paint products, and the like. In some embodiments, utilization of the aldol dehydration products involves one or more chemical transformations, each of which can be independently catalyzed in vivo by a polypeptide (e.g., an enzyme described herein) or can be carried out by conventional non-enzymatic chemical processes. As will be apparent to those skilled in the art, one or more or all of the steps can be carried out in one or more organisms (each of which can independently carry out one or more reactions using substrate(s) produced in the organism itself or substrate(s) derived from the organism) and / or in one or more cultures independently containing one or more types of organisms (each of which can independently carry out one or more reactions using substrate(s) produced in the culture itself or derived from the culture (e.g., feed compounds, compounds produced by another organism, etc.)). In some embodiments, one or more or all of the biosynthetic polypeptides are independently present in a single engineered organism, e.g., a bacterium. In some embodiments, one or more of a set of product-producing biosynthetic polypeptides are expressed in one engineered organism, e.g., a bacterium, and one or more of the other biosynthetic polypeptides in the set are expressed in one or more other engineered organisms, e.g., a bacterium. In some embodiments, the organism, e.g., a bacterium, is engineered to contain one or more exogenous nucleic acids encoding one or more or all of the biosynthetic polypeptides. In some embodiments, production of a product involves multiple reaction steps in a single culture containing one or more bacteria, each of which independently contains one or more or all of the required biosynthetic polypeptides and, together, contains all of the required biosynthetic polypeptides.In some embodiments, production of a product involves multiple reaction steps carried out in two or more cultures, each of which independently contains one or more bacteria, and each of which independently contains one or more or all of the required biosynthetic polypeptides, and together contain all of the required biosynthetic polypeptides.

[0202] For example, in some embodiments, the double bond of the aldol dehydration product is converted to a single bond.

[0203] In some embodiments, the present disclosure provides: contacting an alkene with an alkene reduction product biosynthetic polypeptide to produce an alkene reduction product; the alkene contains a double bond conjugated to a carbonyl group, A method is provided for reducing the double bond conjugated to the carbonyl group in the alkene to a single bond to provide an alkene reduction product.

[0204] In some embodiments, the alkene is an aldol dehydration product.

[0205] In some embodiments, the alkene reduction product biosynthetic polypeptide is or includes an enzyme that catalyzes the reduction of an aldol dehydration product, e.g., 2-oxo-3-enoic acid, as described herein. In some embodiments, such an enzyme is a quinone oxidoreductase, as described herein. In some embodiments, such an enzyme is in the EC 1.6.5 class. In some embodiments, such an enzyme is in the EC 1.6.5.5 class. In some embodiments, such an enzyme is selected from Table 9.

[0206] In some embodiments, the alkene reduction product biosynthetic polypeptide is in an organism, e.g., a microorganism. In some embodiments, the organism expresses an engineered alkene reduction product biosynthetic polypeptide. In some embodiments, the organism expresses an improved level and / or activity of the alkene reduction product biosynthetic polypeptide. In some embodiments, the organism improves the rate and / or yield of alkene reduction product production. In some embodiments, the organism improves substrate utilization in producing the alkene reduction product.

[0207] In some embodiments, the alkene reduction product biosynthetic polypeptide is or comprises an enzyme that is endogenously encoded and / or expressed by an organism without manipulation.

[0208] It will be apparent to those skilled in the art upon reading this disclosure that a variety of aldehydes may be used in accordance with this disclosure. In some embodiments, the aldehyde is a natural or known substrate of a biosynthetic polypeptide, such as a hydratase-aldolase, or an aldol dehydration product biosynthetic polypeptide that includes a hydratase-aldolase. In some embodiments, the aldehyde is not a natural or known substrate. For example, among other things, the present disclosure demonstrates that an aliphatic aldehyde can be used to produce a product using a hydratase-aldolase whose natural or known substrate is an aromatic aldehyde or a conjugated aldehyde.

[0209] In some embodiments, the aldehyde is an aliphatic aldehyde. In some embodiments, the aldehyde has one or two α-hydrogens. In some embodiments, the aldehyde has the structure of Formula A-1 below: R a -L 2 -L 1 -C(O)H A-1 or a salt thereof, wherein R a is R" or -OR" L 1 and L2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 heteroaliphatic groups, one or more of the methylene units of which are optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-; -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; each R" is independently -R', -C(O)R', -CO2R', or -SO2R'; R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Two or more R' groups, together with the intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0-5 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 0-5 heteroatoms.

[0210] In some embodiments, the aldol product has the structure of formula P-1: R a -L 2 -L 1 -CH(OH)-CH2-C(O)-C(O)OH P-1 or a salt thereof, wherein R a is R" or -OR" L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 heteroaliphatic groups, one or more of the methylene units of which are optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-; -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; each R" is independently -R', -C(O)R', -CO2R', or -SO2R'; R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Two or more R' groups, together with the intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0-5 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 0-5 heteroatoms.

[0211] In some embodiments, the aldol dehydration product has the structure of formula P-2: R a -L 2 -L 1-CH=CH-C(O)-C(O)OH P-2 or a salt thereof, wherein R a is R" or -OR" L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 heteroaliphatic groups, one or more of the methylene units of which are optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-; -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; each R" is independently -R', -C(O)R', -CO2R', or -SO2R'; R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Two or more R' groups, together with the intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0-5 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 0-5 heteroatoms.

[0212] In some embodiments, the -CH=CH- of formula P-2 is in the E configuration. In some embodiments, the -CH=CH- of formula P-2 is in the Z configuration.

[0213] In some embodiments, the alkene reduction product has the structure of formula P-3: R a -L 2 -L 1 -CH2-CH2-C(O)-C(O)OH P-3 or a salt thereof, wherein R a is R" or -OR" L 1 and L 2 each independently is a covalent bond or a divalent optionally substituted straight or branched chain C 1-20 Aliphatic group or C 1-20 heteroaliphatic groups, one or more of the methylene units of which are optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-; -Cy- is a divalent optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring, each monocyclic ring independently being an optionally substituted, saturated, partially saturated, or aromatic 3- to 20-membered ring having 0-5 heteroatoms; each R" is independently -R', -C(O)R', -CO2R', or -SO2R'; R' is hydrogen or C 1-10 Aliphatic groups, C with 1 to 5 heteroatoms 1-10 an optionally substituted group selected from a heteroaliphatic group, a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring having 1 to 5 heteroatoms, and a 3- to 10-membered heterocycle having 1 to 5 heteroatoms; or Two or more R' groups, together with the intervening atoms, form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having 0-5 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 0-5 heteroatoms.

[0214] In some embodiments, R a is R″. In some embodiments, R a is -OR”.

[0215] 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'.

[0216] In some embodiments, R' is hydrogen. In some embodiments, R' is not hydrogen.

[0217] 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.

[0218] In some embodiments, L 1 is a covalent bond. In some embodiments, L 1 is not a covalent bond.

[0219] In some embodiments, L 1 is an arbitrarily substituted C 1-6 In some embodiments, L is alkylene. 1 is an optionally substituted linear C 1-6 In some embodiments, L is alkylene. 1 is an optionally substituted —CH—. In some embodiments, L 1is optionally substituted -CHCH-. In some embodiments, L 1 is optionally substituted -CHCHCH-. In some embodiments, L 1 is optionally substituted -CHCHCHCHCH-. In some embodiments, L 1 is optionally substituted -CHCHCHCHCHCH-. 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 -CH-. In some embodiments, L 1 is -CHCH-. In some embodiments, L 1 is -CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2CH2CH2CH2-.

[0220] In some embodiments, L 2 is a covalent bond. In some embodiments, L 2 is not a covalent bond.

[0221] In some embodiments, L 2 is an arbitrarily substituted C 1-6 In some embodiments, L is alkylene. 2 is an optionally substituted linear C 1-6 In some embodiments, L is alkylene. 2 is an optionally substituted —CH—. In some embodiments, L 2is optionally substituted -CHCH-. In some embodiments, L 2 is optionally substituted -CHCHCH-. In some embodiments, L 2 is optionally substituted -CHCHCHCHCH-. In some embodiments, L 2 is optionally substituted -CHCHCHCHCHCH-. 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 -CH-. In some embodiments, L 2 is -CHCH-. 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-.

[0222] In some embodiments, L 1 and L 2 At least one of the is not a covalent bond.

[0223] 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.

[0224] 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 CH2OHCH2CH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH2OHCH2CH2CH(OH)CH2C(O)COOH. In some embodiments, the aldol product is CH2OHCH2CH2CH(OH)CH2C(O)COOH.

[0225] In some embodiments, the aldol dehydration product is CHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHCHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHCHCHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHOHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHOHCHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHOHCHCH=CHC(O)COOH. In some embodiments, the aldol dehydration product is CHOHCHCH=CHC(O)COOH.

[0226] In some embodiments, the alkene reduction product is CH3CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH3CH2CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH3CH2CH2CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH3CH2CH2CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH2OHCH2CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH2OHCH2CH2CH2CH2C(O)COOH. In some embodiments, the alkene reduction product is CH2OHCH2CH2CH2CH2C(O)COOH.

[0227] In some embodiments, the alkene reduction product is converted to a carbonyl reduction product, either enzymatically catalyzed, biosynthetically, or by conventional 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 biosynthetic polypeptide to produce the carbonyl reduction product; The alkene reduction product contains a carbonyl group, The carbonyl group of the alkene reduction product is converted to -CH(OH)-.

[0228] In some embodiments, the carbonyl reduction product biosynthetic polypeptide is or comprises a reductase. In some embodiments, the carbonyl reduction product biosynthetic polypeptide is or comprises a ketoreductase as described herein. In some embodiments, the carbonyl reduction product biosynthetic 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 enzymes are incorporated herein by reference.

[0229] In some embodiments, the conversion of the alkene reduction product to a carbonyl reduction product is catalyzed by a carbonyl reduction product biosynthetic polypeptide.

[0230] As with many other biosynthetic polypeptides, carbonyl reduction product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0231] In some embodiments, the carbonyl reduction product has the structure of formula P-4 below: R a -L 2 -L 1 -CH2-CH2-CH(OH)-C(O)OH P-4 or a salt thereof, wherein each variable is independently as described herein.

[0232] 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.

[0233] In some embodiments, the carbonyl reduction product is converted to a CoA transfer product either by enzyme catalysis, biosynthesis, or conventional organic synthesis without enzyme catalysis. In some embodiments, the CoA transfer product is a compound of formula P-5: R a -L 2 -L 1 -CH2-CH2-CH(OH)-C(O)-S-CoA P-5 or a salt thereof, wherein each variable is independently as described herein.

[0234] In some embodiments, such a conversion is catalyzed by a CoA (CoA = coenzyme A) transfer product biosynthetic polypeptide. In some embodiments, the CoA transfer product biosynthetic polypeptide is or includes a CoA transferase as described herein, such as 2,6-dihydroxyhexanoate CoA transferase. In some embodiments, the CoA transferase is a CoA transferase described in US20170044551, which CoA transferases are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthetic polypeptide.

[0235] As with many other biosynthetic polypeptides, CoA transfer product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0236] 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. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2CH(OH)C(O)S-COA.

[0237] In some embodiments, the CoA transfer product is converted to a dehydration product either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the dehydration product is a compound of formula P-6: R a -L 2 -L 1 -CH2-CH=CH-C(O)-S-CoA P-6 or a salt thereof, wherein each variable is independently as described herein.

[0238] In some embodiments, such conversion is catalyzed by a dehydration product biosynthesis polypeptide. In some embodiments, the dehydration product biosynthesis polypeptide is or includes a dehydratase as described herein. In some embodiments, the dehydratase is or includes a 2,6-dihydroxyhexanoyl-CoA2-dehydratase as described herein. In some embodiments, the dehydratase is described in US20170044551, which is incorporated by reference.

[0239] In some embodiments, such a conversion is catalyzed by a dehydration product biosynthetic polypeptide.

[0240] As with many other biosynthetic polypeptides, dehydration product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0241] In some embodiments, the dehydration product is CH3CH2CH=CHC(O)S-CoA. In some embodiments, the dehydration product is CH3CH2CH2CH=CHC(O)S-COA. In some embodiments, the dehydration product is CH3CH2CH2CH=CHC(O)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH=CHC(O)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH2CH=CHC(O)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH2CH=CHC(O)S-COA. In some embodiments, the dehydration product is CH2OHCH2CH2CH=CHC(O)S-COA.

[0242] In some embodiments, the dehydration product, e.g., a compound of formula P-6 or a salt thereof, is converted to a reduction product either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the reduction product is a compound of formula P-7: R a -L 2 -L 1 -CH2-CH2-CH2-C(O)-S-CoA P-7 or a salt thereof, wherein each variable is independently as described herein.

[0243] In some embodiments, such conversions are catalyzed by a reduction product biosynthetic polypeptide. In some embodiments, the reduction product biosynthetic polypeptide is or includes a 2,3-enoyl-CoA reductase, a 2,3-dehydrocarboxyl-CoA 2'3-reductase, such as a 2,3-dehydrohexanoyl-CoA 2,3-reductase as described herein. In some embodiments, suitable reductases are described in US20170044551, which reductases are incorporated herein by reference. In some embodiments, such conversions are catalyzed by a reduction product biosynthetic polypeptide.

[0244] As with many other biosynthetic polypeptides, reduction product biosynthetic polypeptides may be in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0245] 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. In some embodiments, the reduction product is CH2OHCH2CH2CH2CH2CH2C(O)S-COA.

[0246] 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 either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the CoA transfer product is a compound of formula P-8: R a -L 2 -L 1 -CH2-CH2-CH2-C(O)-OH P-8 or a salt thereof, wherein each variable is independently as described herein.

[0247] In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthetic polypeptide. In some embodiments, the CoA transfer product biosynthetic polypeptide is or includes a CoA transferase as described herein, for example, a 6-hydroxyhexanoyl-CoA transferase as described herein. In some embodiments, the CoA transferase is described in US20170044551, which CoA transferases are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthetic polypeptide.

[0248] As with many other biosynthetic polypeptides, CoA transfer product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0249] 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 CH2OHCH2CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2C(O)OH. In some embodiments, the CoA transfer product is CH2OHCH2CH2CH2CH2CH2C(O)OH.

[0250] In some embodiments, the CoA transfer product, e.g., a compound of formula P-8 or a salt thereof, ais —OH) is converted to an oxidation product either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the oxidation product is a compound of formula P-9: HC(O)-L 2’ -L 1 -CH2-CH2-CH2-C(O)-OH P-9 or a salt thereof, wherein L 2’ is a covalent bond or a divalent optionally substituted straight or branched chain C 1-19 Aliphatic group or C 1-19 A heteroaliphatic group wherein one or more of the methylene units thereof is optionally and independently replaced by -C≡C-, -C(R")2-, -Cy-, -O-, -S-, -SS-, -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-, wherein each variable is independently as described herein.

[0251] In some embodiments, L 2’ is a covalent bond. In some embodiments, L 2’ is not a covalent bond. 1 and L 2’ At least one of the is not a covalent bond.

[0252] In some embodiments, L 2’ is an arbitrarily substituted C 1-6 In some embodiments, L is alkylene. 2’ is an optionally substituted linear C 1-6 In some embodiments, L is alkylene. 2’ is an optionally substituted —CH—. In some embodiments, L 2’ is optionally substituted -CHCH-. In some embodiments, L 2’is optionally substituted -CHCHCH-. In some embodiments, L 2’ is optionally substituted -CHCHCHCHCH-. In some embodiments, L 2’ is optionally substituted -CHCHCHCHCHCH-. 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 -CH-. In some embodiments, L 2’ is -CHCH-. 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-.

[0253] In some embodiments, such conversion is catalyzed by an oxidation product biosynthesis polypeptide. In some embodiments, the oxidation product biosynthesis polypeptide is or includes an alcohol dehydrogenase, for example, a primary alcohol dehydrogenase such as 6-hydroxyhexanoate dehydrogenase as described herein. In some embodiments, the alcohol dehydrogenase is described in US20170044551, which alcohol dehydrogenases are incorporated herein by reference. In some embodiments, such conversion is catalyzed by an oxidation product biosynthesis polypeptide.

[0254] As with many other biosynthetic polypeptides, oxidation product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0255] In some embodiments, the oxidation product is HC(O)CHCHCHC(O)OH. In some embodiments, the oxidation product is HC(O)CHCHCHCHC(O)OH. In some embodiments, the oxidation product is HC(O)CHCHCHCHCHC(O)OH.

[0256] 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 either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the oxidation product is a compound of formula P-10, HO-C(O)-L 2’ -L 1 -CH2-CH2-CH2-C(O)-OH P-10 or a salt thereof, wherein each variable is independently as described herein.

[0257] In some embodiments, such a conversion is catalyzed by an aldehyde oxidation product biosynthetic polypeptide. In some embodiments, the aldehyde oxidation product biosynthetic polypeptide is or includes an aldehyde dehydrogenase, such as a 6-hydroxyhexanoate dehydrogenase as described herein. In some embodiments, the aldehyde dehydrogenase is described in US20170044551, which aldehyde dehydrogenases are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an aldehyde oxidation product biosynthetic polypeptide.

[0258] As with many other biosynthetic polypeptides, aldehyde oxidation product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0259] In some embodiments, the aldehyde oxidation product is HOC(O)CHCHCHC(O)OH. In some embodiments, the oxidation product is HOC(O)CHCHCHCHC(O)OH. In some embodiments, the oxidation product is HOC(O)CHCHCHCHCHC(O)OH.

[0260] 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, either enzymatically catalyzed, biosynthetically, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the carboxyl reduction product is a compound of formula P-9' below: R a -L 2 -L 1 -CH2-CH2-CH2-C(O)-H P-9' or a salt thereof, wherein each variable is independently as described herein.

[0261] In some embodiments, such conversion is catalyzed by a carboxyl reduction product biosynthetic polypeptide. In some embodiments, the carboxyl reduction product biosynthetic polypeptide is or comprises a carboxylic acid reductase or aldehyde dehydrogenase as described herein. In some embodiments, the carboxyl reduction product biosynthetic polypeptide is or comprises a 6-hydroxyhexanoate 1-reductase. In some embodiments, the carboxyl reduction product biosynthetic polypeptide is or comprises a carboxylic acid reductase or aldehyde dehydrogenase as described in US20170044551, which carboxylic acid reductases or aldehyde dehydrogenases are incorporated herein by reference. In some embodiments, such conversion is catalyzed by a carboxyl reduction product biosynthetic polypeptide.

[0262] As with many other biosynthetic polypeptides, the carboxyl reduction product biosynthetic polypeptides may be in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0263] 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. In some embodiments, the carboxyl reduction product is CH2OHCH2CH2CH2CH2CH2C(O)H.

[0264] In some embodiments, the carboxyl reduction product, e.g., a compound of formula P-9' or a salt thereof, is converted to an aldehyde reduction product either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the aldehyde reduction product is a compound of formula P-10': R a -L 2 -L 1 -CH2-CH2-CH2-CH2-OH P-10' or a salt thereof, wherein each variable is independently as described herein.

[0265] In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthetic polypeptide. In some embodiments, the aldehyde reduction product biosynthetic polypeptide is or comprises an aldehyde reductase or 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 reductases and dehydrogenases are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthetic polypeptide.

[0266] As with many other biosynthetic polypeptides, aldehyde reduction product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0267] In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH3CH2CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2CH2OH. In some embodiments, the aldehyde reduction product is CH2OHCH2CH2CH2CH2CH2CH2OH.

[0268] In some embodiments, the alkene reduction product, e.g., a compound of formula P-3 or a salt thereof, is converted to a decarboxylation product, either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the decarboxylation product is a compound of formula P-4' R a -L 2 -L 1 -CH2-CH2-C(O)-H P-4' or a salt thereof, wherein each variable is independently as described herein.

[0269] 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 decarboxylases are incorporated herein by reference. In some embodiments, such conversion is catalyzed by a decarboxylation product biosynthesis polypeptide.

[0270] As with many other biosynthetic polypeptides, decarboxylation product biosynthetic polypeptides may be in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0271] 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. In some embodiments, the decarboxylation product is CH2OHCH2CH2CH2CH2CHO.

[0272] 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, either by enzyme catalysis, by biosynthesis, or by conventional organic synthesis without enzyme catalysis. In some embodiments, the aldehyde reduction product is a compound of formula P-5': R a -L 2 -L 1 -CH2-CH2-CH2-OH P-5' or a salt thereof, wherein each variable is independently as described herein.

[0273] In some embodiments, such conversion is catalyzed by an aldehyde reduction product biosynthetic polypeptide. In some embodiments, the aldehyde reduction product biosynthetic 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 biosynthetic polypeptide.

[0274] As with many other biosynthetic polypeptides, aldehyde reduction product biosynthetic polypeptides may be present in organisms such as bacteria, may be engineered, and / or may be expressed at elevated protein levels and / or activity levels, and their products may be produced at improved rates and / or yields and / or substrate utilization.

[0275] 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 CH3CH2CH2CH2CH2CH2OH. 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 CH2OHCH2CH2CH2CH2CH2OH.

[0276] 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 production organism, such as a bacterium.

[0277] As provided herein, various techniques are available for assessing the activity of a polypeptide with respect to biosynthetic activity. For example, various techniques for assessing the activity of an aldol dehydration product biosynthetic polypeptide (e.g., a hydratase-aldolase) or an alkene reduction product biosynthetic polypeptide (e.g., an enzyme for reducing an aldol dehydration product) are described in the Examples.

[0278] In some embodiments, various biosynthetic polypeptides, e.g., aldol dehydration product biosynthetic polypeptides, are present in an organism, in many embodiments, a microorganism such as a bacterium, fungus, etc. In some embodiments, the biosynthetic polypeptides are expressed from one or more recombinant nucleic acids. In some embodiments, various transformations are performed biosynthetically, e.g., in an organism such as a bacterium. In some embodiments, an organism (e.g., a microorganism such as a bacterium) has been engineered to contain an exogenous nucleic acid encoding a biosynthetic polypeptide, e.g., an aldol dehydration product biosynthetic polypeptide, such as a hydratase-aldolase.

[0279] In some embodiments, an organism, e.g., an organism engineered to produce an aldol dehydration product, expresses an aldol dehydration product biosynthetic polypeptide (e.g., a hydratase-aldolase polypeptide) at a modulated level, typically at an increased level and / or activity.

[0280] 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., various hydratase-aldolases). 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 naturally occurring nucleic acid. In some embodiments, the engineered nucleic acid encodes the same polypeptide or characteristic elements thereof as a reference nucleic acid, e.g., a naturally occurring nucleic acid. In some embodiments, the engineered nucleic acid encodes a polypeptide or characteristic elements thereof that differs from that encoded by the reference nucleic acid. In some embodiments, the engineered polypeptide comprises one or more differences from a reference polypeptide (e.g., a polypeptide encoded by a reference nucleic acid found in nature). In some embodiments, the engineered polypeptide comprises one or more amino acid residues that differ from the reference polypeptide. In some embodiments, the engineered polypeptide is a polypeptide that is not found in the organism into which the polypeptide is being 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 with the reference polypeptide or a characteristic element thereof. In some embodiments, the characteristic element is a domain that catalyzes the relevant reaction. In some embodiments, the characteristic element is a series of amino acid residues. In some embodiments, the characteristic element is a series of amino acid residues that establishes contact with substrates, products, cofactors, etc. and / or facilitates the relevant reaction. As will be apparent to one of skill in the art, the residues in the series of amino acid residues can be adjacent to each other in sequence or can be spaced apart.In some embodiments, two or more amino acid residues in the string of amino acid residues may be spatially close to each other, for example, within the catalytic pocket.

[0281] In some embodiments, for biosynthetic production, the organism may express one or more biosynthetic polypeptides at higher levels and / or with higher activity, hi some embodiments, the organism has improved speed and / or yield in producing the desired product.

[0282] As described herein, in some embodiments, the present disclosure provides high product yields. In some embodiments, for example, the yield of a one or more step process involving one or more biosynthetic polypeptides is about or at least about 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, or 1000 mg / L, or about or at least about 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or 1.6 g / L. g / L, 1.6g / L, 1.7g / L, 1.8g / L, 1.9g / L, 2g / L, 2.1g / L, 2.2g / L, 2.3g / L, 2.4g / L, 2.5g / L, 2 .7g / L, 3g / L, 3.5g / L, 4g / L, 4.5g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 15g / L, 20g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L, 220 g / L, 250 g / L, or 300 g / L. In some embodiments, the provided techniques increase the utilization of a substrate, e.g., pyruvate, in a desired product. In some embodiments, the utilization in the 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%.

[0283] Those skilled in the art will recognize that various compounds of the present 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, are useful as materials for producing a variety of compounds, materials, and products. For example, adipic acid can be used to produce nylon 6,6, polyester polyols, polyester resins, plasticizers, food products, and other materials. 1,5-pentanediol can be used to produce various polyurethanes, polyester polyols, and polyesters. 1,6-hexanediol (HDO) can be used to produce various polyesters, some of which are useful in industrial coating applications. HDO can also be used to produce polyurethanes, which can be used, among other things, in automotive coatings. In some embodiments, HDO is used to produce macrodiols, such as adipates and polycarbonate diols, for example, for use in elastomers and polyurethane dispersions (e.g., for parquet flooring and leather coatings). Through conventional chemical or biosynthetic processes, or a combination thereof, 6-hydroxyhexanoic acid can be cyclized to produce ε-caprolactone, which can then be aminated to produce ε-caprolactam. Through conventional chemical or biosynthetic processes, or a combination thereof, 6-hydroxyhexanoic acid can be aminated to produce 6-aminohexanoic acid, which can then be cyclized to produce ε-caprolactam. ε-Caprolactam can be used, among other things, to produce nylon 6, a polymer widely used in many different industries. ε-Caprolactone can be polymerized to produce polycaprolactone (PCL), a biodegradable polyester with a variety of uses, including the production of specialty polyurethanes. Various 2-ketocarboxylic acids are useful in a variety of industrially relevant chemicals and pharmaceuticals. In some embodiments, such chemicals and pharmaceuticals, or intermediates thereof, are amino acids or α-hydroxycarboxylic acids.In some embodiments, compounds of the present disclosure are used to make polyesters, polyester polyols, polyurethanes, nylons (e.g., derived from adipic acid), polycarbonate diols (e.g., derived from HDO or 1,5-pentanediol), diacrylate esters (e.g., derived from HDO or 1,5-pentanediol), diglycidyl ethers (e.g., derived from HDO or 1,5-pentanediol), and the like.

[0284] In some embodiments, the disclosure provides preparations of the provided processes, such as preparations 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, as well as various compounds, materials, products, etc. prepared from such compounds.

[0285] The provided technology offers many advantages. Among other things, the provided processes can use one or more biosynthetic polypeptides and / or substances derived from renewable resources, thereby improving efficiency and / or reducing pollution. In some embodiments, the preparations 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, and various compounds, substances, products prepared from such compounds, etc.) may contain one or more isotopes, e.g., methyl ... 14 In some embodiments, the preparation using a fossil carbon source is enriched in C levels. 14The C level is 0 or substantially 0. Techniques for assessing the isotopic ratios and / or isotopic levels of various atoms in compounds, compositions, preparations, products, etc. are well known to those of skill in the art, and such techniques 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 accelerator mass spectrometry (AMS) and / or stable isotope ratio mass spectrometry (SIRMS), and / or by site-specific natural isotope fractionation nuclear magnetic resonance spectroscopy (SNIF-NMR).

[0286] As will be apparent to one of skill in the art, the provided methods can be performed in vitro in a system containing one or more biosynthetic polypeptides. In many embodiments, the provided techniques are 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, e.g., a bacterium, that expresses one or more biosynthetic polypeptides as described herein. In some embodiments, such organisms have been engineered. In some embodiments, such organisms are engineered and / or cultured to express one or more biosynthetic polypeptides at improved protein and / or activity levels. In some embodiments, such organisms are engineered and / or cultured to more efficiently produce a desired product using a carbon source.

[0287] In some embodiments, the disclosure provides a microorganism that produces an aldol product of an aliphatic aldehyde, wherein the microorganism has improved expression or activity of an aldol product biosynthetic polypeptide. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium.

[0288] In some embodiments, the disclosure provides organisms that produce aldol dehydration products of aldehydes, wherein the microorganisms have improved expression or activity of aldol product biosynthetic polypeptides, aldol dehydration product biosynthetic polypeptides, dehydration product biosynthetic polypeptides, and combinations thereof. In some embodiments, the disclosure provides organisms that produce aldol dehydration products of aldehydes, wherein the microorganisms have improved expression or activity of aldol dehydration product biosynthetic polypeptides. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium. In some embodiments, the aldehyde is an aliphatic aldehyde.

[0289] In some embodiments, the disclosure provides organisms that produce alkene reduction products, wherein the microorganisms have improved expression or activity of an alkene reduction product biosynthetic polypeptide. In some embodiments, the disclosure provides organisms that produce alkene reduction products from pyruvate and aldehydes, wherein the microorganisms have improved expression or activity of an alkene reduction product biosynthetic polypeptide. In some embodiments, the organism is engineered. In some embodiments, the organism is a bacterium.

[0290] In some embodiments, the disclosure provides a culture medium of an organism as described herein. In some embodiments, the disclosure provides a culture medium of a bacteria. In some embodiments, the culture medium includes one or more products of one or more biosynthetic polypeptides, such as 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 salts thereof.

[0291] As will be apparent to those skilled in the art, pyruvate may be provided as pyruvic acid or its salts.

[0292] In one aspect, the present invention provides a compound of formula I: [ka] wherein R is CHOH, CH or H; or a salt thereof, or a solvate of the compound or salt thereof, the process comprising an enzymatic step.

[0293] In some embodiments, the method includes (a) producing a hydratase-aldolase having the formula: [ka] C N Aldehydes in which R is CHOH, CH or H N The aldehyde and pyruvate are converted to intermediate C. N+3 (b) converting the 3,4-dehydro-2-ketocarboxylic acid to the C using an oxidoreductase having EC number 1.6.5 (e.g., EC number 1.6.5.5.) N+3 3,4-Dehydro-2-ketocarboxylic acid to C N+3 under conditions that convert the compound into a 2-ketocarboxylic acid (i.e., a compound of formula I) or a salt thereof, or a solvate of the compound or salt thereof, N The method may comprise, consist essentially of, or even consist of combining or incubating in solution an aldehyde and pyruvate.

[0294] In some embodiments, the method includes (a) first converting an aldehyde group of formula (I) to an aldehyde group of formula (II) through an aldol addition reaction catalyzed by a hydratase-aldolase (referred to herein as Ads-Hyd) having EC No. 4.1.2.45 or EC No. 4.1.2.34 or EC No. 4.1.1.4: [ka] C NAldehydes in which R is CHOH, CH or H N The aldehyde and pyruvate are converted to intermediate C. N+3 (b) converting the 4-hydroxy-2-ketocarboxylic acid to C using the hydratase-aldolase described above. N+3 3,4-dehydro-2-ketocarboxylic acid, and then (c) using an oxidoreductase having EC number 1.6.5 (e.g., EC number 1.6.5.5) to convert the C N+3 3,4-Dehydro-2-ketocarboxylic acid to C N+3 The method may comprise, consist essentially of, or even consist of combining or incubating in solution the above-described CN aldehyde with pyruvate under conditions to convert the compound into a 2-ketocarboxylic acid (i.e., a compound of Formula I) or a salt thereof, or a solvate of the compound or salt thereof.

[0295] In another aspect, the present invention provides a method for preparing a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxyhexanoic acid, comprising, consisting essentially of, or consisting of: a) converting 3-hydroxypropanal and pyruvate to an intermediate 6-hydroxy-2-ketocarboxylic acid using a combination of a hydratase-aldolase having EC No. 4.1.2.45, EC No. 4.1.2.34, or EC No. 4.1.1.4, and an oxidoreductase having EC No. 1.6.5 (e.g., EC No. 1.6.5.5); and b) converting the intermediate 6-hydroxy-2-ketocarboxylic acid to the compound via an enzymatic process.

[0296] In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase, which has the EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-2'-carboxybenzalpyruvate hydratase-aldolase, which has the EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is an acetoacetate decarboxylase, which has the EC number 4.1.1.4.

[0297] In some embodiments, a microorganism is used as a host for preparing 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 its salt. As used herein, "host" refers to a cell or microorganism that is capable of producing one or more enzymes that can catalyze a reaction, either within the cell or microorganism (e.g., by taking up starting material(s) and optionally secreting the product(s)), or outside the cell or microorganism (e.g., by secreting the enzymes).

[0298] In some embodiments, the method further comprises, consists essentially of, or even 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 thereof from the solution, culture medium, and / or host cell.

[0299] In some embodiments, the conditions of the methods disclosed herein involve heating the components to a temperature of about 10 to about 200°C, or at least (all temperatures are given in degrees Celsius) 10°C, at least 15°C, at least 20°C, at least 25°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 37°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 45°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C, at least 100°C, at least 110°C, at least 120°C, at least 130°C, at least or at least 140°C, at least 150°C, at least 160°C, at least 170°C, at least 180°C, or at least 190°C, or at a temperature of 190°C or less, 180°C or less, 170°C or less, 160°C or less, 150°C or less, 140°C or less, 130°C or less, 120°C or less, 110°C or less, 100°C or less, 90°C or less, 80°C or less, 70°C or less, 60°C or less, 50°C or less, 40°C or less, 39°C or less, 38°C or less, 37°C or less, 36°C or less, 35°C or less, 34°C or less, 33°C or less, 32°C or less, 31°C or less, 30°C or less, 29°C or less, 28°C or less, or 25°C or less (the lower temperature limit being 10°C). In some embodiments, the conditions comprise, consist essentially of, or even consist of an incubation solution having 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, and up to about 12. In some embodiments, the pH is 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7.5 or less, 7 or less, 6.5 or less, 6 or less, 5.5 or less, or 4 or less, with the lower pH limit not being below 2.

[0300] In some embodiments, the conditions include pyruvate and C NThe method may comprise, consist essentially of, or even consist of a molar concentration of the aldehyde of about 0.1 μM to about 5 M. In some embodiments, the concentration is at least about 0.1 μM, 0.5 μM, 1 μM, 10 μM, 100 μM, 500 μM, or 1 M. In some embodiments, the concentration is about 4 M or less, 3 M or less, 2 M or less, 1 M or less, 500 μM or less, 200 μM or less, 100 μM or less, or 10 μM or less. N The concentrations of can independently be the same or different and may vary depending on other incubation conditions.

[0301] In some embodiments, the conditions include the presence of a non-naturally occurring microbial organism 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 carboxylic acid reductases, each of which is a reaction-specific enzyme.

[0302] In some embodiments, the microorganism, i.e., host, is genetically engineered to overexpress the enzyme, or to express the enzyme in greater amounts than its wild-type counterpart. Methods for determining the expression level of an enzyme or expression product are known in the art, such as by PCR.

[0303] In some embodiments, C N The aldehyde is 3-hydroxypropanal.

[0304] In some embodiments, the method further comprises, consists essentially of, or consists of preparing 3-hydroxypropanal and pyruvate from glycerol, C5 sugars, C6 sugars, phosphoglycerate, other carbon sources, intermediates of the glycolytic pathway, intermediates of propanoate metabolism, or combinations thereof.

[0305] In some embodiments, 3-hydroxypropanal is obtained by dehydration of glycerol.

[0306] In some embodiments, the C5 sugar comprises, consists essentially of, or even consists of one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose.

[0307] In some embodiments, the C6 sugar comprises, consists essentially of, or even consists of one or more of allose, altrose, glucose, mannose, gulose, idose, talose, galactose, fructose, psicose, sorbose, and tagatose.

[0308] In some embodiments, the other carbon source is a feedstock suitable as a carbon source for the microorganism, which feedstock comprises, consists essentially of, or even consists of amino acids, lipids, corn stover, miscanthus, municipal solid waste, energy grade sugarcane, sugar grade sugarcane, bagasse, starch stream, dextrose stream, methanol, formate, or combinations thereof.

[0309] In some embodiments, the microorganism is used as a host in preparing 1,5-pentanediol, adipic acid, 1,6-hexanediol, or 6-hydroxyhexanoic acid.

[0310] In some embodiments, the microorganism is capable of converting C5 sugars, C6 sugars, glycerol, other carbon sources, or combinations thereof, to pyruvate.

[0311] In some embodiments, the microorganism is engineered to enhance sugar uptake, for example, C5 sugar uptake, C6 / C5 sugar co-uptake, C6 sugar / glycerol co-uptake, C5 sugar / glycerol co-uptake, or a combination thereof.

[0312] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] 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 microorganisms.

[0313] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] 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.

[0314] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] with a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid, wherein the trans-o-hydroxybenzylidenepyruvate 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.

[0315] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] 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 microorganisms, and the pyruvate and [ka] However, an aldol condensation reaction catalyzed only by the hydratase-aldolase occurs to produce 2-oxo-3-enoic acid, which is then reduced, catalyzed only by the quinone oxidoreductase, to produce a 2-ketocarboxylic acid.

[0316] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] 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 microorganisms, and the method is performed in the presence of the one or more non-naturally occurring microorganisms, and [ka] However, an aldol condensation reaction catalyzed only by the hydratase-aldolase occurs to produce 2-oxo-3-enoic acid, which is then reduced, catalyzed only by the quinone oxidoreductase, to produce a 2-ketocarboxylic acid.

[0317] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms; [ka] 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 two or more non-naturally occurring microorganisms.

[0318] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms; [ka] 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 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.

[0319] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms; [ka] with a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid, wherein the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and the quinone oxidoreductase are expressed by the 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.

[0320] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms; [ka] 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 two or more non-naturally occurring microorganisms, and the pyruvate and [ka] However, an aldol condensation reaction catalyzed only by the hydratase-aldolase occurs to produce 2-oxo-3-enoic acid, which is then reduced, catalyzed only by the quinone oxidoreductase, to produce a 2-ketocarboxylic acid.

[0321] In another aspect, the present invention provides a method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: detecting pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms; [ka] 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 two or more non-naturally occurring microorganisms, and the method is performed in the presence of the two or more non-naturally occurring microorganisms, and [ka] However, this method involves an aldol condensation reaction catalyzed exclusively by hydratase-aldolase to produce 2-oxo-3-enoic acid, which is then reduced exclusively by quinone oxidoreductase to produce a 2-ketocarboxylic acid.

[0322] In some embodiments, [ka] is 3-hydroxypropanal. In some embodiments, the 3-hydroxypropanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by one or more non-naturally occurring microorganisms.

[0323] In some embodiments, the method for producing a 2-keto carboxylic acid further includes separating the 2-keto carboxylic acid from the one or more non-naturally occurring microorganisms or from a culture medium comprising the one or more non-naturally occurring microorganisms.

[0324] In another aspect, the present invention provides a method for producing 1,5-pentanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxypentanal; contacting the 5-hydroxypentanal with a primary alcohol dehydrogenase to produce 1,5-pentanediol; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0325] In another aspect, the present invention provides a method for producing 1,5-pentanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 2-keto acid decarboxylase to produce 5-hydroxypentanal; contacting the 5-hydroxypentanal with a primary alcohol dehydrogenase to produce 1,5-pentanediol; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0326] In another aspect, the present invention provides a method for producing 1,6-hexanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxyhexanal; and contacting the 6-hydroxyhexanal with 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0327] In another aspect, the present invention provides a method for producing 1,6-hexanediol, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with a 6-hydroxyhexanoate 1-reductase to produce 6-hydroxyhexanal; and contacting the 6-hydroxyhexanal with 6-hydroxyhexanal 1-reductase to produce 1,6-hexanediol; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0328] In another aspect, the present invention provides a method for producing 6-hydroxyhexanoate, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0329] In another aspect, the present invention provides a method for producing 6-hydroxyhexanoate, comprising the steps of: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0330] In another aspect, the present invention provides a method for producing adipic acid (AA), comprising: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with 6-hydroxyhexanoate dehydrogenase to produce 6-oxohexanoate; contacting the 6-oxohexanoate with 6-oxohexanoate oxidase to produce adipic acid; Including, The method is carried out in a culture medium containing one or more microorganisms that do not occur in nature.

[0331] In another aspect, the present invention provides a method for producing adipic acid (AA), comprising: contacting pyruvate and 3-hydroxypropanal with a hydratase-aldolase and a quinone oxidoreductase to produce a 2-ketocarboxylic acid of the formula: [ka] wherein R is CHOH. contacting the 2-ketocarboxylic acid with a 6-hydroxy-2-oxohexanoate-2-reductase to produce 2,6-dihydroxyhexanoate; contacting the 2,6-dihydroxyhexanoate with 2,6-dihydroxyhexanoate CoA transferase to produce 2,6-dihydroxyhexanoyl CoA; contacting the 2,6-dihydroxyhexanoyl-CoA with 2,6-dihydroxyhexanoyl-CoA 2-dehydratase to produce 6-hydroxy-2,3-dehydrohexanoyl-CoA; contacting the 6-hydroxy-2,3-dehydrohexanoyl-CoA with 2,3-dehydrohexanoyl-CoA 2,3-reductase to produce 6-hydroxyhexanoyl-CoA; contacting the 6-hydroxyhexanoyl-CoA with 6-hydroxyhexanoyl-CoA transferase to produce 6-hydroxyhexanoate; contacting the 6-hydroxyhexanoate with 6-hydroxyhexanoate dehydrogenase to produce 6-oxohexanoate; contacting the 6-oxohexanoate with 6-oxohexanoate oxidase to produce adipic acid; Including, This method is carried out in a culture medium containing two or more microorganisms that do not occur in nature.

[0332] In some embodiments, the hydratase-aldolase is an enzyme that 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 hydratase-aldolase is an enzyme that has EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase that has EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is an enzyme that has EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is an enzyme that has EC number 4.1.1.4.

[0333] In some embodiments, the hydratase-aldolase is selected from the group consisting of GenBank or RefSeq or Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_1154780 33, 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 selected from the group consisting of GenBank, RefSeq, or Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A 0A370X7D8, 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.

[0334] In some embodiments, the hydratase-aldolase is selected from the group consisting of GenBank or RefSeq or Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A and having 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 0A1G9YWG7, 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 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.

[0335] In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme selected from Tables 1, 5, 6, 7, and 8.

[0336] In some embodiments, the hydratase-aldolase further comprises one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0337] In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme that is in the EC group 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified by GenBank, RefSeq, or Uniprot ID numbers 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 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.

[0338] In some embodiments, the quinone oxidoreductase is selected from the group consisting of GenBank, RefSeq, and Uniprot ID numbers P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, and Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A21 2, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1 or WP_136898000.1, or 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 in 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 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.

[0339] In some embodiments, the quinone oxidoreductase further comprises one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0340] In some embodiments, at least one of the hydratase-aldolase and quinone oxidoreductase is exogenously expressed by one or more non-naturally occurring microbial organisms, hi some embodiments, at least one of the hydratase-aldolase and quinone oxidoreductase is exogenously expressed by two or more non-naturally occurring microbial organisms.

[0341] In some embodiments, at least one of the hydratase-aldolase and quinone oxidoreductase enzymes 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 quinone oxidoreductase enzymes 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 quinone oxidoreductase enzymes 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 quinone oxidoreductase enzymes is expressed by two or more exogenous genes expressed by two or more non-naturally occurring microorganisms. One or more exogenous genes can include 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. Two or more exogenous genes can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more exogenous genes.

[0342] In some embodiments, the hydratase-aldolase is exogenously expressed by one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by two or more non-naturally occurring microorganisms.

[0343] In some embodiments, the quinone oxidoreductase is exogenously expressed by one or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by two or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microorganisms.

[0344] In some embodiments, the hydratase-aldolase is exogenously expressed by one or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by two or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by two or more non-naturally occurring microorganisms.

[0345] In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are expressed by one or more non-naturally occurring microorganisms. In some embodiments, the 2-keto acid decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by one or more non-naturally occurring microorganisms.

[0346] In some embodiments, the 2-keto acid decarboxylase is selected from the group consisting of EC No. 4.1.1.1, EC No. 4.1.1.2, EC No. 4.1.1.3, EC No. 4.1.1.4, EC No. 4.1.1.5, EC No. 4.1.1.6, EC No. 4.1.1.7, EC No. 4.1.1.11, EC No. 4.1.1.12, EC No. 4.1.1.15, EC No. 4.1.1.16, EC No. 4.1.1.17, EC No. 4.1.1.18, E In some embodiments, the 2-keto acid decarboxylase is an enzyme selected from the group of enzymes identified by Uniprot ID Nos. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto acid decarboxylase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more identical to an enzyme selected from the group of enzymes identified by Uniprot ID numbers Q6QBS4, A7M7D6 or P20906.

[0347] In some embodiments, the 2-keto acid decarboxylase further comprises one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0348] In some embodiments, the primary alcohol dehydrogenase is an enzyme having the EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers 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 selected from the group consisting of those identified by Uniprot or GenBank ID numbers 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 ... 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 as BX39192.1, 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.

[0349] In some embodiments, the primary alcohol dehydrogenase further comprises one or more protein tags, hi some embodiments, the protein tags are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0350] In some embodiments, the hydratase-aldolase is an enzyme identified by Uniprot ID number A0A286PH18, the quinone oxidoreductase is an enzyme identified by Uniprot ID number P28304, and the 2-keto acid decarboxylase is an enzyme identified by Uniprot ID number P28304. The enzyme is identified by ID number Q6QBS4, and primary alcohol dehydrogenases are identified by Uniprot or GenBank ID numbers 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, and YP_001703694.In some embodiments, the hydratase-aldolase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more identical to the enzyme identified in Uniprot ID number A0A286PH18; the quinone oxidoreductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more identical to the enzyme identified in Uniprot ID number P28304; and the 2-keto acid decarboxylase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more identical to the enzyme identified in Uniprot ID number P28304. Primary alcohol dehydrogenases with at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the enzyme identified by ID number Q6QBS4, and primary alcohol dehydrogenases with Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, and ANO and have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzymes identified by 04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

[0351] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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.

[0352] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-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.

[0353] 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, and the 2,6-dihydroxyhexanoate CoA transferase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12, and ...yl CoA 2-dehydratase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12. 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44; 6-hydroxyhexanoyl-CoA transferase is an enzyme having the 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 having the EC number 1.2.99.6; and 6-hydroxyhexanal 1-reductase is an enzyme having the EC number 1.1.1.

[0354] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID number T4VW93; the 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme identified by Uniprot ID numbers Q5U924, Q5U925, and Q5U923; and the 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme identified by Uniprot ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409. The enzyme identified by ID number Q73Q47, 6-hydroxyhexanoyl CoA transferase, is a member of the Uniprot The enzyme identified by ID number T4VW93, 6-hydroxyhexanoate 1-reductase, is listed in Uniprot or GenBank ID numbers 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 6-hydroxyhexanal 1-reductase is an enzyme identified by Uniprot or GenBank ID numbers 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.

[0355] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93, A0A0C7GD16, A0A175L1W4, or 0A2X3BTQ9; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme selected from the group of enzymes identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409. The enzymes are identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9. 2,3-Dehydrohexanoyl CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47. 6-Hydroxyhexanoyl CoA transferase is an enzyme identified by Uniprot ID number Q73Q47. The enzymes identified by ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or A0A2X3BTQ9, and 6-hydroxyhexanoate 1-reductase are listed in Uniprot or GenBank ID numbers 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, AN The enzymes identified by A98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7 are 6-hydroxyhexanal 1-reductases with Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, and ANA99315.The enzymes identified are GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, and YP_001703694.

[0356] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is selected from the group consisting of Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.2,6-dihydroxyhexanoate CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified in 1, Uniprot 2,6-dihydroxyhexanoyl CoA2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified by ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or 0A2X3BTQ9. 2,3-dehydrohexanoyl-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% identity to the enzymes identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9, and The 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q73Q47, and the 6-hydroxyhexanoate 1-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID numbers T4VW93, A0A0C7GD16, A0A175L1W4 or A0A2X3BTQ9. Ze has Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.6-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% identity to the enzymes identified in Uniprot or GenBank ID numbers D6Z860, YP_001705436.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7. 407.1, 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, or YP_001703694, and have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the enzymes identified in

[0357] 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-dihydroxyhexanoate 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; and the 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme comprising the sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63. Or it is an enzyme having the sequence of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, the 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the sequence of SEQ ID NO: 65, the 6-hydroxyhexanoyl-CoA transferase is an enzyme having 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 having 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 having the sequence of SEQ ID NO: 70.

[0358] 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, and the 2,6-dihydroxyhexanoate 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 identity is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, and the 2,6-dihydroxyhexanoyl-CoA2-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, and the 2,3-dehydrohexanoyl-CoA2,The 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; 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 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 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.

[0359] In some embodiments, one or more of 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate 1-reductase, and 6-hydroxyhexanal 1-reductase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0360] 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, and the 2,6-dihydroxyhexanoate CoA transferase is selected from the group of enzymes identified by EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme having the EC number 4.2.1.167; 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44; and 6-hydroxyhexanoyl-CoA transferase is an enzyme having the EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

[0361] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; and the 2,6-dihydroxyhexanoyl CoA 2-dehydratase is an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1. The 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47, and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified by Uniprot ID number T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

[0362] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to an enzyme identified by Uniprot or GenBank ID numbers WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1, and the 2,6-dihydroxyhexanoate CoA transfer ... 2,6-dihydroxyhexanoyl CoA2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the enzyme identified by ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16 or A0A175L1W4. 2,3-dehydrohexanoyl-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% identity to the enzymes identified by ID numbers Q5U924, Q5U925 and Q5U923, or A0A2X3BK09, A0A2X3BU19 and A0A1V9IXA9, and The enzyme is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q73Q47, and the 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID numbers T4VW93, A0A2X3BTQ9, A0A0C7GD16 or A0A175L1W4.

[0363] 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-dihydroxyhexanoate 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-dihydroxyhexanoyl-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-dehydrohexanoyl-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.

[0364] 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. and the 2,6-dihydroxyhexanoate CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% 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, and the 2,6-dihydroxyhexanoyl 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% identity to an enzyme comprising the sequence of SEQ ID NO:59, SEQ ID NO:61 and SEQ ID NO:62. The 2,3-dehydrohexanoyl-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% identity to an enzyme comprising the sequence of SEQ ID NO: 63, or SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64, and the 2,3-dehydrohexanoyl-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% identity to an enzyme comprising the sequence of SEQ ID NO: 65. 0%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, 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.

[0365] 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, and the 2,6-dihydroxyhexanoate CoA transferase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12, and ...yl CoA 2-dehydratase is an enzyme having EC Number 2.8.3, EC Number 2.8.3.1, or EC Number 2.8.3.12. 2,3-Dehydrohexanoyl-CoA 2,3-reductase is an enzyme having the EC number 1.3.1.44; 6-hydroxyhexanoyl-CoA transferase is an enzyme having the EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; 6-hydroxyhexanoate dehydrogenase is an enzyme having the EC number 1.1.1.258; and 6-oxohexanoate oxidase is an enzyme having the EC number 1.2.1.63.

[0366] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified by Uniprot ID number Q5FTU6; the 2,6-dihydroxyhexanoate CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxyhexanoyl-CoA 2-dehydratase is an enzyme identified by Uniprot ID numbers Q5U924, Q5U925, and Q5U923, or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydrohexanoyl-CoA 2,3-reductase is an enzyme identified by Uniprot ID number Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified by Uniprot ID numbers T4VW93 or A0A2X3BTQ9. 6-hydroxyhexanoate dehydrogenase is an enzyme identified by Uniprot ID number Q7WVD0 or Q84H78, and 6-oxohexanoate oxidase is an enzyme identified by Uniprot ID number Q9R2F4.

[0367] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to the enzyme identified by Uniprot ID number Q5FTU6, and the 2,6-dihydroxyhexanoate CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more identical to the enzyme identified by Uniprot ID number Q5FTU6. 2,6-dihydroxyhexanoyl CoA2-dehydratase that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID numbers T4VW93 or A0A2X3BTQ9. 2,3-dehydrohexanoyl CoA2, ...The 3-reductase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number T4VW93 or A0A2X3BTQ9; and the 6-hydroxyhexanoate dehydrogenase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number T4VW93 or A0A2X3BTQ9. The 6-oxohexanoate oxidase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by ID number Q7WVD0 or Q84H78, and the 6-oxohexanoate oxidase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the enzyme identified by Uniprot ID number Q9R2F4.

[0368] In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising the sequence of SEQ ID NO:53, the 2,6-dihydroxyhexanoate CoA transferase is an enzyme comprising the sequence of SEQ ID NO:55 or SEQ ID NO:58, the 2,6-dihydroxyhexanoyl-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-dehydrohexanoyl-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 comprising the sequence of SEQ ID NO:71 or SEQ ID NO:72, and the 6-oxohexanoate oxidase is an enzyme comprising the sequence of SEQ ID NO:75.

[0369] 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, and the 2,6-dihydroxyhexanoate 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. 2,6-dihydroxyhexanoyl-CoA2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% 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, and 2,3-dehydrohexanoyl-CoA2,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% 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% identity to an enzyme comprising the sequence of SEQ ID NO: 55 or SEQ ID NO: 58 ... The 6-oxohexanoate 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, and the 6-oxohexanoate 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.

[0370] In some embodiments, one or more of 6-hydroxy-2-oxohexanoate-2-reductase, 2,6-dihydroxyhexanoate CoA transferase, 2,6-dihydroxyhexanoyl-CoA 2-dehydratase, 2,3-dehydrohexanoyl-CoA 2,3-reductase, 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and 6-oxohexanoate oxidase further comprise one or more protein tags, which in some embodiments are selected from a polyhistidine tag, a GST tag (glutathione-S-transferase tag), an HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose-binding protein tag, a chitin-binding protein tag, and a fluorescent tag.

[0371] In some embodiments, pyruvate is produced from a carbon source selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or combinations thereof.

[0372] In some embodiments, 3-hydroxypropanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by one or more non-naturally occurring microorganisms.

[0373] The one or more non-naturally occurring microorganisms include 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 microorganisms. The two or more non-naturally occurring microorganisms include 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 microorganisms. In some embodiments, the methods disclosed herein are performed in the presence of one non-naturally occurring microorganism. In some embodiments, the methods disclosed herein are performed in the presence of two non-naturally occurring microorganisms. In some embodiments, the methods disclosed herein are performed in the presence of three non-naturally occurring microorganisms. In some embodiments, the methods disclosed herein are carried out in the presence of four non-naturally occurring microorganisms. In some embodiments, the methods disclosed herein are carried out in the presence of five non-naturally occurring microorganisms.

[0374] Throughout this application, various publications are referenced, the disclosures of which in their entireties, including the GenBank accession number(s), Uniprot ID number(s) or RefSeq ID number(s) for these publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this disclosure pertains.

[0375] In some embodiments, the present disclosure provides the following embodiments as examples.

[0376] 1. A method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises: culturing pyruvate and ATP in a culture medium comprising one or more naturally occurring microorganisms; [ka] 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.

[0377] 2. The method of embodiment 1, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microorganisms.

[0378] 3. The method of embodiment 1, wherein the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microorganisms.

[0379] 4. The method of embodiment 1, wherein said quinone oxidoreductase is exogenously expressed by said one or more non-naturally occurring microorganisms.

[0380] 5. The method of embodiment 1, wherein the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microorganisms.

[0381] 6. The method of embodiment 1, wherein the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microorganisms.

[0382] 7. [ka] 7. The method of any one of embodiments 1 to 6, wherein is 3-hydroxypropanal.

[0383] 8. The method of embodiment 7, wherein said 3-hydroxypropanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by said one or more non-naturally occurring microorganisms.

[0384] 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 microorganisms or a culture medium comprising the one or more non-naturally occurring microorganisms.

[0385] 10. A method for producing a 2-ketocarboxylic acid of the formula: [ka] wherein R is H, CH or CHOH; The method comprises culturing pyruvate and ATP in a culture medium comprising two or more naturally occurring microorganisms. [ka] 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 two or more non-naturally occurring microorganisms.

[0386] 11. The method of embodiment 10, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microorganisms.

[0387] 12. The method of embodiment 10, wherein the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microorganisms.

[0388] 13. The method of embodiment 10, wherein said quinone oxidoreductase is exogenously expressed by said two or more non-naturally occurring microorganisms.

[0389] 14. The method of embodiment 10, wherein the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microorganisms.

[0390] 15. The method of embodiment 10, wherein the hydratase-aldolase is exogenously expressed by said two or more non-naturally occurring microorganisms and the quinone oxidoreductase is overexpressed by said two or more non-naturally occurring microorganisms.

[0391] 16. [ka] The method of any one of embodiments 10 to 15, wherein is 3-hydroxypropanal.

[0392] 17. The method of embodiment 16, wherein said 3-hydroxypropanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by said two or more non-naturally occurring microorganisms.

[0393] 18. The method of any one of embodiments 10-17, further comprising isolating the 2-ketocarboxylic acid from the two or more non-naturally occurring microorganisms or a culture medium comprising the two or more non-naturally occurring microorganisms.

[0394] 19. The method of any one of embodiments 1 to 18, wherein the hydratase-aldolase is an enzyme having EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4.

[0395] 20. The hydratase-aldolase is selected from the group consisting of GenBank, RefSeq, and Uniprot ID numbers D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, and WP_028222253. 19. The method of any one of embodiments 1 to 18, wherein the enzyme is selected from the group of enzymes identified by 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.

[0396] 21. The method of any one of embodiments 1 to 18, wherein 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.

[0397] 22. The method of any one of embodiments 1 to 18, wherein the hydratase-aldolase has at least 50% identity to 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.

[0398] 23. The method of any one of embodiments 1 to 18, wherein the hydratase-aldolase has at least 70% identity to 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, SE...

Claims

1. Pyruvate and 3-hydroxypropanal (HO-CH ２ -CH ２ —CHO) is contacted with the aldol dehydration product biosynthetic polypeptide to produce 6-hydroxy-2-oxo-3-hexenoic acid (HO—CH ２ -CH ２ 1. A method of producing a compound of formula (I) comprising: The method, wherein the aldol dehydration product biosynthetic polypeptide comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:

27.

2. 2. The method of claim 1, wherein the aldol dehydration product biosynthetic polypeptide comprises the amino acid sequence of SEQ ID NO:

27.

3. 2. The method of claim 1, wherein the aldol dehydration product biosynthetic polypeptide has the amino acid sequence of SEQ ID NO:

27.

4. The method of any one of claims 1 to 3, wherein the aldol dehydration product biosynthetic polypeptide is in a microorganism.

5. 5. The method of claim 4, wherein the microorganism is an engineered microorganism that expresses the aldol dehydration product biosynthetic polypeptide.

6. 6. The method of claim 5, wherein the engineered microorganism comprises improved expression or activity of the aldol dehydration product biosynthetic polypeptide.

7. 6. The method of claim 4 or 5, wherein the engineered microorganism comprises an exogenous nucleic acid encoding the aldol dehydration product biosynthetic polypeptide.

8. 5. The method of claim 4, wherein the microorganism is E. coli.

9. 6. The method of claim 5, wherein the microorganism is E. coli.

10. 7. The method of claim 6, wherein the microorganism is E. coli.

11. 8. The method of claim 7, wherein the microorganism is E. coli.

12. The method according to any one of claims 1 to 11, wherein the contacting is carried out in a culture medium.

13. The method of claim 4 , wherein the contacting is carried out in a culture medium.

14. The method of claim 5 , wherein the contacting is carried out in a culture medium.

15. The method of claim 6 , wherein the contacting is carried out in a culture medium.

16. The method of claim 7 , wherein the contacting is carried out in a culture medium.

17. The method of claim 8 , wherein the contacting is carried out in a culture medium.

18. The method of claim 9 , wherein the contacting is carried out in a culture medium.

19. The method of claim 10 , wherein the contacting is carried out in a culture medium.

20. The method of claim 11 , wherein the contacting is carried out in a culture medium.

21. The method of claim 12, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

22. The method of claim 13, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

23. The method of claim 14, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

24. 16. The method of claim 15, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

25. 17. The method of claim 16, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

26. 18. The method of claim 17, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

27. 19. The method of claim 18, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

28. 20. The method of claim 19, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

29. 21. The method of claim 20, wherein the culture medium comprises the microorganism, pyruvate, and 3-hydroxypropanal.

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