MICROORGANISMS AND METHODS FOR PRODUCING BUTADIENE AND RELATED COMPOUNDS BY FORMATE ASSIMILATION

Abstract

Provided herein are non-naturally occurring microbial organisms having a FaldFP, a FAP and/or metabolic modifications which can further include a MMP, a MOP, a hydrogenase and/or a CODH. These microbial organisms can further include a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway. Additionally provided are methods of using such microbial organisms to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

Description

BACKGROUND OF THE INVENTION

The present invention relates generally to metabolic and biosynthetic processes and microbial organisms capable of producing organic compounds, and more specifically to non-naturally occurring microbial organisms having a formate assimilation pathway and an organic compound pathway, such as butadiene, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol or 3-buten-1-ol.

Over 25 billion pounds of butadiene (1,3-butadiene, “BD”) are produced annually and is applied in the manufacture of polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1,4-butanediol. For example, butadiene can be reacted with numerous other chemicals, such as other alkenes, e.g. styrene, to manufacture numerous copolymers, e.g. acrylonitrile 1,3-butadiene styrene (ABS), styrene-1,3-butadiene (SBR) rubber, styrene-1,3-butadiene latex. These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing. Butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. The ability to manufacture butadiene from alternative and/or renewable feedstocks would represent a major advance in the quest for more sustainable chemical production processes.

One possible way to produce butadiene renewably involves fermentation of sugars or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to butadiene in a second step involving metal-based catalysis. Direct fermentative production of butadiene from renewable feedstocks would obviate the need for dehydration steps and butadiene gas (bp −4.4° C.) would be continuously emitted from the fermenter and readily condensed and collected. Developing a fermentative production process would eliminate the need for fossil-based butadiene and would allow substantial savings in cost, energy, and harmful waste and emissions relative to petrochemically-derived butadiene.

1,3-butanediol (1,3-BDO or 13BDO) is a four carbon diol traditionally produced from acetylene via its hydration. The resulting acetaldehyde is then converted to 3-hydroxybutyraldehdye which is subsequently reduced to form 1,3-BDO. In more recent years, acetylene has been replaced by the less expensive ethylene as a source of acetaldehyde. 1,3-BDO is commonly used as an organic solvent for food flavoring agents. It is also used as a co-monomer for polyurethane and polyester resins and is widely employed as a hypoglycaemic agent. Optically active 1,3-BDO is a useful starting material for the synthesis of biologically active compounds and liquid crystals. A commercial use of 13BDO is subsequent dehydration to afford 1,3-butadiene (Ichikawa et al., J. of Molecular Catalysis A-Chemical, 256:106-112 (2006); Ichikawa et al., J. of Molecular Catalysis A-Chemical, 231:181-189 (2005)), a 25 billion lb/yr petrochemical used to manufacture synthetic rubbers (e.g., tires), latex, and resins. The reliance on petroleum based feedstocks for either acetylene or ethylene warrants the development of a renewable feedstock based route to 13BDO and to butadiene.

Crotyl alcohol (“CrotOH”), also referred to as 2-buten-1-ol, is a valuable chemical intermediate. It serves as a precursor to crotyl halides, esters, and ethers, which in turn are chemical intermediates in the production of monomers, fine chemicals, agricultural chemicals, and pharmaceuticals. Exemplary fine chemical products include sorbic acid, trimethylhydroquinone, crotonic acid and 3-methoxybutanol. CrotOH is also a precursor to 1,3-butadiene. CrotOH is currently produced exclusively from petroleum feedstocks. For example Japanese Patent 47-013009 and U.S. Pat. Nos. 3,090,815, 3,090,816, and 3,542,883 describe a method of producing CrotOH by isomerization of 1,2-epoxybutane. The ability to manufacture CrotOH from alternative and/or renewable feedstocks would represent a major advance in the quest for more sustainable chemical production processes.

3-Buten-2-ol (also referenced to as methyl vinyl carbinol (“MVC”)) is an intermediate that can be used to produce butadiene. There are significant advantages to use of MVC over 1,3-BDO because there are fewer separation steps and only one dehydration step. MVC can also be used as a solvent, a monomer for polymer production, or a precursor to fine chemicals Accordingly, the ability to manufacture MVC from alternative and/or renewable feedstock would again present a significant advantage for sustainable chemical production processes.

3-Buten-1-ol is a raw material used in pharmaceuticals, agrochemicals, perfumes and resins. The palladium-catalyzed coupling of 3-buten-1-ol with aryl halides is a valuable process for the preparation of aryl-substituted aldehydes such as, for example, the antifolate compound Pemetrexed disodium (R. C. Larock et al., Tetrahedron Letters, 30, 6629 (1989) and U.S. Pat. No. 6,262,262). 3-Buten-1-ol is commonly prepared from propylene and formaldehyde in the presence of a catalyst at high temperature and pressure. Alternately, it is prepared from 3,4-epoxy-1-butene. Preparation of 3-buten-1-ol from renewable feedstocks would provide a valuable alternative to existing production techniques.

Thus, there exists a need for alternative methods for effectively producing commercial quantities of compounds such as butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

In one embodiment, provided herein is a non-naturally occurring microbial organism having a formaldehyde fixation pathway (“FaldFP”) and a formate assimilation pathway (“FAP”), wherein the organism includes at least one exogenous nucleic acid encoding a FaldFP enzyme disclosed herein that is expressed in a sufficient amount to produce pyruvate, and wherein the organism includes at least one exogenous nucleic acid encoding a FAP enzyme disclosed herein that is expressed in a sufficient amount to produce formaldehyde, pyruvate or acetyl-CoA. In one aspect, the microbial organism can further include a methanol metabolic pathway (“MMP”), a methanol oxidation pathway (“MOP”), a hydrogenase and/or a carbon monoxide dehydrogenase (“CODH”), wherein the organism includes at least one exogenous nucleic acid encoding a MMP enzyme, a MOP enzyme, the hydrogenase and/or the CODH that is expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents. Such organisms of the invention advantageously enhance the production of substrates and/or pathway intermediates for the production of butadiene (“BD”), 13BDO, CrotOH, MVC or 3-buten-1-ol.

In one embodiment, the organism further includes a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway. In certain embodiments, the organism includes at least one exogenous nucleic acid encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. The invention additionally provides methods of using such microbial organisms to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol by culturing a non-naturally occurring microbial organism containing a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway as described herein under conditions and for a sufficient period of time to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

In one embodiment, provided herein is a non-naturally occurring microbial organism having a butadiene, MVC or 3-buten-1-ol pathway. In certain embodiments, the organism includes at least one exogenous nucleic acid encoding a butadiene, MVC or 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce butadiene, MVC or 3-buten-1-ol. In certain embodiments, the organism can further include a FaldFP, a MMP, a MOP, a hydrogenase and/or a CODH. The invention additionally provides methods of using such microbial organisms to produce butadiene, MVC or 3-buten-1-ol by culturing a non-naturally occurring microbial organism containing a butadiene, MVC or 3-buten-1-ol pathway as described herein under conditions and for a sufficient period of time to produce butadiene, MVC or 3-buten-1-ol.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, wherein the microbial organism further includes attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA, or a gene disruption of one or more endogenous nucleic acids encoding such enzymes. For example, in some aspects, the endogenous enzyme can be selected from DHA kinase, methanol oxidase, PQQ-dependent methanol dehydrogenase, DHA synthase or any combination thereof.

The invention further provides non-naturally occurring microbial organisms that have elevated or enhanced synthesis or yields of acetyl-CoA (e.g. intracellular) or biosynthetic products such as butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway and methods of using those non-naturally occurring organisms to produce such biosynthetic products. The enhanced synthesis of intracellular acetyl-CoA enables enhanced production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol from which acetyl-CoA is an intermediate and further, may have been rate limiting.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a fatty butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, wherein the microbial organism further includes attenuation of one or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway or a gene disruption of one or more endogenous nucleic acids encoding enzymes of a competing formaldehyde assimilation or dissimilation pathway. Examples of these endogenous enzymes are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary metabolic pathways enabling the conversion of CO2, formate, formaldehyde, MeOH, glycerol, and glucose to acetyl-CoA (ACCOA), 13BDO and crotyl-alcohol, and exemplary endogenous enzyme targets for optional attenuation or disruption. The enzymatic transformations shown are carried out by the following enzymes: A) methanol dehydrogenase (“MeDH”), B) 3-hexulose-6-phosphate synthase, C) 6-phospho-3-hexuloisomerase (“6P3HI”), D) dihydroxyacetone synthase (“DHAS”), E) formate reductase, F) formate ligase, formate transferase, or formate synthetase, G) formyl-CoA reductase, H) formyltetrahydrofolate synthetase (“FTHFS”), I) methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase (“MTHFDH”), K) spontaneous or formaldehyde-forming enzyme, L) glycine cleavage system, M) serine hydroxymethyltransferase, N) serine deaminase, O) methylenetetrahydrofolate reductase, P) acetyl-CoA synthase, Q) pyruvate formate lyase, R) pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, or pyruvate:NADP+ oxidoreductase, S) formate dehydrogenase, T) acetyl-CoA carboxylase, U) acetoacetyl-CoA synthase, V) acetyl-CoA:acetyl-CoA acyltransferase, W) acetoacetyl-CoA reductase (“AcAcCoAR”) (ketone reducing), X) 3-hydroxybutyryl-CoA reductase (aldehyde forming), Y) 3-hydroxybutyraldehyde reductase, Z) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, AA) 3-hydroxybutyrate reductase, AB) 3-hydroxybutyryl-CoA dehydratase (or crotonase), AC) crotonyl-CoA reductase (aldehyde forming), AD) crotonaldehyde reductase, AE) crotonyl-CoA transferase, hydrolase, or synthetase, AF) crotonate reductase, AG) CrotOH dehydratase or chemical dehydration. The enzyme targets are indicated by arrows having “X” markings. The endogenous enzyme targets include DHA kinase, methanol oxidase (AOX), PQQ-dependent MeDH(PQQ) and/or DHA synthase. See abbreviation list below for compound names.

FIG. 2 shows exemplary metabolic pathways enabling the conversion of CO2, formate, formaldehyde, MeOH, glycerol, and glucose to acetyl-CoA (ACCOA) and butadiene, and exemplary endogenous enzyme targets for optional attenuation or disruption. The enzymatic transformations shown are carried out by the following enzymes: A) MeDH, B) 3-hexulose-6-phosphate synthase, C) 6P3HI, D) DHAS, E) formate reductase, F) formate ligase, formate transferase, or formate synthetase, G) formyl-CoA reductase, H) FTHFS, I) methenyltetrahydrofolate cyclohydrolase, J) MTHFDH, K) spontaneous or formaldehyde forming enzyme, L) glycine cleavage system, M) serine hydroxymethyltransferase, N) serine deaminase, O) methylenetetrahydrofolate reductase, P) acetyl-CoA synthase, Q) pyruvate formate lyase, R) pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, or pyruvate:NADP+ oxidoreductase, S) formate dehydrogenase (“FDH”), T) acetyl-CoA carboxylase, U) acetoacetyl-CoA synthase, V) acetyl-CoA:acetyl-CoA acyltransferase, W) acetoacetyl-CoA reductase (ketone reducing), X) 3-hydroxybutyryl-CoA dehydratase (or crotonase), Y) crotonyl-CoA transferase, hydrolase, or synthetase, AF) crotonate reductase, Z) crotonate reductase, AA) crotonyl-CoA reductase (aldehyde reductase), AB) crotonaldehyde reductase, AC) Crotyl alcohol kinase (“CrotOH kinase”), AD) crotyl-phosphate kinase, AE) butadiene synthase (“BDS”). The enzyme targets are indicated by arrows having “X” markings. The endogenous enzyme targets include DHA kinase, methanol oxidase (AOX), PQQ-dependent MeDH(PQQ) and/or DHA synthase. See abbreviation list below for compound names.

FIG. 3 shows metabolic pathways enabling the extraction of reducing equivalents from methanol, hydrogen, or carbon monoxide. The enzymatic transformations shown are carried out by the following enzymes: A) methanol methyltransferase, B) methylenetetrahydrofolate reductase, C) MTHFDH, D) methenyltetrahydrofolate cyclohydrolase, E) formyltetrahydrofolate deformylase, F) FTHFS, G) formate hydrogen lyase, H) hydrogenase, I) FDH, J) MeDH, K) spontaneous or formaldehyde activating enzyme, L) formaldehyde dehydrogenase, M) spontaneous or S-(hydroxymethyl)glutathione synthase, N) Glutathione-Dependent Formaldehyde Dehydrogenase, 0) S-formylglutathione hydrolase, P) CODH. See abbreviation list below for compound names.

FIG. 4 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 13BDO from methanol can be increased from 0.167 mol 13BDO/mol methanol (1:6 ratio) to 0.250 mol 13BDO/mol methanol (1:4 ratio) by enabling fixation of formaldehyde with formate reutilization. The upper value of each flux value pair indicates flux distribution for 6.00 mole methanol, and the lower value indicates that for 4 mole methanol when formaldehyde is assimilated with formate reutilization. See abbreviation list below for compound names.

FIG. 5 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 13BDO from glucose can be increased from 1.00 mol 13BDO/mol glucose (upper value of each flux value pair) to 1.09 mol 13BDO/mol glucose (lower value of each flux value pair) by enabling fixation of formaldehyde with formate reutilization. See abbreviation list below for compound names.

FIG. 6 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 13BDO from glycerol can be increased from 0.50 mol 13BDO/mol glycerol (upper value of each flux value pair) to 0.64 mol 13BDO/mol glycerol (lower value of each flux value pair) by enabling fixation of formaldehyde with formate reutilization. See abbreviation list below for compound names.

FIG. 7 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 13BDO from glucose can be increased from 1.00 mol 13BDO/mol glucose (upper value of each flux value pair) to 1.50 mol 13BDO/mol glucose (lower value of each flux value pair) by enabling fixation of formaldehyde with formate reutilization and extraction of reducing equivalents from an external source such as hydrogen. See abbreviation list below for compound names.

FIG. 8 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 13BDO from glycerol can be increased from 0.50 mol 13BDO/mol glycerol (upper value of each flux value pair) to 0.75 mol 13BDO/mol glycerol (lower value of each flux value pair) by enabling fixation of formaldehyde with formate reutilization and extraction of reducing equivalents from an external source such as hydrogen. See abbreviation list below for compound names.

FIG. 9 shows an exemplary flux distribution that demonstrates how CO2 can be converted to 13BDO using the FaldFPs and an external source of redox such as hydrogen. See abbreviation list below for compound names.

FIG. 10 shows exemplary pathways for formation of 13BDO and CrotOH from acetyl-CoA. Enzymes are: A. 3-ketoacyl-ACP synthase, B. Acetoacetyl-ACP reductase, C. 3-hydroxybutyryl-ACP dehydratase, D. acetoacetyl-CoA:ACP transferase, E. acetoacetyl-CoA hydrolase, transferase or synthetase, F. acetoacetate reductase (acid reducing), G. 3-oxobutyraldehyde reductase (aldehyde reducing), H. acetoacetyl-ACP thioesterase, I. AcAcCoAR(CoA-dependent, aldehyde forming), J. acetoacetyl-ACP reductase (aldehyde forming), K. AcAcCoAR(alcohol forming), L. 3-hydroxybutyryl-ACP thioesterase, M. 3-hydroxybutyryl-ACP reductase (aldehyde forming), N. 3-hydroxybutyryl-CoA reductase (aldehyde forming), O. 3-hydroxybutyryl-CoA reductase (alcohol forming), P. AcAcCoAR(ketone reducing), Q. acetoacetate reductase (ketone reducing), R. 3-oxobutyraldehyde reductase (ketone reducing), S. 4-hydroxy-2-butanone reductase, T. crotonyl-ACP thioesterase, U. crotonyl-ACP reductase (aldehyde forming), V. crotonyl-CoA reductase (aldehyde forming), W. crotonyl-CoA (alcohol forming), X. 3-hydroxybutyryl-CoA:ACP transferase, Y. 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, Z. 3-hydroxybutyrate reductase, AA. 3-hydroxybutyraldehyde reductase, AB. 3-hydroxybutyryl-CoA dehydratase, AC. 3-hydroxybutyrate dehydratase, AD. 3-hydroxybutyraldehyde dehydratase, AE. crotonyl-CoA:ACP transferase, AF. crotonyl-CoA hydrolase, transferase or synthetase, AG. crotonate reductase, AH. crotonaldehyde reductase, AS. acetoacetyl-CoA synthase, AT. acetyl-CoA:acetyl-CoA acyltransferase, AU. 4-hydroxybutyryl-CoA dehydratase. ACP is acyl carrier protein.

FIG. 11 shows pathways for conversion of CrotOH to butadiene. Enzymes are: A. CrotOH kinase, B. 2-butenyl-4-phosphate kinase, C. BDS, D. CrotOH diphosphokinase, E. CrotOH dehydratase or chemical dehydration, F. BDS (monophosphate).

FIG. 12 shows an exemplary pathway for production of butadiene from malonyl-CoA plus acetyl-CoA. Enzymes for transformation of the identified substrates to products include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoglutaryl-CoA reductase (ketone-reducing), C. 3-hydroxyglutaryl-CoA reductase (aldehyde forming), D. 3-hydroxy-5-oxopentanoate reductase, E. 3,5-dihydroxypentanoate kinase, F. 3H5PP kinase, G. 3H5PDP decarboxylase, H. butenyl 4-diphosphate isomerase, I. BDS, J. 3-hydroxyglutaryl-CoA reductase (alcohol forming), K. 3-oxoglutaryl-CoA reductase (aldehyde forming), L. 3,5-dioxopentanoate reductase (ketone reducing), M. 3,5-dioxopentanoate reductase (aldehyde reducing), N. 5-hydroxy-3-oxopentanoate reductase, O. 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming). Compound abbreviations include: 3H5PP=3-Hydroxy-5-phosphonatooxypentanoate and 3H5PDP=3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate.

FIG. 13. Pathway for converting 2-butanol to MVC. Step A is catalyzed by 2-butanol desaturase. Step B is catalyzed by MVC dehydratase or chemical dehydration.

FIG. 14. Pathway for converting pyruvate to 2-butanol. Enzymes are A. acetolactate synthase, B. acetolactate decarboxylase, C. butanediol dehydrogenase, D. butanediol dehydratase, E. butanol dehydrogenase.

FIG. 15. Pathway for converting 13BDO to MVC and/or butadiene. Enzymes are A. 13BDO kinase, B. 3-hydroxybutyrylphosphate kinase, C. 3-hydroxybutyryldiphosphate lyase, D. 13BDO diphosphokinase, E. 13BDO dehydratase, F. 3-hydroxybutyrylphosphate lyase, G. MVC dehydratase or chemical reaction.

FIG. 16. Pathway for converting acrylyl-CoA to MVC or butadiene. Enzymes are A. 3-oxopent-4-enoyl-CoA thiolase, B. 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, C. 3-oxopent-4-enoate decarboxylase or spontaneous, D. 3-buten-2-one reductase and E. MVC dehydratase or chemical dehydration.

FIG. 17. Pathways for converting lactoyl-CoA to MVC and/or butadiene. Enzymes are A. 3-Oxo-4-hydroxypentanoyl-CoA thiolase, B. 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, C. 3-oxo-4-hydroxypentanoate reductase, D. 3,4-dihydroxypentanoate decarboxylase, E. 3-oxo-4-hydroxypentanoyl-CoA reductase, F. 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, G. MVC dehydratase or chemical dehydration, H. 3,4-dihydroxypentanoate dehydratase, I. 4-oxopentanoate reductase, J. 4-hyd4-oxoperoxypentanoate decarboxylase.

FIG. 18. Pathways for converting succinyl-CoA to MVC and/or butadiene. Enzymes are A. 3-oxoadipyl-CoA thiolase, B. 3-oxoadipyl-CoA transferase, synthetase or hydrolase, C. 3-oxoadipate decarboxylase or spontaneous reaction (non-enzymatic), D. 4-oxopentanoate reductase, E. 4-hydroxypentanoate decarboxylase, F. MVC dehydratase or chemical dehydration.

FIG. 19 shows exemplary metabolic pathways enabling the conversion of crotonyl-CoA to 3-buten-1-ol and butadiene. The enzymatic transformations shown are carried out by the following enzymes: A) crotonyl-CoA delta-isomerase, B) vinylacetyl-CoA reductase, C) 3-buten-1-al reductase, D) 3-buten-1-ol dehydratase or chemical dehydration.

FIG. 20 shows improved use of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 by E. coli strain MG1655 having a xR mutation (squares) compared to wild-type MG1655 (diamonds).

FIG. 21 shows immediate and complete use of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 by E. coli strain that is a variant of MG1655 modified to express 1,4-butanediol pathway genes and having a xR mutation (Xs) compared to that variant without xR (triangles).

FIG. 22 shows the growth of 11 different xR mutants on Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 compared to wild-type xR.

FIG. 23 shows the utilization rate of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 for 15 different xR mutants compared to wild-type xR.

FIG. 24 shows the amount of residual Sugar 2 at a single time point following 40 minutes of fermentation of 15 different xR mutants compared to wild-type xR in the presence of catabolite-repressing concentrations of Sugar 1.

FIG. 25 shows improved use of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 3 by E. coli strain MG1655 having a xylR mutation (squares) compared to wild-type MG1655 (diamonds).

FIG. 26 shows pathways from 3-hydroxypropanoyl-CoA and/or acrylyl-CoA to butadiene via 2,4-pentadienoate, 3-butene-1-ol or 3-hydroxypent-4-eoate. Enzymes are A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, K. 3-hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Q. 5-hydroxypent-2-enoate dehydratase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, S. 3-hydroxypent-4-enoate dehydratase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase, V. 5-hydroxypent-2-enoate decarboxylase, W. 3-butene-1-ol dehydratase (or chemical conversion), X. 2,4-pentadiene decarboxylase, Y. 3-hydroxypent-4-enoate decarboxylase. 3-HP-CoA is 3-hydroxypropanoyl-CoA.

FIG. 27 shows exemplary pathways for conversion of propionyl-CoA to butadiene via 2,4-pentadienoate. Enzymes are: A. 3-oxopentanoyl-CoA thiolase or synthase, B. 3-oxopentanoyl-CoA reductase, C. 3-hydroxypentanoyl-CoA dehydratase, D. pent-2-enoyl-CoA isomerase, E. pent-3-enoyl-CoA dehydrogenase, F. 2,4-pentadienoyl-CoA hydrolase, transferase or synthetase, G. pent-2-enoyl-CoA dehydrogenase, X. 2,4-pentadienoate decarboxylase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to metabolic and biosynthetic processes and microbial organisms capable of producing butadiene, 13BDO, CrotOH, MVC, or 3-buten-1-ol. Specifically, the non-naturally occurring microbial organisms include a FaldFP and a FAP, which can further include a MMP, a MOP, a hydrogenase and/or a CODH. These microbial organisms can further include a butadiene, 13BDO, CrotOH, MVC, or 3-buten-1-ol pathway.

The following is a list of abbreviations and their corresponding compound or composition names. These abbreviations, which are used throughout the disclosure and the figures. It is understood that one of ordinary skill in the art can readily identify these compounds/compositions by such nomenclature. MeOH or MEOH=methanol; Fald=formaldehyde; GLC=glucose=Sugar 1; G6P=glucose-6-phosphate; H6P=hexulose-6-phosphate; F6P=fructose-6-phosphate; FDP=fructose diphosphate or fructose-1,6-diphosphate; DHA=dihydroxyacetone; DHAP=dihydroxyacetone phosphate; G3P=and glyceraldehyde-3-phosphate; PYR=pyruvate; Sugar 3=arabinose; ACCOA=acetyl-CoA; AACOA=acetoacetyl-CoA; MALCOA=malonyl-CoA; FTHF=formyltetrahydrofolate; THF=tetrahydrofolate; E4P=erythrose-4-phosphate: Xu5P=xyulose-5-phosphate; Sugar 2=xylose; Ru5P=ribulose-5-phosphate; S7P=sedoheptulose-7-phosphate: R5P=ribose-5-phosphate; 3HBCOA=3-hydroxybutryl-CoA; 3HB=3-hydroxybutyrate; 3HBALD=3-hydroxyburylaldehyde-CoA; xylR=xR; Xy1R=XR; 13BDO=13BDO; CROTCOA=crotonyl-CoA or crotyl-CoA; CROT=crotonate; CROTALD=crotonaldehyde; CROTALC=crotyl alcohol or crotonyl alcohol; BD=butadiene; CROT-Pi=crotyl phosphate or 2-butenyl-4-diphosphate; CROT-PPi=crotyl diphosphate or 2-butenyl-4-diphosphate; TCA=tricarboxylic acid

It is also understood that association of multiple steps in a pathway can be indicated by linking their step identifiers with or without spaces or punctuation; for example, the following are equivalent to describe the 4-step pathway comprising Step W, Step X, Step Y and Step Z: steps WXYZ or W,X,Y,Z or W;X;Y;Z or W-X-Y-Z. One of ordinary skill can readily distinguish a single step designator of “AA” or “AB” or “AD” from a multiple step pathway description based on context and use in the description and figures herein.

Methanol is a relatively inexpensive organic feedstock that can be used as a redox, energy, and carbon source for the production of chemicals such as butadiene, 13BDO, CrotOH, MVC and 3-buten-1-ol, and their intermediates, by employing one or more methanol metabolic enzymes as described herein, for example as shown in FIGS. 1, 2, and 3. Methanol can enter central metabolism in most production hosts by employing MeDH(FIG. 1, step A) along with a pathway for formaldehyde assimilation. One exemplary formaldehyde assimilation pathway that can utilize formaldehyde produced from the oxidation of methanol is shown in FIG. 1, which involves condensation of formaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate (H6P) by hexulose-6-phosphate synthase (FIG. 1, step B). The enzyme can use Mg²⁺ or Mn²⁺ for maximal activity, although other metal ions are useful, and even non-metal-ion-dependent mechanisms are contemplated. H6P is converted into fructose-6-phosphate by 6P3HI (FIG. 1, step C). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol proceeds through dihydroxyacetone. DHAS (FIG. 1, step D) is a transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis. The DHA obtained from DHA synthase can be then further phosphorylated to form DHA phosphate by a DHA kinase. DHAP can be assimilated into glycolysis, e.g. via isomerization to G3P, and several other pathways. Alternatively, DHA and G3P can be converted by fructose-6-phosphate aldolase to form fructose-6-phosphate (F6P). The above also applies to FIG. 2.

By combining the pathways for methanol oxidation (FIG. 1, step A) and formaldehyde fixation (FIG. 1, Steps B and C or Step D), molar yields of 0.167 mol product/mol methanol can be achieved for 1,3-BDO, CrotOH, and butadiene, and their intermediates. The same applies to FIG. 2 and when methanol oxidation and FaldFPs are combined with other product synthesis pathways for 13BDO, CrotOH and butadiene such as those described herein. For example, FIG. 4 shows an exemplary flux distribution that will lead to a 0.167 mol 1,3-BDO/mol MeOH yield (see the upper flux value of each flux value pair; 1:6 mole ratio 13BDO:MeOH). The following maximum theoretical yield stoichiometries for 1,3-BDO, CrotOH, and butadiene are thus made possible by combining the steps for methanol oxidation, formaldehyde fixation, and product synthesis.

6CH₄O+3.5O₂→C₄H₁₀O₂+7H₂O+2CO₂ (1,3-BDO on MeOH)

6CH₄O+3.5O₂→C₄H₈O+8H₂O+2CO₂ (CrotOH on MeOH)

6CH₄O+3.5O₂→C₄H₆+9H₂O+2CO₂ (Butadiene on MeOH)

The yield on several substrates, including methanol, can be further increased by capturing some of the carbon lost from the conversion of pathway intermediates, e.g. pyruvate to acetyl-CoA, using one of the formate reutilization pathways shown in FIG. 1. For example, the CO₂ generated by conversion of pyruvate to acetyl-CoA (FIG. 1, step R) can be converted to formate via FDH (FIG. 1, step S). Alternatively, pyruvate formate lyase, which forms formate directly instead of CO₂, can be used to convert pyruvate to acetyl-CoA (FIG. 1, step Q). Formate can be converted to formaldehyde by using: 1) formate reductase (FIG. 1, step E), 2) a formyl-CoA synthetase, transferase, or ligase along with formyl-CoA reductase (FIG. 1, steps F-G), or 3) FTHFS, methenyltetrahydrofolate cyclohydrolase, MTHFDH, and formaldehyde-forming enzyme (FIG. 1, steps H-I-J-K). Conversion of methylene-THF to formaldehyde alternatively will occur spontaneously. Alternatively, formate can be reutilized by converting it to pyruvate or acetyl-CoA using FIG. 1, steps H-I-J-L-M-N or FIG. 1, steps H-I-J-O-P, respectively. Formate reutilization is also useful when formate is an external carbon source. For example, formate can be obtained from organocatalytic, electrochemical, or photoelectrochemical conversion of CO2 to formate. An alternative source of methanol for use in the present methods is organocatalytic, electrochemical, or photoelectrochemical conversion of CO2 to methanol, The above applies to FIG. 2.

By combining the pathways for methanol oxidation (FIG. 1, step A), formaldehyde fixation (FIG. 1, Steps B and C or Step D), and formate reutilization, molar yields as high as 0.250 mol product/mol methanol can be achieved for 1,3-BDO, CrotOH, and butadiene. The same applies to FIG. 2 and when methanol oxidation, formaldehyde fixation and formate reutilization pathways are combined with other product synthesis pathways for 13BDO, CrotOH and butadiene such as those described herein. For example, FIG. 4 shows an exemplary flux distribution that will lead to a 0.250 mol 1,3-BDO/mol MeOH yield (see the lower flux value of each flux value pair; 1:4 mole ratio 13BDO:MeOH). The following maximum theoretical yield stoichiometries for 1,3-BDO, CrotOH, and butadiene are thus made possible by combining the steps for methanol oxidation, formaldehyde fixation, formate reutilization, and product synthesis.

4CH₄O+0.5O₂→C₄H₁₀O₂+3H₂O (1,3-BDO on MeOH)

4CH₄O+0.5O₂→C₄H₈O+4H₂O (CrotOH on MeOH)

4CH₄O+0.5O₂→C₄H₆+5H₂O (Butadiene on MeOH)

By combining pathways for formaldehyde fixation and formate reutilization, yield increases on additional substrates are also available including but not limited to glucose, glycerol, sucrose, fructose, xylose, arabinose and galactose. For example, FIG. 5 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 1,3-BDO from glucose can be increased from 1.00 mol 1,3-BDO/mol glucose to 1.09 mol 1,3-BDO/mol glucose (compare the upper and lower flux value of each flux value pair) by enabling fixation of formaldehyde from generation and utilization of formate. The following maximum theoretical yield stoichiometries for 1,3-BDO, CrotOH, and butadiene on glucose are thus made possible by combining the steps for formaldehyde fixation, formate reutilization, and product synthesis.

11C₆H₁₂O₆→12C₄H₁₀O₂+6H₂O+18CO₂ (1,3-BDO on glucose)

11C₆H₁₂O₆→12C₄H₈O+18H₂O+18CO₂ (CrotOH on glucose)

11C₆H₁₂O₆→12C₄H₆+30H₂O+18CO₂ (Butadiene on glucose)

Similarly, FIG. 6 shows exemplary flux distributions that demonstrate how the maximum theoretical yield of 1,3-BDO from glycerol can be increased from 0.50 mol 1,3-BDO/mol glycerol to 0.64 mol 1,3-BDO/mol glycerol (compare the upper and lower flux value of each flux value pair) by enabling fixation of formaldehyde from generation and utilization of formate. The following maximum theoretical yield stoichiometries for 1,3-BDO, CrotOH, and butadiene on glycerol are thus made possible by combining the steps for formaldehyde fixation, formate reutilization, and product synthesis.

11C₃H₈O₃→7C₄H₁₀O₂+9H₂O+5CO₂ (1,3-BDO on glycerol)

11C₃H₈O₃→7C₄H₈O+16H₂O+5CO₂ (CrotOH on glycerol)

11C₃H₈O₃→7C₄H₆+23H₂O+5CO₂ (Butadiene on glycerol)

In numerous engineered pathways, product yields based on carbohydrate feedstock are hampered by insufficient reducing equivalents or by loss of reducing equivalents to byproducts. Methanol is a relatively inexpensive organic feedstock that can be used to generate reducing equivalents by employing one or more methanol metabolic enzymes as shown in FIG. 3. Reducing equivalents can also be extracted from hydrogen and carbon monoxide by employing hydrogenase and CODH enzymes, respectively, as shown in FIG. 3. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H₂, or water, respectively. Reduced ferredoxin, reduced quinones and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway, reductive TCA cycle, or product pathway enzymes.

The reducing equivalents produced by the metabolism of methanol, hydrogen, and carbon monoxide can be used to power several 1,3-BDO, CrotOH, and butadiene production pathways. For example, FIG. 7 and FIG. 8 show exemplary flux distributions that demonstrate how the maximum theoretical yield of 1,3-BDO from glucose and glycerol, respectively, can be increased by enabling fixation of formaldehyde, formate reutilization, and extraction of reducing equivalents from an external source such as hydrogen. In fact, by combining pathways for formaldehyde fixation, formate reutilization, reducing equivalent extraction, and product synthesis, the following maximum theoretical yield stoichiometries for 1,3-BDO, CrotOH, and butadiene on glucose and glycerol are made possible.

C₆H₂O₆+4.5H₂→1.5C₄H₁₀O₂+3H₂O (1,3-BDO on glucose+external redox)

C₆H₁₂O₆+4.5H₂→1.5C₄H₈O+4.5H₂O (CrotOH on glucose+external redox)

C₆H₁₂O₆+4.5H₂→1.5C₄H₆+6H₂O (Butadiene on glucose+external redox)

C₃H₈O₃+1.25H₂→0.75C₄H₁₀O₂+1.5H₂O (1,3-BDO on glycerol+external redox)

C₃H₈O₃+1.25H₂→0.75C₄H₈O+2.25H₂O (CrotOH on glycerol+external redox)

C₃H₈O₃+1.25H₂→0.75C₄H₆+3H₂O (Butadiene on glycerol+external redox)

In most instances, achieving such maximum yield stoichiometries may require some oxidation of reducing equivalents (e.g., H₂+½O₂→H₂O, CO+½O₂→CO₂, CH₄O+1.5O₂→CO₂+2H₂O, C₆H₁₂O₆+6O₂→6CO₂+6H₂O) to provide sufficient energy for the substrate to product pathways to operate. Nevertheless, if sufficient reducing equivalents are available, enabling pathways for fixation of formaldehyde, formate reutilization, extraction of reducing equivalents, and product synthesis can even lead to production of 1,3-BDO, CrotOH, and butadiene, and their intermediates, directly from CO₂ as demonstrated in FIG. 9.

Pathways identified herein, and particularly pathways exemplified in specific combinations presented herein, are superior over other pathways based in part on the applicant's ranking of pathways based on attributes including maximum theoretical BDO yield, maximal carbon flux, maximal production of reducing equivalents, minimal production of CO2, pathway length, number of non-native steps, thermodynamic feasibility, number of enzymes active on pathway substrates or structurally similar substrates, and having steps with currently characterized enzymes, and furthermore, the latter pathways are even more favored by having in addition at least the fewest number of non-native steps required, the most enzymes known active on pathway substrates or structurally similar substrates, and the fewest total number of steps from central metabolism.

In one embodiment, the invention utilizes in silico stoichiometric models of Escherichia coli metabolism that identify metabolic designs for biosynthetic production of butadiene or 3-buten-1-ol. The results described herein indicate that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis of butadiene or 3-buten-1-ol in Escherichia coli and other cells or organisms. Biosynthetic production of butadiene or 3-buten-1-ol, for example, for the in silico designs can be confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment butadiene biosynthesis, including under conditions approaching theoretical maximum growth.

In certain embodiments, the butadiene or 3-buten-1-ol biosynthesis characteristics of the designed strains make them genetically stable and particularly useful in continuous bioprocesses. Separate strain design strategies were identified with incorporation of different non-native or heterologous reaction capabilities into E. coli or other host organisms leading to butadiene or 3-buten-1-ol producing metabolic pathways from crotonyl-CoA. In silico metabolic designs were identified that resulted in the biosynthesis of butadiene or 3-buten-1-ol in microorganisms from each of these substrates or metabolic intermediates.

Strains identified via the computational component of the platform can be put into actual production by genetically engineering any of the predicted metabolic alterations, which lead to the biosynthetic production of butadiene or 3-buten-1-ol or other intermediate and/or downstream products. In yet a further embodiment, strains exhibiting biosynthetic production of these compounds can be further subjected to adaptive evolution to further augment product biosynthesis. The levels of product biosynthesis yield following adaptive evolution also can be predicted by the computational component of the system.

The maximum theoretical butadiene yield from glucose is 1.09 mol/mol (0.32 g/g).

11C₆H₁₂O₆=12C₄H₆+18CO₂+30H₂O

The pathways presented in FIG. 19 achieve a yield of 1.09 moles butadiene per mole of glucose utilized.

The maximum theoretical 3-buten-1-ol yield from glucose is 1.09 mol/mol (0.437 g/g).

11C₆H₁₂O₆=12C₄H₈O+18CO₂+18H₂O

The pathways presented in FIG. 19 achieve a yield of 1.09 moles 3-buten-1-ol per mole of glucose utilized.

As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway.

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

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

The term “isolated” when used in reference to a nucleic acid molecule is intended to mean a nucleic acid molecule that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

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

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

As used herein, the term “ACP” or “acyl carrier protein” refers to any of the relatively small acidic proteins that are associated with the fatty acid synthase system of many organisms, from bacteria to plants. ACPs can contain one 4′-phosphopantetheine prosthetic group bound covalently by a phosphate ester bond to the hydroxyl group of a serine residue. The sulfhydryl group of the 4′-phosphopantetheine moiety serves as an anchor to which acyl intermediates are (thio)esterified during fatty-acid synthesis. An example of an ACP is Escherichia coli ACP, a separate single protein, containing 77 amino-acid residues (8.85 kDa), wherein the phosphopantetheine group is linked to serine 36.

As used herein, the term “butadiene,” having the molecular formula C₄H₆ and a molecular mass of 54.09 g/mol (see FIGS. 1, 5, 6, 12 and 19) (IUPAC name Buta-1,3-diene), is used interchangeably throughout with 1,3-butadiene, biethylene, erythrene, divinyl, vinylethylene. Butadiene is a colorless, non corrosive liquefied gas with a mild aromatic or gasoline-like odor. Butadiene is both explosive and flammable because of its low flash point.

As used herein, the term “3-buten-1-ol,” having the molecular formula C4H8O and a molecular mass of 72.11 g/mol (see FIG. 19) (IUPAC name But-3-en-1-ol), is used interchangeably throughout with allylcarbinol, 1-buten-4-ol, 3-butenyl alcohol, but-3-en-1-ol, vinylethyl alcohol. 3-buten-1-ol is a colorless and flammable liquid.

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

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

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

As used herein, the term “gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product, for example, replacement of a gene's promoter with a weaker promoter, replacement or insertion of one or more amino acid of the encoded protein to reduce its activity, stability or concentration, or inactivation of a gene's transactivating factor such as a regulatory protein. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring microorganisms of the invention. A gene disruption also includes a null mutation, which refers to a mutation within a gene or a region containing a gene that results in the gene not being transcribed into RNA and/or translated into a functional gene product. Such a null mutation can arise from many types of mutations including, for example, inactivating point mutations, deletion of a portion of a gene, entire gene deletions, or deletion of chromosomal segments.

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

As used herein, the term “attenuate,” or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein. Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given pathway to function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of acetyl-CoA or a bioderived compound of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host microbial organism to survive, reproduce or grow. Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of acetyl-CoA or a bioderived compound of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.

As used herein, the term “xylose” or “Sugar 2,” is intended to refer to a monosaccharide of the aldopentose type having an aldehyde functional group, the chemical formula HOCH₂(CH(OH))₃CHO and a molecular mass of 150.13 g/mol. The term is intended to include both D- and L-forms. Sugar 2 is a sugar component of hemicellulosic biomass.

As used herein, the term “glucose” or “Sugar 1” is intended to refer to a monosaccharide of the aldohexose type having the chemical formula C₆H₁₂O₆ and a molecular weight of mass of 180.16 g/mole. Sugar 1 D-form has the standard name (2R,3S,4R,5R)-2,3,4,5,6-Pentahydroxyhexanal. The term is intended to include both D- and L-forms.

As used herein, Sugar 2 is intended to include both D- and L-forms. Sugar 2 is a sugar component of hemicellulosic biomass.

As used herein, glucose includes both D- and L-forms.

As used herein, Sugar 3 includes both D- and L-forms. Sugar 3 is a sugar component of hemicellulosic biomass.

As used herein, xR(gene) or XR (gene product) refer to the encoding nucleic acid and the gene product, respectively, of a regulator of the Sugar 2 operons, designated herein as operon t2 and operon m2 (see below). Exemplary XR-encoding and XR sequence are E. coli xR and its gene product XR which are known in the art and can be found under NCBI Gene ID number 948086, GenBank number AAB18546.1 and GI number GI: 466707. The E. coli XR is a 392 amino acid protein. XR is a DNA-binding positive regulatory protein, which activates the transcription of operons involved in transport and catabolism of D-Sugar 2. Gene induction occurs when the physiological inducer, D-Sugar 2, binds to XR and when cellular cyclic AMP levels are high. Other exemplary wild-type XR proteins suitable for modification as described herein include any bacterial glucose- or arabinose-catabolite-repressed Sugar 2 operon positive regulatory protein having at least 95% amino acid sequence identity with XR of E. coli, including the following:

Organism	GenBank ID	GI number
Shigella boydii CDC 3083-94	YP_001882235.1	187731584
Escherichia sp. TW15838	WP_000494495.1	446416640
Escherichia albertii	WP_000494491.1	446416636
Salmonella enterica subsp.	YP_001572910.1	161505798
arizonae serovar 62:z4,
z23:- str. RSK2980
Citrobacter rodentium ICC168	YP_003367654.1	283787789
Enterobacter cloacae subsp.	YP_006576712.1	401761705
cloacae ENHKU01
Thauera selenatis	WP_020686535.1	522177986
Kosakonia radicincitans	WP_007369537.1	494611291
Cronobacter universalis	WP_007703139.1	494977115
Yokenella regensburgei	WP_006817716.1	493871203
Raoultella ornithinolytica B6	YP_007876166.1	481851726
Klebsiella pneumoniae	WP_004205023.1	490310307
Cedecea davisae	WP_016538783.1	514236134
Erwinia toletana	WP_017799764.1	516410366
Pantoea agglomerans	WP_010672192.1	498358036
Serratia odorifera	WP_004957135.1	491095531

As used herein, the terms “araE” (gene) or “AraE” (gene product) refer to the encoding nucleic acid and the gene product, respectively, of an Sugar 3 transporter, preferably one that is deregulated. AraE is a proton symporter that acts as a low-affinity high-capacity transporter for Sugar 3. By “deregulated” in this context is meant that the AraE is not regulated, i.e. inhibited, under conditions that regulate or inhibit the AraE of E. coli such as the condition of glucose catabolite repression. As discovered by the present inventors the use of an deregulated AraE in an engineered microorganism permits transport, and thus metabolism, of arabinose even under conditions that would otherwise inhibit arabinose transport and its metabolism, such as the repression of arabinose transport in the presence glucose or its metabolites (i.e. glucose catabolite repression). Exemplary sequences for E. coli araE and its gene product AraE are known in the art and can be found under NP_417318.1 and GI:16130745. While deregulation can be achieved by overexpression of an E. coli AraE or highly related AraE protein that is normally glucose catabolite repressed, or by use of a constitutive promoter or a promoter that is not glucose catabolite repressed may reduce glucose catabolite expression (which can be at the level of gene expression and/or protein modification), preferred is to use an AraE that is deregulated at the protein level. A preferred deregulated AraE is from Corynebacterium glutamicum is a 479 amino acid protein of sequence of GenBank ID: BAH60837.1 and its encoding gene sequence is identified as GI:238231325. Other exemplary deregulated AraE proteins suitable for use as described herein include any bacterial Sugar 3 transporter having at least 95% amino acid sequence identity with the AraE of E. coli, and is deregulated under conditions that regulate, i.e. inhibit, the AraE of E. coli, such as condition of glucose catabolite repression, including the following:

	GenBank ID	GI number
Shigella sonnei Ss046	YP_311830.1	74313411
Escherichia coli	WP_000456407.1	446378552
Shigella boydii CDC 3083-94	YP_001881418.1	187730497
Salmonella enterica	WP_000253646.1	446175791
Citrobacter rodentium ICC168	YP_003366412.1	283786547
Shigella dysenteriae	WP_000256434.1	446178579
Shigella boydii	WP_004993719.1	491135300
Shigella flexneri	WP_005070724.1	491212393

Deregulated AraE include those arabinose transporters that have less than 85% amino acid sequence identity with the Ara E of E. coli. One such transporter is the AraE of Corynebacterium glutamicum, which is a 479 amino acid protein of sequence of GenBank ID: BAH60837.1 and its encoding gene sequence is identified as GI:238231325. This arabinose-transporter protein sequence GI:238231325 has about 31% amino acid sequence identity with the AraE of E. coli MG1655 GI: 16130745, yet in E. coli it is successfully expressed, transports arabinose, and is deregulated allowing arabinose transport in the presence of glucose. Other exemplary deregulated AraE proteins suitable for use as described herein include any bacterial Sugar 3 transporter having less than 85% amino acid sequence identity with the AraE of E. coli, and is deregulated under conditions that regulate, i.e. inhibit, the AraE of E. coli, such as condition of glucose catabolite repression, including the following:

	GenBank ID	GI number
Rahnella sp. Y9602	YP_004213957.1	322833930
Pantoea ananatis LMG 20103	YP_003519386.1	291616644
Pantoea ananatis AJ13355	YP_005933284.1	386015007
Salmonella enterica subsp.	YP_006887493.1	409246789
enterica serovar Weltevreden str.
2007-60-3289-1
Paenibacillus polymyxa M1	YP_005958620.1	386039666
Paenibacillus polymyxa E681	YP_003869380.1	308067775
Bacillus amyloliquefaciens FZB42	YP_003519386.1	291616644

In addition to the AraE of Corynebacterium glutamicum, which is used in the Examples, other exemplary deregulated AraE proteins suitable for use as described herein include any bacterial Sugar 3 transporter having at least 70% amino acid sequence identity with the AraE of Corynebacterium glutamicum and is also deregulated under conditions that regulate, i.e. inhibit, the AraE of E. coli such as the condition of glucose catabolite repression, including the following:

	GenBank ID	GI number
Lactobacillus versmoldensis	WP_010624011.1	498309855
Bacillus sonorensis	WP_006637052.1	493686963
Bacillus licheniformis DSM	YP_079231.2	163119467
Lactobacillus sakei subsp. sakei 2	YP_396471.1	81429470
Bacillus licheniformis 9945A	YP_008078227.1	GI:511062909

As used herein, the terms “operon t2” (gene) or “Operon T2” (gene product) refer to the encoding nucleic acids and the gene products, respectively, of a Sugar 2 transporter. The exemplary E. coli operon t2 encodes three essential components of the binding-protein-mediated transport system that act as a high-affinity ATP-binding cassette (“ABC”)-type transporter for Sugar 2. In particular, the operon t2 F gene product is a Sugar 2-binding protein (GI: number 16131437; NCBI Gene ID: 948090), the G gene product is an ATP-binding protein (GI: number 16131438; NCBI Gene ID: 948127), and the H gene product is a membrane transporter (GI: number 16131439; NCBI Gene ID: 948083). As used herein, the terms “operon m2” (gene) or “Operon M2” (gene product) refer to the encoding nucleic acids and the two gene products, respectively, of Sugar 2 metabolizing enzymes, the A and B gene products. Exemplary sequences for E. coli operon m2 and its gene products Operon M2 and functions are as follows. Sugar 2 is first isomerized by the isomerase, the operon m2 A gene product (440 amino acids; NCBI Reference Sequence: NP_418022.1 and Gene ID: 948141, and then phosphorylated by the kinase, the operon m2 B gene product (484 amino acids; NCBI Reference Sequence: NP_418021.1 and Gene ID: 948133).

Feedstock refers to a substance used as a raw material for the growth of an organism, including an industrial growth process. When used in reference to a culture of microbial organisms such as a fermentation process with cells, the term refers to the raw material used to supply a carbon or other energy source for the cells. A “renewable” feedstock refers to a renewable energy source such as material derived from living organisms or their metabolic byproducts including material derived from biomass, often consisting of underutilized components like chaff Agricultural products specifically grown for use as renewable feedstocks include, for example, corn, soybeans and cotton, primarily in the United States; flaxseed and rapeseed, primarily in Europe; sugar cane in Brazil and palm oil in South-East Asia. Therefore, the term includes the array of carbohydrates, fats and proteins derived from agricultural or animal products across the planet.

Biomass refers to any plant-derived organic matter. In the context of post-fermentation processing, biomass can be used to refer to the microbial cell mass produced during fermentation. Biomass available for energy on a sustainable basis includes herbaceous and woody energy crops, agricultural food and feed crops, agricultural crop wastes and residues, wood wastes and residues, aquatic plants, and other waste materials including some municipal wastes. Biomass feedstock compositions, uses, analytical procedures and theoretical yields are readily available from the U.S. Department of Energy and can be found described, for example, at the URL 1.eere.energy.gov/biomass/information_resources.html, which includes a database describing more than 150 exemplary kinds of biomass sources. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as Sugar 1, Sugar 2, Sugar 3, galactose, mannose, fructose and starch.

The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.

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

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

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

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

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

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly for a gene disruption, evolutionally related genes can also be disrupted or deleted in a host microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.

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

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

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP and a FAP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises 1B, 1C, or 1D or any combination thereof, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1O, or 1P or any combination thereof, wherein 1E is a formate reductase, 1F is a formate ligase, a formate transferase, or a formate synthetase, wherein 1G is a formyl-CoA reductase, wherein 1H is a FTHFS, wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a MTHFDH, wherein 1K is a formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine cleavage system, wherein 1M is a serine hydroxymethyltransferase, wherein 1N is a serine deaminase, wherein 10 is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase.

In one embodiment, the FaldFP comprises 1B. In one embodiment, the FaldFP comprises 1C. In one embodiment, the FaldFP comprises 1D. In one embodiment, the FAPs comprises 1E. In one embodiment, the FAPs comprises 1F, 1G. In one embodiment, the FAPs comprises 1H. In one embodiment, the FAPs comprises 1I. In one embodiment, the FAPs comprises 1J. In one embodiment, the FAPs comprises 1K. In one embodiment, the FAPs comprises 1L. In one embodiment, the FAPs comprises 1M. In one embodiment, the FAPs comprises 1N. In one embodiment, the FAPs comprises 10. In one embodiment, the FAPs comprises 1P. Any combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen pathway enzymes of 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1O, or 1P is also contemplated.

In one aspect, provided herein is a non-naturally occurring microbial organism having a FaldFP and a FAP, wherein said organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, wherein said organism comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P.

In certain embodiments, the FaldFP comprises 1B and 1C. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1E. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1F, and 1G. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1H, 1I, 1J, and 1K. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1E, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1K, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1H, 1I, 1J, 1O, and 1P.

In certain embodiments, the FaldFP comprises 1D. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1E. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1F, and 1G. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1H, 1I, 1J, and 1K. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1E, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1K, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1H, 1I, 1J, 1O, and 1P.

In certain embodiments, the FAP further comprises 1Q, 1R, or 1S or any combination thereof, wherein 1Q is a pyruvate formate lyase, wherein 1R is a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, or a pyruvate:NADP+ oxidoreductase, wherein 1S is a FDH. Thus, in certain embodiments the FAP comprises 1Q. Thus, in certain embodiments the FAP comprises 1R Thus, in certain embodiments the FAP comprises 1S.

In certain embodiments, FAP comprises 1Q, or 1R and 1S, and the FaldFP comprises 1B and 1C. In certain embodiments, FAP comprises 1Q, or 1R and 1S, and the FaldFP comprises 1D. In certain embodiments the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, and 1E. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1F, and 1G. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1H, 1I, 1J, and 1K. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1E, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1K, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1B and 1C, and the FAP comprises 1Q, 1H, 1I, 1J, 1O, and 1P. In certain embodiments the FaldFP comprises 1D, and the FAP comprises 1Q, and 1E. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1F, and 1G. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1H, 1I, 1J, and 1K. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1E, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1K, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, the FaldFP comprises 1D, and the FAP comprises 1Q, 1H, 1I, 1J, 1O, and 1P.

In certain embodiments, the FaldFP or the FAP is a pathway depicted in FIG. 1 or 2.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP and a MMP. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P5, and comprises at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises a pathway selected from: (1) 3J; (2) 3A and 3B; (3) 3A, 3B and 3C; (4) 3J, 3K and 3C; (5) 3J, 3M, and 3N; (6) 3J and 3L; (7) 3A, 3B, 3C, 3D, and 3E; (8) 3A, 3B, 3C, 3D, and 3F; (9) 3J, 3K, 3C, 3D, and 3E; (10) 3J, 3K, 3C, 3D, and 3F; (11) 3J, 3M, 3N, and 30; (12) 3A, 3B, 3C, 3D, 3E, and 3G; (13) 3A, 3B, 3C, 3D, 3F, and 3G; (14) 3J, 3K, 3C, 3D, 3E, and 3G; (15) 3J, 3K, 3C, 3D, 3F, and 3G; (16) 3J, 3M, 3N, 3O, and 3G; (17) 3A, 3B, 3C, 3D, 3E, and 3I; (18) 3A, 3B, 3C, 3D, 3F, and 3I; (19) 3J, 3K, 3C, 3D, 3E, and 3I; (20) 3J, 3K, 3C, 3D, 3F, and 3I; and (21) 3J, 3M, 3N, 3O, and 3I, wherein 3A is a methanol methyltransferase, wherein 3B is a methylenetetrahydrofolate reductase, wherein 3C is a MTHFDH, wherein 3D is a methenyltetrahydrofolate cyclohydrolase, wherein 3E is a formyltetrahydrofolate deformylase, wherein 3F is a FTHFS, wherein 3G is a formate hydrogen lyase, wherein 3H is a hydrogenase, wherein 3I is a FDH, wherein 3J is a MeDH, wherein 3K is a formaldehyde activating enzyme or spontaneous, wherein 3L is a formaldehyde dehydrogenase, wherein 3M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 3N is a glutathione-dependent formaldehyde dehydrogenase, wherein 3O is a S-formylglutathione hydrolase.

In certain embodiments, the MMP comprises 3A. In certain embodiments, the MMP comprises 3B. In certain embodiments, the MMP comprises 3C. In certain embodiments, the MMP comprises 3D. In certain embodiments, the MMP comprises 3E. In certain embodiments, the MMP comprises 3F. In certain embodiments, the MMP comprises 3G. In certain embodiments, the MMP comprises 3H. In certain embodiments, the MMP comprises 31. In certain embodiments, the MMP comprises 3J. In certain embodiments, the MMP comprises 3K. In certain embodiments, the MMP comprises 3L. In certain embodiments, the MMP comprises 3M. In certain embodiments, the MMP comprises 3N. In certain embodiments, the MMP comprises 3O.

In certain embodiments, the MMP comprises 3J. In certain embodiments, the MMP comprises 3A and 3B. In certain embodiments, the MMP comprises 3A, 3B and 3C. In certain embodiments, the MMP comprises 3J, 3K and 3C. In certain embodiments, the MMP comprises 3J, 3M, and 3N. In certain embodiments, the MMP comprises 3J and 3L. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, and 3E. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, and 3F. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, and 3E. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, and 3F. In certain embodiments, the MMP comprises 3J, 3M, 3N, and 3O. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, 3E, and 3G. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, 3F, and 3G. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, 3E, and 3G. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, 3F, and 3G. In certain embodiments, the MMP comprises 3J, 3M, 3N, 3O, and 3G. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, 3E, and 3I. In certain embodiments, the MMP comprises 3A, 3B, 3C, 3D, 3F, and 3I. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, 3E, and 3I. In certain embodiments, the MMP comprises 3J, 3K, 3C, 3D, 3F, and 3I. In certain embodiments, the MMP comprises 3J, 3M, 3N, 3O, and 3I.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP and a MOP. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P5, and comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein 1A a MeDH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP and a MOP. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises: (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, and comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein 1A a MeDH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, and comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises: (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P5, and comprises at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises a pathway selected from: (1) 3J; (2) 3A and 3B; (3) 3A, 3B and 3C; (4) 3J, 3K and 3C; (5) 3J, 3M, and 3N; (6) 3J and 3L; (7) 3A, 3B, 3C, 3D, and 3E; (8) 3A, 3B, 3C, 3D, and 3F; (9) 3J, 3K, 3C, 3D, and 3E; (10) 3J, 3K, 3C, 3D, and 3F; (11) 3J, 3M, 3N, and 3O; (12) 3A, 3B, 3C, 3D, 3E, and 3G; (13) 3A, 3B, 3C, 3D, 3F, and 3G; (14) 3J, 3K, 3C, 3D, 3E, and 3G; (15) 3J, 3K, 3C, 3D, 3F, and 3G; (16) 3J, 3M, 3N, 30, and 3G; (17) 3A, 3B, 3C, 3D, 3E, and 3I; (18) 3A, 3B, 3C, 3D, 3F, and 3I; (19) 3J, 3K, 3C, 3D, 3E, and 3I; (20) 3J, 3K, 3C, 3D, 3F, and 3I; and (21) 3J, 3M, 3N, 3O, and 3I, wherein 3A is a methanol methyltransferase, wherein 3B is a methylenetetrahydrofolate reductase, wherein 3C is a MTHFDH, wherein 3D is a methenyltetrahydrofolate cyclohydrolase, wherein 3E is a formyltetrahydrofolate deformylase, wherein 3F is a FTHFS, wherein 3G is a formate hydrogen lyase, wherein 3H is a hydrogenase, wherein 31 is a FDH, wherein 3J is a MeDH, wherein 3K is a formaldehyde activating enzyme or spontaneous, wherein 3L is a formaldehyde dehydrogenase, wherein 3M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 3N is a glutathione-dependent formaldehyde dehydrogenase, wherein 30 is a S-formylglutathione hydrolase, wherein said microbial organism further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MOP, and comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises: (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P5, and comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein 1A a MeDH, wherein said microbial organism further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH.

In some embodiments, a non-naturally occurring micoribial organism of the invention includes a MOP. Such a pathway can include at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol. An exemplary MOP enzyme is a MeDH. Accordingly, in some embodiments, a non-naturally occurring micoribial organism of the invention includes at least one exogenous nucleic acid encoding a MeDH expressed in a sufficient amount to produce formaldehyde in the presence of methanol.

In some embodiments, the exogenous nucleic acid encoding an MeDH is expressed in a sufficient amount to produce an amount of formaldehyde greater than or equal to 1 μM, 10 μM, 20 μM, or 50 μM, or a range thereof, in culture medium or intracellularly. In other embodiments, the exogenous nucleic acid encoding an MeDH is capable of producing an amount of formaldehyde greater than or equal to 1 μM, 10 μM, 20 μM, or 50 μM, or a range thereof, in culture medium or intracellularly. In some embodiments, the range is from 1 μM to 50 μM or greater. In other embodiments, the range is from 10 μM to 50 μM or greater. In other embodiments, the range is from 20 μM to 50 μM or greater. In other embodiments, the amount of formaldehyde production is 50 μM or greater. In specific embodiments, the amount of formaldehyde production is in excess of, or as compared to, that of a negative control, e.g., the same species of organism that does not comprise the exogenous nucleic acid, such as a wild-type microbial organism or a control microbial organism thereof. In certain embodiments, the MeDH is selected from those provided herein, e.g., as exemplified in Example II (see FIG. 1, Step A, or FIG. 10, Step J). In certain embodiments, the amount of formaldehyde production is determined by a whole cell assay, such as that provided in Example II (see FIG. 1, Step A, or FIG. 10, Step J), or by another assay provided herein or otherwise known in the art. In certain embodiments, formaldehyde utilization activity is absent in the whole cell.

In certain embodiments, the exogenous nucleic acid encoding an MeDH is expressed in a sufficient amount to produce at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100× or more formaldehyde in culture medium or intracellularly. In other embodiments, the exogenous nucleic acid encoding an MeDH is capable of producing an amount of formaldehyde at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, or a range thereof, in culture medium or intracellularly. In some embodiments, the range is from 1× to 100×. In other embodiments, the range is from 2× to 100×. In other embodiments, the range is from 5× to 100× In other embodiments, the range is from 10× to 100×. In other embodiments, the range is from 50× to 100×. In some embodiments, the amount of formaldehyde production is at least 20×. In other embodiments, the amount of formaldehyde production is at least 50×. In specific embodiments, the amount of formaldehyde production is in excess of, or as compared to, that of a negative control, e.g., the same species of organism that does not comprise the exogenous nucleic acid, such as a wild-type microbial organism or a control microbial organism thereof. In certain embodiments, the MeDH is selected from those provided herein, e.g., as exemplified in Example II (see FIG. 1, Step A, or FIG. 10, Step J). In certain embodiments, the amount of formaldehyde production is determined by a whole cell assay, such as that provided in Example II (see FIG. 1, Step A, or FIG. 10, Step J), or by another assay provided herein or otherwise known in the art. In certain embodiments, formaldehyde utilization activity is absent in the whole cell.

In some embodiments, a non-naturally occurring microbial organism of the invention includes one or more enzymes for generating reducing equivalents. For example, the microbial organism can further include a hydrogenase and/or a CODH. In some aspects, the organism comprises an exogenous nucleic acid encoding the hydrogenase or the CODH.

A reducing equivalent can also be readily obtained from a glycolysis intermediate by any of several central metabolic reactions including glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate formate lyase and NAD(P)-dependent FDH, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. Additionally, reducing equivalents can be generated from glucose 6-phosphate-1-dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway. Overall, at most twelve reducing equivalents can be obtained from a C6 glycolysis intermediate (e.g., glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate) and at most six reducing equivalents can be generated from a C3 glycolysis intermediate (e.g., dihydroxyacetone phosphate, glyceraldehyde-3-phosphate).

In some embodiments, the invention provides a non-naturally occurring microbial organism having a butadiene pathway including at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, wherein the butadiene pathway includes a pathway shown in FIGS. 10-11 and 13-19 selected from:

(1) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (2) 10A, 10D, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (3) 10A, 10D, 10K, 10S, 15A, 15B, 15C, and 15G; (4) 10A, 10H, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (5) 10A, 10J, 10G, 10S, 15A, 15B, 15C, and 15G; (6) 10A, 10J, 10R, 10AA, 15A, 15B, 15C, and 15G; (7) 10A, 10H, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (8) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (9) 10A, 10D, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (10) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (11) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (12) 10A, 10D, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (13) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (14) 10A, 10B, 10M, 10AA, 15A, 15B, 15C, and 15G; (15) 10A, 10B, 10L, 10Z, 10AA, 15A, 15B, 15C, and 15G; (16) 10A, 10B, 10X, 10N, 10AA, 15A, 15B, 15C, and 15G; (17) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (18) 10A, 10D, 10P, 10O, 15A, 15B, 15C, and 15G; (19) 10A, 10B, 10X, 10O, 15A, 15B, 15C, and 15G; (20) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (21) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (22) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (23) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (24) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15B, 15C, and 15G; (25) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (26) 10AU, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (27) 10AU, 10AB, 10O, 15A, 15B, 15C, and 15G; (28) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (29) 1T, 10AS, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (30) 1T, 10AS, 10K, 10S, 15A, 15B, 15C, and 15G; (31) 1T, 10AS, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (32) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (33) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (34) 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (35) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (36) 1T, 10AS, 10P, 10O, 15A, 15B, 15C, and 15G; (37) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (38) 10AT, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (39) 10AT, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (40) 10AT, 10K, 10S, 15A, 15B, 15C, and 15G; (41) 10AT, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (42) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (43) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (44) 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (45) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (46) 10AT, 10P, 10O, 15A, 15B, 15C, and 15G; (47) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (48) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (49) 10A, 10D, 10I, 10G, 10S, 15D, and 15G; (50) 10A, 10D, 10K, 10S, 15D, and 15G; (51) 10A, 10H, 10F, 10G, 10S, 15D, and 15G; (52) 10A, 10J, 10G, 10S, 15D, and 15G; (53) 10A, 10J, 10R, 10AA, 15D, and 15G; (54) 10A, 10H, 10F, 10R, 10AA, 15D, and 15G; (55) 10A, 10H, 10Q, 10Z, 10AA, 15D, and 15G; (56) 10A, 10D, 10I, 10R, 10AA, 15D, and 15G; (57) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (58) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (59) 10A, 10D, 10P, 10N, 10AA, 15D, and 15G; (60) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (61) 10A, 10B, 10M, 10AA, 15D, and 15G; (62) 10A, 10B, 10L, 10Z, 10AA, 15D, and 15G; (63) 10A, 10B, 10X, 10N, 10AA, 15D, and 15G; (64) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15D, and 15G; (65) 10A, 10D, 10P, 10O, 15D, and 15G; (66) 10A, 10B, 10X, 10O, 15D, and 15G; (67) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (68) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (69) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (70) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15D, and 15G; (71) 10A, 10B, 10C, 10AE, 10AB, 10O, 15D, and 15G; (72) 10AU, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (73) 10AU, 10AB, 10N, 10AA, 15D, and 15G; (74) 10AU, 10AB, 10O, 15D, and 15G; (75) 1T, 10AS, 10E, 10F, 10G, 10S, 15D, and 15G; (76) 1T, 10AS, 10I, 10G, 10S, 15D, and 15G; (77) 1T, 10AS, 10K, 10S, 15D, and 15G; (78) 1T, 10AS, 10I, 10R, 10AA, 15D, and 15G; (79) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (80) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (81) 1T, 10AS, 10P, 10N, 10AA, 15D, and 15G; (82) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (83) 1T, 10AS, 10P, 10O, 15D, and 15G; (84) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (85) 10AT, 10E, 10F, 10G, 10S, 15D, and 15G; (86) 10AT, 10I, 10G, 10S, 15D, and 15G; (87) 10AT, 10K, 10S, 15D, and 15G; (88) 10AT, 10I, 10R, 10AA, 15D, and 15G; (89) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (90) 10AT, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (91) 10AT, 10P, 10N, 10AA, 15D, and 15G; (92) 10AT, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (93) 10AT, 10P, 10O, 15D, and 15G; (94) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (95) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (96) 10A, 10D, 10I, 10G, 10S, 15E, 15C, and 15G; (97) 10A, 10D, 10K, 10S, 15E, 15C, and 15G; (98) 10A, 10H, 10F, 10G, 10S, 15E, 15C, and 15G; (99) 10A, 10J, 10G, 10S, 15E, 15C, and 15G; (100) 10A, 10J, 10R, 10AA, 15E, 15C, and 15G; (101) 10A, 10H, 10F, 10R, 10AA, 15E, 15C, and 15G; (102) 10A, 10H, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (103) 10A, 10D, 10I, 10R, 10AA, 15E, 15C, and 15G; (104) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (105) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (106) 10A, 10D, 10P, 10N, 10AA, 15E, 15C, and 15G; (107) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (108) 10A, 10B, 10M, 10AA, 15E, 15C, and 15G; (109) 10A, 10B, 10L, 10Z, 10AA, 15E, 15C, and 15G; (110) 10A, 10B, 10X, 10N, 10AA, 15E, 15C, and 15G; (111) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (112) 10A, 10D, 10P, 10O, 15E, 15C, and 15G; (113) 10A, 10B, 10X, 10O, 15E, 15C, and 15G; (114) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (115) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (116) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (117) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15E, 15C, and 15G; (118) 10A, 10B, 10C, 10AE, 10AB, 10O, 15E, 15C, and 15G; (119) 10AU, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (120) 10AU, 10AB, 10N, 10AA, 15E, 15C, and 15G; (121) 10AU, 10AB, 10O, 15E, 15C, and 15G; (122) 1T, 10AS, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (123) 1T, 10AS, 10I, 10G, 10S, 15E, 15C, and 15G; (124) 1T, 10AS, 10K, 10S, 15E, 15C, and 15G; (125) 1T, 10AS, 10I, 10R, 10AA, 15E, 15C, and 15G; (126) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (127) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (128) 1T, 10AS, 10P, 10N, 10AA, 15E, 15C, and 15G; (129) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (130) 1T, 10AS, 10P, 10O, 15E, 15C, and 15G; (131) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (132) 10AT, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (133) 10AT, 10I, 10G, 10S, 15E, 15C, and 15G; (134) 10AT, 10K, 10S, 15E, 15C, and 15G; (135) 10AT, 10I, 10R, 10AA, 15E, 15C, and 15G; (136) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (137) 10AT, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (138) 10AT, 10P, 10N, 10AA, 15E, 15C, and 15G; (139) 10AT, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (140) 10AT, 10P, 10O, 15E, 15C, and 15G; (141) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (142) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (143) 10A, 10D, 10I, 10G, 10S, 15A, 15F, and 15G; (144) 10A, 10D, 10K, 10S, 15A, 15F, and 15G; (145) 10A, 10H, 10F, 10G, 10S, 15A, 15F, and 15G; (146) 10A, 10J, 10G, 10S, 15A, 15F, and 15G; (147) 10A, 10J, 10R, 10AA, 15A, 15F, and 15G; (148) 10A, 10H, 10F, 10R, 10AA, 15A, 15F, and 15G; (149) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (150) 10A, 10D, 10I, 10R, 10AA, 15A, 15F, and 15G; (151) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (152) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (153) 10A, 10D, 10P, 10N, 10AA, 15A, 15F, and 15G; (154) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (155) 10A, 10B, 10M, 10AA, 15A, 15F, and 15G; (156) 10A, 10B, 10L, 10Z, 10AA, 15A, 15F, and 15G; (157) 10A, 10B, 10X, 10N, 10AA, 15A, 15F, and 15G; (158) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (159) 10A, 10D, 10P, 10O, 15A, 15F, and 15G; (160) 10A, 10B, 10X, 10O, 15A, 15F, and 15G; (161) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (162) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (163) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (164) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15F, and 15G; (165) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15F, and 15G; (166) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (167) 10AU, 10AB, 10N, 10AA, 15A, 15F, and 15G; (168) 10AU, 10AB, 10O, 15A, 15F, and 15G; (169) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (170) 1T, 10AS, 10I, 10G, 10S, 15A, 15F, and 15G; (171) 1T, 10AS, 10K, 10S, 15A, 15F, and 15G; (172) 1T, 10AS, 10I, 10R, 10AA, 15A, 15F, and 15G; (173) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (174) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (175) 1T, 10AS, 10P, 10N, 10AA, 15A, 15F, and 15G; (176) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (177) 1T, 10AS, 10P, 10O, 15A, 15F, and 15G; (178) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (179) 10AT, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (180) 10AT, 10I, 10G, 10S, 15A, 15F, and 15G; (181) 10AT, 10K, 10S, 15A, 15F, and 15G; (182) 10AT, 10I, 10R, 10AA, 15A, 15F, and 15G; (183) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (184) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (185) 10AT, 10P, 10N, 10AA, 15A, 15F, and 15G; (186) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (187) 10AT, 10P, 10O, 15A, 15F, and 15G; (188) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (189) 14A, 14B, 14C, 14D, 14E, 13A, and 13B; (190) 15A, 15B, 15C, and 15G; (191) 15D, and 15G; (192) 15E, 15C, and 15G; (193) 15A, 15F, and 15G; (194) 16A, 16B, 16C, 16D, and 16E; (195) 17A, 17B, 17C, 17D, and 17G; (196) 17A, 17E, 17F, 17D, and 17G; (197) 17A, 17B, 17C, 17H, 17I, 17J, and 17G; (198) 18A, 18B, 18C, 18D, 18E, and 18F; (199) 13A, and 13B; (200) 17A, 17E, 17F, 17H, 17I, 17J, and 17G; (201) 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D; (202) 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D; (203) 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D; (204) 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D; (205) 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D; (206) 10P, 10AB, 19A, 19B, 19C, and 19D; (207) 10AU, 19A, 19B, 19C, and 19D; and (208) 19A, 19B, 19C, and 19D, (209) 11A and 11F; (210) 10A, 10J, 10R, 10AD, 10AH, 11A, and 11F; (211) 10A, 10H, 10F, 10R, 10AD, 10AH, 11A, and 11F; (212) 10A, 10H, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (213) 10A, 10H, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (214) 10A, 10D, 10I, 10R, 10AD, 10AH, 11A, and 11F; (215) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (216) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (217) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (218) 10A, 10D, 10P, 10N, 10AD, 10AH, 11A, and 11F; (219) 10A, 10D, 10I), 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (220) 10A, 10D, 10I), 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (221) 10A, 10D, 10P, 10AB, 10V, 10AH, 11A, and 11F; (222) 10A, 10D, 10P, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (223) 10A, 10B, 10M, 10AD, 10AH, 11A, and 11F; (224) 10A, 10B, 10L, 10Z, 10AD, 10AH, 11A, and 11F; (225) 10A, 10B, 10L, 10AC, 10AG, 10AH, 11A, and 11F; (226) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (227) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (228) 10A, 10B, 10X, 10AB, 10V, 10AH, 11A, and 11F; (229) 10A, 10B, 10X, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (230) 10A, 10B, 10C, 10U, 10AH, 11A, and 11F; (231) 10A, 10B, 10C, 10T, 10AG, 10AH, 11A, and 11F; (232) 10A, 10B, 10C, 10AE, LOAF, 10AG, 10AH, 11A, and 11F; (233) 10A, 10D, 10P, 10AB, 10W, 11A, and 11F; (234) 10A, 10B, 10X, 10AB, 10W, 11A, and 11F; (235) 10A, 10B, 10C, 10AE, 10W, 11A, and 11F; (236) 10A, 10B, 10C, 10AE, 10V, 10AH, 11A, and 11F; (237) 10I, 10R, 10AD, 10AH, 11A, and 11F; (238) 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (239) 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (240) 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (241) 10P, 10N, 10AD, 10AH, 11A, and 11F; (242) 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (243) 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (244) 10P, 10AB, 10V, 10AH, 11A, and 11F; (245) 10P, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (246) 10P, 10AB, 10W, 11A, and 11F; (247) 1T, 10AS, 10I, 10R, 10AD, 10AH, 11A, and 11F; (248) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (249) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (250) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (251) 1T, 10AS, 10P, 10N, 10AD, 10AH, 11A, and 11F; (252) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (253) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (254) 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, and 11F; (255) 1T, 10AS, 10P, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (256) 1T, 10AS, 10P, 10AB, 10W, 11A, and 11F; (257) 10AT, 10I, 10R, 10AD, 10AH, 11A, and 11F; (258) 10AT, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (259) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (260) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (261) 10AT, 10P, 10N, 10AD, 10AH, 11A, and 11F; (262) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (263) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (264) 10AT, 10P, 10AB, 10V, 10AH, 11A, and 11F; (265) 10AT, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (266) 10AT, 10P, 10AB, 10W, 11A, and 11F; (267) 10AU, LOAF, 10AG, 10AH, 11A, and 11F; (268) 10AU, 10W, 11A, and 11F; (269) 10AU, 10V, 10AH, 11A, and 11F; (270) 10A, 10B, 10X, 10N, 10AD, 10AH, 11A, and 11F; and (271) 10A, 10B, 10X, 10N, 10AD, 10AH, and 11E, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 10O is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 11E is a CrotOH dehydratase, wherein 11F is a BDS (monophosphate), wherein 13A is a 2-butanol desaturase, wherein 13B is a MVC dehydratase, wherein 14A is an acetolactate synthase, wherein 14B is an acetolactate decarboxylase, wherein 14C is a butanediol dehydrogenase, wherein 14D is a butanediol dehydratase, wherein 14E is a butanol dehydrogenase, wherein 15A is a 13BDO kinase, wherein 15B is a 3-hydroxybutyrylphosphate kinase, 15C is a 3-hydroxybutyryldiphosphate lyase, wherein 15D is a 13BDO diphosphokinase, wherein 15E is a 13BDO dehydratase, wherein 15F is a 3-hydroxybutyrylphosphate lyase, wherein 15G is a MVC dehydratase, wherein 16A is a 3-oxopent-4-enoyl-CoA thiolase, wherein 16B is a 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, wherein 16C is a 3-oxopent-4-enoate decarboxylase or spontaneous, wherein 16D is a 3-buten-2-one reductase, wherein 16E is a MVC dehydratase, wherein 17A is a 3-oxo-4-hydroxypentanoyl-CoA thiolase, wherein 17B is a 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17C is a 3-oxo-4-hydroxypentanoate reductase, wherein 17D is a 3,4-dihydroxypentanoate decarboxylase, wherein 17E is a 3-oxo-4-hydroxypentanoyl-CoA reductase, wherein 17F is a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17G is a MVC dehydratase, wherein 17H is a 3,4-dihydroxypentanoate dehydratase, wherein 171 is a 4-oxopentanoate reductase, wherein 17J is a 4-hyd4-oxoperoxypentanoate decarboxylase, wherein 18A is a 3-oxoadipyl-CoA thiolase, wherein 18B is a 3-oxoadipyl-CoA transferase, synthetase or hydrolase, wherein 18C is a 3-oxoadipate decarboxylase or spontaneous, wherein 18D is a 4-oxopentanoate reductase, wherein 18E is a 4-hydroxypentanoate decarboxylase, wherein 18F is a MVC dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase, wherein 19D is a 3-buten-1-ol dehydratase.

In some aspects, the microbial organism can includes one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve exogenous nucleic acids each encoding a butadiene pathway enzyme. In some aspects, microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the butadiene pathways selected from (1)-(271). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In one aspect, the non-naturally occurring microbial organism a butadiene pathway described above further comprises a FaldFP comprising at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS.

In one aspect, the non-naturally occurring microbial organism having a butadiene pathway described above further comprises a MMP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises a pathway selected from: (1) 3J; (2) 3A and 3B; (3) 3A, 3B and 3C; (4) 3J, 3K and 3C; (5) 3J, 3M, and 3N; (6) 3J and 3L; (7) 3A, 3B, 3C, 3D, and 3E; (8) 3A, 3B, 3C, 3D, and 3F; (9) 3J, 3K, 3C, 3D, and 3E; (10) 3J, 3K, 3C, 3D, and 3F; (11) 3J, 3M, 3N, and 30; (12) 3A, 3B, 3C, 3D, 3E, and 3G; (13) 3A, 3B, 3C, 3D, 3F, and 3G; (14) 3J, 3K, 3C, 3D, 3E, and 3G; (15) 3J, 3K, 3C, 3D, 3F, and 3G; (16) 3J, 3M, 3N, 30, and 3G; (17) 3A, 3B, 3C, 3D, 3E, and 3I; (18) 3A, 3B, 3C, 3D, 3F, and 3I; (19) 3J, 3K, 3C, 3D, 3E, and 3I; (20) 3J, 3K, 3C, 3D, 3F, and 3I; and (21) 3J, 3M, 3N, 30, and 31, wherein 3A is a methanol methyltransferase, wherein 3B is a methylenetetrahydrofolate reductase, wherein 3C is a MTHFDH, wherein 3D is a methenyltetrahydrofolate cyclohydrolase, wherein 3E is a formyltetrahydrofolate deformylase, wherein 3F is a FTHFS, wherein 3G is a formate hydrogen lyase, wherein 3H is a hydrogenase, wherein 31 is a FDH, wherein 3J is a MeDH, wherein 3K is a formaldehyde activating enzyme or spontaneous, wherein 3L is a formaldehyde dehydrogenase, wherein 3M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 3N is a glutathione-dependent formaldehyde dehydrogenase, wherein 30 is a S-formylglutathione hydrolase,

In one aspect, the non-naturally occurring microbial organism having a butadiene pathway described above further comprises a MOP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein 1A a MeDH.

In one aspect, the non-naturally occurring microbial organism having a butadiene pathway described above further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH. In certain embodiments, the organism comprises an exogenous nucleic acid encoding said hydrogenase or said CODH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described above, wherein the organism further comprises a butadiene pathway. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, wherein said butadiene pathway as shown in FIGS. 1, 2, and 10-19 comprises a pathway selected from: (1) 10A, 10J, 10R, 10AD, 10AH, 11A, 11B, and 11C; (2) 10A, 10H, 10F, 10R, 10AD, 10AH, 11A, 11B, and 11C; (3) 10A, 10H, 10Q, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (4) 10A, 10H, 10Q, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (5) 10A, 10D, 10I, 10R, 10AD, 10AH, 11A, 11B, and 11C; (6) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, 11A, 11B, and 11C; (7) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (8) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (9) 10A, 10D, 10P, 10N, 10AD, 10AH, 11A, 11B, and 11C; (10) 10A, 10D, 10P, 10Y, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (11) 10A, 10D, 10P, 10Y, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (12) 10A, 10D, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; (13) 10A, 10D, 10P, 10AB, LOAF, 10AG, 10AH, 11A, 11B, and 11C; (14) 10A, 10B, 10M, 10AD, 10AH, 11A, 11B, and 11C; (15) 10A, 10B, 10L, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (16) 10A, 10B, 10L, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (17) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (18) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (19) 10A, 10B, 10X, 10AB, 10V, 10AH, 11A, 11B, and 11C; (20) 10A, 10B, 10X, 10AB, LOAF, 10AG, 10AH, 11A, 11B, and 11C; (21) 10A, 10B, 10C, 10U, 10AH, 11A, 11B, and 11C; (22) 10A, 10B, 10C, 10T, 10AG, 10AH, 11A, 11B, and 11C; (23) 10A, 10B, 10C, 10AE, 10AF, 10AG, 10AH, 11A, 11B, and 11C; (24) 10A, 10D, 10P, 10AB, 10W, 11A, 11B, and 11C; (25) 10A, 10B, 10X, 10AB, 10W, 11A, 11B, and 11C; (26) 10A, 10B, 10C, 10AE, 10W, 11A, 11B, and 11C; (27) 10A, 10B, 10C, 10AE, 10V, 10AH; 11A, 11B, and 11C (28) 10A, 10J, 10R, 10AD, 10AH, 11D, and 11C; (29) 10A, 10H, 10F, 10R, 10AD, 10AH, 11D, and 11C; (30) 10A, 10H, 10Q, 10Z, 10AD, 10AH, 11D, and 11C; (31) 10A, 10H, 10Q, 10AC, 10AG, 10AH, 11D, and 11C; (32) 10A, 10D, 10I, 10R, 10AD, 10AH, 11D, and 11C; (33) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, 11D, and 11C; (34) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, 11D, and 11C; (35) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, 11D, and 11C; (36) 10A, 10D, 10P, 10N, 10AD, 10AH, 11D, and 11C; (37) 10A, 10D, 10P, 10Y, 10Z, 10AD, 10AH, 11D, and 11C; (38) 10A, 10D, 10P, 10Y, 10AC, 10AG, 10AH, 11D, and 11C; (39) 10A, 10D, 10P, 10AB, 10V, 10AH, 11D, and 11C; (40) 10A, 10D, 10P, 10AB, 10AF, 10AG, 10AH, 11D, and 11C; (41) 10A, 10B, 10M, 10AD, 10AH, 11D, and 11C; (42) 10A, 10B, 10L, 10Z, 10AD, 10AH, 11D, and 11C; (43) 10A, 10B, 10L, 10AC, 10AG, 10AH, 11D, and 11C; (44) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, 11D, and 11C; (45) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, 11D, and 11C; (46) 10A, 10B, 10X, 10AB, 10V, 10AH, 11D, and 11C; (47) 10A, 10B, 10X, 10AB, 10AF, 10AG, 10AH, 11D, and 11C; (48) 10A, 10B, 10C, 10U, 10AH, 11D, and 11C; (49) 10A, 10B, 10C, 10T, 10AG, 10AH, 11D, and 11C; (50) 10A, 10B, 10C, 10AE, 10AF, 10AG, 10AH, 11D, and 11C; (51) 10A, 10D, 10P, 10AB, 10W, 11D, and 11C; (52) 10A, 10B, 10X, 10AB, 10W, 11D, and 11C; (53) 10A, 10B, 10C, 10AE, 10W, 11D, and 11C; (54) 10A, 10B, 10C, 10AE, 10V, 10AH, 11D, and 11C; (55) 10I, 10R, 10AD, 10AH, 11A, 11B, and 11C; (56) 10E, 10F, 10R, 10AD, 10AH, 11A, 11B, and 11C; (57) 10E, 10Q, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (58) 10E, 10Q, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (59) 10P, 10N, 10AD, 10AH, 11A, 11B, and 11C; (60) 10P, 10Y, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (61) 10P, 10Y, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (62) 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; (63) 10P, 10AB, 10AF, 10AG, 10AH, 11A, 11B, and 11C; (64) 10P, 10AB, 10W, 11A, 11B, and 11C; (65) 10I, 10R, 10AD, 10AH, 11D, and 11C; (66) 10E, 10F, 10R, 10AD, 10AH, 11D, and 11C; (67) 10E, 10Q, 10Z, 10AD, 10AH, 11D, and 11C; (68) 10E, 10Q, 10AC, 10AG, 10AH, 11D, and 11C; (69) 10P, 10N, 10AD, 10AH, 11D, and 11C; (70) 10P, 10Y, 10Z, 10AD, 10AH, 11D, and 11C; (71) 10P, 10Y, 10AC, 10AG, 10AH, 11D, and 11C; (72) 10P, 10AB, 10V, 10AH, 11D, and 11C; (73) 10P, 10AB, 10AF, 10AG, 10AH, 11D, and 11C; (74) 10P, 10AB, 10W, 11D, and 11C; (75) 1T, 10AS, 10I, 10R, 10AD, 10AH, 11A, 11B, and 11C; (76) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, 11A, 11B, and 11C; (77) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (78) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (79) 1T, 10AS, 10P, 10N, 10AD, 10AH, 11A, 11B, and 11C; (80) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (81) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (82) 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; (83) 1T, 10AS, 10P, 10AB, 10AF, 10AG, 10AH, 11A, 11B, and 11C; (84) 1T, 10AS, 10P, 10AB, 10W, 11A, 11B, and 11C; (85) 1T, 10AS, 10I, 10R, 10AD, 10AH, 11D, and 11C; (86) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, 11D, and 11C; (87) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, 11D, and 11C; (88) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, 11D, and 11C; (89) 1T, 10AS, 10P, 10N, 10AD, 10AH, 11D, and 11C; (90) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, 11D, and 11C; (91) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, 11D, and 11C; (92) 1T, 10AS, 10P, 10AB, 10V, 10AH, 11D, and 11C; (93) 1T, 10AS, 10P, 10AB, 10AF, 10AG, 10AH, 11D, and 11C; (94) 1T, 10AS, 10P, 10AB, 10W, 11D, and 11C; (95) 10AT, 10I, 10R, 10AD, 10AH, 11A, 11B, and 11C; (96) 10AT, 10E, 10F, 10R, 10AD, 10AH, 11A, 11B, and 11C; (97) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (98) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (99) 10AT, 10P, 10N, 10AD, 10AH, 11A, 11B, and 11C; (100) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, 11A, 11B, and 11C; (10P) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, 11A, 11B, and 11C; (102) 10AT, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; (103) 10AT, 10P, 10AB, LOAF, 10AG, 10AH, 11A, 11B, and 11C; (104) 10AT, 10P, 10AB, 10W, 11A, 11B, and 11C; (105) 10AT, 10I, 10R, 10AD, 10AH, 11D, and 11C; (106) 10AT, 10E, 10F, 10R, 10AD, 10AH, 11D, and 11C; (107) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, 11D, and 11C; (108) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, 11D, and 11C; (109) 10AT, 10P, 10N, 10AD, 10AH, 11D, and 11C; (110) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, 11D, and 11C; (111) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, 11D, and 11C; (112) 10AT, 10P, 10AB, 10V, 10AH, 11D, and 11C; (113) 10AT, 10P, 10AB, 10AF, 10AG, 10AH, 11D, and 11C; (114) 10AT, 10P, 10AB, 10W, 11D, and 11C; (115) 10AU, 10AF, 10AG, 10AH, 11A, 11B, and 11C; (116) 10AU, 10W, 11A, 11B, and 11C; (117) 10AU, 10V, 10AH; 11A, 11B, and 11C; (118) 10AU, LOAF, 10AG, 10AH, 11D, and 11C; (119) 10AU, 10W, 11D, and 11C; (120) 10AU, 10V, 10AH, 11D, and 11C; (121) 10A, 10J, 10R, 10AD, 10AH, and 11E; (122) 10A, 10H, 10F, 10R, 10AD, 10AH, and 11E; (123) 10A, 10H, 10Q, 10Z, 10AD, 10AH, and 11E; (124) 10A, 10H, 10Q, 10AC, 10AG, 10AH, and 11E; (125) 10A, 10D, 10I, 10R, 10AD, 10AH, and 11E; (126) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, and 11E; (127) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, and 11E; (128) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, and 11E; (129) 10A, 10D, 10P, 10N, 10AD, 10AH, and 11E; (130) 10A, 10D, 10P, 10Y, 10Z, 10AD, 10AH, and 11E; (131) 10A, 10D, 10P, 10Y, 10AC, 10AG, 10AH, and 11E; (132) 10A, 10D, 10P, 10AB, 10V, 10AH, and 11E; (133) 10A, 10D, 10P, 10AB, LOAF, 10AG, 10AH, and 11E; (134) 10A, 10B, 10M, 10AD, 10AH, and 11E; (135) 10A, 10B, 10L, 10Z, 10AD, 10AH, and 11E; (136) 10A, 10B, 10L, 10AC, 10AG, 10AH, and 11E; (137) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, and 11E; (138) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, and 11E; (139) 10A, 10B, 10X, 10AB, 10V, 10AH, and 11E; (140) 10A, 10B, 10X, 10AB, LOAF, 10AG, 10AH, and 11E; (141) 10A, 10B, 10C, 10U, 10AH, and 11E; (142) 10A, 10B, 10C, 10T, 10AG, 10AH, and 11E; (143) 10A, 10B, 10C, 10AE, LOAF, 10AG, 10AH, and 11E; (144) 10A, 10D, 10P, 10AB, 10W, and 11E; (145) 10A, 10B, 10X, 10AB, 10W, and 11E; (146) 10A, 10B, 10C, 10AE, 10W, and 11E; (147) 10A, 10B, 10C, 10AE, 10V, 10AH, and 11E; (148) 10I, 10R, 10AD, 10AH, and 11E; (149) 10E, 10F, 10R, 10AD, 10AH, and 11E; (150) 10E, 10Q, 10Z, 10AD, 10AH, and 11E; (151) 10E, 10Q, 10AC, 10AG, 10AH, and 11E; (152) 10P, 10N, 10AD, 10AH, and 11E; (153) 10P, 10Y, 10Z, 10AD, 10AH, and 11E; (154) 10P, 10Y, 10AC, 10AG, 10AH, and 11E; (155) 10P, 10AB, 10V, 10AH, and 11E; (156) 10P, 10AB, LOAF, 10AG, 10AH, and 11E; (157) 10P, 10AB, 10W, and 11E; (158) 1T, 10AS, 10I, 10R, 10AD, 10AH, and 11E; (159) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, and 11E; (160) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, and 11E; (161) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, and 11E; (162) 1T, 10AS, 10P, 10N, 10AD, 10AH, and 11E; (163) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, and 11E; (164) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, and 11E; (165) 1T, 10AS, 10P, 10AB, 10V, 10AH, and 11E; (166) 1T, 10AS, 10P, 10AB, 10AF, 10AG, 10AH, and 11E; (167) 1T, 10AS, 10P, 10AB, 10W, and 11E; (168) 10AT, 10I, 10R, 10AD, 10AH, and 11E; (169) 10AT, 10E, 10F, 10R, 10AD, 10AH, and 11E; (170) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, and 11E; (171) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, and 11E; (172) 10AT, 10P, 10N, 10AD, 10AH, and 11E; (173) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, and 11E; (174) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, and 11E; (175) 10AT, 10P, 10AB, 10V, 10AH, and 11E; (176) 10AT, 10P, 10AB, 10AF, 10AG, 10AH, and 11E; (177) 10AT, 10P, 10AB, 10W, and 11E; (178) 10AU, 10AF, 10AG, 10AH, and 11E; (179) 10AU, 10W, and 11E; (180) 10AU, 10V, 10AH, and 11E; (181) 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, and 12I; (182) 12A, 12K, 12M, 12N, 12E, 12F, 12G, 12H, and 12I; (183) 12A, 12K, 12L, 12D, 12E, 12F, 12G, 12H, and 12I; (184) 12A, 120, 12N, 12E, 12F, 12G, 12H, and 12I; (185) 12A, 12B, 12J, 12E, 12F, 12G, 12H, and 12I; (186) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (187) 10A, 10D, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (188) 10A, 10D, 10K, 10S, 15A, 15B, 15C, and 15G; (189) 10A, 10H, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (190) 10A, 10J, 10G, 10S, 15A, 15B, 15C, and 15G; (191) 10A, 10J, 10R, 10AA, 15A, 15B, 15C, and 15G; (192) 10A, 10H, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (193) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (194) 10A, 10D, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (195) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (196) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (197) 10A, 10D, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (198) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (199) 10A, 10B, 10M, 10AA, 15A, 15B, 15C, and 15G; (200) 10A, 10B, 10L, 10Z, 10AA, 15A, 15B, 15C, and 15G; (201) 10A, 10B, 10X, 10N, 10AA, 15A, 15B, 15C, and 15G; (202) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (203) 10A, 10D, 10P, 10O, 15A, 15B, 15C, and 15G; (204) 10A, 10B, 10X, 10O, 15A, 15B, 15C, and 15G; (205) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (206) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (207) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (208) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (209) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15B, 15C, and 15G; (210) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (211) 10AU, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (212) 10AU, 10AB, 10O, 15A, 15B, 15C, and 15G; (213) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (214) 1T, 10AS, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (215) 1T, 10AS, 10K, 10S, 15A, 15B, 15C, and 15G; (216) 1T, 10AS, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (217) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (218) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (219) 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (220) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (221) 1T, 10AS, 10P, 10O, 15A, 15B, 15C, and 15G; (222) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (223) 10AT, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (224) 10AT, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (225) 10AT, 10K, 10S, 15A, 15B, 15C, and 15G; (226) 10AT, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (227) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (228) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (229) 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (230) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (231) 10AT, 10P, 10O, 15A, 15B, 15C, and 15G; (232) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (233) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (234) 10A, 10D, 10I, 10G, 10S, 15D, and 15G; (235) 10A, 10D, 10K, 10S, 15D, and 15G; (236) 10A, 10H, 10F, 10G, 10S, 15D, and 15G; (237) 10A, 10J, 10G, 10S, 15D, and 15G; (238) 10A, 10J, 10R, 10AA, 15D, and 15G; (239) 10A, 10H, 10F, 10R, 10AA, 15D, and 15G; (240) 10A, 10H, 10Q, 10Z, 10AA, 15D, and 15G; (241) 10A, 10D, 10I, 10R, 10AA, 15D, and 15G; (242) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (243) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (244) 10A, 10D, 10P, 10N, 10AA, 15D, and 15G; (245) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (246) 10A, 10B, 10M, 10AA, 15D, and 15G; (247) 10A, 10B, 10L, 10Z, 10AA, 15D, and 15G; (248) 10A, 10B, 10X, 10N, 10AA, 15D, and 15G; (249) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15D, and 15G; (250) 10A, 10D, 10P, 10O, 15D, and 15G; (251) 10A, 10B, 10X, 10O, 15D, and 15G; (252) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (253) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (254) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (255) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15D, and 15G; (256) 10A, 10B, 10C, 10AE, 10AB, 10O, 15D, and 15G; (257) 10AU, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (258) 10AU, 10AB, 10N, 10AA, 15D, and 15G; (259) 10AU, 10AB, 10O, 15D, and 15G; (260) 1T, 10AS, 10E, 10F, 10G, 10S, 15D, and 15G; (261) 1T, 10AS, 10I, 10G, 10S, 15D, and 15G; (262) 1T, 10AS, 10K, 10S, 15D, and 15G; (263) 1T, 10AS, 10I, 10R, 10AA, 15D, and 15G; (264) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (265) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (266) 1T, 10AS, 10P, 10N, 10AA, 15D, and 15G; (267) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (268) 1T, 10AS, 10P, 10O, 15D, and 15G; (269) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (270) 10AT, 10E, 10F, 10G, 10S, 15D, and 15G; (271) 10AT, 10I, 10G, 10S, 15D, and 15G; (272) 10AT, 10K, 10S, 15D, and 15G; (273) 10AT, 10I, 10R, 10AA, 15D, and 15G; (274) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (275) 10AT, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (276) 10AT, 10P, 10N, 10AA, 15D, and 15G; (277) 10AT, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (278) 10AT, 10P, 10O, 15D, and 15G; (279) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (280) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (281) 10A, 10D, 10I, 10G, 10S, 15E, 15C, and 15G; (282) 10A, 10D, 10K, 10S, 15E, 15C, and 15G; (283) 10A, 10H, 10F, 10G, 10S, 15E, 15C, and 15G; (284) 10A, 10J, 10G, 10S, 15E, 15C, and 15G; (285) 10A, 10J, 10R, 10AA, 15E, 15C, and 15G; (286) 10A, 10H, 10F, 10R, 10AA, 15E, 15C, and 15G; (287) 10A, 10H, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (288) 10A, 10D, 10I, 10R, 10AA, 15E, 15C, and 15G; (289) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (290) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (291) 10A, 10D, 10P, 10N, 10AA, 15E, 15C, and 15G; (292) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (293) 10A, 10B, 10M, 10AA, 15E, 15C, and 15G; (294) 10A, 10B, 10L, 10Z, 10AA, 15E, 15C, and 15G; (295) 10A, 10B, 10X, 10N, 10AA, 15E, 15C, and 15G; (296) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (297) 10A, 10D, 10P, 10O, 15E, 15C, and 15G; (298) 10A, 10B, 10X, 10O, 15E, 15C, and 15G; (299) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (300) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (301) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (302) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15E, 15C, and 15G; (303) 10A, 10B, 10C, 10AE, 10AB, 10O, 15E, 15C, and 15G; (304) 10AU, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (305) 10AU, 10AB, 10N, 10AA, 15E, 15C, and 15G; (306) 10AU, 10AB, 10O, 15E, 15C, and 15G; (307) 1T, 10AS, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (308) 1T, 10AS, 10I, 10G, 10S, 15E, 15C, and 15G; (309) 1T, 10AS, 10K, 10S, 15E, 15C, and 15G; (310) 1T, 10AS, 10I, 10R, 10AA, 15E, 15C, and 15G; (311) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (312) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (313) 1T, 10AS, 10P, 10N, 10AA, 15E, 15C, and 15G; (314) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (315) 1T, 10AS, 10P, 10O, 15E, 15C, and 15G; (316) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (317) 10AT, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (318) 10AT, 10I, 10G, 10S, 15E, 15C, and 15G; (319) 10AT, 10K, 10S, 15E, 15C, and 15G; (320) 10AT, 10I, 10R, 10AA, 15E, 15C, and 15G; (321) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (322) 10AT, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (323) 10AT, 10P, 10N, 10AA, 15E, 15C, and 15G; (324) 10AT, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (325) 10AT, 10P, 10O, 15E, 15C, and 15G; (326) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (327) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (328) 10A, 10D, 10I, 10G, 10S, 15A, 15F, and 15G; (329) 10A, 10D, 10K, 10S, 15A, 15F, and 15G; (330) 10A, 10H, 10F, 10G, 10S, 15A, 15F, and 15G; (331) 10A, 10J, 10G, 10S, 15A, 15F, and 15G; (332) 10A, 10J, 10R, 10AA, 15A, 15F, and 15G; (333) 10A, 10H, 10F, 10R, 10AA, 15A, 15F, and 15G; (334) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (335) 10A, 10D, 10I, 10R, 10AA, 15A, 15F, and 15G; (336) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (337) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (338) 10A, 10D, 10P, 10N, 10AA, 15A, 15F, and 15G; (339) 10A, 10D, 10I), 10Y, 10Z, 10AA, 15A, 15F, and 15G; (340) 10A, 10B, 10M, 10AA, 15A, 15F, and 15G; (341) 10A, 10B, 10L, 10Z, 10AA, 15A, 15F, and 15G; (342) 10A, 10B, 10X, 10N, 10AA, 15A, 15F, and 15G; (343) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (344) 10A, 10D, 10I), 10O, 15A, 15F, and 15G; (345) 10A, 10B, 10X, 10O, 15A, 15F, and 15G; (346) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (347) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (348) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (349) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15F, and 15G; (350) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15F, and 15G; (351) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (352) 10AU, 10AB, 10N, 10AA, 15A, 15F, and 15G; (353) 10AU, 10AB, 10O, 15A, 15F, and 15G; (354) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (355) 1T, 10AS, 10I, 10G, 10S, 15A, 15F, and 15G; (356) 1T, 10AS, 10K, 10S, 15A, 15F, and 15G; (357) 1T, 10AS, 10I, 10R, 10AA, 15A, 15F, and 15G; (358) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (359) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (360) 1T, 10AS, 10P, 10N, 10AA, 15A, 15F, and 15G; (361) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (362) 1T, 10AS, 10P, 10O, 15A, 15F, and 15G; (363) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (364) 10AT, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (365) 10AT, 10I, 10G, 10S, 15A, 15F, and 15G; (366) 10AT, 10K, 10S, 15A, 15F, and 15G; (367) 10AT, 10I, 10R, 10AA, 15A, 15F, and 15G; (368) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (369) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (370) 10AT, 10P, 10N, 10AA, 15A, 15F, and 15G; (371) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (372) 10AT, 10P, 10O, 15A, 15F, and 15G; (373) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (374) 14A, 14B, 14C, 14D, 14E, 13A, and 13B; (375) 16A, 16B, 16C, 16D, and 16E; (376) 17A, 17B, 17C, 17D, and 17G; (377) 17A, 17E, 17F, 17D, and 17G; (378) 17A, 17B, 17C, 17H, 17I, 17J, and 17G; (379) 18A, 18B, 18C, 18D, 18E, and 18F; (380) 13A and 13B; (381) 7A, 17E, 17F, 17H, 17I, 17J, and 17G; (382) 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D; (383) 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D; (384) 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D; (385) 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D; (386) 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D; (387) 10P, 10AB, 19A, 19B, 19C, and 19D; (388) 10AU, 19A, 19B, 19C, and 19D; and (389) 19A, 19B, 19C, and 19D, (390) 11A and 11F; (391) 10A, 10J, 10R, 10AD, 10AH, 11A, and 11F; (392) 10A, 10H, 10F, 10R, 10AD, 10AH, 11A, and 11F; (393) 10A, 10H, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (394) 10A, 10H, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (395) 10A, 10D, 10I, 10R, 10AD, 10AH, 11A, and 11F; (396) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (397) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (398) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (399) 10A, 10D, 10P, 10N, 10AD, 10AH, 11A, and 11F; (400) 10A, 10D, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (401) 10A, 10D, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (402) 10A, 10D, 10P, 10AB, 10V, 10AH, 11A, and 11F; (403) 10A, 10D, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (404) 10A, 10B, 10M, 10AD, 10AH, 11A, and 11F; (405) 10A, 10B, 10L, 10Z, 10AD, 10AH, 11A, and 11F; (406) 10A, 10B, 10L, 10AC, 10AG, 10AH, 11A, and 11F; (407) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (408) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (409) 10A, 10B, 10X, 10AB, 10V, 10AH, 11A, and 11F; (410) 10A, 10B, 10X, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (411) 10A, 10B, 10C, 10U, 10AH, 11A, and 11F; (412) 10A, 10B, 10C, 10T, 10AG, 10AH, 11A, and 11F; (413) 10A, 10B, 10C, 10AE, LOAF, 10AG, 10AH, 11A, and 11F; (414) 10A, 10D, 10P, 10AB, 10W, 11A, and 11F; (415) 10A, 10B, 10X, 10AB, 10W, 11A, and 11F; (416) 10A, 10B, 10C, 10AE, 10W, 11A, and 11F; (417) 10A, 10B, 10C, 10AE, 10V, 10AH, 11A, and 11F; (418) 10I, 10R, 10AD, 10AH, 11A, and 11F; (419) 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (420) 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (421) 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (422) 10P, 10N, 10AD, 10AH, 11A, and 11F; (423) 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (424) 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (425) 10P, 10AB, 10V, 10AH, 11A, and 11F; (426) 10P, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (427) 10P, 10AB, 10W, 11A, and 11F; (428) 1T, 10AS, 10I, 10R, 10AD, 10AH, 11A, and 11F; (429) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (430) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (431) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (432) 1T, 10AS, 10P, 10N, 10AD, 10AH, 11A, and 11F; (433) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (434) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (435) 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, and 11F; (436) 1T, 10AS, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (437) 1T, 10AS, 10P, 10AB, 10W, 11A, and 11F; (438) 10AT, 10I, 10R, 10AD, 10AH, 11A, and 11F; (439) 10AT, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (440) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (441) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (442) 10AT, 10P, 10N, 10AD, 10AH, 11A, and 11F; (443) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (444) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (445) 10AT, 10P, 10AB, 10V, 10AH, 11A, and 11F; (446) 10AT, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (447) 10AT, 10P, 10AB, 10W, 11A, and 11F; (448) 10AU, LOAF, 10AG, 10AH, 11A, and 11F; (449) 10AU, 10W, 11A, and 11F; (450) 10AU, 10V, 10AH, 11A, and 11F; (451) 10A, 10B, 10X, 10N, 10AD, 10AH, 11A, and 11F; and (452) 10A, 10B, 10X, 10N, 10AD, 10AH, and 11E, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 10O is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10T is a crotonyl-ACP thioesterase, wherein 10U is a crotonyl-ACP reductase (aldehyde forming), wherein 10V is a crotonyl-CoA reductase (aldehyde forming), wherein 10W is a crotonyl-CoA (alcohol forming), wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AC is a 3-hydroxybutyrate dehydratase, wherein LOAD is a 3-hydroxybutyraldehyde dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein LOAF is a crotonyl-CoA hydrolase, transferase or synthetase, wherein 10AG is a crotonate reductase, wherein 10AH is a crotonaldehyde reductase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 11A is a CrotOH kinase, wherein 11B is a 2-butenyl-4-phosphate kinase, wherein 11C is a BDS, wherein 11D is a CrotOH diphosphokinase, wherein 11E is a CrotOH dehydratase, wherein 11F is a BDS (monophosphate), wherein 12A is a malonyl-CoA:acetyl-CoA acyltransferase, wherein 12B is a 3-oxoglutaryl-CoA reductase (ketone-reducing), wherein 12C is a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), wherein 12D is a 3-hydroxy-5-oxopentanoate reductase, wherein 12E is a 3,5-dihydroxypentanoate kinase, wherein 12F is a 3-hydroxy-5-phosphonatooxypentanoate kinase, wherein 12G is a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, wherein 12H is a butenyl 4-diphosphate isomerase, wherein 12I is a BDS, wherein 12J is a 3-hydroxyglutaryl-CoA reductase (alcohol forming), wherein 12K is a 3-oxoglutaryl-CoA reductase (aldehyde forming), wherein 12L is a 3,5-dioxopentanoate reductase (ketone reducing), wherein 12M is a 3,5-dioxopentanoate reductase (aldehyde reducing), wherein 12N is a 5-hydroxy-3-oxopentanoate reductase, wherein 120 is a 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming), wherein 13A is a 2-butanol desaturase, wherein 13B is a MVC dehydratase, wherein 14A is an acetolactate synthase, wherein 14B is an acetolactate decarboxylase, wherein 14C is a butanediol dehydrogenase, wherein 14D is a butanediol dehydratase, wherein 14E is a butanol dehydrogenase, wherein 15A is a 13BDO kinase, wherein 15B is a 3-hydroxybutyrylphosphate kinase, 15C is a 3-hydroxybutyryldiphosphate lyase, wherein 15D is a 13BDO diphosphokinase, wherein 15E is a 13BDO dehydratase, wherein 15F is a 3-hydroxybutyrylphosphate lyase, wherein 15G is a MVC dehydratase, wherein 16A is a 3-oxopent-4-enoyl-CoA thiolase, wherein 16B is a 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, wherein 16C is a 3-oxopent-4-enoate decathoxylase or spontaneous, wherein 16D is a 3-buten-2-one reductase, wherein 16E is a MVC dehydratase, wherein 17A is a 3-oxo-4-hydroxypentanoyl-CoA thiolase, wherein 17B is a 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17C is a 3-oxo-4-hydroxypentanoate reductase, wherein 17D is a 3,4-dihydroxypentanoate decathoxylase, wherein 17E is a 3-oxo-4-hydroxypentanoyl-CoA reductase, wherein 17F is a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17G is a MVC dehydratase, wherein 17H is a 3,4-dihydroxypentanoate dehydratase, wherein 171 is a 4-oxopentanoate reductase, wherein 17J is a 4-hyd4-oxoperoxypentanoate decathoxylase, wherein 18A is a 3-oxoadipyl-CoA thiolase, wherein 18B is a 3-oxoadipyl-CoA transferase, synthetase or hydrolase, wherein 18C is a 3-oxoadipate decathoxylase or spontaneous, wherein 18D is a 4-oxopentanoate reductase, wherein 18E is a 4-hydroxypentanoate decarboxylase, wherein 18F is a MVC dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase, and wherein 19D is a 3-buten-1-ol dehydratase.

In some aspects, the microbial organism can include one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve exogenous nucleic acids each encoding a butadiene pathway enzyme. In some aspects, microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the butadiene pathways selected from (1)-(452). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described above, wherein the organism further comprises a CrotOH pathway. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a CrotOH pathway enzyme expressed in a sufficient amount to produce CrotOH, wherein said CrotOH pathway comprises a pathway as shown in FIGS. 1, 2, and 10 selected from: (1) 10A, 10J, 10R, 10AD, and 10AH; (2) 10A, 10H, 10F, 10R, 10AD, and 10AH; (3) 10A, 10H, 10Q, 10Z, 10AD, and 10AH; (4) 10A, 10H, 10Q, 10AC, 10AG, and 10AH; (5) 10A, 10D, 10I, 10R, 10AD, and 10AH; (6) 10A, 10D, 10E, 10F, 10R, 10AD, and 10AH; (7) 10A, 10D, 10E, 10Q, 10Z, 10AD, and 10AH; (8) 10A, 10D, 10E, 10Q, 10AC, 10AG, and 10AH; (9) 10A, 10D, 10P, 10N, 10AD, and 10AH; (10) 10A, 10D, 10P, 10Y, 10Z, 10AD, and 10AH; (11) 10A, 10D, 10P, 10Y, 10AC, 10AG, and 10AH; (12) 10A, 10D, 10P, 10AB, 10V, and 10AH; (13) 10A, 10D, 10P, 10AB, LOAF, 10AG, and 10AH; (14) 10A, 10B, 10M, 10AD, and 10AH; (15) 10A, 10B, 10L, 10Z, 10AD, and 10AH; (16) 10A, 10B, 10L, 10AC, 10AG, and 10AH; (17) 10A, 10B, 10X, 10Y, 10Z, 10AD, and 10AH; (18) 10A, 10B, 10X, 10Y, 10AC, 10AG, and 10AH; (19) 10A, 10B, 10X, 10AB, 10V, and 10AH; (20) 10A, 10B, 10X, 10AB, 10AF, 10AG, and 10AH; (21) 10A, 10B, 10C, 10U, and 10AH; (22) 10A, 10B, 10C, 10T, 10AG, and 10AH; (23) 10A, 10B, 10C, 10AE, 10AF, 10AG, and 10AH; (24) 10A, 10D, 10P, 10AB, and 10W; (25) 10A, 10B, 10X, 10AB, and 10W; (26) 10A, 10B, 10C, 10AE, and 10W; (27) 10A, 10B, 10C, 10AE, 10V, and 10AH; (28) 10I, 10R, 10AD, and 10AH; (29) 10E, 10F, 10R, 10AD, and 10AH; (30) 10E, 10Q, 10Z, 10AD, and 10AH; (31) 10E, 10Q, 10AC, 10AG, and 10AH; (32) 10P, 10N, 10AD, and 10AH; (33) 10P, 10Y, 10Z, 10AD, and 10AH; (34) 10P, 10Y, 10AC, 10AG, and 10AH; (35) 10P, 10AB, 10V, and 10AH; (36) 10P, 10AB, LOAF, 10AG, and 10AH; (37) 10P, 10AB, and 10W; (38) 1T, 10AS, 10I, 10R, 10AD, and 10AH; (39) 1T, 10AS, 10E, 10F, 10R, 10AD, and 10AH; (40) 1T, 10AS, 10E, 10Q, 10Z, 10AD, and 10AH; (41) 1T, 10AS, 10E, 10Q, 10AC, 10AG, and 10AH; (42) 1T, 10AS, 10P, 10N, 10AD, and 10AH; (43) 1T, 10AS, 10P, 10Y, 10Z, 10AD, and 10AH; (44) 1T, 10AS, 10P, 10Y, 10AC, 10AG, and 10AH; (45) 1T, 10AS, 10P, 10AB, 10V, and 10AH; (46) 1T, 10AS, 10P, 10AB, 10AF, 10AG, and 10AH; (47) 1T, 10AS, 10P, 10AB, and 10W; (48) 10AT, 10I, 10R, 10AD, and 10AH; (49) 10AT, 10E, 10F, 10R, 10AD, and 10AH; (50) 10AT, 10E, 10Q, 10Z, 10AD, and 10AH; (51) 10AT, 10E, 10Q, 10AC, 10AG, and 10AH; (52) 10AT, 10P, 10N, 10AD, and 10AH; (53) 10AT, 10P, 10Y, 10Z, 10AD, and 10AH; (54) 10AT, 10P, 10Y, 10AC, 10AG, and 10AH; (55) 10AT, 10P, 10AB, 10V, and 10AH; (56) 10AT, 10P, 10AB, 10AF, 10AG, and 10AH; (57) 10AT, 10P, 10AB, and 10W; (58) 10AU, 10AF, 10AG, and 10AH; (59) 10AU, and 10W; and (60) 10AU, 10V, and 10AH,

wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10T is a crotonyl-ACP thioesterase, wherein 10U is a crotonyl-ACP reductase (aldehyde forming), wherein 10V is a crotonyl-CoA reductase (aldehyde forming), wherein 10W is a crotonyl-CoA (alcohol forming), wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AC is a 3-hydroxybutyrate dehydratase, wherein LOAD is a 3-hydroxybutyraldehyde dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein LOAF is a crotonyl-CoA hydrolase, transferase or synthetase, wherein 10AG is a crotonate reductase, wherein 10AH is a crotonaldehyde reductase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described above, wherein the organism further comprises a 13BDO pathway. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a 13BDO pathway enzyme expressed in a sufficient amount to produce 13BDO, wherein said 13BDO pathway comprises a pathway shown in FIGS. 1 and 10 selected from: (1) 10A, 10D, 10E, 10F, 10G, and 10S; (2) 10A, 10D, 10I, 10G, and 10S; (3) 10A, 10D, 10K, and 10S; (4) 10A, 10H, 10F, 10G, and 10S; (5) 10A, 10J, 10G, and 10S; (6) 10A, 10J, 10R, and 10AA; (7) 10A, 10H, 10F, 10R, and 10AA; (8) 10A, 10H, 10Q, 10Z, and 10AA; (9) 10A, 10D, 10I, 10R, and 10AA; (10) 10A, 10D, 10E, 10F, 10R, and 10AA; (11) 10A, 10D, 10E, 10Q, 10Z, and 10AA; (12) 10A, 10D, 10P, 10N, and 10AA; (13) 10A, 10D, 10P, 10Y, 10Z, and 10AA; (14) 10A, 10B, 10M, and 10AA; (15) 10A, 10B, 10L, 10Z, and 10AA; (16) 10A, 10B, 10X, 10N, and 10AA; (17) 10A, 10B, 10X, 10Y, 10Z, and 10AA; (18) 10A, 10D, 10P, and 10O; (19) 10A, 10B, 10X, and 10O; (20) 10A, 10D, 10E, 10F, 10R, and 10AA; (21) 10A, 10D, 10E, 10F, 10G, and 10S; (22) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, and 10AA; (23) 10A, 10B, 10C, 10AE, 10AB, 10N, and 10AA; (24) 10A, 10B, 10C, 10AE, 10AB, and 10O; (25) 10AU, 10AB, 10Y, 10Z, and 10AA; (26) 10AU, 10AB, 10N, and 10AA; (27) 10AU, 10AB, and 100; (28) 1T, 10AS, 10E, 10F, 10G, and 10S; (29) 1T, 10AS, 10I, 10G, and 10S; (30) 1T, 10AS, 10K, and 10S; (31) 1T, 10AS, 10I, 10R, and 10AA; (32) 1T, 10AS, 10E, 10F, 10R, and 10AA; (33) 1T, 10AS, 10E, 10Q, 10Z, and 10AA; (34) 1T, 10AS, 10P, 10N, and 10AA; (35) 1T, 10AS, 10P, 10Y, 10Z, and 10AA; (36) 1T, 10AS, 10P, and 10O; (37) 1T, 10AS, 10E, 10F, 10R, and 10AA; (38) 10AT, 10E, 10F, 10G, and 10S; (39) 10AT, 10I, 10G, and 10S; (40) 10AT, 10K, and 10S; (41) 10AT, 10I, 10R, and 10AA; (42) 10AT, 10E, 10F, 10R, and 10AA; (43) 10AT, 10E, 10Q, 10Z, and 10AA; (44) 10AT, 10P, 10N, and 10AA; (45) 10AT, 10P, 10Y, 10Z, and 10AA; (46) 10AT, 10P, and 10O; and (47) 10AT, 10E, 10F, 10R, and 10AA, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 10O is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a MVC pathway including at least one exogenous nucleic acid encoding a MVC pathway enzyme expressed in a sufficient amount to produce MVC, wherein the MVC pathway includes a pathway shown in FIGS. 1, 10, and 13-18 selected from: (1) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (2) 10A, 10D, 10I, 10G, 10S, 15A, 15B, and 15C; (3) 10A, 10D, 10K, 10S, 15A, 15B, and 15C; (4) 10A, 10H, 10F, 10G, 10S, 15A, 15B, and 15C; (5) 10A, 10J, 10G, 10S, 15A, 15B, and 15C; (6) 10A, 10J, 10R, 10AA, 15A, 15B, and 15C; (7) 10A, 10H, 10F, 10R, 10AA, 15A, 15B, and 15C; (8) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (9) 10A, 10D, 10I, 10R, 10AA, 15A, 15B, and 15C; (10) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (11) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (12) 10A, 10D, 10P, 10N, 10AA, 15A, 15B, and 15C; (13) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (14) 10A, 10B, 10M, 10AA, 15A, 15B, and 15C; (15) 10A, 10B, 10L, 10Z, 10AA, 15A, 15B, and 15C; (16) 10A, 10B, 10X, 10N, 10AA, 15A, 15B, and 15C; (17) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (18) 10A, 10D, 10P, 10O, 15A, 15B, and 15C; (19) 10A, 10B, 10X, 10O, 15A, 15B, and 15C; (20) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (21) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (22) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (23) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15B, and 15C; (24) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15B, and 15C; (25) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (26) 10AU, 10AB, 10N, 10AA, 15A, 15B, and 15C; (27) 10AU, 10AB, 10O, 15A, 15B, and 15C; (28) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (29) 1T, 10AS, 10I, 10G, 10S, 15A, 15B, and 15C; (30) 1T, 10AS, 10K, 10S, 15A, 15B, and 15C; (31) 1T, 10AS, 10I, 10R, 10AA, 15A, 15B, and 15C; (32) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (33) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (34) 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, and 15C; (35) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (36) 1T, 10AS, 10P, 10O, 15A, 15B, and 15C; (37) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (38) 10AT, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (39) 10AT, 10I, 10G, 10S, 15A, 15B, and 15C; (40) 10AT, 10K, 10S, 15A, 15B, and 15C; (41) 10AT, 10I, 10R, 10AA, 15A, 15B, and 15C; (42) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (43) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (44) 10AT, 10P, 10N, 10AA, 15A, 15B, and 15C; (45) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (46) 10AT, 10P, 10O, 15A, 15B, and 15C; (47) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (48) 10A, 10D, 10E, 10F, 10G, 10S, and 15D; (49) 10A, 10D, 10I, 10G, 10S, and 15D; (50) 10A, 10D, 10K, 10S, and 15D; (51) 10A, 10H, 10F, 10G, 10S, and 15D; (52) 10A, 10J, 10G, 10S, and 15D; (53) 10A, 10J, 10R, 10AA, and 15D; (54) 10A, 10H, 10F, 10R, 10AA, and 15D; (55) 10A, 10H, 10Q, 10Z, 10AA, and 15D; (56) 10A, 10D, 10I, 10R, 10AA, and 15D; (57) 10A, 10D, 10E, 10F, 10R, 10AA, and 15D; (58) 10A, 10D, 10E, 10Q, 10Z, 10AA, and 15D; (59) 10A, 10D, 10P, 10N, 10AA, and 15D; (60) 10A, 10D, 10P, 10Y, 10Z, 10AA, and 15D; (61) 10A, 10B, 10M, 10AA, and 15D; (62) 10A, 10B, 10L, 10Z, 10AA, and 15D; (63) 10A, 10B, 10X, 10N, 10AA, and 15D; (64) 10A, 10B, 10X, 10Y, 10Z, 10AA, and 15D; (65) 10A, 10D, 10P, 10O, and 15D; (66) 10A, 10B, 10X, 10O, and 15D; (67) 10A, 10D, 10E, 10F, 10R, 10AA, and 15D; (68) 10A, 10D, 10E, 10F, 10G, 10S, and 15D; (69) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, and 15D; (70) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, and 15D; (71) 10A, 10B, 10C, 10AE, 10AB, 10O, and 15D; (72) 10AU, 10AB, 10Y, 10Z, 10AA, and 15D; (73) 10AU, 10AB, 10N, 10AA, and 15D; (74) 10AU, 10AB, 10O, and 15D; (75) 1T, 10AS, 10E, 10F, 10G, 10S, and 15D; (76) 1T, 10AS, 10I, 10G, 10S, and 15D; (77) 1T, 10AS, 10K, 10S, and 15D; (78) 1T, 10AS, 10I, 10R, 10AA, and 15D; (79) 1T, 10AS, 10E, 10F, 10R, 10AA, and 15D; (80) 1T, 10AS, 10E, 10Q, 10Z, 10AA, and 15D; (81) 1T, 10AS, 10P, 10N, 10AA, and 15D; (82) 1T, 10AS, 10P, 10Y, 10Z, 10AA, and 15D; (83) 1T, 10AS, 10P, 10O, and 15D; (84) 1T, 10AS, 10E, 10F, 10R, 10AA, and 15D; (85) 10AT, 10E, 10F, 10G, 10S, and 15D; (86) 10AT, 10I, 10G, 10S, and 15D; (87) 10AT, 10K, 10S, and 15D; (88) 10AT, 10I, 10R, 10AA, and 15D; (89) 10AT, 10E, 10F, 10R, 10AA, and 15D; (90) 10AT, 10E, 10Q, 10Z, 10AA, and 15D; (91) 10AT, 10P, 10N, 10AA, and 15D; (92) 10AT, 10P, 10Y, 10Z, 10AA, and 15D; (93) 10AT, 10P, 10O, and 15D; (94) 10AT, 10E, 10F, 10R, 10AA, and 15D; (95) 10A, 10D, 10E, 10F, 10G, 10S, 15E, and 15C; (96) 10A, 10D, 10I, 10G, 10S, 15E, and 15C; (97) 10A, 10D, 10K, 10S, 15E, and 15C; (98) 10A, 10H, 10F, 10G, 10S, 15E, and 15C; (99) 10A, 10J, 10G, 10S, 15E, and 15C; (100) 10A, 10J, 10R, 10AA, 15E, and 15C; (10P) 10A, 10H, 10F, 10R, 10AA, 15E, and 15C; (102) 10A, 10H, 10Q, 10Z, 10AA, 15E, and 15C; (103) 10A, 10D, 10I, 10R, 10AA, 15E, and 15C; (104) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, and 15C; (105) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (106) 10A, 10D, 10P, 10N, 10AA, 15E, and 15C; (107) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (108) 10A, 10B, 10M, 10AA, 15E, and 15C; (109) 10A, 10B, 10L, 10Z, 10AA, 15E, and 15C; (110) 10A, 10B, 10X, 10N, 10AA, 15E, and 15C; (111) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15E, and 15C; (112) 10A, 10D, 10I), 10O, 15E, and 15C; (113) 10A, 10B, 10X, 10O, 15E, and 15C; (114) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, and 15C; (115) 10A, 10D, 10E, 10F, 10G, 10S, 15E, and 15C; (116) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15E, and 15C; (117) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15E, and 15C; (118) 10A, 10B, 10C, 10AE, 10AB, 10O, 15E, and 15C; (119) 10AU, 10AB, 10Y, 10Z, 10AA, 15E, and 15C; (120) 10AU, 10AB, 10N, 10AA, 15E, and 15C; (121) 10AU, 10AB, 10O, 15E, and 15C; (122) 1T, 10AS, 10E, 10F, 10G, 10S, 15E, and 15C; (123) 1T, 10AS, 10I, 10G, 10S, 15E, and 15C; (124) 1T, 10AS, 10K, 10S, 15E, and 15C; (125) 1T, 10AS, 10I, 10R, 10AA, 15E, and 15C; (126) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, and 15C; (127) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (128) 1T, 10AS, 10P, 10N, 10AA, 15E, and 15C; (129) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (130) 1T, 10AS, 10P, 10O, 15E, and 15C; (131) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, and 15C; (132) 10AT, 10E, 10F, 10G, 10S, 15E, and 15C; (133) 10AT, 10I, 10G, 10S, 15E, and 15C; (134) 10AT, 10K, 10S, 15E, and 15C; (135) 10AT, 10I, 10R, 10AA, 15E, and 15C; (136) 10AT, 10E, 10F, 10R, 10AA, 15E, and 15C; (137) 10AT, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (138) 10AT, 10P, 10N, 10AA, 15E, and 15C; (139) 10AT, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (140) 10AT, 10P, 10O, 15E, and 15C; (141) 10AT, 10E, 10F, 10R, 10AA, 15E, and 15C; (142) 10A, 10D, 10E, 10F, 10G, 10S, 15A, and 15F; (143) 10A, 10D, 10I, 10G, 10S, 15A, and 15F; (144) 10A, 10D, 10K, 10S, 15A, and 15F; (145) 10A, 10H, 10F, 10G, 10S, 15A, and 15F; (146) 10A, 10J, 10G, 10S, 15A, and 15F; (147) 10A, 10J, 10R, 10AA, 15A, and 15F; (148) 10A, 10H, 10F, 10R, 10AA, 15A, and 15F; (149) 10A, 10H, 10Q, 10Z, 10AA, 15A, and 15F; (150) 10A, 10D, 10I, 10R, 10AA, 15A, and 15F; (151) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, and 15F; (152) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (153) 10A, 10D, 10P, 10N, 10AA, 15A, and 15F; (154) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (155) 10A, 10B, 10M, 10AA, 15A, and 15F; (156) 10A, 10B, 10L, 10Z, 10AA, 15A, and 15F; (157) 10A, 10B, 10X, 10N, 10AA, 15A, and 15F; (158) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, and 15F; (159) 10A, 10D, 10P, 10O, 15A, and 15F; (160) 10A, 10B, 10X, 10O, 15A, and 15F; (161) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, and 15F; (162) 10A, 10D, 10E, 10F, 10G, 10S, 15A, and 15F; (163) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, and 15F; (164) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, and 15F; (165) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, and 15F; (166) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, and 15F; (167) 10AU, 10AB, 10N, 10AA, 15A, and 15F; (168) 10AU, 10AB, 10O, 15A, and 15F; (169) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, and 15F; (170) 1T, 10AS, 10I, 10G, 10S, 15A, and 15F; (171) 1T, 10AS, 10K, 10S, 15A, and 15F; (172) 1T, 10AS, 10I, 10R, 10AA, 15A, and 15F; (173) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, and 15F; (174) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (175) 1T, 10AS, 10P, 10N, 10AA, 15A, and 15F; (176) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (177) 1T, 10AS, 10P, 10O, 15A, and 15F; (178) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, and 15F; (179) 10AT, 10E, 10F, 10G, 10S, 15A, and 15F; (180) 10AT, 10I, 10G, 10S, 15A, and 15F; (181) 10AT, 10K, 10S, 15A, and 15F; (182) 10AT, 10I, 10R, 10AA, 15A, and 15F; (183) 10AT, 10E, 10F, 10R, 10AA, 15A, and 15F; (184) 10AT, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (185) 10AT, 10P, 10N, 10AA, 15A, and 15F; (186) 10AT, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (187) 10AT, 10P, 10O, 15A, and 15F; (188) 10AT, 10E, 10F, 10R, 10AA, 15A, and 15F; (189) 14A, 14B, 14C, 14D, 14E, and 13A; (190) 16A, 16B, 16C, and 16D; (191) 17A, 17B, 17C, and 17D; (192) 17A, 17E, 17F, and 17D; (193) 17A, 17B, 17C, 17H, 17I, and 17J; (194) 18A, 18B, 18C, 18D, and 18E; and (195) 17A, 17E, 17F, 17H, 17I, and 17J, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 100 is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 13A is a 2-butanol desaturase, wherein 14A is an acetolactate synthase, wherein 14B is an acetolactate decarboxylase, wherein 14C is a butanediol dehydrogenase, wherein 14D is a butanediol dehydratase, wherein 14E is a butanol dehydrogenase, wherein 15A is a 13BDO kinase, wherein 15B is a 3-hydroxybutyrylphosphate kinase, 15C is a 3-hydroxybutyryldiphosphate lyase, wherein 15D is a 13BDO diphosphokinase, wherein 15E is a 13BDO dehydratase, wherein 15F is a 3-hydroxybutyrylphosphate lyase, wherein 16A is a 3-oxopent-4-enoyl-CoA thiolase, wherein 16B is a 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, wherein 16C is a 3-oxopent-4-enoate decarboxylase or spontaneous, wherein 16D is a 3-buten-2-one reductase, wherein 17A is a 3-oxo-4-hydroxypentanoyl-CoA thiolase, wherein 17B is a 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17C is a 3-oxo-4-hydroxypentanoate reductase, wherein 17D is a 3,4-dihydroxypentanoate decarboxylase, wherein 17E is a 3-oxo-4-hydroxypentanoyl-CoA reductase, wherein 17F is a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17H is a 3,4-dihydroxypentanoate dehydratase, wherein 171 is a 4-oxopentanoate reductase, wherein 17I is a 4-hyd4-oxoperoxypentanoate decarboxylase, wherein 18A is a 3-oxoadipyl-CoA thiolase, wherein 18B is a 3-oxoadipyl-CoA transferase, synthetase or hydrolase, wherein 18C is a 3-oxoadipate decarboxylase or spontaneous, wherein 18D is a 4-oxopentanoate reductase, wherein 18E is a 4-hydroxypentanoate decarboxylase.

In one aspect, the non-naturally occurring microbial organism a MVC pathway described above further comprises a FaldFP comprising at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS.

In one aspect, the non-naturally occurring microbial organism having a MVC pathway described above further comprises a MMP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises a pathway selected from: (1) 3J; (2) 3A and 3B; (3) 3A, 3B and 3C; (4) 3J, 3K and 3C; (5) 3J, 3M, and 3N; (6) 3J and 3L; (7) 3A, 3B, 3C, 3D, and 3E; (8) 3A, 3B, 3C, 3D, and 3F; (9) 3J, 3K, 3C, 3D, and 3E; (10) 3J, 3K, 3C, 3D, and 3F; (11) 3J, 3M, 3N, and 30; (12) 3A, 3B, 3C, 3D, 3E, and 3G; (13) 3A, 3B, 3C, 3D, 3F, and 3G; (14) 3J, 3K, 3C, 3D, 3E, and 3G; (15) 3J, 3K, 3C, 3D, 3F, and 3G; (16) 3J, 3M, 3N, 30, and 3G; (17) 3A, 3B, 3C, 3D, 3E, and 3I; (18) 3A, 3B, 3C, 3D, 3F, and 3I; (19) 3J, 3K, 3C, 3D, 3E, and 3I; (20) 3J, 3K, 3C, 3D, 3F, and 3I; and (21) 3J, 3M, 3N, 30, and 31, wherein 3A is a methanol methyltransferase, wherein 3B is a methylenetetrahydrofolate reductase, wherein 3C is a MTHFDH, wherein 3D is a methenyltetrahydrofolate cyclohydrolase, wherein 3E is a formyltetrahydrofolate deformylase, wherein 3F is a FTHFS, wherein 3G is a formate hydrogen lyase, wherein 3H is a hydrogenase, wherein 31 is a FDH, wherein 3J is a MeDH, wherein 3K is a formaldehyde activating enzyme or spontaneous, wherein 3L is a formaldehyde dehydrogenase, wherein 3M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 3N is a glutathione-dependent formaldehyde dehydrogenase, wherein 30 is a S-formylglutathione hydrolase,

In one aspect, the non-naturally occurring microbial organism having a MVC pathway described above further comprises a MOP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein lA a MeDH.

In one aspect, the non-naturally occurring microbial organism having a MVC pathway described above further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH. In certain embodiments, the organism comprises an exogenous nucleic acid encoding said hydrogenase or said CODH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described above, wherein the organism further comprises a MVC pathway. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a MVC pathway enzyme expressed in a sufficient amount to produce MVC, wherein said MVC pathway comprises a pathway as shown in FIGS. 1, 10 and 13-18 selected from: (1) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (2) 10A, 10D, 10I, 10G, 10S, 15A, 15B, and 15C; (3) 10A, 10D, 10K, 10S, 15A, 15B, and 15C; (4) 10A, 10H, 10F, 10G, 10S, 15A, 15B, and 15C; (5) 10A, 10J, 10G, 10S, 15A, 15B, and 15C; (6) 10A, 10J, 10R, 10AA, 15A, 15B, and 15C; (7) 10A, 10H, 10F, 10R, 10AA, 15A, 15B, and 15C; (8) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (9) 10A, 10D, 10I, 10R, 10AA, 15A, 15B, and 15C; (10) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (11) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (12) 10A, 10D, 10P, 10N, 10AA, 15A, 15B, and 15C; (13) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (14) 10A, 10B, 10M, 10AA, 15A, 15B, and 15C; (15) 10A, 10B, 10L, 10Z, 10AA, 15A, 15B, and 15C; (16) 10A, 10B, 10X, 10N, 10AA, 15A, 15B, and 15C; (17) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (18) 10A, 10D, 10P, 10O, 15A, 15B, and 15C; (19) 10A, 10B, 10X, 10O, 15A, 15B, and 15C; (20) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (21) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (22) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (23) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15B, and 15C; (24) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15B, and 15C; (25) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (26) 10AU, 10AB, 10N, 10AA, 15A, 15B, and 15C; (27) 10AU, 10AB, 10O, 15A, 15B, and 15C; (28) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (29) 1T, 10AS, 10I, 10G, 10S, 15A, 15B, and 15C; (30) 1T, 10AS, 10K, 10S, 15A, 15B, and 15C; (31) 1T, 10AS, 10I, 10R, 10AA, 15A, 15B, and 15C; (32) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (33) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (34) 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, and 15C; (35) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (36) 1T, 10AS, 10P, 10O, 15A, 15B, and 15C; (37) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (38) 10AT, 10E, 10F, 10G, 10S, 15A, 15B, and 15C; (39) 10AT, 10I, 10G, 10S, 15A, 15B, and 15C; (40) 10AT, 10K, 10S, 15A, 15B, and 15C; (41) 10AT, 10I, 10R, 10AA, 15A, 15B, and 15C; (42) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (43) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15B, and 15C; (44) 10AT, 10P, 10N, 10AA, 15A, 15B, and 15C; (45) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15B, and 15C; (46) 10AT, 10P, 10O, 15A, 15B, and 15C; (47) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, and 15C; (48) 10A, 10D, 10E, 10F, 10G, 10S, and 15D; (49) 10A, 10D, 10I, 10G, 10S, and 15D; (50) 10A, 10D, 10K, 10S, and 15D; (51) 10A, 10H, 10F, 10G, 10S, and 15D; (52) 10A, 10J, 10G, 10S, and 15D; (53) 10A, 10J, 10R, 10AA, and 15D; (54) 10A, 10H, 10F, 10R, 10AA, and 15D; (55) 10A, 10H, 10Q, 10Z, 10AA, and 15D; (56) 10A, 10D, 10I, 10R, 10AA, and 15D; (57) 10A, 10D, 10E, 10F, 10R, 10AA, and 15D; (58) 10A, 10D, 10E, 10Q, 10Z, 10AA, and 15D; (59) 10A, 10D, 10P, 10N, 10AA, and 15D; (60) 10A, 10D, 10P, 10Y, 10Z, 10AA, and 15D; (61) 10A, 10B, 10M, 10AA, and 15D; (62) 10A, 10B, 10L, 10Z, 10AA, and 15D; (63) 10A, 10B, 10X, 10N, 10AA, and 15D; (64) 10A, 10B, 10X, 10Y, 10Z, 10AA, and 15D; (65) 10A, 10D, 10P, 10O, and 15D; (66) 10A, 10B, 10X, 10O, and 15D; (67) 10A, 10D, 10E, 10F, 10R, 10AA, and 15D; (68) 10A, 10D, 10E, 10F, 10G, 10S, and 15D; (69) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, and 15D; (70) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, and 15D; (71) 10A, 10B, 10C, 10AE, 10AB, 10O, and 15D; (72) 10AU, 10AB, 10Y, 10Z, 10AA, and 15D; (73) 10AU, 10AB, 10N, 10AA, and 15D; (74) 10AU, 10AB, 10O, and 15D; (75) 1T, 10AS, 10E, 10F, 10G, 10S, and 15D; (76) 1T, 10AS, 10I, 10G, 10S, and 15D; (77) 1T, 10AS, 10K, 10S, and 15D; (78) 1T, 10AS, 10I, 10R, 10AA, and 15D; (79) 1T, 10AS, 10E, 10F, 10R, 10AA, and 15D; (80) 1T, 10AS, 10E, 10Q, 10Z, 10AA, and 15D; (81) 1T, 10AS, 10P, 10N, 10AA, and 15D; (82) 1T, 10AS, 10P, 10Y, 10Z, 10AA, and 15D; (83) 1T, 10AS, 10P, 10O, and 15D; (84) 1T, 10AS, 10E, 10F, 10R, 10AA, and 15D; (85) 10AT, 10E, 10F, 10G, 10S, and 15D; (86) 10AT, 10I, 10G, 10S, and 15D; (87) 10AT, 10K, 10S, and 15D; (88) 10AT, 10I, 10R, 10AA, and 15D; (89) 10AT, 10E, 10F, 10R, 10AA, and 15D; (90) 10AT, 10E, 10Q, 10Z, 10AA, and 15D; (91) 10AT, 10P, 10N, 10AA, and 15D; (92) 10AT, 10P, 10Y, 10Z, 10AA, and 15D; (93) 10AT, 10P, 10O, and 15D; (94) 10AT, 10E, 10F, 10R, 10AA, and 15D; (95) 10A, 10D, 10E, 10F, 10G, 10S, 15E, and 15C; (96) 10A, 10D, 10I, 10G, 10S, 15E, and 15C; (97) 10A, 10D, 10K, 10S, 15E, and 15C; (98) 10A, 10H, 10F, 10G, 10S, 15E, and 15C; (99) 10A, 10J, 10G, 10S, 15E, and 15C; (100) 10A, 10J, 10R, 10AA, 15E, and 15C; (101) 10A, 10H, 10F, 10R, 10AA, 15E, and 15C; (102) 10A, 10H, 10Q, 10Z, 10AA, 15E, and 15C; (103) 10A, 10D, 10I, 10R, 10AA, 15E, and 15C; (104) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, and 15C; (105) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (106) 10A, 10D, 10P, 10N, 10AA, 15E, and 15C; (107) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (108) 10A, 10B, 10M, 10AA, 15E, and 15C; (109) 10A, 10B, 10L, 10Z, 10AA, 15E, and 15C; (110) 10A, 10B, 10X, 10N, 10AA, 15E, and 15C; (111) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15E, and 15C; (112) 10A, 10D, 10P, 10O, 15E, and 15C; (113) 10A, 10B, 10X, 10O, 15E, and 15C; (114) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, and 15C; (115) 10A, 10D, 10E, 10F, 10G, 10S, 15E, and 15C; (116) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15E, and 15C; (117) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15E, and 15C; (118) 10A, 10B, 10C, 10AE, 10AB, 10O, 15E, and 15C; (119) 10AU, 10AB, 10Y, 10Z, 10AA, 15E, and 15C; (120) 10AU, 10AB, 10N, 10AA, 15E, and 15C; (121) 10AU, 10AB, 10O, 15E, and 15C; (122) 1T, 10AS, 10E, 10F, 10G, 10S, 15E, and 15C; (123) 1T, 10AS, 10I, 10G, 10S, 15E, and 15C; (124) 1T, 10AS, 10K, 10S, 15E, and 15C; (125) 1T, 10AS, 10I, 10R, 10AA, 15E, and 15C; (126) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, and 15C; (127) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (128) 1T, 10AS, 10P, 10N, 10AA, 15E, and 15C; (129) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (130) 1T, 10AS, 10P, 100, 15E, and 15C; (131) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, and 15C; (132) 10AT, 10E, 10F, 10G, 10S, 15E, and 15C; (133) 10AT, 10I, 10G, 10S, 15E, and 15C; (134) 10AT, 10K, 10S, 15E, and 15C; (135) 10AT, 10I, 10R, 10AA, 15E, and 15C; (136) 10AT, 10E, 10F, 10R, 10AA, 15E, and 15C; (137) 10AT, 10E, 10Q, 10Z, 10AA, 15E, and 15C; (138) 10AT, 10P, 10N, 10AA, 15E, and 15C; (139) 10AT, 10P, 10Y, 10Z, 10AA, 15E, and 15C; (140) 10AT, 10P, 10O, 15E, and 15C; (141) 10AT, 10E, 10F, 10R, 10AA, 15E, and 15C; (142) 10A, 10D, 10E, 10F, 10G, 10S, 15A, and 15F; (143) 10A, 10D, 10I, 10G, 10S, 15A, and 15F; (144) 10A, 10D, 10K, 10S, 15A, and 15F; (145) 10A, 10H, 10F, 10G, 10S, 15A, and 15F; (146) 10A, 10J, 10G, 10S, 15A, and 15F; (147) 10A, 10J, 10R, 10AA, 15A, and 15F; (148) 10A, 10H, 10F, 10R, 10AA, 15A, and 15F; (149) 10A, 10H, 10Q, 10Z, 10AA, 15A, and 15F; (150) 10A, 10D, 10I, 10R, 10AA, 15A, and 15F; (151) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, and 15F; (152) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (153) 10A, 10D, 10P, 10N, 10AA, 15A, and 15F; (154) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (155) 10A, 10B, 10M, 10AA, 15A, and 15F; (156) 10A, 10B, 10L, 10Z, 10AA, 15A, and 15F; (157) 10A, 10B, 10X, 10N, 10AA, 15A, and 15F; (158) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, and 15F; (159) 10A, 10D, 10P, 10O, 15A, and 15F; (160) 10A, 10B, 10X, 10O, 15A, and 15F; (161) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, and 15F; (162) 10A, 10D, 10E, 10F, 10G, 10S, 15A, and 15F; (163) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, and 15F; (164) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, and 15F; (165) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, and 15F; (166) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, and 15F; (167) 10AU, 10AB, 10N, 10AA, 15A, and 15F; (168) 10AU, 10AB, 10O, 15A, and 15F; (169) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, and 15F; (170) 1T, 10AS, 10I, 10G, 10S, 15A, and 15F; (171) 1T, 10AS, 10K, 10S, 15A, and 15F; (172) 1T, 10AS, 10I, 10R, 10AA, 15A, and 15F; (173) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, and 15F; (174) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (175) 1T, 10AS, 10P, 10N, 10AA, 15A, and 15F; (176) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (177) 1T, 10AS, 10P, 10O, 15A, and 15F; (178) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, and 15F; (179) 10AT, 10E, 10F, 10G, 10S, 15A, and 15F; (180) 10AT, 10I, 10G, 10S, 15A, and 15F; (181) 10AT, 10K, 10S, 15A, and 15F; (182) 10AT, 10I, 10R, 10AA, 15A, and 15F; (183) 10AT, 10E, 10F, 10R, 10AA, 15A, and 15F; (184) 10AT, 10E, 10Q, 10Z, 10AA, 15A, and 15F; (185) 10AT, 10P, 10N, 10AA, 15A, and 15F; (186) 10AT, 10P, 10Y, 10Z, 10AA, 15A, and 15F; (187) 10AT, 10P, 10O, 15A, and 15F; (188) 10AT, 10E, 10F, 10R, 10AA, 15A, and 15F; (189) 14A, 14B, 14C, 14D, 14E, and 13A; (190) 16A, 16B, 16C, and 16D; (191) 17A, 17B, 17C, and 17D; (192) 17A, 17E, 17F, and 17D; (193) 17A, 17B, 17C, 17H, 17I, and 17J; (194) 18A, 18B, 18C, 18D, and 18E; (195) 13A; and (196) 17A, 17E, 17F, 17H, 17I, and 17J, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 101 is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 100 is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 13A is a 2-butanol desaturase, wherein 14A is an acetolactate synthase, wherein 14B is an acetolactate decarboxylase, wherein 14C is a butanediol dehydrogenase, wherein 14D is a butanediol dehydratase, wherein 14E is a butanol dehydrogenase, wherein 15A is a 13BDO kinase, wherein 15B is a 3-hydroxybutyrylphosphate kinase, 15C is a 3-hydroxybutyryldiphosphate lyase, wherein 15D is a 13BDO diphosphokinase, wherein 15E is a 13BDO dehydratase, wherein 15F is a 3-hydroxybutyrylphosphate lyase, wherein 16A is a 3-oxopent-4-enoyl-CoA thiolase, wherein 16B is a 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, wherein 16C is a 3-oxopent-4-enoate decarboxylase or spontaneous, wherein 16D is a 3-buten-2-one reductase, wherein 17A is a 3-oxo-4-hydroxypentanoyl-CoA thiolase, wherein 17B is a 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17C is a 3-oxo-4-hydroxypentanoate reductase, wherein 17D is a 3,4-dihydroxypentanoate decarboxylase, wherein 17E is a 3-oxo-4-hydroxypentanoyl-CoA reductase, wherein 17F is a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17H is a 3,4-dihydroxypentanoate dehydratase, wherein 171 is a 4-oxopentanoate reductase, wherein 17J is a 4-hyd4-oxoperoxypentanoate decarboxylase, wherein 18A is a 3-oxoadipyl-CoA thiolase, wherein 18B is a 3-oxoadipyl-CoA transferase, synthetase or hydrolase, wherein 18C is a 3-oxoadipate decarboxylase or spontaneous, wherein 18D is a 4-oxopentanoate reductase, wherein 18E is a 4-hydroxypentanoate decarboxylase.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a 3-buten-1-ol pathway including at least one exogenous nucleic acid encoding a 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce 3-buten-1-ol, wherein the 3-buten-1-ol pathway includes a pathway shown in FIGS. 1, 10 and 19 selected from: (1) 10A, 10B, 10C, 10AE, 19A, 19B, and 19C; (2) 10A, 10B, 10X, 10AB, 19A, 19B, and 19C; (3) 10A, 10D, 10P, 10AB, 19A, 19B, and 19C; (4) 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C; (5) 10AT, 10P, 10AB, 19A, 19B, and 19C; (6) 10P, 10AB, 19A, 19B, and 19C; (7) 10AU, 19A, 19B, and 19C; and (8) 19A, 19B, and 19C, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10P is an AcAcCoAR(ketone reducing), wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase.

In some aspects, the microbial organism can include one, two, three, four, five, six or seven exogenous nucleic acids each encoding a butadiene pathway enzyme. In some aspects, microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the butadiene pathways selected from (1)-(8). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In one aspect, the non-naturally occurring microbial organism a 3-buten-1-ol pathway described above further comprises a FaldFP comprising at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS.

In one aspect, the non-naturally occurring microbial organism having a 3-buten-1-ol pathway described above further comprises a MMP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount to produce formaldehyde or produce or enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises a pathway selected from: (1) 3J; (2) 3A and 3B; (3) 3A, 3B and 3C; (4) 3J, 3K and 3C; (5) 3J, 3M, and 3N; (6) 3J and 3L; (7) 3A, 3B, 3C, 3D, and 3E; (8) 3A, 3B, 3C, 3D, and 3F; (9) 3J, 3K, 3C, 3D, and 3E; (10) 3J, 3K, 3C, 3D, and 3F; (11) 3J, 3M, 3N, and 30; (12) 3A, 3B, 3C, 3D, 3E, and 3G; (13) 3A, 3B, 3C, 3D, 3F, and 3G; (14) 3J, 3K, 3C, 3D, 3E, and 3G; (15) 3J, 3K, 3C, 3D, 3F, and 3G; (16) 3J, 3M, 3N, 30, and 3G; (17) 3A, 3B, 3C, 3D, 3E, and 3I; (18) 3A, 3B, 3C, 3D, 3F, and 3I; (19) 3J, 3K, 3C, 3D, 3E, and 3I; (20) 3J, 3K, 3C, 3D, 3F, and 3I; and (21) 3J, 3M, 3N, 30, and 31, wherein 3A is a methanol methyltransferase, wherein 3B is a methylenetetrahydrofolate reductase, wherein 3C is a MTHFDH, wherein 3D is a methenyltetrahydrofolate cyclohydrolase, wherein 3E is a formyltetrahydrofolate deformylase, wherein 3F is a FTHFS, wherein 3G is a formate hydrogen lyase, wherein 3H is a hydrogenase, wherein 31 is a FDH, wherein 3J is a MeDH, wherein 3K is a formaldehyde activating enzyme or spontaneous, wherein 3L is a formaldehyde dehydrogenase, wherein 3M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 3N is a glutathione-dependent formaldehyde dehydrogenase, wherein 30 is a S-formylglutathione hydrolase,

In one aspect, the non-naturally occurring microbial organism having a 3-buten-1-ol pathway described above further comprises a MOP. In certain embodiments, the organism comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises 1A, wherein 1A a MeDH.

In one aspect, the non-naturally occurring microbial organism having a 3-buten-1-ol pathway described above further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a CODH. In certain embodiments, the organism comprises an exogenous nucleic acid encoding said hydrogenase or said CODH.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described above, wherein the organism further comprises a 3-buten-1-ol pathway. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce 3-buten-1-ol, wherein said 3-buten-1-ol pathway comprises a pathway as shown in FIGS. 1, 10 and 19 selected from: (1) 10A, 10B, 10C, 10AE, 19A, 19B, and 19C; (2) 10A, 10B, 10X, 10AB, 19A, 19B, and 19C; (3) 10A, 10D, 10P, 10AB, 19A, 19B, and 19C; (4) 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C; (5) 10AT, 10P, 10AB, 19A, 19B, and 19C; (6) 10P, 10AB, 19A, 19B, and 19C; (7) 10AU, 19A, 19B, and 19C; and (8) 19A, 19B, and 19C, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10P is an AcAcCoAR(ketone reducing), wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase.

In some aspects, the microbial organism can include one, two, three, four, five, six or seven exogenous nucleic acids each encoding a butadiene pathway enzyme. In some aspects, microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the butadiene pathways selected from (1)-(8). In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

In certain embodiments, provided herein is a non-naturally occurring microbial organism having a FaldFP, a FAP, a MOP, and a butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway. In some aspects, the organism comprises at least one exogenous nucleic acid encoding a FaldFP enzyme expressed in a sufficient amount to produce pyruvate, wherein said FaldFP comprises. (1) 1B and 1C; or (2) 1D, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6P3HI, wherein 1D is a DHAS, comprises at least one exogenous nucleic acid encoding a FAP enzyme expressed in a sufficient amount to produce formaldehyde, pyruvate, or acetyl-CoA, wherein said FAP comprises a pathway selected from: (3) 1E; (4) 1F, and 1G; (5) 1H, 1I, 1J, and 1K; (6) 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (10) 1H, 1I, 1J, 1O, and 1P5, comprises at least one exogenous nucleic acid encoding a MOP enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol, wherein said MOP comprises a methanol dehydrdogenase, and comprises at least one exogenous nucleic acid encoding a butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol, wherein said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises a pathway selected from: steps 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; or steps 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; or steps 14A, 14B, 14C, 14D, 14E, 13A, and 13B; or steps 17A, 17B, 17C, 17D, and 17G; or steps 17A, 17E, 17F, 17D, and 17G; or steps 18A, 18B, 18C, 18D, 18E, and 18F; or steps 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D; or steps 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D; or steps 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10AU, 19A, 19B, 19C, and 19D; or steps 19A, 19B, 19C, and 19D; or steps 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; or steps 10AT, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; or steps 13A and 13B; or steps 1T, 10AS, 10P, 10AB, 10V, and 10AH; 10AS, 10P, 10AB, LOAF, 10AG, and 10AH; or steps 1T, 10AS, 10P, 10AB, and 10W; or steps 10AT, 10P, 10AB, 10V, and 10AH; or steps 10AT, 10P, 10AB, 10AF, 10AG, and 10AH; or steps 10AT, 10P, 10AB, and 10W; or steps 1T, 10AS, 10P, 10N, and 10AA; or steps 1T, 10AS, 10P, 10Y, 10Z, and 10AA; or steps 10AT, 10P, 10N, and 10AA; or steps 10AT, 10P, 10Y, 10Z, and 10AA; or steps 10AS, 10P, 10N, 10AA, 15A, 15B, and 15C; or steps 10AT, 10P, 10N, 10AA, 15A, 15B; or steps 14A, 14B, 14C, 14D, 14E, and 13A; or steps 17A, 17B, 17C, and 17D; or steps 17A, 17E, 17F, and 17D; or steps 18A, 18B, 18C, 18D, and 18E; or steps 10A, 10B, 10C, 10AE, 19A, 19B, and 19C; or steps 10A, 10B, 10X, 10AB, 19A, 19B, and 19C; or steps 10A, 10D, 10P, 10AB, 19A, 19B, and 19C; or steps 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C; or steps 10AT, 10P, 10AB, 19A, 19B, and 19C; or steps 10P, 10AB, 19A, 19B, and 19C; or steps 10AU, 19A, 19B, and 19C; or steps 19A, 19B, and 19C. In certain embodiments, said FaldFP comprises. (1) 1B and 1C. In certain embodiments, said FaldFP comprises: (2) 1D. In certain embodiments, said FAP comprises: (3) 1E. In certain embodiments, said FAP comprises: (4) 1F, and 1G. In certain embodiments, said FAP comprises. (5) 1H, 1I, 1J, and 1K. In certain embodiments, said FAP comprises: (6) 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, said FAP comprises: (7) 1E, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, said FAP comprises. (8) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, said FAP comprises: (9) 1K, 1H, 1I, 1J, 1L, 1M, and 1N. In certain embodiments, said FAP comprises: (10) 1H, 1I, 1J, 1O, and 1P5. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 14A, 14B, 14C, 14D, 14E, 13A, and 13B. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 17A, 17B, 17C, 17D, and 17G. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 17A, 17E, 17F, 17D, and 17G. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 18A, 18B, 18C, 18D, 18E, and 18F. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10P, 10AB, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AU, 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 19A, 19B, 19C, and 19D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 13A and 13B; or steps 1T, 10AS, 10P, 10AB, 10V, and 10AH. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10AS, 10P, 10AB, LOAF, 10AG, and 10AH. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 1T, 10AS, 10P, 10AB, and 10W. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10AB, 10V, and 10AH. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10AT, 10P, 10AB, 10AF, 10AG, and 10AH. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10AT, 10P, 10AB, and 10W. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 1T, 10AS, 10P, 10N, and 10AA. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 1T, 10AS, 10P, 10Y, 10Z, and 10AA. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10N, and 10AA. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10AT, 10P, 10Y, 10Z, and 10AA. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10AS, 10P, 10N, 10AA, 15A, 15B, and 15C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10N, 10AA, 15A, 15B. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 14A, 14B, 14C, 14D, 14E, and 13A. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 17A, 17B, 17C, and 17D. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 17A, 17E, 17F, and 17D; or steps 18A, 18B, 18C, 18D, and 18E. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 10A, 10B, 10C, 10AE, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10A, 10B, 10X, 10AB, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10A, 10D, 10P, 10AB, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises. 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AT, 10P, 10AB, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10P, 10AB, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 10AU, 19A, 19B, and 19C. In certain embodiments, said butadiene, CrotOH, 13BDO, MVC or 3-buten-1-ol pathway comprises: 19A, 19B, and 19C.

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of MeOH to Fald, Fald to H6P, Fald to DHA and G3P, PYR to formate and ACCOA, PYR to CO2 and ACCOA, CO2 to formate, formate to Fald, formate to Formyl-CoA, Formyl-CoA to Fald, Formate to FTHF, FTHF to methenyl-THF, methenyl-THF to methylene-THF, methylene-THF to Fald, methylene-THF to glycine, glycine to serine, serine to PYR, methylene-THF to methyl-THF, methyl-THF to ACCOA, ACCOA to MALCOA, methanol to methyl-THF, methyl-THF to methylene-THF, formaldehyde to methylene-THF, methylene-THF to methenyl-THF, formyl-THF to formate, formate to CO2, formaldehyde to S-hydroxymethylglutathione, S-hydroxymethylglutathione to S-formylglutathione to formate, formaldehyde to formate, malonyl-ACP and acetyl-CoA or acetyl-ACP to acetoacetyl-ACP, acetoacetyl-ACP to 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-ACP to crotonyl-ACP, acetoacetyl-ACP to acetoacetyl-CoA, malonyl-CoA and acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to acetoacetate, acetoacetate to 3-oxobutyraldehyde, 3-oxobutyraldehyde to 4-hydroxy-2-butanone, acetoacetyl-ACP to acetoacetate, acetoacetyl-CoA to 3-oxobutyraldehyde, acetoacetyl-ACP to 3-oxobutyraldehyde, acetoacetyl-CoA to 4-hydroxy-2-butanone, 3-hydroxybutyryl-ACP to 3-hydroxybutyrate, 3-hydroxybutyryl-ACP to 3-hydroxybutyraldehyde, 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, 3-hydroxybutyryl-CoA to 13BDO, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, acetoacetate to 3-hydroxybutyrate, 3-oxobutyraldehyde to 3-hydroxybutyraldehyde, 4-hydroxy-2-butanone to 13BDO, crotonyl-ACP to crotonate, crotonyl-ACP to crotonaldehyde, crotonyl-CoA to crotonaldehyde, crotonyl-CoA to CrotOH, 3-hydroxybutyryl-ACP to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to 3-hydroxybutyrate, 3-hydroxybutyrate to 3-hydroxybutyraldehyde, 3-hydroxybutyraldehyde to 13BDO, 3-hydroxybutyryl-CoA to crotonyl-CoA, 3-hydroxybutyrate to crotonate, 3-hydroxybutyraldehyde to crotonaldehyde, crotonyl-ACP to crotonyl-CoA, crotonyl-CoA to crotonate, crotonate to crotonaldehyde, crotonaldehyde to CrotOH, CrotOH to 2-butenyl-4-phosphate, 2-butenyl-4-phosphate to 2-butenyl-4-diphosphate, CrotOH to 2-butenyl-4-diphosphate, 2-butenyl-4-diphosphate to butadiene, CrotOH to butadiene, malonyl-CoA and acetyl-CoA to 3-oxoglutaryl-CoA, 3-oxoglutaryl-CoA to 3-hydroxyglutaryl-CoA to 3-hydroxy-5-oxopentanoate, 3-hydroxy-5-oxopentanoate to 3,5-dihydroxy pentanoate, 3,5-dihydroxy pentanoate to 3-hydroxy-5-phosphonatooxypentanoate, 3-hydroxy-5-phosphonatooxypentanoate to 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate to butenyl 4-biphosphate, butenyl 4-biphosphate to 2-butenyl 4-diphosphate, 2-butenyl 4-diphosphate to butadiene, 2-butanol to MVC, MVC to butadiene, pyruvate to acetolactate, acetolactate to acetoin, acetoin to 2,3-butanediol, 2,3-butanediol to 2-butanal, 2-butanal to 2-butanol, 13BDO to 3-hydroxybutyryl phosphate, 3-hydroxybutyryl phosphate to 3-hydroxybutyryl diphosphate, 3-hydroxybutyryl diphosphate to MVC, 13BDO to 3-hydroxybutyryl diphosphate, 13BDO to MVC, acrylyl-CoA and acetyl-CoA to 3-oxopent-4-enoyl-CoA, 3-oxopent-4-enoyl-CoA to 3-oxopent-4-enoate, 3-oxopent-4-enoate to 3-buten-2-one, 3-buten-2-one to MVC, lactoyl-CoA and acetyl-CoA to 3-oxo-4-hydroxy pentanoyl-CoA, 3-oxo-4-hydroxy pentanoyl-CoA to 3-oxo-4-hydroxy pentanoate, 3-oxo-4-hydroxy pentanoate to 3,4-dihydroxypentanoate, 3,4-dihydroxypentanoate to MVC, 3-oxo-4hydroxy pentanoyl-CoA to 3,4-dihydroxypentanoyl-CoA, 3,4-dihydroxypentanoyl-CoA to 3,4-dihydroxypentanoate, 3,4-dihydroxypentanoate to 4-oxopentanoate, 4-oxopentanoate to 4-hydroxypentanoate, 4-hydroxypentanoate to MVC, succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to 3-oxoadipate, 3-oxoadipate to 4-oxopentanoate, 4-oxopentanoate to 4-hydroxypentanoate, 4-hydroxypentanoate to 3-butene-2-ol, crotonyl-CoA to vinylacetyl-CoA, vinylacetyl-CoA to 3-buten-1-al, 3-buten-1-al to 3-buten-1-ol, 3-buten-1-ol to butadiene, 3-HP-CoA to acrylyl-CoA, acrylyl-CoA to 3-HP-CoA, 3-HP-CoA to 3-oxo-5-hydroxypentanoyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA to 3,5-dihydroxypentanoyl-CoA, 3,5-dihydroxypentanoyl-CoA to 5-hydroxypent-2-enoyl-CoA, 5-hydroxypent-2-enoyl-CoA to pent-2,4-dienoyl-CoA, pent-2,4-dienoyl-CoA, to 2,4-pentadienoate, 2,4-pentadienoate to butadiene, 3-oxo-5-hydroxypentanoyl-CoA to 3-oxo-5-hydroxypentanoate, 3-oxo-5-hydroxypentanoate to 3-oxo-5-hydroxypentanoyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA to 3-oxopent-4-enoyl-CoA, 3-oxopent-4-enoyl-CoA to 3-oxo-5-hydroxypentanoyl-CoA, 3,5-dihydroxypentanoyl-CoA to 3,5-dihydroxypentanoate, 3,5-dihydroxypentanoate to 3,5-dihydroxypentanoyl-CoA, 3-oxo-5-hydroxypentanoate to 3,5-dihydroxypentanoyl-CoA, 3,5-dihydroxypentanoyl-CoA to 3-butene-1-ol, 3,5-dihydroxypentanoyl-CoA to 5-hydroxypenta-2-enoate, 5-hydroxypenta-2-enoate to 3-butene-1-ol, 5-hydroxypent-2-enoyl-CoA to 5-hydroxypenta-2-enoate, 5-hydroxypenta-2-enoate to 5-hydroxypent-2-enoyl-CoA, 5-hydroxypent-2-enoyl-CoA to 2,4-pentadienoate, acrylyl-CoA to 3-oxopent-4-enoyl-CoA, 3-oxopent-4-enoyl-CoA to 3-hydroxypent-4-enoyl-CoA, 3-hydroxypent-4-enoyl-CoA to pent-2,4-dienoyl-CoA, 3-hydroxypent-4-enoyl-CoA3-hydroxypent-4-enoate, 3-oxopent-4-enoyl-CoA to 3-oxopent-4-enoate, 3-oxopent-4-enoate to 3-hydroxypent-4-enoate, 3-hydroxypent-4-enoate to 2,4-pentadienoate, 3-hydroxypent-4-enoate to butadiene, propionyl-CoA to 3-oxopentanoyl-CoA, 3-oxopentanoyl-CoA to 3-hydroxypentanoyl-CoA, 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoA, pent-2-enoyl-CoA to pent-3-enoyl-CoA, pent-3-enoyl-CoA to 2,4-pentadienoyl-CoA. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, such as that shown in FIGS. 1-19, 26 and 27.

In some embodiments, the present invention also provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway can include enzymes selected from any of the numerous pathways shown in FIG. 26 starting from 3-HP-CoA or acryloyl-CoA. In some embodiments, the non-naturally occurring microbial organism having a 2,4-pentadienoate pathway, further includes a FaldFP, a FAP, a MMP, a MOP, a hydrogenase and/or a CODH, attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA, a gene disruption of one or more endogenous nucleic acids encoding such enzymes or any combination thereof as described herein.

It is also understood that enzymes and the corresponding encoding nucleic acids for conversion of actyl-CoA to 3-HP-CoA, acryloyl-CoA, or propionyl-CoA are well known in the art and can be readily identified and included in the microbial organisms described herein.

Exemplary pathways from 3-HP-CoA include the following enzyme sets (A) 1) 3-hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA reductase, 3) 3,5-dihydroxypentanoyl-CoA dehydratase, 4) 5-hydroxypent-2-enoyl-CoA dehydratase, and 5) pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps A-E of FIG. 26, and (B) 1) 3-hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxo-5-hydroxypentanoate reductase, 4) 3,5-dihydroxypentanoate dehydratase, and 5) 5-hydroxypent-2-enoate dehydratase, as shown in steps A, F, I, J, and Q of FIG. 26. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3-HP-CoA can be intermingled via reversible enzymes 3,5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, as shown by step G in FIG. 26, and 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, as shown by step H in FIG. 26. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include the enzymes in steps A, B, G, J, and Q, or steps A, B, C, H, and Q, or steps A, B, G, J, H, D, and E, or steps A, F, I, G, C, D, and E, or steps, A, F, I, G, C, H, and Q, or steps A, F, I, J, H, D, and E, each shown in FIG. 26.

Exemplary pathways from acryloyl-CoA include the following enzyme sets (C) 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxopent-4-enoate reductase, 4) 3-hydroxypent-4-enoate dehydratase, as shown in steps M, O, P, and S in FIG. 26 and (D), 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA reductase, 3) 3-hydroxypent-4-enoyl-CoA dehydratase, and 4) pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps M, N, R, and E. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3-HP-CoA and (C) and (D) from acryloyl-CoA can be intermingled via reversible enzymes 3-hydroxypropanoyl-CoA dehydratase, as shown in step K of FIG. 26, and 3-oxo-5-hydroxypentanoyl-CoA dehydratase, as shown in step L of FIG. 26. Thus, step K can be added to any of the enumerated pathways from acryloyl-CoA to 2,4-pentadienoate providing 2,4-pentadienoate pathways such as steps K, M, N, R, and E or steps K, M, O, P, and S. Step K can also be used a shuttle alternative to step A to provide 3-oxo-5-hydroxypentanoyl-CoA from 3-HP-CoA via steps K, M, and L. Thus, any of the aforementioned pathways utilizing the enzyme of step A can utilize the enzymes of steps K, M, and L, in its place. The same 3-oxo-5-hydroxypentanoyl-CoA intermediate can be accessed from acryloyl-CoA by pathways via the enzymes of steps K and A or M and L of FIG. 26. Thus, acryloyl-CoA can be used to access all the enumerated pathways that would be accessible from 3-HP-CoA. Thus, for example, an acryloyl-CoA to 2,4-pentadienoate pathway can include enzymes from steps K, A, B, C, D, and E, or steps K, A, F, I, J and Q, or steps K, A, B, G, J, and Q, or steps K, A, B, G, J, H, D, and E, or steps K, A, B, C, H, and Q, or steps K, A, F, I, G, C, D, and E, or steps K, A, F, I, G, C, H, Q, or steps K, A, F, I, J, H, D and E, or steps M, L, B, C, D, and E, or steps M, L, F, I, J and Q, or steps M, L, B, G, J, and Q, or steps M, L, B, G, J, H, D, and E, or steps M, L, B, C, H, and Q, or steps M, L, F, I, G, C, D, and E, or steps M, L, F, I, G, C, H, Q, or steps M, L, F, I, J, H, D and E, all as shown in FIG. 26. Similarly, 3-HP-CoA can feed into the enumerated acryloyl-CoA pathways via intermediate 3-oxopent-4-enoyl-CoA using the enzyme of step L. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include enzymes from steps A, L, N, R, and E or steps A, L, O, P, and S, each pathway being shown in FIG. 26.

In some embodiments, the invention provides a non-naturally occurring microbial organism, having a microbial organism having a 2,4-pentadienoate pathway having at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, wherein the 2,4-pentadienoate pathway includes a pathway shown in FIG. 27 selected from: (1) 27A, 27B, 27C, 27D, 27E and 27F, wherein 27A is a 3-oxopentanoyl-CoA thiolase or 3-oxopentanoyl-CoA synthase, wherein 27B is a 3-oxopentanoyl-CoA reductase, wherein 27C is a 3-hydroxypentanoyl-CoA dehydratase, wherein 27D is a pent-2-enoyl-CoA isomerase, wherein 27E is a pent-3-enoyl-CoA dehydrogenase, wherein 27F is a 2,4-pentadienoyl-CoA hydrolase, a 2,4-pentadienoyl-CoA transferase or a 2,4-pentadienoyl-CoA synthetase.

In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism of the invention further includes a 2,4-pentadieneoate decarboxylase to convert 2,4-pentadienoate to butadiene (FIG. 26 or 27, step X). Accordingly, in some aspects the microbial organism of the invention includes at least one exogenous nucleic acid encoding a 2,4-pentadieneoate decarboxylase expressed in a sufficient amount to produce butadiene.

In some embodiments, the invention provides a non-naturally occurring microbial organism having a butadiene pathway as depicted in FIG. 26, which includes at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene. The butadiene pathway can include a set of enzymes selected from: 1) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 2) M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; 3) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 4) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 6) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase;

In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, or five exogenous nucleic acids each encoding a butadiene pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention includes at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism having a butadiene pathway depicted in FIG. 26, further includes a FaldFP, a FAP, a MMP, a MOP, a hydrogenase and/or a CODH, attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA, a gene disruption of one or more endogenous nucleic acids encoding such enzymes or any combination thereof as described herein.

In some embodiments, the present invention provides a non-naturally occurring microbial organism having a butadiene pathway as depicted in FIG. 26, which includes at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol. The 3-butene-1-ol pathway can include a set of enzymes selected from: 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 6) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 7) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 8) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 11) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 12) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 13) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 14) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 15) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 16) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 17) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 18) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 19) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 20) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase.

In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, five, six, or seven, exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism of the invention further includes a 3-butene-1-ol dehydratase to convert 3-butene-1-ol to butadiene as depicted in FIG. 26. In some embodiments, the non-naturally occurring microbial organism having a 3-butene-1-ol pathway, further includes a FaldFP, a FAP, a MMP, a MOP, a hydrogenase and/or a CODH, attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA, a gene disruption of one or more endogenous nucleic acids encoding such enzymes or any combination thereof as described herein.

While generally described herein as a microbial organism that contains a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce an intermediate of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway. For example, as disclosed herein, a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway is exemplified in FIG. 1-19, 26 or 27. Therefore, in addition to a microbial organism containing a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway that produces butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme, where the microbial organism produces a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate, for example, acetoacetyl-CoA, acetoacetate, 3-oxobutyraldehyde, acetoacetyl-ACP, acetoacetyl-CoA, acetoacetyl-ACP, acetoacetyl-CoA, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA, acetoacetyl-CoA, acetoacetate, 3-oxobutyraldehyde, 4-hydroxy-2-butanone, crotonyl-ACP, crotonyl-CoA, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-CoA, 3-hydroxybutyrate, 3-hydroxybutyraldehyde, crotonaldehyde, crotonyl-ACP, crotonyl-CoA, crotonate, crotonaldehyde, 2-butenyl-4-phosphate, 2-butenyl-4-diphosphate, 3-oxoglutaryl-CoA, 3-hydroxy-5-oxopentanoate, 3,5-dihydroxypentanoate, 3-hydroxy-5-phosphonatooxypentanoate, 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, butenyl 4-biphosphate, 2-butenyl 4-diphosphate, 2-butanol, acetolactate, acetoin, 2,3-butanediol, 3-hydroxybutyryl phosphate, 3-hydroxybutyryl diphosphate, 3-oxopent-4-enoyl-CoA, 3-oxopent-4-enoate, 3-buten-2-one, 3-oxo-4-hydroxy pentanoyl-CoA, 3-oxo-4-hydroxy pentanoate, 3,4-dihydroxypentanoate, 3,4-dihydroxypentanoyl-CoA, 3,4-dihydroxypentanoate, 4-oxopentanoate, 4-hydroxypentanoate, 3-oxoadipyl-CoA, 3-oxoadipate, 4-oxopentanoate, 4-hydroxypentanoate, vinylacetyl-CoA, 3-buten-1-al, 3-oxopent-4-enoyl-CoA, 3-hydroxypent-4-enoyl-CoA, 3-oxopent-4-enoate, 3-hydroxypent-4-eonoate, 3-oxo-5-hydroxypentanoyl-CoA, 3,5-dihydroxypentanoyl-CoA, 5-hydroxypent-2-enoyl-CoA, pent-2,4-dienoyl-CoA, 2,4-pentadienoate, 3-oxo-5-hydroxypentanoate, 3,5-dihydroxypentanoate, 5-hydroxypent-2-enoate, 3-oxopentanoyl-CoA, 3-hydroxypentanoyl-CoA, pent-2-enoyl-CoA, or pent-3-enoyl-CoA. In certain embodiments, the microbial organisms of the invention do not include the production of a product other than butadiene, 13BDO, CrotOH, 3-butene-2-ol or 3-buten-1-ol, such as, but not limited to ethanol.

It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of FIGS. 1-19, 26 and 27, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate can be utilized to produce the intermediate as a desired product.

The invention further provides non-naturally occurring microbial organisms that have elevated or enhanced synthesis or yields of acetyl-CoA (e.g. intracellular) or biosynthetic products such as butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol and methods of using those non-naturally occurring organisms to produce such biosynthetic products. The enhanced synthesis of intracellular acetyl-CoA enables enhanced production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol from which acetyl-CoA is an intermediate and further, may have been rate limiting.

The non-naturally occurring microbial organisms having enhanced yields of a biosynthetic product include one or more of the various pathway configurations employing a MeDH for methanol oxidation and/or a FaldFP and/or an acetyl-CoA enhancing pathway for directing the carbon from methanol into acetyl-CoA and other desired products via formaldehyde fixation. The various different methanol oxidation and formaldehyde fixation configurations exemplified below can be engineered in conjunction with any or each of the various methanol oxidation, formaldehyde fixation, formate reutilization, butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol pathways exemplified previously and herein. The metabolic modifications exemplified below increase biosynthetic product yields over, for example, endogenous methanol utilization pathways because they further focus methanol derived carbon into the assimilation pathways described herein, decrease inefficient use of methanol carbon through competing methanol utilization and/or FaldFPs and/or increase the production of reducing equivalents.

In this regard, methylotroph microbial organisms utilize methanol as the sole source of carbon and energy. In such methylotrophic organisms, the oxidation of methanol to formaldehyde is catalyzed by one of three different enzymes: NADH dependent MeDH(MeDH), PQQ-dependent MeDH(MeDH-PQQ) and alcohol oxidase (AOX).

Methanol oxidase is a specific type of AOX with activity on methanol. Gram positive bacterial methylotrophs such as Bacillus methanolicus utilize a cytosolic MeDH which generates reducing equivalents in the form of NADH. Gram negative bacterial methylotrophs utilize periplasmic PQQ-containing MeDH enzymes which transfer electrons from methanol to specialized cytochromes CL, and subsequently to a cytochrome oxidase (Afolabi et al, Biochem 40:9799-9809 (2001)). Eukaryotic methylotrophs employ a peroxisomal oxygen-consuming and hydrogen-peroxide producing alcohol oxidase.

Bacterial methylotrophs are found in in the genera Bacillus, Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis and Hyphomicrobium. These organisms utilize either the serine cycle (type II) or the RUMP cycle (type I) to further assimilate formaldehyde into central metabolism (Hanson and Hanson, Microbiol Rev 60:439-471 (1996)). As described previously, the RUMP pathway combines formaldehyde with ribulose monophosphate to form hexulose-6-phosphate, which is further converted to fructose-6-phosphate (see FIG. 1, step C). In the serine cycle formaldehyde is initially converted to 5,10-methylene-THF, which is combined with glycine to form serine. Overall, the reactions of the serine cycle produce one equivalent of acetyl-CoA from three equivalents of methanol (Anthony, Science Prog 94:109-37 (2011)). The RUMP cycle also yields one equivalent of acetyl-CoA from three equivalents methanol in the absence of a FAP. Genetic tools are available for numerous prokaryotic methylotrophs and methanotrophs.

Eukaryotic methylotrophs are found in the genera Candida, Pichia, Ogataea, Kuraishia and Komagataella. Particularly useful methylotrophic host organisms are those with well-characterized genetic tools and gene expression systems such as Hansenula polymorpha, Pichia pastoris, Candida boidinii and Pichia methanolica (for review see Yurimoto et al, Int J Microbiol (2011)). The initial step of methanol assimilation in eukaryotic methylotrophs occurs in the peroxisomes, where methanol and oxygen are oxidized to formaldehyde and hydrogen peroxide by alcohol oxidase (AOX). Formaldehyde assimilation with xylulose-5-phosphate via DHA synthase also occurs in the peroxisomes. During growth on methanol, the two enzymes DHA synthase and AOX together comprise 80% of the total cell protein (Horiguchi et al, J Bacteriol 183:6372-83 (2001)). DHA synthase products, DHA and glyceraldehyde-3-phosphate, are secreted into the cytosol where they undergo a series of rearrangements catalyzed by pentose phosphate pathway enzymes, and are ultimately converted to cellular constituents and xylulose-5-phosphate, which is transported back into the peroxisomes. The initial step of formaldehyde dissimilation, catalyzed by S-(hydroxymethyl)-glutathione synthase, also occurs in the peroxisomes. Like the bacterial methylotrophic pathways described above, eukaryotic methylotrophic pathways convert three equivalents of methanol to at most one equivalent of acetyl-CoA because they lack a FAP.

As exemplified further below, the various configurations of metabolic modifications disclosed herein for enhancing product yields via methanol derived carbon include enhancing methanol oxidation and production of reducing equivalents using either an endogenous NADH dependent MeDH, an exogenous NADH dependent MeDH, both an endogenous NADH dependent MeDH and exogenous NADH dependent MeDH alone or in combination with one or more metabolic modifications that attenuate, for example, DHA synthase and/or AOX. In addition, other metabolic modifications as exemplified below that reduce carbon flux away from methanol oxidation and formaldehyde fixation also can be included, alone or in combination, with the methanol oxidation and FaldFP configurations disclosed herein that enhance carbon flux into product precursors such as acetyl-CoA and, therefore, enhance product yields.

Accordingly, the microbial organisms of the invention having one or more of any of the above and/or below metabolic modifications to a methanol utilization pathway and/or formaldehyde assimilation pathway configurations for enhancing product yields can be combined with any one or more, including all of the previously described methanol oxidation, formaldehyde fixation, formate reutilization, butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol pathways to enhance the yield and/or production of a product such as any of the butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein.

Given the teachings and guidance provided herein, the methanol oxidation and FaldFP configurations can be equally engineered into both prokaryotic and eukaryotic organisms. In prokaryotic microbial organisms, for example, one skilled in the art will understand that utilization of an endogenous MOP enzyme or expression of an exogenous nucleic acid encoding a MOP enzyme will naturally occur cytosolically because prokaryotic organisms lack peroxisomes. In eukaryotic microbial organisms one skilled in the art will understand that certain MOPs occur in the peroxisome as described above and that cytosolic expression of the MOP or pathways described herein to enhance product yields can be beneficial. The peroxisome located pathways and competing pathways remain or, alternatively, attenuated as described below to further enhance methanol oxidation and formaldehyde fixation.

With respect to eukaryotic microbial host organisms, those skilled in the art will know that yeasts and other eukaryotic microorganisms exhibit certain characteristics distinct from prokaryotic microbial organisms. When such characteristics are desirable, one skilled in the art can choose to use such eukaryotic microbial organisms as a host for engineering the various different methanol oxidation and formaldehyde fixation configurations exemplified herein for enhancing product yields. For example, yeast are robust organisms, able to grow over a wide pH range and able to tolerate more impurities in the feedstock Yeast also ferment under low growth conditions and are not susceptible to infection by phage. Less stringent aseptic design requirements can also reduce production costs. Cell removal, disposal and propagation are also cheaper, with the added potential for by-product value for animal feed applications. The potential for cell recycle and semi-continuous fermentation offers benefits in increased overall yields and rates. Other benefits include: potential for extended fermentation times under low growth conditions, lower viscosity broth (vs E. coli) with insoluble hydrophobic products, the ability to employ large fermenters with external loop heat exchangers.

Eukaryotic host microbial organisms suitable for engineering carbon efficient methanol utilization capability can be selected from, and the non-naturally occurring microbial organisms generated in, for example, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. As described previously, exemplary yeasts or fungi include species selected from the genera Saccharomyces, Schizosaccharomyces, Schizochytrium, Rhodotorula, Thraustochytrium, Aspergillus, Kluyveromyces, Issatchenkia, Yarrowia, Candida, Pichia, Ogataea, Kuraishia, Hansenula and Komagataella. Useful host organisms include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, Issatchenkia orientalis and the like.

The methanol oxidation and/or formaldehyde assimilation pathway configurations described herein for enhancing product synthesis or yields include, for example, a NADH-dependent MeDH(MeDH) and/or one or more formaldehyde assimilation pathways. Such engineered pathways provide a synthesis or yield enhancement over endogenous pathways found in methylotrophic organisms. For example, methanol assimilation via MeDH provides reducing equivalents in the useful form of NADH, whereas alcohol oxidase and PQQ-dependent MeDH do not. Several product pathways described herein have several NADH-dependent enzymatic steps. In addition, deletion of redox-inefficient methanol oxidation enzymes as described further below, combined with increased cytosolic or peroxisomal expression of an NADH-dependent MeDH, improves the ability of the organism to extract useful reducing equivalents from methanol. In some aspects, if NADH-dependent MeDH is engineered into the peroxisome, an efficient means of shuttling redox in the form of NADH out of the peroxisome and into the cytosol can be included. Further employment of a formaldehyde assimilation pathway in combination with a FAP enables high synthesis or yield conversion of methanol to acetyl-CoA, and subsequently to acetyl-CoA derived products.

Metabolic modifications for enabling redox- and carbon-efficient cytosolic methanol utilization in a eukaryotic or prokaryotic organism are exemplified in further detail below.

In one embodiment, the invention provides cytosolic expression of one or more methanol oxidation and/or formaldehyde assimilation pathways Engineering into a host microbial organism carbon- and redox-efficient cytosolic formaldehyde assimilation can be achieved by expression of one or more endogenous or exogenous MOPs and/or one or more endogenous or exogenous formaldehyde assimilation pathway enzymes in the cytosol. An exemplary pathway for methanol oxidation includes NADH dependent MeDH as shown in FIGS. 1 and 2.

Exemplary pathways for converting cytosolic formaldehyde into glycolytic intermediates also are shown in FIGS. 1 and 2. Such pathways include methanol oxidation via expression of an cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic DHA synthase, both methanol oxidation via expression of an cytosolic NADH dependent MeDH and formaldehyde fixation via expression of cytosolic DHA synthase alone or together with the metabolic modifications exemplified below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase (e.g., when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

For example, in the carbon-efficient DHA pathway of formaldehyde assimilation shown in FIGS. 1 and 2, step D, formaldehyde is converted to dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (GAP) by DHA synthase (FIGS. 1D and 2D). DHA and G3P are then converted to fructose-6-phosphate in one step by F6P aldolase (FIGS. 1C and 2C) or in three steps by DHA kinase, FBP aldolase and fructose-1,6-bisphosphatase (not shown). Formation of F6P from DHA and G3P by F6P aldolase is more ATP-efficient than using DHA kinase, FBP aldolase and fructose-1,6-bisphosphatase. Rearrangement of F6P and E4P by enzymes of the pentose phosphate pathway (transaldolase, transketolase, R5P epimerase and Ru5P epimerase) regenerates xylulose-5-phosphate, the DHA synthase substrate. Conversion of F6P to G3P and E4P followed by conversion of G3P to pyruvate results in the carbon-efficient generation of cytosolic acetyl-CoA by further conversion of pyruvate to acetyl-CoA (FIGS. 1 and 2, step R or Q). Exemplary enzymes catalyzing each step of the carbon efficient DHA pathway are described elsewhere herein.

An alternate carbon efficient pathway for formaldehyde assimilation proceeding through ribulose-5-phosphate (Ru5P) is shown in FIGS. 1 and 2, step B. The formaldehyde assimilation enzyme of this pathway is 3-hexulose-6-phosphate synthase, which combines ru5p and formaldehyde to form hexulose-6-phosphate (FIGS. 1B and 2B). 6P3HI converts H6P to F6P (FIGS. 1C and 2C). Regeneration of Ru5P from F6P proceeds by pentose phosphate pathway enzymes. Conversion of F6P to G3P and E4P followed by conversion of G3P to pyruvate results in the carbon-efficient generation of cytosolic acetyl-CoA by further conversion of pyruvate to acetyl-CoA (FIGS. 1 and 2, step R or Q). Exemplary enzymes catalyzing step of the carbon efficient RUMP pathway are described elsewhere herein.

Thus, in this embodiment, conversion of cytosolic formaldehyde into glycolytic intermediates can occur via expression of a cytosolic 3-hexulose-6-phosphate (3-Hu6P) synthase and 6P3HI. Thus, exemplary pathways that can be engineered into a microbial organism of the invention can include methanol oxidation via expression of a cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic 3-Hu6P synthase and 6P3HI, both methanol oxidation via expression of an cytosolic NADH dependent MeDH and formaldehyde fixation via expression of cytosolic 3-Hu6P synthase and 6P3HI alone or together with the metabolic modifications exemplified below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

In yet another embodiment increased product yields can be accomplished by engineering into the host microbial organism of the invention both the RUMP and DHA pathways as shown in FIGS. 1 and 2. In this embodiment, the microbial organisms can have cytosolic expression of one or more methanol oxidation and/or formaldehyde assimilation pathways. The formaldehyde assimilation pathways can include both assimilation through cytosolic DHA synthase and 3-Hu6P synthase. Such pathways include methanol oxidation via expression of a cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic DHA synthase and 3-Hu6P synthase, both methanol oxidation via expression of an cytosolic NADH dependent dehydrogenase and formaldehyde fixation via expression of cytosolic DHA synthase and 3-Hu6P synthase alone or together with the metabolic modifications exemplified previously and also below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

Increasing the expression and/or activity of one or more formaldehyde assimilation pathway enzymes in the cytosol can be utilized to assimilate formaldehyde at a high rate. Increased activity can be achieved by increased expression, altering the ribosome binding site, altering the enzyme activity, or altering the sequence of the gene to ensure, for example, that codon usage is balanced with the needs of the host organism, or that the enzyme is targeted to the cytosol as disclosed herein.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA. For example, in some aspects, the endogenous enzyme can be selected from DHA kinase, methanol oxidase, PQQ-dependent MeDH, DHA synthase or any combination thereof. Accordingly, in some aspects, the attenuation is of the endogenous enzyme DHA kinase. In some aspects, the attenuation is of the endogenous enzyme methanol oxidase. In some aspects, the attenuation is of the endogenous enzyme PQQ-dependent MeDH. In some aspects, the attenuation is of the endogenous enzyme DHA synthase. The invention also provides a microbial organism wherein attenuation is of any combination of two or three endogenous enzymes described herein. For example, a microbial organism of the invention can include attenuation of DHA kinase and DHA synthase, or alternatively methanol oxidase and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and DHA synthase. The invention also provides a microbial organism wherein attenuation is of all endogenous enzymes described herein. For example, in some aspects, a microbial organism described herein includes attenuation of DHA kinase, methanol oxidase, PQQ-dependent MeDH and DHA synthase.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes attenuation of one or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway. Examples of these endogenous enzymes are disclosed in FIGS. 1 and 2 and described in Example XXII. It is understood that a person skilled in the art would be able to readily identify enzymes of such competing pathways. Competing pathways can be dependent upon the host microbial organism and/or the exogenous nucleic acid introduced into the microbial organism as described herein. Accordingly, in some aspects of the invention, the microbial organism includes attenuation of one, two, three, four, five, six, seven, eight, nine, ten or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes a gene disruption of one or more endogenous nucleic acids encoding enzymes, which enhances carbon flux through acetyl-CoA. For example, in some aspects, the endogenous enzyme can be selected from DHA kinase, methanol oxidase, PQQ-dependent MeDH, DHA synthase or any combination thereof. According, in some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme DHA kinase. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme methanol oxidase. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme PQQ-dependent MeDH. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme DHA synthase. The invention also provides a microbial organism wherein the gene disruption is of any combination of two or three nucleic acids encoding endogenous enzymes described herein. For example, a microbial organism of the invention can include a gene disruption of DHA kinase and DHA synthase, or alternatively methanol oxidase and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and DHA synthase. The invention also provides a microbial organism wherein all endogenous nucleic acids encoding enzymes described herein are disrupted. For example, in some aspects, a microbial organism described herein includes disruption of DHA kinase, methanol oxidase, PQQ-dependent MeDH and DHA synthase.

In some embodiments, the invention provides a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes a gene disruption of one or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway. Examples of these endogenous enzymes are disclosed in FIGS. 1 and 2 and described in Example XXII. It is understood that a person skilled in the art would be able to readily identify enzymes of such competing pathways. Competing pathways can be dependent upon the host microbial organism and/or the exogenous nucleic acid introduced into the microbial organism as described herein. Accordingly, in some aspects of the invention, the microbial organism includes a gene disruption of one, two, three, four, five, six, seven, eight, nine, ten or more endogenous nucleic acids encoding enzymes of a competing formaldehyde assimilation or dissimilation pathway.

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

The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable or suitable to fermentation processes. Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobiospirillum; the order Pasteurellales, family Pasteurellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas. Non-limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary bacterial methylotrophs include, for example, Bacillus, Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis and Hyphomicrobium.

Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, and the like. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae and yeasts or fungi selected from the genera Saccharomyces, Schizosaccharomyces, Schizochytrium, Rhodotorula, Thraustochytrium, Aspergillus, Kluyveromyces, Issatchenkia, Yarrowia, Candida, Pichia, Ogataea, Kuraishia, Hansenula and Komagataella. Useful host organisms include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, Issatchenkia orientalis and the like. Exemplary eukaryotic methylotrophs include, for example, eukaryotic methylotrophs found in the genera Candida, Pichia, Ogataea, Kuraishia and Komagataella. Particularly useful methylotrophic host organisms include, for example, Hansenula polymorpha, Pichia pastoris, Candida boidinii and Pichia methanolica. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.

Depending on the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathways. For example, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be included, such as steps 1B, 1C, 1F, 1G and 1Q in combination with any one of steps 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; or steps 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; or steps 14A, 14B, 14C, 14D, 14E, 13A, and 13B; or steps 17A, 17B, 17C, 17D, and 17G; or steps 17A, 17E, 17F, 17D, and 17G; or steps 18A, 18B, 18C, 18D, 18E, and 18F; or steps 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D; or steps 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D; or steps 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10P, 10AB, 19A, 19B, 19C, and 19D; or steps 10AU, 19A, 19B, 19C, and 19D; or steps 19A, 19B, 19C, and 19D; or steps 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; or steps 10AT, 10P, 10AB, 10V, 10AH, 11A, 11B, and 11C; or steps 13A and 13B; or steps 1T, 10AS, 10P, 10AB, 10V, and 10AH; 10AS, 10P, 10AB, 10AF, 10AG, and 10AH; or steps 1T, 10AS, 10P, 10AB, and 10W; or steps 10AT, 10P, 10AB, 10V, and 10AH; or steps 10AT, 10P, 10AB, 10AF, 10AG, and 10AH; or steps 10AT, 10P, 10AB, and 10W; or steps 1T, 10AS, 10P, 10N, and 10AA; or steps 1T, 10AS, 10P, 10Y, 10Z, and 10AA; or steps 10AT, 10P, 10N, and 10AA; or steps 10AT, 10P, 10Y, 10Z, and 10AA; or steps 10AS, 10P, 10N, 10AA, 15A, 15B, and 15C; or steps 10AT, 10P, 10N, 10AA, 15A, 15B; or steps 14A, 14B, 14C, 14D, 14E, and 13A; or steps 17A, 17B, 17C, and 17D; or steps 17A, 17E, 17F, and 17D; or steps 18A, 18B, 18C, 18D, and 18E; or steps 10A, 10B, 10C, 10AE, 19A, 19B, and 19C; or steps 10A, 10B, 10X, 10AB, 19A, 19B, and 19C; or steps 10A, 10D, 10P, 10AB, 19A, 19B, and 19C; or steps 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C; or steps 10AT, 10P, 10AB, 19A, 19B, and 19C; or steps 10P, 10AB, 19A, 19B, and 19C; or steps 10AU, 19A, 19B, and 19C; or steps 19A, 19B, and 19C, as depicted in FIGS. 1, and 10-19.

Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty up to all nucleic acids encoding the enzymes or proteins constituting a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway precursors such as pyruvate, formate, acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, malonyl-ACP, acetoacetyl-CoA, succinyl-CoA, crotonyl-CoA, vinylacetyl-CoA, and 3-buten-1-al.

Generally, a host microbial organism is selected such that it produces the precursor of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, pyruvate, formate, acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, malonyl-ACP, acetoacetyl-CoA, succinyl-CoA, crotonyl-CoA, vinylacetyl-CoA, and 3-buten-1-al are produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway.

In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. In this specific embodiment it can be useful to increase the synthesis or accumulation of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway product to, for example, drive butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway reactions toward butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzymes or proteins. Overexpression of the enzyme or enzymes and/or protein or proteins of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, through overexpression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, that is, up to all nucleic acids encoding butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway.

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

It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic capability. For example, a non-naturally occurring microbial organism having a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of a formate reductase and a MVC dehydratase, or alternatively, a MeDH and CrotOH dehydratase, or alternatively a formaldehyde dehydrogenase and a 3-hydroxybutraldehyde reductase, or alternatively a crotonyl-CoA delta-isomerase and a vinylacetyl-CoA reductase, or alternatively a crotonyl-CoA delta-isomerase and a 3-buten-1-al reductase, or alternatively a crotonyl-CoA delta-isomerase and a 3-buten-1-ol dehydratase, or alternatively a vinylacetyl-CoA reductase and a 3-buten-1-al reductase, or alternatively a vinylacetyl-CoA reductase and a 3-buten-1-ol dehydratase, or alternatively a 3-buten-1-al reductase and a 3-buten-1-ol dehydratase, and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, a pyruvate formate lyase, a formyl-CoA reductase, and a crotonaldehyde reductase, or alternatively a FDH, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase, or alternatively a 3-dexulose-6-phosphate synthase, a 6P3HI, and aAcAcCoAR (ketone reduceing), or alternatively a crotonyl-CoA delta-isomerase, a vinylacetyl-CoA reductase, and a 3-buten-1-al reductase; or alternatively a crotonyl-CoA delta-isomerase, a vinylacetyl-CoA reductase, and a 3-buten-1-ol dehydratase, or alternatively a crotonyl-CoA delta-isomerase, a 3-buten-1-al reductase, and a 3-buten-1-ol dehydratase, or alternatively a vinylacetyl-CoA reductase, a 3-buten-1-al reductase, and a 3-buten-1-ol dehydratase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

In addition to the biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and/or with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol other than use of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producers is through addition of another microbial organism capable of converting a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate to butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. One such procedure includes, for example, the fermentation of a microbial organism that produces a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate. The butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate can then be used as a substrate for a second microbial organism that converts the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate to butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. The butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate can be added directly to another culture of the second organism or the original culture of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-01 pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol intermediate and the second microbial organism converts the intermediate to butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase production of acetyl-CoA or a bioderived compound. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase acetyl-CoA or a bioderived compound biosynthesis. In a particular embodiment, the increased production couples biosynthesis of acetyl-CoA or a bioderived compound to growth of the organism, and can obligatorily couple production of acetyl-CoA or a bioderived compound to growth of the organism if desired and as disclosed herein.

Sources of encoding nucleic acids for a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Abies grandis, Achromobacter xylosoxidans AXX-A, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1, Allochromatium vinosum DSM 180, Amycolicicoccus subflavus DQS3-9A1, Anabaena variabilis ATCC 29413, Anaerotruncus colihominis, Aquincola tertiaricarbonis L108, Arabidopsis thaliana, Arabidopsis thaliana col, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Arthrobacter globiformis, Aspergillus niger, Aspergillus terreus NIH2624, Azotobacter vinelandii DJ, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coahuilensis, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus pseudofirmus, Bacillus selenitireducens MLS10, Bacillus sphaericus, Bacillus subtilis, Bacteroides capillosus, Bordetella bronchiseptica KU1201, Bordetella bronchiseptica MO149, Bordetella parapertussis 12822, Bos taurus, Brassica napsus, Burkholderia ambifaria AMMD, Burkholderia phymatum, Burkholderia stabilis, Burkholderia xenovorans, Campylobacter curvus 525.92, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Candida parapsilosis, Candida tropicalis, Carboxydothermus hydrogenoformans, Carpoglyphus lactis, Carthamus tinctorius, Castellaniella defragrans, Chlamydomonas reinhardtii, Chlorobium phaeobacteroides DSM 266, Chloroflexus aurantiacus, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae ATCC 29220, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridium aminobutyricum, Clostridium beijerinckii, Clostridium beijerinckii NRRL B593, Clostridium botulinum, Clostridium botulinum C str. Eklund, Clostridium butyricum, Clostridium carboxidivorans P7, Clostridium cellulolyticum H10, Clostridium cellulovorans 743B, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahlii, Clostridium ljungdahlii DSM 13528, Clostridium novyi NT, Clostridium pasteuranum, Clostridium perfringens, Clostridium phytofermentans ISDg, Clostridium propionicum, Clostridium saccharoperbutylacetonicum, Comamonas sp. CNB-1, Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium sp., Corynebacterium sp. U-96, Cryptosporidium parvum Iowa II, Cucumis sativus, Cuphea hookeriana, Cuphea palustris, Cupriavidus taiwanensis, Cyanobium PCC7001, Cyanothece sp. PCC 7424, Cyanothece sp. PCC 7425, Cyanothece sp. PCC 7822, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafinense, Desulfovibrio africanus, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Desulfovibrio fructosovorans JJ, Dictyostelium discoideum AX4, Elizabethkingia meningoseptica, Enterococcus faecalis, Erythrobacter sp. NAP1, Escherichia coli C, Escherichia coli K12, Escherichia coli K-12 MG1655, Escherichia coli W, Eubacterium barkeri, Eubacterium rectale ATCC 33656, Euglena gracilis, Fusobacterium nucleatum, Geobacillus thermoglucosidasius, Geobacter metallireducens GS-15, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Haematococcus pluvialis, Haliangium ochraceum DSM 14365, Haloarcula marismortui, Haloarcula marismortui ATCC 43049, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hyphomicrobium denitnficans ATCC 51888, Hyphomicrobium zavarzinii, Jeotgalicoccus sp. ATCC8456, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella pneumonia ATCC 25955, Klebsiella pneumonia L4M1063, Klebsiella pneumoniae, Klebsiella terrigena, Kluyveromyces lactis, Lactobacillus acidophilus, Lactobacillus brevis ATCC 367, Lactobacillus collinoides, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc mesenteroides, Lycopersicon hirsutum f. glabratum, Lyngbya majuscule 3L, Lyngbya sp. PCC 8106, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Macrococcus caseolyticus, Malus x domestica, marine gamma proteobacterium HTCC2080, Mesorhizobium loti MAFF303099, Metallosphaera sedula, Metarhizium acridum CQMa 102, Methanocaldococcus jannaschii, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanothermobacter thermautotrophicus, Methylibium petroleiphilum PM1, Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens AM1, Methylococcus capsulatas, Methylococcus capsulatis, Methylomonas aminofaciens, Moorella thermoacetica, Mus musculus, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri, Mycobacterium marinum M, Mycobacterium smegmatis MC2 155, Mycobacterium tuberculosis, Mycoplasma pneumoniae M129, Natranaerobius thermophilus, Nectria haematococca mpVI 77-13-4, Neurospora crassa, Nicotiana tabacum, Nocardia brasiliensis, Nocardia farcinica IFM 10152, Nocardia iowensis, Nocardia iowensis (sp. NRRL 5646), Nodularia spumigena CCY9414, Nostoc azollae, Nostoc sp. PCC 7120, Ocimum basilicum, Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Otyctolagus cuniculus, Oxalobacter formigenes, Paenibacillus polymyxa, Paracoccus denitrificans, Pelobacter carbinolicus DSM 2380, Pelotomaculum thermopropionicum, Penicillium chrysogenum, Perkinsus marinus ATCC 50983, Picea abies, Pichia pastoris, Pinus sabiniana, Plasmodium falciparum, Populus alba, Populus tremula x Populus alba, Polphyromonas gingivalis, Porphyromonas gingivalis ATCC 33277, Polphyromonas gingivalis W83, Prochlorococcus marinus MIT 9312, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas knackmussii, Pseudomonas knackmussii (B13), Pseudomonas mendocina, Pseudomonas putida, Pseudomonas sp, Pseudomonas sp. CF600, Psychroflexus torquis ATCC 700755, Pueraria montana, Pyrobaculum aerophilum str. IM2, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha H16, Ralstonia metallidurans, Ralstonia pickettii, Rattus norvegicus, Rhizobium leguminosarum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodococcus opacus B4, Rhodococcus ruber, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodospirillum rubrum, Roseburia intestinalis L1-82, Roseburia inulinivorans, Roseburia sp. A2-183, Rosefflexus castenholzii, Rubrivivax gelatinosus, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp. arizonae serovar, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica Typhimurium, Salmonella typhimurium, Salmonella typhimurium LT2, Schizosaccharomyces pombe, Simmondsia chinensis, Sinorhizobium meliloti 1021, Solanum lycopersicum, Solibacillus silvestris, Sporosarcina newyorkensis, Staphylococcus aureus, Staphylococcus pseudintermedius, Stereum hirsutum FP-91666 SS1, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes ATCC 10782, Streptomyces anulatus, Streptomyces avermitillis, Streptomyces cinnamonensis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces griseus subsp. griseus NBRC 13350, Streptomyces sp CL190, Streptomyces sp. ACT-1, Streptomyces sp. KO-3988, Sulfolobus acidocalarius, Sulfolobus shibatae, Sulfolobus solfataricus, Sulfolobus tokodaii, Synechococcus elongatus PCC 6301, Synechococcus elongatus PCC7942, Synechococcus sp. PCC 7002, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Syntrophus aciditrophicus, Thauera aromatica, Thermoanaerobacter brockii HTD4, Thermoanaerobacter tengcongensis MB4, Thermococcus kodakaraensis, Thermococcus litoralis, Thermomyces lanuginosus, Thermoproteus neutrophilus, Thermotoga maritime MSB8, Thermus thermophilus, Thiocapsa roseopersicina, Trichomonas vaginalis G3, Trypsonoma brucei, Tsukamurella paurometabola DSM 20162, Umbellularia californica, Xanthobacter autotrophicus Py2, Yarrowia lipolytica, Yersinia intermedia ATCC 29909, Zea mays, Zoogloea ramigera, Zymomonas mobilis, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

In some instances, such as when an alternative butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway exists in an unrelated species, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

A nucleic acid molecule encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein of the invention or other nucleic acid or protein of the invention can also include a nucleic acid molecule that hybridizes to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a nucleic acid molecule that can be used in the invention can be described as having a certain percent sequence identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. For example, the nucleic acid molecule can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleic acid described herein.

Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (T_m) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleic acid sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65° C., for example, if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleic acid sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

A nucleic acid molecule encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein of the invention can have at least a certain sequence identity to a nucleotide sequence disclosed herein. According, in some aspects of the invention, a nucleic acid molecule encoding a butadiene or 3-buten-1-ol pathway enzyme or protein has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number.

Sequence identity (also known as homology or similarity) refers to sequence similarity between two nucleic acid molecules or between two polypeptides. Identity can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information.

Methods for constructing and testing the expression levels of a non-naturally occurring butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffineister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one or more butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

In some embodiments, the invention provides a method for producing butadiene. In some aspects, the method for producing butadiene includes culturing the non-naturally occurring microbial organism of having a butadiene pathway as described herein under conditions and for a sufficient period of time to produce butadiene. Accordingly, in certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene. In some aspects, the microbial organism can include one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve exogenous nucleic acids each encoding a butadiene pathway enzyme. In some aspects, the microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the butadiene pathways provided herein. In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the organism is cultured in a substantially anaerobic culture medium.

In some aspects, the method for producing butadiene includes culturing the non-naturally occurring microbial organism as described herein under conditions and for a sufficient to produce 3-buten-1-ol, and chemically dehydrating the 3-buten-1-ol to produce butadiene. Accordingly, in certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In some aspects, the non-naturally occurring microbial organism used in a method of the invention for producing butadiene includes a non-naturally occurring microbial organism having a 3-buten-1-ol pathway and at least one exogenous nucleic acid encoding a 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce 3-buten-1-ol. In some aspects, the microbial organism can include one, two, three, four, five, six or seven exogenous nucleic acids each encoding a 3-buten-1-ol pathway enzyme. In some aspects, the microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the 3-buten-1-ol pathways provided herein. In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.

The dehydration of alcohols are known in the art and can include various thermal processes, both catalyzed and non-catalyzed. In some embodiments, a catalyzed thermal dehydration employs a metal oxide catalyst or silica.

Dehydration can be achieved via activation of the alcohol group and subsequent elimination by standard elimination mechanisms such as E1 or E2 elimination. Activation can be achieved by way of conversion of the alcohol group to a halogen such as iodide, chloride, or bromide. Activation can also be accomplished by way of a sulfonyl, phosphate or other activating functionality that convert the alcohol into a good leaving group. In some embodiments, the activating group is a sulfate or sulfate ester selected from a tosylate, a mesylate, a nosylate, a brosylate, and a triflate. In some embodiments, the leaving group is a phosphate or phosphate ester. In some such embodiments, the dehydrating agent is phosphorus pentoxide.

In another aspect, provided herein is a method for producing CrotOH comprising culturing the non-naturally occurring microbial organism of having a CrotOH pathway as described herein under conditions and for a sufficient period of time to produce CrotOH. In certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a CrotOH pathway enzyme expressed in a sufficient amount to produce CrotOH. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium.

In another aspect, provided herein is a method for producing 13BDO comprising culturing the non-naturally occurring microbial organism of having a 13BDO pathway as described herein under conditions and for a sufficient period of time to produce 13BDO. In certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a 13BDO pathway enzyme expressed in a sufficient amount to produce 13BDO. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium.

In another aspect, provided herein is a method for producing MVC comprising culturing the non-naturally occurring microbial organism of having a MVC pathway as described herein under conditions and for a sufficient period of time to produce MVC. In certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In certain embodiments, the microbial organism comprises at least one exogenous nucleic acid encoding a MVC pathway enzyme expressed in a sufficient amount to produce MVC. In certain embodiments, the organism is cultured in a substantially anaerobic culture medium.

In some embodiments, the invention provides a method for producing 3-buten-1-ol. In some aspects, the method includes culturing the non-naturally occurring microbial organism as described herein under conditions and for a sufficient period of time to produce 3-buten-1-ol. Accordingly, in certain embodiments, the microbial organism has a FaldFP, a FAP, a MMP, a MOP, a hydrogenase, a CODH or any combination described herein. In some aspects, the non-naturally occurring microbial organism used in a method of the invention for producing 3-buten-1-ol includes a non-naturally occurring microbial organism having a 3-buten-1-ol pathway and at least one exogenous nucleic acid encoding a 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce 3-buten-1-ol. In some aspects, the microbial organism can include one, two, three, four, five, six or seven exogenous nucleic acids each encoding a 3-buten-1-ol pathway enzyme. In some aspects, the microbial organism can include exogenous nucleic acids encoding each of the enzymes of at least one of the 3-buten-1-ol pathways provided herein. In some aspects, the at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.

In some embodiments, access to butadiene can be accomplished by biosynthetic production of CrotOH and subsequent chemical dehydration to butadiene. In some embodiments, the invention provides a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces CrotOH as described herein; and (b) converting CrotOH produced by culturing the non-naturally occurring microbial organism to butadiene. In some aspects, the converting CrotOH to butadiene is performed by chemical dehydration in the presence of a catalyst.

In some embodiments, access to butadiene can be accomplished by biosynthetic production of 13BDO and subsequent chemical dehydration to butadiene. In some embodiments, the invention provides a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces 13BDO as described herein; and (b) converting 13BDO produced by culturing the non-naturally occurring microbial organism to butadiene. In some aspects, the converting 13BDO to butadiene is performed by chemical dehydration in the presence of a catalyst.

In some embodiments, access to butadiene can be accomplished by biosynthetic production of MVC and subsequent chemical dehydration to butadiene. In some embodiments, the invention provides a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces MVC as described herein; and (b) converting MVC produced by culturing the non-naturally occurring microbial organism to butadiene. In some aspects, the converting MVC to butadiene is performed by chemical dehydration in the presence of a catalyst.

In other aspects, the invention further provides methods for producing elevated or enhanced synthesis or yields of biosynthetic products such as a butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol.

The methods for producing enhanced synthesis or yields of butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein include using a non-naturally occurring microbial organisms having one or more of the various pathway configurations employing a MeDH for methanol oxidation, a FaldFP, and/or an acetyl-CoA enhancing pathway for directing the carbon from methanol into acetyl-CoA and other desired products via formaldehyde fixation as described previously. The methods include using a non-naturally occurring microbial organism of the invention having one or more of the various different methanol oxidation and formaldehyde fixation configurations exemplified previously and below engineered in conjunction with any or each of the various methanol oxidation, formaldehyde fixation, formate reutilization, butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol pathway exemplified previously. Accordingly, the methods of the invention can use a microbial organism having one or more of the metabolic modifications exemplified previously and also below that increase biosynthetic product yields over, for example, endogenous methanol utilization pathways because they further focus methanol derived carbon into the assimilation pathways described herein, decrease inefficient use of methanol carbon through competing methanol utilization and/or FaldFPs and/or increase the production of reducing equivalents.

In some aspects, the methods of the invention can use microbial organisms containing or engineered to contain one or more of the various configurations of metabolic modifications disclosed herein for enhancing product yields via methanol derived carbon include enhancing methanol oxidation and production of reducing equivalents using either an endogenous NADH dependent MeDH, an exogenous NADH dependent MeDH, both an endogenous NADH dependent MeDH and exogenous NADH dependent MeDH alone or in combination with one or more metabolic modifications that attenuate, for example, DHA synthase and/or AOX. In addition, other metabolic modifications as exemplified previously and further below that reduce carbon flux away from methanol oxidation and formaldehyde fixation also can be included, alone or in combination, with the methanol oxidation and FaldFP configurations disclosed herein that enhance carbon flux into product precursors such as acetyl-CoA and, therefore, enhance product yields.

Accordingly, in some embodiments, the microbial organisms used in a method of the invention can include one or more of any of the above and/or below metabolic modifications to a methanol utilization pathway and/or formaldehyde assimilation pathway configurations for enhancing product yields can be combined with any one or more, including all of the previously described methanol oxidation, formaldehyde fixation, formate reutilization, fatty butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-01 pathway to enhance the yield and/or production of a product such as any of the butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein.

Given the teachings and guidance provided herein, both prokaryotic and eukaryotic microbial organisms engineered to have methanol oxidation and/or FaldFP configurations for enhancing product yields can be used in the methods of the invention. As exemplified herein and well known in the art, those skilled in the art will know which organism to select for a particular application. For example, with respect to eukaryotic microbial host organisms, those skilled in the art will know that yeasts and other eukaryotic microorganisms exhibit certain characteristics distinct from prokaryotic microbial organisms. When such characteristics are desirable, one skilled in the art can choose to use such eukaryotic microbial organisms having one or more of the various different methanol oxidation and formaldehyde fixation configurations exemplified herein for enhancing product yields in a method of the invention. Such characteristics have been described previously.

In some embodiments, the microbial organism used in a method of the invention and having a methanol oxidation and/or formaldehyde assimilation pathway configurations described herein for enhancing product yields can include, for example, a NADH-dependent MeDH(MeDH) and/or one or more formaldehyde assimilation pathways.

In one embodiment, the methods of the invention use microbial organisms that have cytosolic expression of one or more methanol oxidation and/or formaldehyde assimilation pathways. As described previously, exemplary pathways for converting cytosolic formaldehyde into glycolytic intermediates are shown in FIGS. 1 and 2. Such pathways include methanol oxidation via expression of a cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic DHA synthase, both methanol oxidation via expression of an cytosolic NADH dependent MeDH and formaldehyde fixation via expression of cytosolic DHA synthase alone or together with the metabolic modifications exemplified previously and also below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

In another embodiment, conversion of cytosolic formaldehyde into glycolytic intermediates can occur via expression of a cytosolic 3-hexulose-6-phosphate (3-Hu6P) synthase. Thus, exemplary pathways that can be engineered into a microbial organism used in a method of the invention can include methanol oxidation via expression of a cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic 3-Hu6P synthase, both methanol oxidation via expression of an cytosolic NADH dependent dehydrogenase and formaldehyde fixation via expression of cytosolic 3-Hu6P synthase alone or together with the metabolic modifications exemplified previously and also below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

In yet another embodiment, the methods of the invention use microbial organisms that have cytosolic expression of one or more methanol oxidation and/or formaldehyde assimilation pathways. The formaldehyde assimilation pathways can include both assimilation through cytosolic DHA synthase and 3-Hu6P synthase. In this specific embodiment, such pathways include methanol oxidation via expression of a cytosolic NADH dependent MeDH, formaldehyde fixation via expression of cytosolic DHA synthase and 3-Hu6P synthase, both methanol oxidation via expression of an cytosolic NADH dependent dehydrogenase and formaldehyde fixation via expression of cytosolic DHA synthase and 3-Hu6P synthase alone or together with the metabolic modifications exemplified previously and also below that attenuate less beneficial methanol oxidation and/or FaldFPs. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and/or attenuation of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation is utilized).

In some embodiments, the method for producing butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein includes using a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes attenuation of one or more endogenous enzymes, which enhances carbon flux through acetyl-CoA. For example, in some aspects, the endogenous enzyme can be selected from DHA kinase, methanol oxidase, PQQ-dependent MeDH, DHA synthase or any combination thereof. Accordingly, in some aspects, the attenuation is of the endogenous enzyme DHA kinase. In some aspects, the attenuation is of the endogenous enzyme methanol oxidase. In some aspects, the attenuation is of the endogenous enzyme PQQ-dependent MeDH. In some aspects, the attenuation is of the endogenous enzyme DHA synthase. The invention also provides a method wherein the microbial organism used includes attenuation of any combination of two or three endogenous enzymes described herein. For example, a microbial organism can include attenuation of DHA kinase and DHA synthase, or alternatively methanol oxidase and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and DHA synthase. The invention also provides a method wherein the microbial organism used includes attenuation of all endogenous enzymes described herein. For example, in some aspects, a microbial organism includes attenuation of DHA kinase, methanol oxidase, PQQ-dependent MeDH and DHA synthase.

In some embodiments, the method for producing butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein includes using a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes attenuation of one or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway. Examples of these endogenous enzymes are disclosed in FIGS. 1 and 2 and described in Example XXIII. It is understood that a person skilled in the art would be able to readily identify enzymes of such competing pathways. Competing pathways can be dependent upon the host microbial organism and/or the exogenous nucleic acid introduced into the microbial organism as described herein. Accordingly, in some aspects of the invention, the method includes a microbial organism having attenuation of one, two, three, four, five, six, seven, eight, nine, ten or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway.

In some embodiments, the method for producing butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein includes using a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes a gene disruption of one or more endogenous nucleic acids encoding enzymes, which enhances carbon flux through acetyl-CoA. For example, in some aspects, the endogenous enzyme can be selected from DHA kinase, methanol oxidase, PQQ-dependent MeDH, DHA synthase or any combination thereof. According, in some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme DHA kinase. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme methanol oxidase. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme PQQ-dependent MeDH. In some aspects, the gene disruption is of an endogenous nucleic acid encoding the enzyme DHA synthase. The invention also provides a method wherein the microbial organism used includes the gene disruption of any combination of two or three nucleic acids encoding endogenous enzymes described herein. For example, a microbial organism of the invention can include a gene disruption of DHA kinase and DHA synthase, or alternatively methanol oxidase and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and PQQ-dependent MeDH, or alternatively DHA kinase, methanol oxidase, and DHA synthase. The invention also provides a method wherein the microbial organism used includes wherein all endogenous nucleic acids encoding enzymes described herein are disrupted. For example, in some aspects, a microbial organism described herein includes disruption of DHA kinase, methanol oxidase, PQQ-dependent MeDH and DHA synthase.

In some embodiments, the method for producing butadiene, 13BDO, CrotOH, MVC and/or 3-buten-1-ol described herein includes using a non-naturally occurring microbial organism as described herein, wherein the microbial organism further includes a gene disruption of one or more endogenous enzymes of a competing formaldehyde assimilation or dissimilation pathway. Examples of these endogenous enzymes are disclosed in FIGS. 1 and 2 and described in Example XXII. It is understood that a person skilled in the art would be able to readily identify enzymes of such competing pathways. Competing pathways can be dependent upon the host microbial organism and/or the exogenous nucleic acid introduced into the microbial organism as described herein. Accordingly, in some aspects of the invention, the microbial organism used in the method includes a gene disruption of one, two, three, four, five, six, seven, eight, nine, ten or more endogenous nucleic acids encoding enzymes of a competing formaldehyde assimilation or dissimilation pathway.

Suitable purification and/or assays to test for the production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art.

The butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producers can be cultured for the biosynthetic production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. Accordingly, in some embodiments, the invention provides culture medium having the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate described herein. In some aspects, the culture mediums can also be separated from the non-naturally occurring microbial organisms of the invention that produced the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate. Methods for separating a microbial organism from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.

For the production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol yields.

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

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microbial organism of the invention. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In one embodiment, H2, CO, CO2 or any combination thereof can be supplied as the sole or supplemental feedstock to the other sources of carbon disclosed herein. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is a sugar-containing biomass. In some embodiments, the sugar is glucose. In one embodiment, the sugar is xylose. In another embodiment, the sugar is arabinose. In one embodiment, the sugar is galactose. In another embodiment, the sugar is fructose. In other embodiments, the sugar is sucrose. In one embodiment, the sugar is starch. In certain embodiments, the carbon source is glycerol. In some embodiments, the carbon source is crude glycerol. In one embodiment, the carbon source is crude glycerol without treatment. In other embodiments, the carbon source is glycerol and glucose. In another embodiment, the carbon source is methanol and glycerol. In one embodiment, the carbon source is carbon dioxide. In one embodiment, the carbon source is formate. In one embodiment, the carbon source is methane. In one embodiment, the carbon source is methanol. In one embodiment, the carbon source is chemoelectro-generated carbon (see, e.g., Liao et al. (2012) Science 335:1596). In one embodiment, the chemoelectro-generated carbon is methanol. In one embodiment, the chemoelectro-generated carbon is formate. In one embodiment, the chemoelectro-generated carbon is formate and methanol. In one embodiment, the carbon source is a sugar and methanol. In another embodiment, the carbon source is a sugar and glycerol. In other embodiments, the carbon source is a sugar and crude glycerol. In yet other embodiments, the carbon source is a sugar and crude glycerol without treatment. In one embodiment, the carbon source is a sugar-containing biomass and methanol. In another embodiment, the carbon source is a sugar-containing biomass and glycerol. In other embodiments, the carbon source is a sugar-containing biomass and crude glycerol. In other embodiments, the carbon source is a methanol and crude glycerol. In other embodiments, the carbon source is a methanol and glycerol. In yet other embodiments, the carbon source is a sugar-containing biomass and crude glycerol without treatment Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms provided herein for the production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol and other pathway intermediates.

In one embodiment, the carbon source is glycerol. In certain embodiments, the glycerol carbon source is crude glycerol or crude glycerol without further treatment. In a further embodiment, the carbon source comprises glycerol or crude glycerol, and also sugar or a sugar-containing biomass, such as glucose. In a specific embodiment, the concentration of glycerol in the fermentation broth is maintained by feeding crude glycerol, or a mixture of crude glycerol and sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of glycerol to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass. In certain other embodiments of the ratios provided above, the glycerol is a crude glycerol or a crude glycerol without further treatment. In other embodiments of the ratios provided above, the sugar is a sugar-containing biomass, and the glycerol is a crude glycerol or a crude glycerol without further treatment.

Crude glycerol can be a by-product produced in the production of biodiesel, and can be used for fermentation without any further treatment. Biodiesel production methods include (1) a chemical method wherein the glycerol-group of vegetable oils or animal oils is substituted by low-carbon alcohols such as methanol or ethanol to produce a corresponding fatty acid methyl esters or fatty acid ethyl esters by transesterification in the presence of acidic or basic catalysts; (2) a biological method where biological enzymes or cells are used to catalyze transesterification reaction and the corresponding fatty acid methyl esters or fatty acid ethyl esters are produced; and (3) a supercritical method, wherein transesterification reaction is carried out in a supercritical solvent system without any catalysts. The chemical composition of crude glycerol can vary with the process used to produce biodiesel, the transesterification efficiency, recovery efficiency of the biodiesel, other impurities in the feedstock, and whether methanol and catalysts were recovered. For example, the chemical compositions of eleven crude glycerol collected from seven Australian biodiesel producers reported that glycerol content ranged between 38% and 96%, with some samples including more than 14% methanol and 29% ash. In certain embodiments, the crude glycerol comprises from 5% to 99% glycerol. In some embodiments, the crude glycerol comprises from 10% to 90% glycerol. In some embodiments, the crude glycerol comprises from 10% to 80% glycerol. In some embodiments, the crude glycerol comprises from 10% to 70% glycerol. In some embodiments, the crude glycerol comprises from 10% to 60% glycerol. In some embodiments, the crude glycerol comprises from 10% to 50% glycerol. In some embodiments, the crude glycerol comprises from 10% to 40% glycerol. In some embodiments, the crude glycerol comprises from 10% to 30% glycerol. In some embodiments, the crude glycerol comprises from 10% to 20% glycerol. In some embodiments, the crude glycerol comprises from 80% to 90% glycerol. In some embodiments, the crude glycerol comprises from 70% to 90% glycerol. In some embodiments, the crude glycerol comprises from 60% to 90% glycerol. In some embodiments, the crude glycerol comprises from 50% to 90% glycerol. In some embodiments, the crude glycerol comprises from 40% to 90% glycerol. In some embodiments, the crude glycerol comprises from 30% to 90% glycerol. In some embodiments, the crude glycerol comprises from 20% to 90% glycerol. In some embodiments, the crude glycerol comprises from 20% to 40% glycerol. In some embodiments, the crude glycerol comprises from 40% to 60% glycerol. In some embodiments, the crude glycerol comprises from 60% to 80% glycerol. In some embodiments, the crude glycerol comprises from 50% to 70% glycerol. In one embodiment, the glycerol comprises 5% glycerol. In one embodiment, the glycerol comprises 10% glycerol. In one embodiment, the glycerol comprises 15% glycerol. In one embodiment, the glycerol comprises 20% glycerol. In one embodiment, the glycerol comprises 25% glycerol. In one embodiment, the glycerol comprises 30% glycerol. In one embodiment, the glycerol comprises 35% glycerol. In one embodiment, the glycerol comprises 40% glycerol. In one embodiment, the glycerol comprises 45% glycerol. In one embodiment, the glycerol comprises 50% glycerol. In one embodiment, the glycerol comprises 55% glycerol. In one embodiment, the glycerol comprises 60% glycerol. In one embodiment, the glycerol comprises 65% glycerol. In one embodiment, the glycerol comprises 70% glycerol. In one embodiment, the glycerol comprises 75% glycerol. In one embodiment, the glycerol comprises 80% glycerol. In one embodiment, the glycerol comprises 85% glycerol. In one embodiment, the glycerol comprises 90% glycerol. In one embodiment, the glycerol comprises 95% glycerol. In one embodiment, the glycerol comprises 99% glycerol.

In one embodiment, the carbon source is methanol or formate. In certain embodiments, methanol is used as a carbon source in the formaldehyde assimilation pathways provided herein. In one embodiment, the carbon source is methanol or formate. In other embodiments, formate is used as a carbon source in the formaldehyde assimilation pathways provided herein. In specific embodiments, methanol is used as a carbon source in the MMPs provided herein, either alone or in combination with the product pathways provided herein.

In one embodiment, the carbon source comprises methanol, and sugar (e.g., glucose) or a sugar-containing biomass. In another embodiment, the carbon source comprises formate, and sugar (e.g., glucose) or a sugar-containing biomass. In one embodiment, the carbon source comprises methanol, formate, and sugar (e.g., glucose) or a sugar-containing biomass. In specific embodiments, the methanol or formate, or both, in the fermentation feed is provided as a mixture with sugar (e.g., glucose) or sugar-comprising biomass. In certain embodiments, sugar is provided for sufficient strain growth.

In certain embodiments, the carbon source comprises methanol and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar (e.g., glucose). In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of formate to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture of methanol and formate, and a sugar (e.g., glucose). In certain embodiments, sugar is provided for sufficient strain growth. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 70:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:20. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is provided at a molar concentration ratio of methanol and formate to sugar of 1:2. In certain embodiments of the ratios provided above, the sugar is a sugar-containing biomass.

In addition to renewable feedstocks such as those exemplified above, the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H₂ and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H₂ and CO, syngas can also include CO₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO₂ and CO₂/H₂ mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H₂-dependent conversion of CO₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2CO₂+4H₂+nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for the production of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyltetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, FDH, FTHFS, methenyltetrahydrofolate cyclodehydratase, MTHFDH and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, CODH and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with CODH and/or hydrogenase activities can also be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, CODH, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H₂ by CODH and hydrogenase are utilized to fix CO₂ via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains a reductive TCA pathway can confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol and any of the intermediate metabolites in the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway when grown on a carbohydrate or other carbon source. The butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, acetoacetyl-CoA, acetoacetate, 3-oxobutyraldehyde, acetoacetyl-ACP, acetoacetyl-CoA, acetoacetyl-ACP, acetoacetyl-CoA, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA, acetoacetyl-CoA, acetoacetate, 3-oxobutyraldehyde, 4-hydroxy-2-butanone, crotonyl-ACP, crotonyl-CoA, 3-hydroxybutyryl-ACP, 3-hydroxybutyryl-CoA, 3-hydroxybutyrate, 3-hydroxybutyraldehyde, crotonaldehyde, crotonyl-ACP, crotonyl-CoA, crotonate, crotonaldehyde, 2-butenyl-4-phosphate, 2-butenyl-4-diphosphate, 3-oxoglutaryl-CoA, 3-hydroxy-5-oxopentanoate, 3,5-dihydroxy pentanoate, 3-hydroxy-5-phosphonatooxypentanoate, 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, butenyl 4-biphosphate, 2-butenyl 4-diphosphate, 2-butanol, acetolactate, acetoin, 2,3-butanediol, 3-hydroxybutyryl phosphate, 3-hydroxybutyryl diphosphate, 3-oxopent-4-enoyl-CoA, 3-oxopent-4-enoate, 3-buten-2-one, 3-oxo-4-hydroxy pentanoyl-CoA, 3-oxo-4-hydroxypentanoate, 3,4-dihydroxypentanoate, 3,4-dihydroxypentanoyl-CoA, 3,4-dihydroxypentanoate, 4-oxopentanoate, 4-hydroxypentanoate, 3-oxoadipyl-CoA, 3-oxoadipate, 4-oxopentanoate, 4-hydroxypentanoate, vinylacetyl-CoA, 3-buten-1-al, 3-oxopent-4-enoyl-CoA, 3-hydroxypent-4-enoyl-CoA, 3-oxopent-4-enoate, 3-hydroxypent-4-eonoate, 3-oxo-5-hydroxypentanoyl-CoA, 3,5-dihydroxypentanoyl-CoA, 5-hydroxypent-2-enoyl-CoA, pent-2,4-dienoyl-CoA, 2,4-pentadienoate, 3-oxo-5-hydroxypentanoate, 3,5-dihydroxypentanoate, 5-hydroxypent-2-enoate, 3-oxopentanoyl-CoA, 3-hydroxypentanoyl-CoA, pent-2-enoyl-CoA, or pent-3-enoyl-CoA.

The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein in sufficient amounts to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producers can synthesize butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producing microbial organisms can produce butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol intracellularly and/or secrete the product into the culture medium.

Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N₂/CO₂ mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C., but the temperature can be maintained at a higher or lower temperature depending on the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired. Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art.

In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM.

In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or any butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as “uptake sources.” Uptake sources can provide isotopic enrichment for any atom present in the product butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate, or for side products generated in reactions diverging away from a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

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

In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon-14, or an environmental or atmospheric carbon source, such as CO₂, which can possess a larger amount of carbon-14 than its petroleum-derived counterpart.

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

Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.

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

The biobased content of a compound is estimated by the ratio of carbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and M represent the ¹⁴C/¹²C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to δ¹³C_VPDB=−19 per mil (Olsson, The use of Oxalic acid as a Standard. in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to δ¹³C_VPDB=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geoftsik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴, and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm=100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a “modern” source includes biobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a “pre-bomb” standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50% water would be considered to have a Biobased Content=100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content=66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum-based product would be considered to have a Biobased Content=0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene terephthalate (PP 1) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is CO₂. In some embodiments, the present invention provides butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically produced butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment. For example, in some aspects the invention provides bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol intermediate having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product Methods of chemically modifying a bioderived product of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, agrochemical, or perfume having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, wherein the polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume are generated directly from or in combination with bioderived butadiene or 3-buten-1-ol or a bioderived butadiene or 3-buten-1-ol pathway intermediate as disclosed herein.

Butadiene is a chemical commonly used in many commercial and industrial applications. Provided herein are a bioderived butadiene and biobased products comprising one or more bioderived butadiene or bioderived butadiene intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. Also provided herein are uses for bioderived butadiene and the biobased products. Non-limiting examples are described herein and include the following. Biobased products comprising all or a portion of bioderived butadiene include polymers, including synthetic rubbers and ABS resins, and chemicals, including hexamethylenediamine (HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam, chloroprene, sulfalone, n-octanol and octene-1. The biobased polymers, including co-polymers, and resins include those where butadiene is reacted with one or more other chemicals, such as other alkenes, e.g. styrene, to manufacture numerous copolymers, including acrylonitrile 1,3-butadiene styrene (ABS), styrene-1,3-butadiene rubber (styrene butadiene rubber; SBR), styrene-1,3-butadiene latex. Products comprising biobased butadiene in the form of polymer synthetic rubber (SBR) include synthetic rubber articles, including tires, adhesives, seals, sealants, coatings, hose and shoe soles, and in the form of synthetic ruber polybutadiene (polybutadiene rubber, PBR or BR) which is used in synthetic rubber articles including tires, seals, gaskets and adhesives and as an intermediate in production of thermoplastic resin including acrylonitrile-butadiene-styrene (ABS) and in production of high impact modifier of polymers such as high impact polystyrene (HIPS). ABS is used in molded articles, including pipe, telephone, computer casings, mobile phones, radios, and appliances. Other biobased BD polymers include a latex, including styrene-butadiene latex (SB), used for example in paper coatings, carpet backing, adhesives, and foam mattresses; nitrile rubber, used in for example hoses, fuel lines, gasket seals, gloves and footwear; and styrene-butadiene block copolymers, used for example in asphalt modifiers (for road and roofing construction applications), adhesives, footwear and toys. Chemical intermediates made from butadiene include adiponitrile, HMDA, lauryl lactam, and caprolactam, used for example in production of nylon, including nylon-6,6 and other nylon-6,X, and chloroprene used for example in production of polychloroprene (neoprene). Butanediol produced from butadiene is used for example in production of speciality polymer resins including thermoplastic including polybutylene terephthalate (PBT), used in molded articles including parts for automotive, electrical, water systems and small appliances. Butadiene is also a co-monomer for polyurethane and polyurethane-polyurea copolymers. Butadiene is a co-monomer for biodegradable polymers, including PBAT (poly(butylene adipate-co-terephthalate)) and PBS (poly(butylene succinate)). Tetrahydrofuran produced from butadiene finds use as a solvent and in production of elastic fibers. Conversion of butadiene to THF, and subsequently to polytetramethylene ether glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide and PTHF, poly(tetrahydrofuran)), provides an intermediate used to manufacture elastic fibers, e.g. spandex fiber, used in products such as LYCRA® fibers or elastane, for example when combined with polyurethane-polyurea copolymers. THF also finds use as an industrial solvent and in pharmaceutical production. PTMEG is also combined with in the production of specialty thermoplastic elastomers (TPE), including thermoplastic elastomer polyester (TPE-E or TPEE) and copolyester ethers (COPE). COPEs are high modulus elastomers with excellent mechanical properties and oil/environmental resistance, allowing them to operate at high and low temperature extremes. PTMEG and butadiene also make thermoplastic polyurethanes (e.g. TPE-U or TPEU) processed on standard thermoplastic extrusion, calendaring, and molding equipment, and are characterized by their outstanding toughness and abrasion resistance. Other biobased products of bioderived BD include styrene block copolymers used for example in bitumen modification, footwear, packaging, and molded extruded products; methylmethacrylate butadiene styrene and methacrylate butadiene styrene (MBS) resins—clear resins—used as impact modifier for transparent thermoplastics including polycarbonate (PC), polyvinyl carbonate (PVC) and poly)methyl methacrylate (PMMA); sulfalone used as a solvent or chemical; n-octanol and octene-1. Accordingly, in some embodiments, the invention provides a biobased product comprising one or more bioderived butadiene or bioderived butadiene intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

CrotOH, also referred to as 2-buten-1-ol, is a valuable chemical intermediate. CrotOH is a chemical commonly used in many commercial and industrial applications. Non-limiting examples of such applications include production of crotyl halides, esters, and ethers, which in turn are chemical are chemical intermediates in the production of monomers, fine chemicals, such as sorbic acid, trimethylhydroquinone, crotonic acid and 3-methoxybutanol, agricultural chemicals, and pharmaceuticals. Exemplary fine chemical products include sorbic acid, trimethylhydroquinone, crotonic acid and 3-methoxybutanol. CrotOH is also a precursor to 1,3-butadiene. CrotOH is currently produced exclusively from petroleum feedstocks. For example Japanese Patent 47-013009 and U.S. Pat. Nos. 3,090,815, 3,090,816, and 3,542,883 describe a method of producing CrotOH by isomerization of 1,2-epoxybutane. The ability to manufacture CrotOH from alternative and/or renewable feedstocks would represent a major advance in the quest for more sustainable chemical production processes. Accordingly, in some embodiments, the invention provides a biobased monomer, fine chemical, agricultural chemical, or pharmaceutical comprising one or more bioderived CrotOH or bioderived CrotOH intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

13BDO is a chemical commonly used in many commercial and industrial applications. Non-limiting examples of such applications include its use as an organic solvent for food flavoring agents or as a hypoglycaemic agent and its use in the production of polyurethane and polyester resins. Moreover, optically active 13BDO is also used in the synthesis of biologically active compounds and liquid crystals. Still further, 13BDO can be used in commercial production of 1,3-butadiene, a compound used in the manufacture of synthetic rubbers (e.g., tires), latex, and resins. 13BDO can also be sued to synthesize (R)-3-hydroxybutyryl-(R)-13BDO monoester or (R)-3-ketobutyryl-(R)-13BDO. Accordingly, in some embodiments, the invention provides a biobased organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin comprising one or more bioderived 13BDO or bioderived 13BDO intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

MVC is a chemical commonly used in many commercial and industrial applications. Non-limiting examples of such applications include it use as a solvent, e.g. as a viscosity adjustor, a monomer for polymer production, or a precursor to a fine chemical such as in production of contrast agents for imaging (see US20110091374) or production of glycerol (see US20120302800A1). MVC can also be used as a precursor in the production of 1,3-butadiene. Accordingly, in some embodiments, the invention provides a biobased solvent, polymer (or plastic or resin made from that polymer), or fine chemical comprising one or more bioderived MVC or bioderived MVC intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

3-Buten-1-ol is a chemical commonly used in many commercial and industrial applications. Non-limiting examples of such applications include production of pharmaceuticals, agrochemicals, perfumes and resins. Accordingly, in some embodiments, the invention provides a biobased pharmaceutical, agrochemical, perfume or resin comprising one or more bioderived 3-buten-1-ol or bioderived 3-buten-1-ol intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

Further, the present invention relates to the biologically produced butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a pathway intermediate thereof as disclosed herein, and to the products derived therefrom, including non-biosynthetic enzymatic or chemical conversion of 13BDO, CrotOH, MVC or 3-buten-1-ol to butadiene, wherein the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a pathway intermediate thereof has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment. For example, in some aspects the invention provides: bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a pathway intermediate thereof having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or a bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product Methods of chemically modifying a bioderived product of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, and are described herein. For each of the bioderived compounds described herein, the invention further provides a biobased product including biobased product and its uses as described herein, and further where the biobased product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, and wherein the biobased product is generated directly from or in combination with bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, preferably bioderived butadiene made completely bio-synthetically or by enzymatic or chemical conversion of 13BDO, CrotOH, MVC or 3-buten-1-ol to butadiene, or with bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol intermediate as disclosed herein. Non-limiting examples of such biobased products include those described for each bioderived chemical, e.g. bioderived butadiene, including a plastic, thermoplastic, elastomer, polyester, polyurethane, polymer, co-polymer, synthetic rubber, resin, chemical, polymer intermediate, a molded product, a resin, organic solvent, hypoglycaemic agent, polyester resin, latex, monomer, fine chemical, agricultural chemical, pharmaceutical, cosmetic, personal care product, or perfume.

In some embodiments, the invention provides polymer, synthetic rubber, resin, or chemical comprising bioderived butadiene or bioderived butadiene pathway intermediate, wherein the bioderived butadiene or bioderived butadiene pathway intermediate includes all or part of the butadiene or butadiene pathway intermediate used in the production of polymer, synthetic rubber, resin, or chemical, or other biobased products described herein (for example hexamethylenediamine (HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG, COPE). Thus, in some aspects, the invention provides a biobased polymer, synthetic rubber, resin, or chemical or other biobased product described herein comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived butadiene or bioderived butadiene pathway intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased polymer, synthetic rubber, resin, or chemical or other biobased product described herein (for example hexamethylenediamine (HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG, COPE), wherein the butadiene or butadiene pathway intermediate used in its production is a combination of bioderived and petroleum derived butadiene or butadiene pathway intermediate. For example, a biobased polymer, synthetic rubber, resin, or chemical or other biobased product described herein (for example hexamethylenediamine (HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG, COPE) can be produced using 50% bioderived butadiene and 50% petroleum derived butadiene or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing polymer, synthetic rubber, resin, or chemical or other biobased product described herein (for example hexamethylenediamine (HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG, COPE) using the bioderived butadiene or bioderived butadiene pathway intermediate of the invention are well known in the art.

In some embodiments, the invention provides organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin comprising bioderived 13BDO or bioderived 13BDO pathway intermediate, wherein the bioderived 13BDO or bioderived 13BDO pathway intermediate includes all or part of the 13BDO or 13BDO pathway intermediate used in the production of organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin. Thus, in some aspects, the invention provides a biobased organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived 13BDO or bioderived 13BDO pathway intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin wherein the 13BDO or 13BDO pathway intermediate used in its production is a combination of bioderived and petroleum derived 13BDO or 13BDO pathway intermediate. For example, a biobased organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin can be produced using 50% bioderived 13BDO and 50% petroleum derived 13BDO or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing organic solvent, hypoglycaemic agent, polyurethane, polyester resin, synthetic rubber, latex, or resin using the bioderived 13BDO or bioderived 13BDO pathway intermediate of the invention are well known in the art.

In some embodiments, the invention provides monomer, fine chemical, agricultural chemical, or pharmaceutical comprising bioderived CrotOH or bioderived CrotOH pathway intermediate, wherein the bioderived CrotOH or bioderived CrotOH pathway intermediate includes all or part of the CrotOH or CrotOH pathway intermediate used in the production of monomer, fine chemical, agricultural chemical, or pharmaceutical. Thus, in some aspects, the invention provides a biobased monomer, fine chemical, agricultural chemical, or pharmaceutical comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived CrotOH or bioderived CrotOH pathway intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased monomer, fine chemical, agricultural chemical, or pharmaceutical wherein the CrotOH or CrotOH pathway intermediate used in its production is a combination of bioderived and petroleum derived CrotOH or CrotOH pathway intermediate. For example, a biobased monomer, fine chemical, agricultural chemical, or pharmaceutical can be produced using 50% bioderived CrotOH and 50% petroleum derived CrotOH or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing monomer, fine chemical, agricultural chemical, or pharmaceutical using the bioderived CrotOH or bioderived CrotOH pathway intermediate of the invention are well known in the art.

In some embodiments, the invention provides solvent (or solvent-containing composition), polymer (or plastic or resin made from that polymer), or a fine chemical, comprising bioderived MVC or bioderived MVC pathway intermediate, wherein the bioderived MVC or bioderived MVC pathway intermediate includes all or part of the MVC or MVC pathway intermediate used in the production of the solvent (or composition containing the solvent), polymer (or plastic or resin made from that polymer) or fine chemical. Thus, in some aspects, the invention provides a biobased solvent (or composition containing the solvent), polymer (or plastic or resin made from that polymer) or fine chemical comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived MVC or bioderived MVC pathway intermediate as disclosed herein. Additionally, in some aspects, the invention provides the biobased solvent (or composition containing the solvent), polymer (or plastic or resin made from that polymer) or fine chemical wherein the MVC or MVC pathway intermediate used in its production is a combination of bioderived and petroleum derived MVC or MVC pathway intermediate. For example, the biobased the solvent (or composition containing the solvent), polymer (or plastic or resin made from that polymer) or fine chemical can be produced using 50% bioderived MVC and 50% petroleum derived MVC or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing the solvent (or composition containing the solvent), polymer (or plastic or resin made from that polymer) or fine chemical using the bioderived MVC or bioderived MVC pathway intermediate of the invention are well known in the art.

As used herein, the term “bioderived” means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term “biobased” means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides a biobased product comprising bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate, wherein the bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate includes all or part of the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate used in the production of the biobased product. For example, the final biobased product can contain the bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, butadiene, 13BDO, CrotOH, MVC or 3-buten-1-olpathway intermediate, or a portion thereof that is the result of the manufacturing of biobased product. Such manufacturing can include chemically reacting the bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final biobased product. Thus, in some aspects, the invention provides a biobased product comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate as disclosed herein.

Additionally, in some embodiments, the invention provides a composition having a bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate disclosed herein and a compound other than the bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate. For example, in some aspects, the invention provides a biobased product wherein the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate used in its production is a combination of bioderived and petroleum derived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate. For example, a biobased product can be produced using 50% bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol and 50% petroleum derived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing a biobased product using the bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol or bioderived butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway intermediate of the invention are well known in the art.

In some embodiments, the invention provides polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume comprising bioderived butadiene or 3-buten-1-ol or bioderived butadiene or 3-buten-1-ol pathway intermediate, wherein the bioderived butadiene or 3-buten-1-ol or bioderived butadiene or 3-buten-1-ol pathway intermediate includes all or part of the butadiene or 3-buten-1-ol or butadiene or 3-buten-1-ol pathway intermediate used in the production of polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume. For example, the final polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume can contain the bioderived butadiene or 3-buten-1-ol, butadiene or 3-buten-1-ol pathway intermediate, or a portion thereof that is the result of the manufacturing of polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume. Such manufacturing can include chemically reacting the bioderived butadiene or 3-buten-1-ol or bioderived butadiene or 3-buten-1-ol pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume. Thus, in some aspects, the invention provides a biobased polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived butadiene or 3-buten-1-ol or bioderived butadiene or 3-buten-1-ol pathway intermediate as disclosed herein.

Additionally, in some embodiments, the invention provides a composition having a bioderived butadiene or 3-buten-1-ol or butadiene or 3-buten-1-ol pathway intermediate disclosed herein and a compound other than the bioderived butadiene or 3-buten-1-ol or butadiene or 3-buten-1-ol pathway intermediate. For example, in some aspects, the invention provides a biobased polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (TPE), elastomer polyester, monomer, agrochemical, or perfume wherein the butadiene or 3-buten-1-ol or butadiene or 3-buten-1-ol pathway intermediate used in its production is a combination of bioderived and petroleum derived butadiene or 3-buten-1-ol or butadiene or 3-buten-1-ol pathway intermediate. For example, a biobased polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume can be produced using 50% bioderived butadiene or 3-buten-1-ol and 50% petroleum derived butadiene or 3-buten-1-ol or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing polymer, synthetic rubber, resin, chemical, copolymer, latex, nylon, thermoplastic, polyurethane, fiber, industrial solvent, thermoplastic elastomer (PE), elastomer polyester, monomer, agrochemical, or perfume using the bioderived butadiene or 3-buten-1-ol or bioderived butadiene or 3-buten-1-01 pathway intermediate of the invention are well known in the art.

In some aspects, the invention provides a biobased product that includes a portion of the bioderived butadiene or 3-buten-1-ol as a repeating unit. In some aspects, the invention provides a molded product obtained by molding a biobased product that includes the bioderived butadiene or 3-buten-1-ol disclosed herein. In some aspects, the invention provides a process for producing a biobased product that includes reacting the bioderived butadiene or 3-buten-1-ol disclosed herein, including chemically reacting the bioderived butadiene or 3-buten-1-ol, with itself or another compound in a reaction that produces a biobased product disclosed herein.

The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achieving biosynthesis of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, an anaerobic condition refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol will include culturing a non-naturally occurring butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producers of the invention for continuous production of substantial quantities of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol, the butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol producers also can be, for example, simultaneously subjected to chemical synthesis and/or enzymatic procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical an/or enzymatic conversion to convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol.

Biomass contains lignocelluloses and hemicelluloses that require treatment (saccharification) to release monosaccharides. Biomass sugar comprises primarily Sugar 2, Sugar 3 and Sugar 1, as well as various incompletely digested di-, tri-, and larger oligo-saccharides. For efficient, cost-effective fermentation at commercial scale, simultaneous use of the biomass' fermentable sugars is desirable. However, many microbial organisms, including E. coli, are susceptible to Sugar 1 catabolite repression of the fermentation of other sugars. When Sugar 1 is present, Sugar 1 is the preferred and essentially exclusive carbon source, repressing the catabolism of other sugars, including Sugar 3 and Sugar 2. In addition, fermentation of Sugar 3 can catabolite repress the fermentation of Sugar 2.

Uptake and preparation of a particular sugar for fermentation is controlled by specific sugar permease and/or transport proteins, as well as sugar modification proteins, such as isomerases, kinases and phosphatases. For example, in E. coli, these proteins are encoded by genes that are located in proximity to each other and under similar regulatory control. The Sugar 2 operon t2 and operon m2 contain genes under transcriptional control of XR, a DNA-binding positive regulatory protein. In the presence of Sugar 2, XR activates these operons to enhance uptake and metabolism of Sugar 2. However, when either Sugar 1 or Sugar 3 is present, Sugar 2-inducible transcription of these operons is repressed. Fermentation of Sugar 2 will not occur until after both Sugar 1 and Sugar 3 are fermented, which leads to inefficient industrial scale fermentation of biomass.

The invention provides engineered microbial organisms, compositions and methods for the co-utilization of Sugar 2 and other sugars with a second, different type of sugar, including for example, Sugar 1 and Sugar 3. Accordingly, the microbial organisms of the invention are relieved from diauxie, or the sequential utilization of different types of sugar, and are able to co-utilize two or more types of sugar simultaneously. Exemplary sugars for co-utilization include Sugar 1, Sugar 3 and/or Sugar 2.

The invention provides an isolated nucleic acid molecule, including: (a) a nucleic acid molecule encoding an amino acid sequence of XR, wherein the amino acid sequence comprises an amino acid substitution at position 121 as set forth in Table 1; (b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes an amino acid substitution at position 121 as set forth in Table 1, or (c) a nucleic acid molecule that is complementary to (a) or (b).

The isolated nucleic acid encodes a XR polypeptide having a mutation that reduces or eliminates catabolite repression of XR from other monosaccharides such as Sugar 1 and Sugar 3. The mutation corresponds to amino acid position 121 of the E. coli XR polypeptide. Table 1 in Example XVIII below lists the amino acid substitutions at position 121 that reduce or eliminate catabolite repression of XR In total there are at least 15 amino acids at position 121 that reduce or eliminate catabolite repression when substituted for the wild-type Arg residue. The invention provides encoding nucleic acids for a XR mutant having any one of the at least 15 amino acid substitutions at position 121. The codon corresponding to position 121 can therefore include a codon corresponding to alanine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine and, in some instances, tryptophane.

The invention additionally provides a xR mutant nucleic acid that includes the degeneracy of the genetic code or that corresponds to a related xR homologue from the same or different species so long as it contains a codon corresponding to position 121 of the reference xR mutant and encoding one of the amino acid substitutions set forth in Table 1 below. The amino acid substitution at position 121 can be engineered into a wild-type reference sequence to produce the xR mutant nucleic acid encoding a XR polypeptide having reduced or eliminated catabolite repression. The xR mutant nucleic acid will hybridize under stringent or highly stringent conditions. Thus, a xR mutant nucleic acid of the invention includes a nucleic acid encoding the same amino acid sequence as a reference mutant XR polypeptide of the invention, but having a different nucleic acid sequence. Also provided is a nucleic acid complementary to the above described xR mutant nucleic acids.

The invention also provides an isolated nucleic acid molecule corresponding to xR, wherein the encoded amino acid sequence other than the amino acid substitution at position 121 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of XR.

The xR mutant nucleic acid and the XR mutant polypeptide are described herein with reference to the E. coli xR nuleic acid. One skilled in the art will readily understand that xR sequences from species other than E. coli can be analyzed with routine and well known methods for aligning sequences (for example BLAST, blast.ncbi.nlm.nih.gov; Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Such alignments can provide information on conserved residues that can be utilized to identify a consensus sequence for preserving enzyme activity as well as for identifying positions is such other species that correspond to position 121 in the E. coli xR nucleic acid. The amino acid substitutions identified in above and in Table 1 below can be engineered into the position corresponding to position 121 of the E. coli xR gene to generate an nucleic acid that encodes a mutant XR product that has reduced or eliminated catabolite repression. Such other nucleic acids can be used in all embodiments described herein with respect to the exemplary E. coli xR mutant encoding nucleic acid and XR polypeptide for the co-utilization of two or more monosaccharides, including expressing the nucleic acid for the production of a target polypeptide. Thus, a xR mutant nucleic acid of the invention includes a nucleic acid encoding a different amino acid sequence as a reference mutant XR polypeptide of the invention, but exhibiting Sugar 2 operons regulatory activity (xR activity) and having reduced or eliminated catabolite repression from a second monosaccharide.

The invention further provides a vector containing a xR mutant nucleic acid molecule of the invention. Nucleic acid vectors, their construction and use have been previously described above and further described below with reference to nucleic acids encoding one or more FaldFP enzyme, FAP enzyme, butadiene pathway enzyme, 13BDO pathway enzyme, CrotOH pathway enzyme, MVC pathway enzyme, MOP enzyme, 3-buten-1-ol pathway enzyme or combinations thereof. As is understood by those skilled in the art, such teachings and guidance are equally applicable to the manipulation, propagation and expression of xR mutant nucleic acids of the invention and for the generation of microbial organisms capable of co-utilizing or co-metabolizing Sugar 2 and a second monosaccharide such as Sugar 1 or Sugar 3 or both. Accordingly, in some embodiments, the vector can be an expression vector having expression and/or regulatory elements, or other genetic elements, operable linked to a xR mutant nucleic acid of the invention as disclosed herein.

The invention additionally provides a non-naturally occurring microbial organism, including: (a) an exogenous nucleic acid molecule encoding an amino acid sequence of XR, wherein said amino acid sequence comprises an amino acid substitution at position 121 as set forth in Table 1; (b) an exogenous nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes an amino acid substitution at position 121 as set forth in Table 1, and (c) an exogenous nucleic acid molecule that is complementary to (a) or (b). The encoded amino acid sequence of XR other than the amino acid substitution at position 121 has can be at least at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of XR.

Any of the xR mutant nucleic acids described above can be introduced into a host to produce a non-naturally occurring microbial organism having a xR mutant nucleic acid of the invention. AxR mutant nucleic acid also can be introduced and expressed to produce a mutant XR polypeptide that exhibits reduced or eliminated catabolite repression and, therefore, confer the ability upon the host to co-metabolize Sugar 2 and a second monosaccharide. The second monosaccharide can be, for example, Sugar 1 or Sugar 3. Methods for introducing the xR mutant nucleic acids described herein with respect to pathway enzymes for the production of various bioderived compounds of the invention are well known in the art can be used to, for example, transform a host or stably integrate an expressible xR mutant nucleic acid of the invention.

The invention further provides the ability to enhance co-metabolism of two or more monosaccharides by a microbial organism. Removal of catabolite repression by expression of an xR mutant nucleic acid of the invention allows simultaneous utilization of one, two, three or more monosaccharides in addition to Sugar 2. Accordingly, increasing the cellular uptake and/or intracellular availability of those other monosaccharides enhances the simultaneous utilization of multiple monosaccharides.

One embodiment of the invention for increasing the uptake or intracellular availability of a monosaccharide is to constitutively express one or more nucleic acids encoding a monosaccharide transporter protein. Another embodiment is to overexpress one or more nucleic acids encoding a monosaccharide transporter protein. As described above and below, nucleic acids encoding such transporter proteins can be exogenously introduced into a microbial organism of the invention to augment uptake or intracellular availability of a monosaccharide. Monosaccharides include, for example, Sugar 1, Sugar 3, Sugar 2, and fructose. Transporter proteins include, for example, AraE, AraFGH, and/or OperonT2. AraE is a proton symporter that acts as a low-affinity high-capacity transporter for Sugar 3. AraFGH is a high-affinity ABC transporter for Sugar 3. Operon T2, i.e. F, G and H proteins, is a high-affinity ABC transporter for Sugar 3.

The arabinose operon is an inducible operon that requires the presence of arabinose for its induction of its encoded enzymes and permeases beyond minimal basal levels. This adaptive mechanism ensures the enzymes needed to catabolize arabinose are produced in sufficient amounts only when arabinose is present in the environment. The araC gene encodes a positive regulatory protein required for arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP-requiring receptor protein and under negative control by its protein product (autoregulation). The arabinose operon also exhibits catabolite repression.

Glucose in the environment will repress the arabinose operon due to low levels of the cAMP molecule. As demonstrated herein, use of an AraE of the present invention, e.g. from C. glutacicum or one that is evolutionarily distant from the AraE of E. coli, that is also under a non-AraC controlled promoter, allows arabinose uptake and use by escaping from need for arabinose positive regulation and glucose catabolite repression in a bacteria that normally is subject to such repression. Without being bound by theory it is believed the AraE protein of the invention is one that is also free from any allosteric or direct inhibition by glucose or its metabolites and/or is not dependent on or controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) system in the bacterial membrane. Accordingly, disclosed herein is a non-naturally-occurring microorganism comprising an enzymatic pathway for a product of interest, e.g. butadiene, 1,4-butanediol, 13BDO, that comprises an deregulated AraE to increase arabinose transport under conditions that inhibit an non-dergualted AraE. A method of co-use of glucose and arabinose as carbon sources to produce the product of interest is provided using the non-naturally-occurring microorganism having a deregulated AraE. The AraE can be one that is deregulated by being overexpressed at the protein level or under a consitituive promoter or promoter that is not subject to glucose catabolite represssion. The AraE can be one that is deregulated at the protein level by not being subject to post-translational inhibition by glucose catoblite repression system in the microorganism.

In some embodiments, the invention includes a microbial organism of the invention having an exogenous xR mutant nucleic acid of the invention. The exogenous xR mutant nucleic acid can be expressed by a variety of modes well known to those in the art and described herein, including for example, constitutive expression, inducible expression and/or overexpression. The microbial organism having an exogenous xR mutant nucleic acid of the invention can further have an exogenous nucleic acid encoding AraE. The microbial organism having an exogenous xR mutant nucleic acid of the invention can further have an exogenous nucleic acid encoding Operon T2 or AraFGH, and further Operon M2. The microbial organism having an exogenous xR mutant nucleic acid of the invention and an exogenous nucleic acid encoding AraE can further have an exogenous nucleic acid encoding Operon T2 or AraFGH, and further Operon M2. In some aspects, the microbial organism having an exogenous xR mutant nucleic acid of the invention can include multiple copies of a Sugar 2 operon regulated by an XR polypeptide, such as operon t2 and operon m2, or a gene therein. Any of the encoding araE, operon t2, operon m2 or araFGH nucleic acids can similarly be expressed by a variety of modes well known to those in the art and described herein, including for example, constitutive expression, inducible expression and/or overexpression. Expression of one, two, three, four or more, including some or all of the exogenous encoding nucleic acids can be following integration into a chromosome or episomally using methods well known in the art and as described herein with reference to expression of other nucleic acids of the invention.

In some embodiments, the invention further provides a culture medium including any of the non-naturally occurring microbial organisms described above. Accordingly, the culture medium can include a non-naturally occurring microbial organism having an exogenous nucleic acid encoding a XR mutant of the invention; two exogenous nucleic acids encoding a XR mutant of the invention and AraE; two or more exogenous nucleic acids encoding a XR mutant of the invention and one or more of Operon T2, Operon M2 or AraFGH; three or more exogenous nucleic acids encoding a XR mutant of the invention, AraE and Operon T2, Operon M2 or AraFGH.

The invention provides an isolated polypeptide having an amino acid sequence of XR, wherein said amino acid sequence includes an amino acid substitution at position 121 as set forth in Table 1. Also provides is an isolated polypeptide that includes an amino acid sequence of XR, wherein said amino acid sequence other than said amino acid substitution at position 121 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to an amino acid sequence of XR. Methods of using an isolated polypeptide having an amino acid sequence of XR that includes an amino acid substitution at position 121 as set forth in Table 1 are also provided. A composition including a isolated polypeptide that includes an amino acid sequence of XR, wherein said amino acid sequence includes an amino acid substitution at position 121 as set forth in Table 1 and at least one substrate for said polypeptide.

In some embodiments, the invention provides an isolated polypeptide having an amino acid sequence of XR, wherein the amino acid sequence comprises a substitution set forth in Table 1 of Example XVIII. In other aspects, the isolated polypeptide of the invention has an amino acid sequence, including a substitution set forth in Table 1 of Example XVIII and has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acids sequence of XR.

The polypeptides of the invention can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present invention can be obtained using well-known recombinant methods (see, for example, Sambrook et al., supra, 1989; Ausubel et al., supra, 1999). The methods and conditions for biochemical purification of a polypeptide of the invention can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

One non-limiting example of a method for preparing the invention polypeptide is to express nucleic acids encoding the polypeptide in a suitable host cell, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known purification methods, so described herein. Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors as described herein. Recombinantly expressed polypeptides of the invention can also be expressed as fusion proteins with appropriate affinity tags, such as glutathione S transferase (GST) or poly His, and affinity purified. Accordingly, in some embodiments, the invention provides a host cell expressing a polypeptide of the invention disclosed herein. An invention polypeptide can also be produced by chemical synthesis using a method of polypeptide synthesis well know to one of skill in the art.

In some embodiments, the invention provides using a polypeptide disclosed herein for screening or structural studies, such as by three-dimensional crystallography.

In some embodiments, the invention provides a composition having a polypeptide disclosed herein and at least one substrate for the polypeptide. Substrate for each of the polypeptides disclosed herein is Sugar 2, as described herein and exemplified in the Figures. The polypeptide within the composition of the invention can react with a substrate under in vitro conditions. In this context, an in vitro condition refers to a reaction in the absence of or outside of a microorganism of the invention.

The invention provides a method for co-utilization of Sugar 2 and a second monosaccharide for production of cell mass. The method includes contacting a non-naturally occurring microbial organism, containing: (a) an exogenous nucleic acid molecule encoding an amino acid sequence of XR, wherein the amino acid sequence includes an amino acid substitution at position 121 as set forth in Table 1; (b) an exogenous nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and includes a nucleic acid sequence that encodes an amino acid substitution at position 121 as set forth in Table 1, or (c) an exogenous nucleic acid molecule that is complementary to (a) or (b). The non-naturally occurring microbial organism is contacted in the presence of Sugar 2 and a second monosaccharide under conditions and for a sufficient period of time to simultaneously metabolize Sugar 2 and the second monosaccharide. Also provided is a method for the co-utilization of Sugar 2 and a second monosaccharide wherein the non-naturally occurring microbial organism contains an exogenous nucleic acid encoding an mutant XR polypeptide of the invention wherein the encoded amino acid sequence other than the amino acid substitution at position 121 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of XR

As describe above, exogenous expression of a xR mutant nucleic acid of the invention enables the co-utilization or co-metabolism of Sugar 2 and a second, different monosaccharide. This result can be harnessed for a variety of useful outcomes including the production, as well as the enhanced production compared to a wild-type microbial organisms that do not express a xR mutant nucleic acid of the invention, of cell mass for a non-naturally occurring microbial organism of the invention and/or for the biosynthesis or production, including the enhanced biosynthesis or production, of a bioderived compound.

As described previously, any of the xR mutant nucleic acids described above can be exogenously introduced into a host and expressed to produce a non-naturally occurring microbial organism to produce a mutant XR polypeptide that exhibits reduced or eliminated catabolite repression. The reduction or elimination of catabolite repression confers onto the host cell the ability to co-metabolize Sugar 2 and a second monosaccharide. The second monosaccharide can be, for example, Sugar 1 or Sugar 3. Methods for introducing the xR mutant nucleic acids have been described herein and are well known in the art. Such methods include, for example, transform a host or stable integration of an expressible xR mutant nucleic acid of the invention. Reduction or elimination of catabolite repression allows for more efficient and simultaneous utilization of two or more, including all, monosaccharides in the culture or fermentation broth. The simultaneous utilization of more than one monosaccharide enhances the generation of cellular mass and the biosynthesis of a bioderived compound.

The invention further provides the ability to enhance co-metabolism of two or more monosaccharides by a microbial organism of the invention and, therefore, the biosynthesis or production of a bioderived compound. Removal of catabolite repression by expression of an xR mutant nucleic acid of the invention allows simultaneous utilization of one, two, three or more monosaccharides other than Sugar 2. Accordingly, increasing the cellular uptake and/or intracellular availability of these other monosaccharides enhances the simultaneous utilization of multiple monosaccharides which can be harnessed by the cellular machinery to generate greater cell mass and/or to enhance the biosynthesis of a bioderived compound.

One embodiment of the invention for increasing cell mass or the production of a bioderived compound includes constitutive expression of one or more nucleic acids encoding a monosaccharide transporter protein. Another embodiment is to overexpress one or more nucleic acids encoding a monosaccharide transporter protein. As described above and below, nucleic acids encoding such transporter proteins can be exogenously introduced into a microbial organism of the invention to augment uptake or intracellular availability of a monosaccharide. Monosaccharides include, for example, Sugar 1, Sugar 3 Sugar 2 and fructose. Transporter proteins include, for example, AraE, Operon T2, Operon M2 and AraFGH. As described previously, AraE is a proton symporter that acts as a low-affinity high-capacity transporter for Sugar 3.

In some embodiments, the invention includes a microbial organism having an exogenous xR mutant nucleic acid of the invention for the production of cell mass or for the production of a bioderived compound. As described above and elsewhere throughout this description, the exogenous xR mutant nucleic acid can be expressed by a variety of modes well known to those in the art and described herein, including for example, constitutive expression, inducible expression and/or overexpression. The microbial organism having an exogenous xR mutant nucleic acid of the invention can further have an exogenous nucleic acid encoding AraE. The microbial organism having an exogenous xR mutant nucleic acid of the invention can further have an exogenous nucleic acid encoding Operon T2, Operon M2 or AraFGH. The microbial organism having an exogenous xR mutant nucleic acid of the invention and an exogenous nucleic acid encoding AraE can further have an exogenous nucleic acid encoding Operon T2, Operon M2 or AraFGH. Any of the encoding araE, operon t2, operon m2 or araFGH nucleic acids can similarly be expressed by a variety of modes well known to those in the art and described herein, including for example, constitutive expression, inducible expression and/or overexpression. Expression of one, two, three, four or more, including some or all of the exogenous encoding nucleic acids can be following integration into a chromosome or episomally using methods well known in the art and as described herein with reference to expression of other nucleic acids of the invention. All of such modes enable the enhanced production of cell mass and/or the enhanced production of a bioderived compound of the invention.

Accordingly, in some embodiments, the invention includes a microbial organism having an exogenous xR mutant nucleic acid of the invention and/or other mutant nucleic acid described herein and further having a bioderived compound pathway. For example, the bioderived compound pathway can be a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway as described herein. Moreover, in some embodiments, the invention includes a microbial organism having an exogenous xR mutant nucleic acid of the invention and/or other mutant nucleic acid described herein and further having a bioderived compound pathway well known in the art. For example, the bioderived compound pathway can be a succinate (U.S. publication 2007/0111294, WO 2007/030830, WO 2013/003432), 3-hydroxypropionic acid (3-hydroxypropionate) (U.S. publication 2008/0199926, WO 2008/091627, U.S. publication 2010/0021978), 1,4-butanediol (U.S. Pat. No. 8,067,214, WO 2008/115840, U.S. Pat. No. 7,947,483, WO 2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S. publication 2011/0003355, WO 2010/141780, U.S. Pat. No. 8,129,169, WO 2010/141920, U.S. publication 2011/0201068, WO 2011/031897, U.S. Pat. No. 8,377,666, WO 2011/047101, U.S. publication 2011/0217742, WO 2011/066076, U.S. publication 2013/0034884, WO 2012/177943), 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-hydroxybutryate) (U.S. Pat. No. 8,067,214, WO 2008/115840, U.S. Pat. No. 7,947,483, WO 2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S. publication 2011/0003355, WO 2010/141780, U.S. Pat. No. 8,129,155, WO 2010/071697), γ-butyrolactone (U.S. Pat. No. 8,067,214, WO 2008/115840, U.S. patent 7947483, WO 2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S. publication 2011/0003355, WO 2010/141780, U.S. publication 2011/0217742, WO 2011/066076), 4-hydroxybutyryl-CoA (U.S. publication 2011/0003355, WO 2010/141780, U.S. publication 2013/0034884, WO 2012/177943), 4-hydroxybutanal (U.S. publication 2011/0003355, WO 2010/141780, U.S. publication 2013/0034884, WO 2012/177943), putrescine (U.S. publication 2011/0003355, WO 2010/141780, U.S. publication 2013/0034884, WO 2012/177943), Olefins (such as acrylic acid and acrylate ester) (U.S. Pat. No. 8,026,386, WO 2009/045637), acetyl-CoA (U.S. Pat. No. 8,323,950, WO 2009/094485), methyl tetrahydrofolate (U.S. Pat. No. 8,323,950, WO 2009/094485), ethanol (U.S. Pat. No. 8,129,155, WO 2010/071697), isopropanol (U.S. Pat. No. 8,129,155, WO 2010/071697, U.S. publication 2010/0323418, WO 2010/127303, U.S. publication 2011/0201068, WO 2011/031897), n-butanol (U.S. Pat. No. 8,129,155, WO 2010/071697), isobutanol (U.S. Pat. No. 8,129,155, WO 2010/071697), n-propanol (U.S. publication 2011/0201068, WO 2011/031897), methylacrylic acid (methylacrylate) (U.S. publication 2011/0201068, WO 2011/031897), primary alcohol (U.S. Pat. No. 7,977,084, WO 2009/111672, WO 2012/177726), long chain alcohol (U.S. Pat. No. 7,977,084, WO 2009/111672, WO 2012/177726), adipate (adipic acid) (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721), 6-aminocaproate (6-aminocaproic acid) (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721), caprolactam (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721), hexamethylenediamine (U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721), levulinic acid (U.S. Pat. No. 8,377,680, WO 2010/129936), 2-hydroxyisobutyric acid (2-hydroxyisobutyrate) (U.S. Pat. No. 8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO 2012/135789), 3-hydroxyisobutyric acid (3-hydroxyisobutyrate) (U.S. Pat. No. 8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO 2012/135789), methacrylic acid (methacrylate) (U.S. Pat. No. 8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO 2012/135789), methacrylate ester (U.S. publication 2013/0065279, WO 2012/135789), fumarate (fumaric acid) (U.S. Pat. No. 8,129,154, WO 2009/155382), malate (malic acid) (U.S. Pat. No. 8,129,154, WO 2009/155382), acrylate (carboxylic acid) (U.S. Pat. No. 8,129,154, WO 2009/155382), methyl ethyl ketone (U.S. publication 2010/0184173, WO 2010/057022, U.S. Pat. No. 8,420,375, WO 2010/144746), 2-butanol (U.S. publication 2010/0184173, WO 2010/057022, U.S. Pat. No. 8,420,375, WO 2010/144746), 13BDO (U.S. publication 2010/0330635, WO 2010/127319, U.S. publication 2011/0201068, WO 2011/031897, U.S. Pat. No. 8,268,607, WO 2011/071682, U.S. publication 2013/0109064, WO 2013/028519, U.S. publication 2013/0066035, WO 2013/036764), cyclohexanone (U.S. publication 2011/0014668, WO 2010/132845), terephthalate (terephthalic acid) (U.S. publication 2011/0124911, WO 2011/017560, U.S. publication 2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO 2012/018624), muconate (muconic acid) (U.S. publication 2011/0124911, WO 2011/017560), aniline (U.S. publication 2011/0097767, WO 2011/050326), p-toluate (p-toluic acid) (U.S. publication 2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO 2012/018624), (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (U.S. publication 2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO 2012/018624), ethylene glycol (U.S. publication 2011/0312049, WO 2011/130378, WO 2012/177983), propylene (U.S. publication 2011/0269204, WO 2011/137198, U.S. publication 2012/0329119, U.S. publication 2013/0109064, WO 2013/028519), butadiene (1,3-butadiene) (U.S. publication 2011/0300597, WO 2011/140171, U.S. publication 2012/0021478, WO 2012/018624, U.S. publication 2012/0225466, WO 2012/106516, U.S. publication 2013/0011891, WO 2012/177710, U.S. publication 2013/0109064, WO 2013/028519), toluene (U.S. publication 2012/0021478, WO 2012/018624), benzene (U.S. publication 2012/0021478, WO 2012/018624), (2-hydroxy-4-oxobutoxy)phosphonate (U.S. publication 2012/0021478, WO 2012/018624), benzoate (benzoic acid) (U.S. publication 2012/0021478, WO 2012/018624), styrene (U.S. publication 2012/0021478, WO 2012/018624), 2,4-pentadienoate (U.S. publication 2012/0021478, WO 2012/018624, U.S. publication 2013/0109064, WO 2013/028519), 3-butene-1-ol (U.S. publication 2012/0021478, WO 2012/018624, U.S. publication 2013/0109064, WO 2013/028519), MVC (U.S. publication 2013/0109064, WO 2013/028519), 1,4-cyclohexanedimethanol (U.S. publication 2012/0156740, WO 2012/082978), CrotOH (U.S. publication 2013/0011891, WO 2012/177710, U.S. publication 2013/0109064, WO 2013/028519), alkene (U.S. publication 2013/0122563, WO 2013/040383), or caprolactone (U.S. publication 2013/0144029, WO 2013/067432) pathway. The patents and patent application publications listed above that disclose bioderived compound pathways are herein incorporated herein by reference.

Furthermore, in some embodiments, the invention provides a culture medium having one or more host cells of the invention. In some aspect, the culture medium can be purified or substantially purified from a host cell of the invention following culturing of the host cell for metabolism of Sugar 2. Methods of purifying or substantially purifying culture medium are well known to one skilled in the art and any one of which can be used to generate the culture medium of the invention, including those methods disclosed herein.

The invention also provides for a method for co-utilization of Sugar 2 and a second monosaccharide for production of a bioderived compound. The method includes contacting a non-naturally occurring microbial organism having: (a) an exogenous nucleic acid molecule encoding an amino acid sequence of XR, wherein the amino acid sequence includes an amino acid substitution at position 121 as set forth in Table 1; (b) an exogenous nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and includes a nucleic acid sequence that encodes an amino acid substitution at position 121 as set forth in Table 1, or (c) an exogenous nucleic acid molecule that is complementary to (a) or (b); with at least one exogenous nucleic acid encoding a target polypeptide. The non-naturally occurring microbial organism can be contacted in the presence of Sugar 2 and a second monosaccharide under conditions and for a sufficient period of time to simultaneously metabolize Sugar 2 and the second monosaccharide.

A target polypeptide of the invention can include any polypeptide desirable to be expressed by the non-naturally occurring microbial organisms of the invention. Such target polypeptides include, for example, cytosolic polypeptides, nuclear polypeptides and/or extracellular polypeptides. Particularly useful target polypeptides include polypeptides encoding enzymes within a biosynthetic pathway of the invention. Such enzymes include, for example, a FaldFP enzyme, FAP enzyme, butadiene (1,3-butadiene) pathway enzyme, 13BDO pathway enzyme, CrotOH pathway enzyme, MVC pathway enzyme, a MOP enzyme, 3-buten-1-ol pathway enzyme, succinate pathway enzyme, 3-hydroxypropionic acid (3-hydroxypropionate) pathway enzyme, 1,4-butanediol pathway enzyme, 4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-hydroxybutryate) pathway enzyme, γ-butyrolactone pathway enzyme, 4-hydroxybutyryl-CoA pathway enzyme, 4-hydroxybutanal pathway enzyme, putrescine pathway enzyme, Olefins (such as acrylic acid and acrylate ester) pathway enzyme, acetyl-CoA pathway enzyme, methyl tetrahydrofolate pathway enzyme, ethanol pathway enzyme, isopropanol pathway enzyme, n-butanol pathway enzyme, isobutanol pathway enzyme, n-propanol pathway enzyme, methylacrylic acid (methylacrylate) pathway enzyme, primary alcohol pathway enzyme, long chain alcohol pathway enzyme, adipate (adipic acid) pathway enzyme, 6-aminocaproate (6-aminocaproic acid) pathway enzyme, caprolactam pathway enzyme, hexamethylenediamine pathway enzyme, levulinic acid pathway enzyme, 2-hydroxyisobutyric acid (2-hydroxyisobutyrate) pathway enzyme, 3-hydroxyisobutyric acid (3-hydroxyisobutyrate) pathway enzyme, methacrylic acid (methacrylate) pathway enzyme, methacrylate ester pathway enzyme, fumarate (fumaric acid) pathway enzyme, malate (malic acid) pathway enzyme, acrylate (carboxylic acid) pathway enzyme, methyl ethyl ketone pathway enzyme, 2-butanol pathway enzyme, cyclohexanone pathway enzyme, terephthalate (terephthalic acid) pathway enzyme, muconate (muconic acid) pathway enzyme, aniline pathway enzyme, p-toluate (p-toluic acid) pathway enzyme, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme, ethylene glycol pathway enzyme, propylene pathway enzyme, toluene pathway enzyme, benzene pathway enzyme, (2-hydroxy-4-oxobutoxy)phosphonate pathway enzyme, benzoate (benzoic acid) pathway enzyme, styrene pathway enzyme, 2,4-pentadienoate pathway enzyme, 1,4-cyclohexanedimethanol pathway enzyme, alkene pathway enzyme, or caprolactone pathway enzyme or a combination thereof as described herein. Other target polypeptides include, for example, any of the polypeptides that reduce or eliminate catabolite repression for simultaneous metabolism of Sugar 2, Sugar 3 and/or Sugar 1, for example.

One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication 2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed Jun. 14, 2002, and in International Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

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

An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein to increase production of butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >10⁴). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Often and Quax. Biomol. Eng 22:1-9 (2005)₄ and Sen et al., Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K_m), including broadening substrate binding to include non-natural substrates; inhibition (K_i), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a butadiene, 13BDO, CrotOH, MVC or 3-buten-1-ol pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Theor. Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode “all genetic diversity in targets” and allows a very high diversity for the shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase Ill, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego Calif.), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein.

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

Example I

FAPs

This example describes enzymatic pathways for converting pyruvate to formaldehyde, and optionally in combination with producing acetyl-CoA and/or reproducing pyruvate.

Step E, FIG. 1: Formate Reductase

The conversion of formate to formaldehyde can be carried out by a formate reductase (step E, FIG. 1). A suitable enzyme for these transformations is the aryl-aldehyde dehydrogenase, or equivalently a carboxylic acid reductase, from Nocardia iowensis. Carboxylic acid reductase catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). This enzyme, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, Fla. (2006)). Information related to these proteins and genes is shown below.

	Protein	GenBank ID	GI number	Organism
	Car	AAR91681.1	40796035	Nocardia iowensis
				(sp. NRRL 5646)
	Npt	ABI83656.1	114848891	Nocardia iowensis
				(sp. NRRL 5646)

Additional car and npt genes can be identified based on sequence homology.

Protein	GenBank ID	GI number	Organism
fadD9	YP_978699.1	121638475	Mycobacterium bovis BCG
BCG_2812c	YP_978898.1	121638674	Mycobacterium bovis BCG
nfa20150	YP_118225.1	54023983	Nocardia farcinica IFM 10152
nfa40540	YP_120266.1	54026024	Nocardia farcinica IFM 10152
SGR_6790	YP_001828302.1	182440583	Streptomyces griseus subsp. griseus NBRC 13350
SGR_665	YP_001822177.1	182434458	Streptomyces griseus subsp. griseus NBRC 13350
MSMEG_2956	YP_887275.1	118473501	Mycobacterium smegmatis MC2 155
MSMEG_5739	YP_889972.1	118469671	Mycobacterium smegmatis MC2 155
MSMEG_2648	YP_886985.1	118471293	Mycobacterium smegmatis MC2 155
MAP1040c	NP_959974.1	41407138	Mycobacterium avium subsp. paratuberculosis K-10
MAP2899c	NP_961833.1	41408997	Mycobacterium avium subsp. paratuberculosis K-10
MMAR_2117	YP_001850422.1	183982131	Mycobacterium marinum M
MMAR_2936	YP_001851230.1	183982939	Mycobacterium marinum M
MMAR_1916	YP_001850220.1	183981929	Mycobacterium marinum M
TpauDRAFT_33060	ZP_04027864.1	227980601	Tsukamurella paurometabola DSM 20162
TpauDRAFT_20920	ZP_04026660.1	227979396	Tsukamurella paurometabola DSM 20162
CPCC7001_1320	ZP_05045132.1	254431429	Cyanobium PCC7001
DDBDRAFT_0187729	XP_636931.1	66806417	Dictyostelium discoideum AX4

An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot. 60(6):380-387 (2007)). Co-expression ofgriC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial. Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
griC	YP_001825755.1	182438036	Streptomyces griseus subsp.
			griseus NBRC 13350
grid	YP_001825756.1	182438037	Streptomyces griseus subsp.
			griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date. Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
LYS2	AAA34747.1	171867	Saccharomyces cerevisiae
LYS5	P50113.1	1708896	Saccharomyces cerevisiae
LYS2	AAC02241.1	2853226	Candida albicans
LYS5	AAO26020.1	28136195	Candida albicans
Lys1p	P40976.3	13124791	Schizosaccharomyces pombe
Lys7p	Q10474.1	1723561	Schizosaccharomyces pombe
Lys2	CAA74300.1	3282044	Penicillium chrysogenum

Tani et al (Agric Biol Chem, 1978, 42: 63-68; Agric Biol Chem, 1974, 38: 2057-2058) showed that purified enzymes from Escherichia coli strain B could reduce the sodium salts of different organic acids (e.g. formate, glycolate, acetate, etc.) to their respective aldehydes (e.g. formaldehyde, glycoaldehyde, acetaldehyde, etc.). Of three purified enzymes examined by Tani et al (1978), only the “A” isozyme was shown to reduce formate to formaldehyde. Collectively, this group of enzymes was originally termed glycoaldehyde dehydrogenase; however, their novel reductase activity led the authors to propose the name glycolate reductase as being more appropriate (Morita et al, Agric Biol Chem, 1979, 43: 185-186). Morita et al (Agric Biol Chem, 1979, 43: 185-186) subsequently showed that glycolate reductase activity is relatively widespread among microorganisms, being found for example in: Pseudomonas, Agrobacterium, Escherichia, Flavobacterium, Micrococcus, Staphylococcus, Bacillus, and others. Without wishing to be bound by any particular theory, it is believed that some of these glycolate reductase enzymes are able to reduce formate to formaldehyde.

Any of these CAR or CAR-like enzymes can exhibit formate reductase activity or can be engineered to do so.

Step F, Figure Formate Ligase, Formate Transferase, Formate Synthetase

The acylation of formate to formyl-CoA is catalyzed by enzymes with formate transferase, synthetase, or ligase activity (Step F, FIG. 1). Formate transferase enzymes have been identified in several organisms including Escherichia coli (Toyota, et al., J Bacteriol. 2008 April; 190(7):2556-64), Oxalobacter formigenes (Toyota, et al., J Bacteriol. 2008 April; 190(7):2556-64; Baetz et al., J Bacteriol. 1990 July; 172(7):3537-40; Ricagno, et al., EMBO J. 2003 Jul. 1; 22(13):3210-9)), and Lactobacillus acidophilus (Azcarate-Peril, et al., Appl. Environ. Microbiol. 2006 72(3) 1891-1899). Homologs exist in several other organisms. Enzymes acting on the CoA-donor for formate transferase may also be expressed to ensure efficient regeneration of the CoA-donor. For example, if oxalyl-CoA is the CoA donor substrate for formate transferase, an additional transferase, synthetase, or ligase may be required to enable efficient regeneration of oxalyl-CoA from oxalate. Similarly, if succinyl-CoA or acetyl-CoA is the CoA donor substrate for formate transferase, an additional transferase, synthetase, or ligase may be required to enable efficient regeneration of succinyl-CoA from succinate or acetyl-CoA from acetate, respectively.

Protein	GenBank ID	GI number	Organism
YfdW	NP_416875.1	16130306	Escherichia coli
frc	O06644.3	21542067	Oxalobacter formigenes
frc	ZP_04021099.1	227903294	Lactobacillus acidophilus

Suitable CoA-donor regeneration or formate transferase enzymes are encoded by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri. These enzymes have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880 (1996)) Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). Yet another transferase capable of the desired conversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplary enzymes can be found in Fusobacterium nucleatum (Barker et al., J. Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J. Biol. Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum (Wiesenbom et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)). Although specific gene sequences were not provided for butyryl-CoA:acetoacetate CoA-transferase in these references, the genes FN0272 and FN0273 have been annotated as a butyrate-acetoacetate CoA-transferase (Kapatral et al., J. Bact. 184(7) 2005-2018 (2002)). Homologs in Fusobacterium nucleatum such as FN1857 and FN1856 also likely have the desired acetoacetyl-CoA transferase activity. FN1857 and FN1856 are located adjacent to many other genes involved in lysine fermentation and are thus very likely to encode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)). Additional candidates from Porphyrmonas gingivalis and Thermoanaerobacter tengcongensis can be identified in a similar fashion (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)). Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
Cat1	P38946.1	729048	Clostridium kluyveri
Cat2	P38942.2	1705614	Clostridium kluyveri
Cat3	EDK35586.1	146349050	Clostridium kluyveri
TVAG_395550	XP_001330176	123975034	Trichomonas vaginalis G3
Tb11.02.0290	XP_828352	71754875	Trypanosoma brucei
FN0272	NP_603179.1	19703617	Fusobacterium nucleatum
FN0273	NP_603180.1	19703618	Fusobacterium nucleatum
FN1857	NP_602657.1	19705162	Fusobacterium nucleatum
FN1856	NP_602656.1	19705161	Fusobacterium nucleatum
PG1066	NP_905281.1	34540802	Porphyromonas gingivalis W83
PG1075	NP_905290.1	34540811	Porphyromonas gingivalis W83
TTE0720	NP_622378.1	20807207	Thermoanaerobacter tengcongensis MB4
TTE0721	NP_622379.1	20807208	Thermoanaerobacter tengcongensis MB4

Additional transferase enzymes of interest include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77:1219-1224 (2008)), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
AtoA	P76459.1	2492994	Escherichia coli
AtoD	P76458.1	2492990	Escherichia coli
CtfA	NP_149326.1	15004866	Clostridium acetobutylicum
CtfB	NP_149327.1	15004867	Clostridium acetobutylicum
CtfA	AAP42564.1	31075384	Clostridium saccharoperbutylacetonicum
CtfB	AAP42565.1	31075385	Clostridium saccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid. Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics 68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)). Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
HPAG1_0676	YP_627417	108563101	Helicobacter pylori
HPAG1_0677	YP_627418	108563102	Helicobacter pylori
ScoA	NP_391778	16080950	Bacillus subtilis
ScoB	NP_391777	16080949	Bacillus subtilis
OXCT1	NP_000427	4557817	Homo sapiens
OXCT2	NP_071403	11545841	Homo sapiens

Two additional enzymes that catalyze the activation of formate to formyl-CoA reaction are AMP-forming formyl-CoA synthetase and ADP-forming formyl-CoA synthetase. Exemplary enzymes, known to function on acetate, are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431 (2004)). Such enzymes may also acylate formate naturally or can be engineered to do so.

Protein	GenBank ID	GI Number	Organism
acs	AAC77039.1	1790505	Escherichia coli
acoE	AAA21945.1	141890	Ralstonia eutropha
acs1	ABC87079.1	86169671	Methanothermobacter thermautotrophicus
acs1	AAL23099.1	16422835	Salmonella enterica
ACS1	Q01574.2	257050994	Saccharomyces cerevisiae

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra (2004)). The enzymes from A. fulgidus, H. marismontui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Additional candidates include the succinyl-CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154 (1993)). Such enzymes may also acylate formate naturally or can be engineered to do so. Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
AF1211	NP_070039.1	11498810	Archaeoglobus fulgidus DSM4304
AF1983	NP_070807.1	11499565	Archaeoglobus fulgidus DSM4304
scs	YP_135572.1	55377722	Haloarcula marismortui ATCC43049
PAE3250	NP_560604.1	18313937	Pyrobaculum aerophilum str. IM2
sucC	NP_415256.1	16128703	Escherichia coli
sucD	AAC73823.1	1786949	Escherichia coli
paaF	AAC24333.2	22711873	Pseudomonas putida

An alternative method for adding the CoA moiety to formate is to apply a pair of enzymes such as a phosphate-transferring acyltransferase and a kinase. These activities enable the net formation of formyl-CoA with the simultaneous consumption of ATP. An exemplary phosphate-transferring acyltransferase is phosphotransacetylase, encoded by pta. The pta gene from E. coli encodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T. Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilize propionyl-CoA instead of acetyl-CoA forming propionate in the process (Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii. Such enzymes may also phosphorylate formate naturally or can be engineered to do so.

Protein	GenBank ID	GI number	Organism
Pta	NP_416800.1	16130232	Escherichia coli
Pta	NP_461280.1	16765665	Salmonella enterica subsp.
			enterica serovar Typhimurium str. LT2
PAT2	XP_001694504.1	159472743	Chlamydomonas reinhardtii
PAT1	XP_001691787.1	159467202	Chlamydomonas reinhardtii

An exemplary acetate kinase is the E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)). Homologs exist in several other organisms including Salmonella enterica and Chlamydomonas reinhardtii. It is likely that such enzymes naturally possess formate kinase activity or can be engineered to have this activity. Information related to these proteins and genes is shown below:

Protein	GenBank ID	GI number	Organism
AckA	NP_416799.1	16130231	Escherichia coli
AckA	NP_461279.1	16765664	Salmonella enterica subsp.
			enterica serovar Typhimurium
			str. LT2
ACK1	XP_001694505.1	159472745	Chlamydomonas reinhardtii
ACK2	XP_001691682.1	159466992	Chlamydomonas reinhardtii

The acylation of formate to formyl-CoA can also be carried out by a formate ligase. For example, the product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA ligase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No. 5,958,745, filed Sep. 28, 1999). Such enzymes may also acylate formate naturally or can be engineered to do so. Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
SucC	NP_415256.1	16128703	Escherichia coli
SucD	AAC73823.1	1786949	Escherichia coli
LSC1	NP_014785	6324716	Saccharomyces cerevisiae
LSC2	NP_011760	6321683	Saccharomyces cerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical J. 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturally catalyze the ATP-dependent conversion of acetoacetate into acetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)). This function has been tentatively assigned to the Msed_1422 gene. Such enzymes may also acylate formate naturally or can be engineered to do so. Information related to these proteins and genes is shown below.

Protein	GenBank ID	GI number	Organism
Phl	CAJ15517.1	77019264	Penicillium chrysogenum
PhlB	ABS19624.1	152002983	Penicillium chrysogenum
PaaF	AAC24333.2	22711873	Pseudomonas putida
BioW	NP_390902.2	50812281	Bacillus subtilis
AACS	NP_084486.1	21313520	Mus musculus
AACS	NP_076417.2	31982927	Homo sapiens
Msed_1422	YP_001191504	146304188	Metallosphaera sedula

Step G, FIG. 1: Formyl-CoA Reductase

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA (e.g., formyl-CoA) to its corresponding aldehyde (e.g., formaldehyde) (Steps F, FIG. 1). Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase (Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002), and a CoA- and NADP-dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996); Sohling and Gottschalk, J. Bacteriol. 1778:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:45-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem. 71:58-68 (2007)). Additional aldehyde dehydrogenase enzyme candidates are found in Desulfatibacillum alkenivorans, Citrobacter koseri, Salmonella enterica, Lactobacillus brevis and Bacillus selenitireducens. Such enzymes may be capable of naturally converting formyl-CoA to formaldehyde or can be engineered to do so.

Protein	GenBank ID	GI number	Organism
acr1	YP_047869.1	50086355	Acinetobacter calcoaceticus
acr1	AAC45217	1684886	Acinetobacter baylyi
acr1	BAB85476.1	18857901	Acinetobacter sp. Strain M-1
sucD	P38947.1	172046062	Clostridium kluyveri
sucD	NP_904963.1	34540484	Porphyromonas gingivalis
bphG	BAA03892.1	425213	Pseudomonas sp
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
Bld	AAP42563.1	31075383	Clostridium saccharoperbutylacetonicum
Ald	ACL06658.1	218764192	Desulfatibacillum alkenivorans AK-01
Ald	YP_001452373	157145054	Citrobacter koseri ATCC BAA-895
pduP	NP_460996.1	16765381	Salmonella enterica Typhimurium
pduP	ABJ64680.1	116099531	Lactobacillus brevis ATCC 367
BselDRAFT_1651	ZP_02169447	163762382	Bacillus selenitireducens MLS10

An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786 (2007); Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., supra (2006); Berg et al., Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO 2007/141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra). Such enzymes may be capable of naturally converting formyl-CoA to formaldehyde or can be engineered to do so.

Protein	GenBank ID	GI number	Organism
Msed_0709	YP_001190808.1	146303492	Metallosphaera sedula
Mcr	NP_378167.1	15922498	Sulfolobus tokodaii
asd-2	NP_343563.1	15898958	Sulfolobus solfataricus
Saci_2370	YP_256941.1	70608071	Sulfolobus acidocaldarius
Ald	AAT66436	9473535	Clostridium beijerinckii
eutE	AAA80209	687645	Salmonella typhimurium
eutE	P77445	2498347	Escherichia coli

Step H, FIG. 1: FTHFS

FTHFS, formyltetrahydrofolate synthetase, ligates formate to tetrahydrofolate at the expense of one ATP. This reaction is catalyzed by the gene product of Moth 0109 in M. thermoacetica (O'brien et al., Experientia Suppl. 26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988); Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986); Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), and CHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005). Homologs exist in C. carboxidivorans P7. This enzyme is found in several other organisms as listed below.

Protein	GenBank ID	GI number	Organism
Moth_0109	YP_428991.1	83588982	Moorella thermoacetica
CHY_2385	YP_361182.1	78045024	Carboxydothermus hydrogenoformans
FHS	P13419.1	120562	Clostridium acidurici
CcarbDRAFT_1913	ZP_05391913.1	255524966	Clostridium carboxidivorans P7
CcarbDRAFT_2946	ZP_05392946.1	255526022	Clostridium carboxidivorans P7
Dhaf_0555	ACL18622.1	219536883	Desulfitobacterium hafniense
fhs	YP_001393842.1	153953077	Clostridium kluyveri DSM 555
fhs	YP_003781893.1	300856909	Clostridium ljungdahlii DSM 13528
MGA3_08300	EIJ83208.1	387590889	Bacillus methanolicus MGA3
PB1_13509	ZP_10132113.1	387929436	Bacillus methanolicus PB1

Steps I and J, FIG. 1: Methenyltetrahydrofolate Cyclohydrolase and MTHFDH

In M. thermoacetica, E. coli, and C. hydrogenoformans, methenyltetrahydrofolate cyclohydrolase and MTHFDH are carried out by the bi-functional gene products of Moth_1516, folD, and CHY_1878, respectively (Pierce et al., Environ. Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. 1:e65 (2005); D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homolog exists in C. carboxidivorans P7 Several other organisms also encode for this bifunctional protein as tabulated below.

Protein	GenBank ID	GI number	Organism
Moth_1516	YP_430368.1	83590359	Moorella thermoacetica
folD	NP_415062.1	16128513	Escherichia coli
CHY_1878	YP_360698.1	78044829	Carboxydothermus hydrogenoformans
CcarbDRAFT_2948	ZP_05392948.1	255526024	Clostridium carboxidivorans P7
folD	ADK16789.1	300437022	Clostridium ljungdahlii DSM 13528
folD-2	NP_951919.1	39995968	Geobacter sulfurreducens PCA
folD	YP_725874.1	113867385	Ralstonia eutropha H16
folD	NP_348702.1	15895353	Clostridium acetobutylicum ATCC 824
folD	YP_696506.1	110800457	Clostridium perfringens
MGA3_09460	EIJ83438.1	387591119	Bacillus methanolicus MGA3
PB1_14689	ZP_10132349.1	387929672	Bacillus methanolicus PB1

Steps K, FIG. 1: Formaldehyde-Forming Enzyme or Spontaneous

Methylene-THF, or active formaldehyde, will spontaneously decompose to formaldehyde and THF (Thorndike and Beck, Cancer Res. 1977, 37(4) 1125-32; Ordonez and Caraballo, Psychopharmacol Commun. 1975 1(3) 253-60; Kallen and Jencks, 1966, J Biol Chem 241(24) 5851-63). To achieve higher rates, a formaldehyde-forming enzyme can be applied. Such an activity can be obtained by engineering an enzyme that reversibly forms methylene-THF from THF and a formaldehyde donor, to release free formaldehyde. Such enzymes include glycine cleavage system enzymes which naturally transfer a formaldehyde group from methylene-THF to glycine (see Step L, FIG. 1 for candidate enzymes). Additional enzymes include serine hydroxymethyltransferase (see Step M, FIG. 1 for candidate enzymes), dimethylglycine dehydrogenase (Porter, et al., Arch Biochem Biophys. 1985, 243(2) 396-407; Brizio et al., 2004, (37) 2, 434-442), sarcosine dehydrogenase (Porter, et al., Arch Biochem Biophys. 1985, 243(2) 396-407), and dimethylglycine oxidase (Leys, et al., 2003, The EMBO Journal 22(16) 4038-4048).

Protein	GenBank ID	GI number	Organism
dmgo	ZP_09278452.1	359775109	Arthrobacter globiformis
dmgo	YP_002778684.1	226360906	Rhodococcus opacus B4
dmgo	EFY87157.1	322695347	Metarhizium acridum
			CQMa 102
shd	AAD53398.2	5902974	Homo sapiens
shd	NP_446116.1	GI:25742657	Rattus norvegicus
dmgdh	NP_037523.2	24797151	Homo sapiens
dmgdh	Q63342.1	2498527	Rattus norvegicus

Step L, FIG. 1: Glycine Cleavage System

The reversible NAD(P)H-dependent conversion of 5,10-methylenetetrahydrofolate and CO₂ to glycine is catalyzed by the glycine cleavage complex, also called glycine cleavage system, composed of four protein components; P, H, T and L. The glycine cleavage complex is involved in glycine catabolism in organisms such as E. coli and glycine biosynthesis in eukaryotes (Kikuchi et al, Proc Jpn Acad Ser 84:246 (2008)). The glycine cleavage system of E. coli is encoded by four genes: gcvPHT and lpdA (Okamura et al, Eur J Biochem 216:539-48 (1993); Heil et al, Microbiol 148:2203-14 (2002)). Activity of the glycine cleavage system in the direction of glycine biosynthesis has been demonstrated in vivo in Saccharomyces cerevisiae (Maaheimo et al, Eur J Biochem 268:2464-79 (2001)). The yeast GCV is encoded by GCV1, GCV2, GCV3 and LPD1.

Protein	GenBank ID	GI Number	Organism
gcvP	AAC75941.1	1789269	Escherichia coli
gcvT	AAC75943.1	1789272	Escherichia coli
gcvH	AAC75942.1	1789271	Escherichia coli
lpdA	AAC73227.1	1786307	Escherichia coli
GCV1	NP_010302.1	6320222	Saccharomyces cerevisiae
GCV2	NP_013914.1	6323843	Saccharomyces cerevisiae
GCV3	NP_009355.3	269970294	Saccharomyces cerevisiae
LPD1	NP_116635.1	14318501	Saccharomyces cerevisiae

Step M, FIG. 1: Serine Hydroxymethyltransferase

Conversion of glycine to serine is catalyzed by serine hydroxymethyltransferase, also called glycine hydroxymethyltranferase. This enzyme reversibly converts glycine and 5,10-methylenetetrahydrofolate to serine and THF. Serine methyltransferase has several side reactions including the reversible cleavage of 3-hydroxyacids to glycine and an aldehyde, and the hydrolysis of 5,10-methenyl-THF to 5-formyl-THF. This enzyme is encoded by glyA of E. coli (Plamann et al, Gene 22:9-18 (1983)). Serine hydroxymethyltranferase enzymes of S. cerevisiae include SHM1 (mitochondrial) and SHM2 (cytosolic) (McNeil et al, J Biol Chem 269:9155-65 (1994)). Similar enzymes have been studied in Corynebacterium glutamicum and Methylobacterium extorquens (Chistoserdova et al, J Bacteriol 176:6759-62 (1994); Schweitzer et al, J Biotechnol 139:214-21 (2009)).

Protein	GenBank ID	GI Number	Organism
glyA	AAC75604.1	1788902	Escherichia coli
SHM1	NP_009822.2	37362622	Saccharomyces cerevisiae
SHM2	NP_013159.1	6323087	Saccharomyces cerevisiae
glyA	AAA64456.1	496116	Methylobacterium extorquens
glyA	AAK60516.1	14334055	Corynebacterium glutamicum

Step N, FIG. 1: Serine Deaminase

Serine can be deaminated to pyruvate by serine deaminase. Serine deaminase enzymes are present in several organisms including Clostridium acidurici (Carter, et al., 1972, J Bacteriol., 109(2) 757-763), Escherichia coli (Cicchillo et al., 2004, J Biol Chem., 279(31) 32418-25), and Corneybacterium sp. (Netzer et al., Appl Environ Microbiol. 2004 December; 70(12):7148-55).

Protein	GenBank ID	GI Number	Organism
sdaA	YP_490075.1	388477887	Escherichia coli
sdaB	YP_491005.1	388478813	Escherichia coli
tdcG	YP_491301.1	388479109	Escherichia coli
tdcB	YP_491307.1	388479115	Escherichia coli
sdaA	YP_225930.1	62390528	Corynebacterium sp.

Step O, FIG. 1: Methylenetetrahydrofolate Reductase

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzed by methylenetetrahydrofolate reductase. In M. thermoacetica, this enzyme is oxygen-sensitive and contains an iron-sulfur cluster (Clark and Ljungdahl, J Biol. Chem. 259:10845-10849 (1984). This enzyme is encoded by metF in E. coli (Sheppard et al., J. Bacteriol. 181:718-725 (1999) and CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005). The M. thermoacetica genes, and its C. hydrogenoformans counterpart, are located near the CODH/ACS gene cluster, separated by putative hydrogenase and heterodisulfide reductase genes. Some additional gene candidates found bioinformatically are listed below. In Acetobacterium woodii metF is coupled to the Rnf complex through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are found in other organisms by blast search. The Rnf complex is known to be a reversible complex (Fuchs (2011) Annu. Rev. Microbiol. 65:631-658).

Protein	GenBank ID	GI number	Organism
Moth_1191	YP_430048.1	83590039	Moorella thermoacetica
Moth_1192	YP_430049.1	83590040	Moorella thermoacetica
metF	NP_418376.1	16131779	Escherichia coli
CHY_1233	YP_360071.1	78044792	Carboxydothermus hydrogenoformans
CLJU_c37610	YP_003781889.1	300856905	Clostridium ljungdahlii DSM 13528
DesfrDRAFT_3717	ZP_07335241.1	303248996	Desulfovibrio fructosovorans JJ
CcarbDRAFT_2950	ZP_05392950.1	255526026	Clostridium carboxidivoransP7
Ccel74_010100023124	ZP_07633513.1	307691067	Clostridium cellulovorans 743B
Cphy_3110	YP_001560205.1	160881237	Clostridium phytofermentans ISDg

Step P, FIG. 1: Acetyl-CoA Synthase

Acetyl-CoA synthase is the central enzyme of the carbonyl branch of the Wood-Ljungdahl pathway. It catalyzes the synthesis of acetyl-CoA from carbon monoxide, coenzyme A, and the methyl group from a methylated corrinoid-iron-sulfur protein. The corrinoid-iron-sulfur-protein is methylated by methyltetrahydrofolate via a methyltransferase. Expression in a foreign host entails introducing one or more of the following proteins and their corresponding activities: Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), CODH (AcsA), and Nickel-protein assembly protein (CooC).

The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthase activity typically reside in a limited region of the native genome that can be an extended operon (Ragsdale, S. W., Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004); Morton et al., J. Biol. Chem. 266:23824-23828 (1991); Roberts et al., Proc. Natl. Acad. Sci. U.S.A. 86:32-36 (1989). Each of the genes in this operon from the acetogen, M. thermoacetica, has already been cloned and expressed actively in E. coli (Morton et al. supra; Roberts et al. supra; Lu et al., J. Biol. Chem. 268:5605-5614 (1993). The protein sequences of these genes can be identified by the following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
AcsE	YP_430054	83590045	Moorella thermoacetica
AcsD	YP_430055	83590046	Moorella thermoacetica
AcsF	YP_430056	83590047	Moorella thermoacetica
Orf7	YP_430057	83590048	Moorella thermoacetica
AcsC	YP_430058	83590049	Moorella thermoacetica
AcsB	YP_430059	83590050	Moorella thermoacetica
AcsA	YP_430060	83590051	Moorella thermoacetica
CooC	YP_430061	83590052	Moorella thermoacetica

The hydrogenic bacterium, Carboxydothermus hydrogenoformans, can utilize carbon monoxide as a growth substrate by means of acetyl-CoA synthase (Wu et al., PLoS Genet. 1:e65 (2005)). In strain Z-2901, the acetyl-CoA synthase enzyme complex lacks CODH due to a frameshift mutation (Wu et al. supra (2005)), whereas in strain DSM 6008, a functional unframeshifted full-length version of this protein has been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci. U.S.A. 101:446-451 (2004)). The protein sequences of the C. hydrogenoformans genes from strain Z-2901 can be identified by the following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
AcsE	YP_360065	78044202	Carboxydothermus hydrogenoformans
AcsD	YP_360064	78042962	Carboxydothermus hydrogenoformans
AcsF	YP_360063	78044060	Carboxydothermus hydrogenoformans
Orf7	YP_360062	78044449	Carboxydothermus hydrogenoformans
AcsC	YP_360061	78043584	Carboxydothermus hydrogenoformans
AcsB	YP_360060	78042742	Carboxydothermus hydrogenoformans
CooC	YP_360059	78044249	Carboxydothermus hydrogenoformans

Homologous ACS/CODH genes can also be found in the draft genome assembly of Clostridium carboxidivorans P7.

Protein	GenBank ID	GI Number	Organism
AcsA	ZP_05392944.1	255526020	Clostridium carboxidivorans P7
CooC	ZP_05392945.1	255526021	Clostridium carboxidivorans P7
AcsF	ZP_05392952.1	255526028	Clostridium carboxidivorans P7
AcsD	ZP_05392953.1	255526029	Clostridium carboxidivorans P7
AcsC	ZP_05392954.1	255526030	Clostridium carboxidivorans P7
AcsE	ZP_05392955.1	255526031	Clostridium carboxidivorans P7
AcsB	ZP_05392956.1	255526032	Clostridium carboxidivorans P7
Orf7	ZP_05392958.1	255526034	Clostridium carboxidivorans P7

The methanogenic archaeon, Methanosarcina acetivorans, can also grow on carbon monoxide, exhibits acetyl-CoA synthase/CODH activity, and produces both acetate and formate (Lessner et al., Proc. Natl. Acad Sci. U.S.A. 103:17921-17926 (2006)). This organism contains two sets of genes that encode ACS/CODH activity (Rother and Metcalf, Proc. Natl. Acad. Sci. U.S.A. 101:16929-16934 (2004)). The protein sequences of both sets of M. acetivorans genes are identified by the following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
AcsC	NP_618736	20092661	Methanosarcina acetivorans
AcsD	NP_618735	20092660	Methanosarcina acetivorans
AcsF, CooC	NP_618734	20092659	Methanosarcina acetivorans
AcsB	NP_618733	20092658	Methanosarcina acetivorans
AcsEps	NP_618732	20092657	Methanosarcina acetivorans
AcsA	NP_618731	20092656	Methanosarcina acetivorans
AcsC	NP_615961	20089886	Methanosarcina acetivorans
AcsD	NP_615962	20089887	Methanosarcina acetivorans
AcsF, CooC	NP_615963	20089888	Methanosarcina acetivorans
AcsB	NP_615964	20089889	Methanosarcina acetivorans
AcsEps	NP_615965	20089890	Methanosarcina acetivorans
AcsA	NP_615966	20089891	Methanosarcina acetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred to as the gamma, delta, beta, epsilon, and alpha subunits of the methanogenic CODH/ACS. Homologs to the epsilon encoding genes are not present in acetogens such as M. thermoacetica or hydrogenogenic bacteria such as C. hydrogenoformans. Hypotheses for the existence of two active CODH/ACS operons in M. acetivorans include catalytic properties (i.e., K_m, V_max, k_cat) that favor carboxidotrophic or aceticlastic growth or differential gene regulation enabling various stimuli to induce CODH/ACS expression (Rother et al., Arch. Microbiol. 188:463-472 (2007)).

Step Q, FIG. 1: Pyruvate Formate Lyase

Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflB in E. coli, can convert pyruvate into acetyl-CoA and formate. The activity of PFL can be enhanced by an activating enzyme encoded by pflA (Knappe et al., Proc. Natl. Acad. Sci U.S.A 81:1332-1335 (1984); Wong et al., Biochemistry 32:14102-14110 (1993)). Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvate formate-lyase 4, is the gene product of tdcE in E. coli. This enzyme catalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation, and can also substitute for pyruvate formate-lyase in anaerobic catabolism (Simanshu et al., J Biosci. 32:1195-1206 (2007)). The enzyme is oxygen-sensitive and, like PflB, can require post-translational modification by PFL-AE to activate a glycyl radical in the active site (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). A pyruvate formate-lyase from Archaeglubus fulgidus encoded by pflD has been cloned, expressed in E. coli and characterized (Lehtio et al., Protein Eng Des Sel 17:545-552 (2004)). The crystal structures of the A. fulgidus and E. coli enzymes have been resolved (Lehtio et al., J Mol. Biol. 357:221-235 (2006); Leppanen et al., Structure. 7:733-744 (1999)). Additional PFL and PFL-AE candidates are found in Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al., Oral. Microbiol Immunol. 18:293-297 (2003)), Chlamydomonas reinhardtii (Hemschemeier et al., Eukaryot. Cell 7:518-526 (2008b); Atteia et al., J. Biol. Chem. 281:9909-9918 (2006)) and Clostridium pasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)).

Protein	GenBank ID	GI Number	Organism
pflB	NP_415423	16128870	Escherichia coli
pflA	NP_415422.1	16128869	Escherichia coli
tdcE	AAT48170.1	48994926	Escherichia coli
pflD	NP_070278.1	11499044	Archaeglubus fulgidus
Pfl	CAA03993	2407931	Lactococcus lactis
Pfl	BAA09085	1129082	Streptococcus mutans
PFL1	XP_001689719.1	159462978	Chlamydomonas reinhardtii
pflA1	XP_001700657.1	159485246	Chlamydomonas reinhardtii
Pfl	Q46266.1	2500058	Clostridium pasteurianum
Act	CAA63749.1	1072362	Clostridium pasteurianum

Step R, FIG. 1: Pyruvate Dehydrogenase, Pyruvate Ferredoxin Oxidoreductase, Pyruvate:nadp+ Oxidoreductase

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion of pyruvate to acetyl-CoA (FIG. 1R). The E. coli PDH complex is encoded by the genes aceEF and lpdA. Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., 56:135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., 98:14802-14807 (2001)) and the E2 catalytic domain from Azotobacter vinelandii are available (Mattevi et al., Science. 255:1544-1550 (1992)). Some mammalian PDH enzymes complexes can react on alternate substrates such as 2-oxobutanoate. Comparative kinetics of Rattus norvegicus PDH and BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton et al., Biochem. J. 234:295-303 (1986)). The S. cerevisiae PDH complex canconsist of an E2 (LAT1) core that binds E1 (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk et al., Yeast 12:1607-1633 (1996)). The PDH complex of S. cerevisiae is regulated by phosphorylation of E1 involving PKP1 (PDH kinase I), PTC5 (PDH phosphatase I), PKP2 and PTC6. Modification of these regulators may also enhance PDH activity. Coexpression of lipoyl ligase (LplA of E. coli and AIM22 in S. cerevisiae) with PDH in the cytosol may be necessary for activating the PDH enzyme complex. Increasing the supply of cytosolic lipoate, either by modifying a metabolic pathway or media supplementation with lipoate, may also improve PDH activity.

Gene	Accession No.	GI Number	Organism
aceE	NP_414656.1	16128107	Escherichia coli
aceF	NP_414657.1	16128108	Escherichia coli
lpd	NP_414658.1	16128109	Escherichia coli
lplA	NP_418803.1	16132203	Escherichia coli
pdhA	P21881.1	3123238	Bacillus subtilis
pdhB	P21882.1	129068	Bacillus subtilis
pdhC	P21883.2	129054	Bacillus subtilis
pdhD	P21880.1	118672	Bacillus subtilis
aceE	YP_001333808.1	152968699	Klebsiella pneumoniae
aceF	YP_001333809.1	152968700	Klebsiella pneumoniae
lpdA	YP_001333810.1	152968701	Klebsiella pneumoniae
Pdha1	NP_001004072.2	124430510	Rattus norvegicus
Pdha2	NP_446446.1	16758900	Rattus norvegicus
Dlat	NP_112287.1	78365255	Rattus norvegicus
Dld	NP_955417.1	40786469	Rattus norvegicus
LAT1	NP_014328	6324258	Saccharomyces cerevisiae
PDA1	NP_011105	37362644	Saccharomyces cerevisiae
PDB1	NP_009780	6319698	Saccharomyces cerevisiae
LPD1	NP_116635	14318501	Saccharomyces cerevisiae
PDX1	NP_011709	6321632	Saccharomyces cerevisiae
AIM22	NP_012489.2	83578101	Saccharomyces cerevisiae

As an alternative to the large multienzyme PDH complexes described above, some organisms utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze acylating oxidative decarboxylation of 2-keto-acids. Unlike the PDH complexes, PFOR enzymes contain iron-sulfur clusters, utilize different cofactors and use ferredoxin or flavodixin as electron acceptors in lieu of NAD(P)H. Pyruvate ferredoxin oxidoreductase (PFOR) can catalyze the oxidation of pyruvate to form acetyl-CoA (FIG. 1R). The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. The M. thermoacetica PFOR is also well characterized (Menon et al., Biochemistry 36:8484-8494 (1997)) and was even shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui et al., J Biol Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, that encodes a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982)). Several additional PFOR enzymes are described in Ragsdale, Chem. Rev. 103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB from Helicobacter pylori or Campylobacter jejuni (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007))) or Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
Por	CAA70873.1	1770208	Desulfovibrio africanus
Por	YP_428946.1	83588937	Moorella thermoacetica
ydbK	NP_415896.1	16129339	Escherichia coli
fqrB	NP_207955.1	15645778	Helicobacter pylori
fqrB	YP_001482096.1	157414840	Campylobacter jejuni
RnfC	EDK33306.1	146346770	Clostridium kluyveri
RnfD	EDK33307.1	146346771	Clostridium kluyveri
RnfG	EDK33308.1	146346772	Clostridium kluyveri
RnfE	EDK33309.1	146346773	Clostridium kluyveri
RnfA	EDK33310.1	146346774	Clostridium kluyveri
RnfB	EDK33311.1	146346775	Clostridium kluyveri

Pyruvate:NADP oxidoreductase (PNO) catalyzes the conversion of pyruvate to acetyl-CoA. This enzyme is encoded by a single gene and the active enzyme is a homodimer, in contrast to the multi-subunit PDH enzyme complexes described above. The enzyme from Euglena gracilis is stabilized by its cofactor, thiamin pyrophosphate (Nakazawa et al, Arch Biochem Biophys 411:183-8 (2003)). The mitochondrial targeting sequence of this enzyme should be removed for expression in the cytosol. The PNO protein of E. gracilis and other NADP-dependent pyruvate:NADP+ oxidoreductase enzymes are listed in the table below.

Protein	GenBank ID	GI Number	Organism
PNO	Q94IN5.1	33112418	Euglena gracilis
cgd4_690	XP_625673.1	66356990	Cryptosporidium
			parvum Iowa II
TPP_PFOR_PNO	XP_002765111.11	294867463	Perkinsus marinus
			ATCC 50983

Step S, FIG. 1: FDH

FDH, formate dehydrogenase, catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity utilize various electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33). FDH enzymes have been characterized from Moorella thermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873 (1973); Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., J Biol Chem. 258:1826-1832 (1983). The loci, Moth 2312 is responsible for encoding the alpha subunit of FDH while the beta subunit is encoded by Moth_2314 (Pierce et al., Environ Microbiol (2008)). Another set of genes encoding FDH activity with a propensity for CO₂ reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658 (2008)). A similar set of genes presumed to carry out the same function are encoded by CHY_0731, CHY_0732, and CHY_0733 in C. hydrogenoformans (Wu et al., PLoS Genet 1:e65 (2005)). FDHs are also found many additional organisms including C. carboxidivorans P7, Bacillus methanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073, Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c. The soluble FDH from Ralstonia eutropha reduces NAD⁺ (fdsG, -B, -A, -C, -D) (Oh and Bowien, 1998). Several FDHs have been identified that have higher specificity for NADP as the cofactor as compared to NAD. This enzyme has been deemed as the NADP-dependent FDH and has been reported from 5 species of the Burkholderia cepacia complex. It was tested and verified in multiple strains of Burkhoideria multivorans, Burkholderia stabilis, Burkholderia pyrrocinia, and Burkholderia cenocepacia (Hatrongjit et al., Enzyme and Microbial Tech., 46: 557-561 (2010)). The enzyme from Burkholderia stabilis has been characterized and the apparent K_m of the enzyme were reported to be 55.5 mM, 0.16 mM and 1.43 mM for formate, NADP, and NAD respectively. More gene candidates can be identified using sequence homology of proteins deposited in Public databases such as NCBI, JGI and the metagenomic databases.

Protein	GenBank ID	GI Number	Organism
Moth_2312	YP_431142	148283121	Moorella thermoacetica
Moth_2314	YP_431144	83591135	Moorella thermoacetica
Sfum_2703	YP_846816.1	116750129	Syntrophobacter fumaroxidans
Sfum_2704	YP_846817.1	116750130	Syntrophobacter fumaroxidans
Sfum_2705	YP_846818.1	116750131	Syntrophobacter fumaroxidans
Sfum_2706	YP_846819.1	116750132	Syntrophobacter fumaroxidans
CHY_0731	YP_359585.1	78044572	Carboxydothermus hydrogenoformans
CHY_0732	YP_359586.1	78044500	Carboxydothermus hydrogenoformans
CHY_0733	YP_359587.1	78044647	Carboxydothermus hydrogenoformans
CcarbDRAFT_0901	ZP_05390901.1	255523938	Clostridium carboxidivorans P7
CcarbDRAFT_4380	ZP_05394380.1	255527512	Clostridium carboxidivorans P7
fdhA, MGA3_06625	EIJ82879.1	387590560	Bacillus methanolicus MGA3
fdhA, PB1_11719	ZP_10131761.1	387929084	Bacillus methanolicus PB1
fdhD, MGA3_06630	EIJ82880.1	387590561	Bacillus methanolicus MGA3
fdhD, PB1_11724	ZP_10131762.1	387929085	Bacillus methanolicus PB1
fdh	ACF35003.	194220249	Burkholderia stabilis
FDH1	AAC49766.1	2276465	Candida boidinii
Fdh	CAA57036.1	1181204	Candida methylica
FDH2	P0CF35.1	294956522	Saccharomyces cerevisiae S288c
FDH1	NP_015033.1	6324964	Saccharomyces cerevisiae S288c
fdsG	YP_725156.1	113866667	Ralstonia eutropha
fdsB	YP_725157.1	113866668	Ralstonia eutropha
fdsA	YP_725158.1	113866669	Ralstonia eutropha
fdsC	YP_725159.1	113866670	Ralstonia eutropha
fdsD	YP_725160.1	113866671	Ralstonia eutropha

Example II

Production of Reducing Equivalents

This example describes MMPs and other additional enzymes generating reducing equivalents as shown in FIG. 3.

FIG. 3, Step A—Methanol Methyltransferase

A complex of 3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC, perform the desired methanol methyltransferase activity (Sauer et al., Eur. J. Biochem. 243:670-677 (1997); Naidu and Ragsdale, J. Bacteriol. 183:3276-3281 (2001); Tallant and Krzycki, J. Biol. Chem. 276:4485-4493 (2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301 (1996); Ragsdale, S. W., Crit. Rev. Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that can catalyze the transfer of a methyl group from methanol to MtaC, a corrinoid protein. Exemplary genes encoding MtaB and MtaC can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931 (2006) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Das et al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genes are adjacent to one another on the chromosome as their activities are tightly interdependent. The protein sequences of various MtaB and MtaC encoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum can be identified by their following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
MtaB1	YP_304299	73668284	Methanosarcina barkeri
MtaC1	YP_304298	73668283	Methanosarcina barkeri
MtaB2	YP_307082	73671067	Methanosarcina barkeri
MtaC2	YP_307081	73671066	Methanosarcina barkeri
MtaB3	YP_304612	73668597	Methanosarcina barkeri
MtaC3	YP_304611	73668596	Methanosarcina barkeri
MtaB1	NP_615421	20089346	Methanosarcina acetivorans
MtaB1	NP_615422	20089347	Methanosarcina acetivorans
MtaB2	NP_619254	20093179	Methanosarcina acetivorans
MtaC2	NP_619253	20093178	Methanosarcina acetivorans
MtaB3	NP_616549	20090474	Methanosarcina acetivorans
MtaC3	NP_616550	20090475	Methanosarcina acetivorans
MtaB	YP_430066	83590057	Moorella thermoacetica
MtaC	YP_430065	83590056	Moorella thermoacetica
MtaA	YP_430064	83590056	Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from M. barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem. 243:670-677 (1997)). The crystal structure of this methanol-cobalamin methyltransferase complex is also available (Hagemeier et al., Proc. Natl. Acad. Sci. U.S.A. 103:18917-18922 (2006)). The MtaB genes, YP_307082 and YP304612, in M. barkeri were identified by sequence homology to YP_304299. In general, homology searches are an effective means of identifying methanol methyltransferases because MtaB encoding genes show little or no similarity to methyltransferases that act on alternative substrates such as trimethylamine, dimethylamine, monomethylamine, or dimethylsulfide. The MtaC genes, YP_307081 and YP_304611 were identified based on their proximity to the MtaB genes and also their homology to YP_304298. The three sets of MtaB and MtaC genes from M. acetivorans have been genetically, physiologically, and biochemically characterized (Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194 (2005)). Mutant strains lacking two of the sets were able to grow on methanol, whereas a strain lacking all three sets of MtaB and MtaC genes sets could not grow on methanol. This suggests that each set of genes plays a role in methanol utilization. The M. thermoacetica MtaB gene was identified based on homology to the methanogenic MtaB genes and also by its adjacent chromosomal proximity to the methanol-induced corrinoid protein, MtaC, which has been crystallized (Zhou et al., Acta Crystallogr. Sect. F. Struct. Biol. Cyst. Commun. 61:537-540 (2005) and further characterized by Northern hybridization and Western Blotting ((Das et al., Proteins 67:167-176 (2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl group from MtaC to either Coenzyme M in methanogens or methyltetrahydrofolate in acetogens. MtaA can also utilize methylcobalamin as the methyl donor. Exemplary genes encoding MtaA can be found in methanogenic archaea such as Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931 (2006) and Methanosarcina acetivorans (Galagan et al., Genome Res. 12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Das et al., Proteins 67:167-176 (2007)). In general, MtaA proteins that catalyze the transfer of the methyl group from CH₃—MtaC are difficult to identify bioinformatically as they share similarity to other corrinoid protein methyltransferases and are not oriented adjacent to the MtaB and MtaC genes on the chromosomes. Nevertheless, a number of MtaA encoding genes have been characterized. The protein sequences of these genes in M. barkeri and M. acetivorans can be identified by the following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
MtaA	YP_304602	73668587	Methanosarcina barkeri
MtaA1	NP_619241	20093166	Methanosarcina acetivorans
MtaA2	NP_616548	20090473	Methanosarcina acetivorans

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, and functionally overexpressed in E. coli (Harms and Thauer, Eur. J. Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required for growth on methanol, whereas MtaA2 is dispensable even though methane production from methanol is reduced in MtaA2 mutants (Bose et al., J. Bacteriol. 190:4017-4026 (2008)). There are multiple additional MtaA homologs in M. barkeri and M. acetivorans that are as yet uncharacterized, but may also catalyze corrinoid protein methyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified by their sequence similarity to the characterized methanogenic MtaA genes. Specifically, three M. thermoacetica genes show high homology (>30% sequence identity) to YP_304602 from M. barkeri. Unlike methanogenic MtaA proteins that naturally catalyze the transfer of the methyl group from CH₃—MtaC to Coenzyme M, an M. thermoacetica MtaA is likely to transfer the methyl group to methyltetrahydrofolate given the similar roles of methyltetrahydrofolate and Coenzyme M in methanogens and acetogens, respectively. The protein sequences of putative MtaA encoding genes from M. thermoacetica can be identified by the following GenBank accession numbers.

Protein	GenBank ID	GI number	Organism
MtaA	YP_430937	83590928	Moorella thermoacetica
MtaA	YP_431175	83591166	Moorella thermoacetica
MtaA	YP_430935	83590926	Moorella thermoacetica
MtaA	YP_430064	83590056	Moorella thermoacetica

FIG. 3, Step B—Methylenetetrahydrofolate Reductase

The conversion of methyl-THF to methylenetetrahydrofolate is catalyzed by methylenetetrahydrofolate reductase. Enzyme candidates are described herein and are those described for Step O, FIG. 1.

FIG. 3, Steps C and D—MTHFDH, Methenyltetrahydrofolate Cyclohydrolase

In M. thermoacetica, E. coli, and C. hydrogenoformans, methenyltetrahydrofolate cyclohydrolase and MTHFDH are carried out by the bi-functional gene products. Suitable enzymes for this step are described herein and are those described for FIG. 1, Steps I and J.

FIG. 3, Step E—Formyltetrahydrofolate Deformylase

This enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-THF) to THF and formate. In E. coli, this enzyme is encoded by purU and has been overproduced, purified, and characterized (Nagy, et al., J. Bacteriol. 3:1292-1298 (1995)). Homologs exist in Corynebacterium sp. U-96 (Suzuki, et al., Biosci. Biotechnol. Biochem. 69(5):952-956 (2005)), Corynebacterium glutamicum ATCC 14067, Salmonella enterica, and several additional organisms

Protein	GenBank ID	GI number	Organism
purU	AAC74314.1	1787483	Escherichia coli K-12 MG1655
purU	BAD97821.1	63002616	Corynebacterium sp. U-96
purU	EHE84645.1	354511740	Corynebacterium glutamicum ATCC 14067
purU	NP_460715.1	16765100	Salmonella enterica subsp.
			enterica serovar Typhimurium str. LT2

FIG. 3, Step F—FTHFS

FTHFS, formyltetrahydrofolate synthetase, ligates formate to tetrahydrofolate at the expense of one ATP. This reaction is catalyzed by the gene product of Moth 0109 in M. thermoacetica (O'brien et al., Experientia Suppl. 26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988); Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986); Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), and CHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005). Homologs exist in C. carboxidivorans P7. This enzyme is found in several other organisms as listed below.

Protein	GenBank ID	GI number	Organism
Moth_0109	YP_428991.1	83588982	Moorella thermoacetica
CHY_2385	YP_361182.1	78045024	Carboxydothermus hydrogenoformans
FHS	P13419.1	120562	Clostridium acidurici
CcarbDRAFT_1913	ZP_05391913.1	255524966	Clostridium carboxidivorans P7
CcarbDRAFT_2946	ZP_05392946.1	255526022	Clostridium carboxidivorans P7
Dhaf_0555	ACL18622.1	219536883	Desulfitobacterium hafniense
fhs	YP_001393842.1	153953077	Clostridium kluyveri DSM 555
fhs	YP_003781893.1	300856909	Clostridium ljungdahlii DSM 13528
MGA3_08300	EIJ83208.1	387590889	Bacillus methanolicus MGA3
PB1_13509	ZP_10132113.1	387929436	Bacillus methanolicus PB1

FIG. 3, Step G—Formate Hydrogen Lyase

A formate hydrogen lyase enzyme can be employed to convert formate to carbon dioxide and hydrogen. An exemplary formate hydrogen lyase enzyme can be found in Escherichia coli. The E. coli formate hydrogen lyase consists of hydrogenase 3 and FDH-H (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). It is activated by the gene product of fhlA. (Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). The addition of the trace elements, selenium, nickel and molybdenum, to a fermentation broth has been shown to enhance formate hydrogen lyase activity (Soini et al., Microb. Cell Fact. 7:26 (2008)). Various hydrogenase 3, FDH and transcriptional activator genes are shown below.

Protein	GenBank ID	GI number	Organism
hycA	NP_417205	16130632	Escherichia coli K-12 MG1655
hycB	NP_417204	16130631	Escherichia coli K-12 MG1655
hycC	NP_417203	16130630	Escherichia coli K-12 MG1655
hycD	NP_417202	16130629	Escherichia coli K-12 MG1655
hycE	NP_417201	16130628	Escherichia coli K-12 MG1655
hycF	NP_417200	16130627	Escherichia coli K-12 MG1655
hycG	NP_417199	16130626	Escherichia coli K-12 MG1655
hycH	NP_417198	16130625	Escherichia coli K-12 MG1655
hycI	NP_417197	16130624	Escherichia coli K-12 MG1655
fdhF	NP_418503	16131905	Escherichia coli K-12 MG1655
fhlA	NP_417211	16130638	Escherichia coli K-12 MG1655

A formate hydrogen lyase enzyme also exists in the hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88 (2008)).

Protein	GenBank ID	GI number	Organism
mhyC	ABW05543	157954626	Thermococcus litoralis
mhyD	ABW05544	157954627	Thermococcus litoralis
mhyE	ABW05545	157954628	Thermococcus litoralis
myhF	ABW05546	157954629	Thermococcus litoralis
myhG	ABW05547	157954630	Thermococcus litoralis
myhH	ABW05548	157954631	Thermococcus litoralis
fdhA	AAB94932	2746736	Thermococcus litoralis
fdhB	AAB94931	157954625	Thermococcus litoralis

Additional formate hydrogen lyase systems have been found in Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacterium formicicum (Vardar-Schara et al., Microbial Biotechnology 1:107-125 (2008)).

FIG. 3, Step H—Hydrogenase

Hydrogenase enzymes can convert hydrogen gas to protons and transfer electrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an “O2-tolerant” hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an O₂-tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132 (2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

Protein	GenBank ID	GI Number	Organism
HoxF	NP_942727.1	38637753	Ralstonia eutropha H16
HoxU	NP_942728.1	38637754	Ralstonia eutropha H16
HoxY	NP_942729.1	38637755	Ralstonia eutropha H16
HoxH	NP_942730.1	38637756	Ralstonia eutropha H16
HoxW	NP_942731.1	38637757	Ralstonia eutropha H16
HoxI	NP_942732.1	38637758	Ralstonia eutropha H16
HoxE	NP_953767.1	39997816	Geobacter sulfurreducens
HoxF	NP_953766.1	39997815	Geobacter sulfurreducens
HoxU	NP_953765.1	39997814	Geobacter sulfurreducens
HoxY	NP_953764.1	39997813	Geobacter sulfurreducens
HoxH	NP_953763.1	39997812	Geobacter sulfurreducens
GSU2717	NP_953762.1	39997811	Geobacter sulfurreducens
HoxE	NP_441418.1	16330690	Synechocystis str. PCC 6803
HoxF	NP_441417.1	16330689	Synechocystis str. PCC 6803
Unknown function	NP_441416.1	16330688	Synechocystis str. PCC 6803
HoxU	NP_441415.1	16330687	Synechocystis str. PCC 6803
HoxY	NP_441414.1	16330686	Synechocystis str. PCC 6803
Unknown function	NP_441413.1	16330685	Synechocystis str. PCC 6803
Unknown function	NP_441412.1	16330684	Synechocystis str. PCC 6803
HoxH	NP_441411.1	16330683	Synechocystis str. PCC 6803
HypF	NP_484737.1	17228189	Nostoc sp. PCC 7120
HypC	NP_484738.1	17228190	Nostoc sp. PCC 7120
HypD	NP_484739.1	17228191	Nostoc sp. PCC 7120
Unknown function	NP_484740.1	17228192	Nostoc sp. PCC 7120
HypE	NP_484741.1	17228193	Nostoc sp. PCC 7120
HypA	NP_484742.1	17228194	Nostoc sp. PCC 7120
HypB	NP_484743.1	17228195	Nostoc sp. PCC 7120
Hox1E	AAP50519.1	37787351	Thiocapsa roseopersicina
Hox1F	AAP50520.1	37787352	Thiocapsa roseopersicina
Hox1U	AAP50521.1	37787353	Thiocapsa roseopersicina
Hox1Y	AAP50522.1	37787354	Thiocapsa roseopersicina
Hox1H	AAP50523.1	37787355	Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to four hydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol. 164:1324-1331 (1985); Sawers and Boxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. Endogenous hydrogen-lyase enzymes of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451 (1992); Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). The M. thermoacetica and Clostridium ljungdahli hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica and C. ljungdahli can grow with CO₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. These protein sequences encoded for by these genes are identified by the following GenBank accession numbers. In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and C. ljungdahli (see for example US 2012/0003652).

	Protein	GenBank ID	GI Number	Organism
	HypA	NP_417206	16130633	Escherichia coli
	HypB	NP_417207	16130634	Escherichia coli
	HypC	NP_417208	16130635	Escherichia coli
	HypD	NP_417209	16130636	Escherichia coli
	HypE	NP_417210	226524740	Escherichia coli
	HypF	NP_417192	16130619	Escherichia coli
	HycA	NP_417205	16130632	Escherichia coli
	HycB	NP_417204	16130631	Escherichia coli
	HycC	NP_417203	16130630	Escherichia coli
	HycD	NP_417202	16130629	Escherichia coli
	HycE	NP_417201	16130628	Escherichia coli
	HycF	NP_417200	16130627	Escherichia coli
	HycG	NP_417199	16130626	Escherichia coli
	HycH	NP_417198	16130625	Escherichia coli
	HycI	NP_417197	16130624	Escherichia coli
	HyfA	NP_416976	90111444	Escherichia coli
	HyfB	NP_416977	16130407	Escherichia coli
	HyfC	NP_416978	90111445	Escherichia coli
	HyfD	NP_416979	16130409	Escherichia coli
	HyfE	NP_416980	16130410	Escherichia coli
	HyfF	NP_416981	16130411	Escherichia coli
	HyfG	NP_416982	16130412	Escherichia coli
	HyfH	NP_416983	16130413	Escherichia coli
	HyfI	NP_416984	16130414	Escherichia coli
	HyfJ	NP_416985	90111446	Escherichia coli
	HyfR	NP_416986	90111447	Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. coli hydrogenase genes are shown below.

Protein	GenBank ID	GI Number	Organism
Moth_2175	YP_431007	83590998	Moorella thermoacetica
Moth_2176	YP_431008	83590999	Moorella thermoacetica
Moth_2177	YP_431009	83591000	Moorella thermoacetica
Moth_2178	YP_431010	83591001	Moorella thermoacetica
Moth_2179	YP_431011	83591002	Moorella thermoacetica
Moth_2180	YP_431012	83591003	Moorella thermoacetica
Moth_2181	YP_431013	83591004	Moorella thermoacetica
Moth_2182	YP_431014	83591005	Moorella thermoacetica
Moth_2183	YP_431015	83591006	Moorella thermoacetica
Moth_2184	YP_431016	83591007	Moorella thermoacetica
Moth_2185	YP_431017	83591008	Moorella thermoacetica
Moth_2186	YP_431018	83591009	Moorella thermoacetica
Moth_2187	YP_431019	83591010	Moorella thermoacetica
Moth_2188	YP_431020	83591011	Moorella thermoacetica
Moth_2189	YP_431021	83591012	Moorella thermoacetica
Moth_2190	YP_431022	83591013	Moorella thermoacetica
Moth_2191	YP_431023	83591014	Moorella thermoacetica
Moth_2192	YP_431024	83591015	Moorella thermoacetica
Moth_0439	YP_429313	83589304	Moorella thermoacetica
Moth_0440	YP_429314	83589305	Moorella thermoacetica
Moth_0441	YP_429315	83589306	Moorella thermoacetica
Moth_0442	YP_429316	83589307	Moorella thermoacetica
Moth_0809	YP_429670	83589661	Moorella thermoacetica
Moth_0810	YP_429671	83589662	Moorella thermoacetica
Moth_0811	YP_429672	83589663	Moorella thermoacetica
Moth_0812	YP_429673	83589664	Moorella thermoacetica
Moth_0814	YP_429674	83589665	Moorella thermoacetica
Moth_0815	YP_429675	83589666	Moorella thermoacetica
Moth_0816	YP_429676	83589667	Moorella thermoacetica
Moth_1193	YP_430050	83590041	Moorella thermoacetica
Moth_1194	YP_430051	83590042	Moorella thermoacetica
Moth_1195	YP_430052	83590043	Moorella thermoacetica
Moth_1196	YP_430053	83590044	Moorella thermoacetica
Moth_1717	YP_430562	83590553	Moorella thermoacetica
Moth_1718	YP_430563	83590554	Moorella thermoacetica
Moth_1719	YP_430564	83590555	Moorella thermoacetica
Moth_1883	YP_430726	83590717	Moorella thermoacetica
Moth_1884	YP_430727	83590718	Moorella thermoacetica
Moth_1885	YP_430728	83590719	Moorella thermoacetica
Moth_1886	YP_430729	83590720	Moorella thermoacetica
Moth_1887	YP_430730	83590721	Moorella thermoacetica
Moth_1888	YP_430731	83590722	Moorella thermoacetica
Moth_1452	YP_430305	83590296	Moorella thermoacetica
Moth_1453	YP_430306	83590297	Moorella thermoacetica
Moth_1454	YP_430307	83590298	Moorella thermoacetica

Genes encoding hydrogenase enzymes from C. ljungdahli are shown below.

Protein	GenBank ID	GI Number	Organism
CLJU_c20290	ADK15091.1	300435324	Clostridium ljungdahli
CLJU_c07030	ADK13773.1	300434006	Clostridium ljungdahli
CLJU_c07040	ADK13774.1	300434007	Clostridium ljungdahli
CLJU_c07050	ADK13775.1	300434008	Clostridium ljungdahli
CLJU_c07060	ADK13776.1	300434009	Clostridium ljungdahli
CLJU_c07070	ADK13777.1	300434010	Clostridium ljungdahli
CLJU_c07080	ADK13778.1	300434011	Clostridium ljungdahli
CLJU_c14730	ADK14541.1	300434774	Clostridium ljungdahli
CLJU_c14720	ADK14540.1	300434773	Clostridium ljungdahli
CLJU_c14710	ADK14539.1	300434772	Clostridium ljungdahli
CLJU_c14700	ADK14538.1	300434771	Clostridium ljungdahli
CLJU_c28670	ADK15915.1	300436148	Clostridium ljungdahli
CLJU_c28660	ADK15914.1	300436147	Clostridium ljungdahli
CLJU_c28650	ADK15913.1	300436146	Clostridium ljungdahli
CLJU_c28640	ADK15912.1	300436145	Clostridium ljungdahli

In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H₂O to CO₂ and H₂ (Fox et al., J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and CO₂ reduction activities when linked to an electrode (Parkin et al., J Am. Chem. Soc. 129:10328-10329 (2007)).

Protein	GenBank ID	GI Number	Organism
CooL	AAC45118	1515468	Rhodospirillum rubrum
CooX	AAC45119	1515469	Rhodospirillum rubrum
CooU	AAC45120	1515470	Rhodospirillum rubrum
CooH	AAC45121	1498746	Rhodospirillum rubrum
CooF	AAC45122	1498747	Rhodospirillum rubrum
CODH	AAC45123	1498748	Rhodospirillum rubrum
(CooS)
CooC	AAC45124	1498749	Rhodospirillum rubrum
CooT	AAC45125	1498750	Rhodospirillum rubrum
CooJ	AAC45126	1498751	Rhodospirillum rubrum
CODH-I	YP_360644	78043418	Carboxydothermus hydrogenoformans
(CooS-I)
CooF	YP_360645	78044791	Carboxydothermus hydrogenoformans
HypA	YP_360646	78044340	Carboxydothermus hydrogenoformans
CooH	YP_360647	78043871	Carboxydothermus hydrogenoformans
CooU	YP_360648	78044023	Carboxydothermus hydrogenoformans
CooX	YP_360649	78043124	Carboxydothermus hydrogenoformans
CooL	YP_360650	78043938	Carboxydothermus hydrogenoformans
CooK	YP_360651	78044700	Carboxydothermus hydrogenoformans
CooM	YP_360652	78043942	Carboxydothermus hydrogenoformans
CooC	YP_360654.1	78043296	Carboxydothermus hydrogenoformans
CooA-1	YP_360655.1	78044021	Carboxydothermus hydrogenoformans

Some hydrogenase and CODH enzymes transfer electrons to ferredoxins. Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a [4Fe-45]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-45] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7, Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed below.

Protein	GenBank ID	GI Number	Organism
fdx1	BAE02673.1	68163284	Hydrogenobacter thermophilus
M11214.1	AAA83524.1	144806	Clostridium pasteurianum
Zfx	AAY79867.1	68566938	Sulfolobus acidocalarius
Fdx	AAC75578.1	1788874	Escherichia coli
hp_0277	AAD07340.1	2313367	Helicobacter pylori
fdxA	CAL34484.1	112359698	Campylobacter jejuni
Moth_0061	ABC18400.1	83571848	Moorella thermoacetica
Moth_1200	ABC19514.1	83572962	Moorella thermoacetica
Moth_1888	ABC20188.1	83573636	Moorella thermoacetica
Moth_2112	ABC20404.1	83573852	Moorella thermoacetica
Moth_1037	ABC19351.1	83572799	Moorella thermoacetica
CcarbDRAFT_4383	ZP_05394383.1	255527515	Clostridium carboxidivorans P7
CcarbDRAFT_2958	ZP_05392958.1	255526034	Clostridium carboxidivorans P7
CcarbDRAFT_2281	ZP_05392281.1	255525342	Clostridium carboxidivorans P7
CcarbDRAFT_5296	ZP_05395295.1	255528511	Clostridium carboxidivorans P7
CcarbDRAFT_1615	ZP_05391615.1	255524662	Clostridium carboxidivorans P7
CcarbDRAFT_1304	ZP_05391304.1	255524347	Clostridium carboxidivorans P7
cooF	AAG29808.1	11095245	Carboxydothermus hydrogenoformans
fdxN	CAA35699.1	46143	Rhodobacter capsulatus
Rru_A2264	ABC23064.1	83576513	Rhodospirillum rubrum
Rru_A1916	ABC22716.1	83576165	Rhodospirillum rubrum
Rru_A2026	ABC22826.1	83576275	Rhodospirillum rubrum
cooF	AAC45122.1	1498747	Rhodospirillum rubrum
fdxN	AAA26460.1	152605	Rhodospirillum rubrum
Alvin_2884	ADC63789.1	288897953	Allochromatium vinosum DSM 180
Fdx	YP_002801146.1	226946073	Azotobacter vinelandii DJ
CKL_3790	YP_001397146.1	153956381	Clostridium kluyveri DSM 555
fer1	NP_949965.1	39937689	Rhodopseudomonas palustris CGA009
Fdx	CAA12251.1	3724172	Thauera aromatica
CHY_2405	YP_361202.1	78044690	Carboxydothermus hydrogenoformans
Fer	YP_359966.1	78045103	Carboxydothermus hydrogenoformans
Fer	AAC83945.1	1146198	Bacillus subtilis
fdx1	NP_249053.1	15595559	Pseudomonas aeruginosa PA01
yfhL	AP_003148.1	89109368	Escherichia coli K-12
CLJU_c00930	ADK13195.1	300433428	Clostridium ljungdahli
CLJU_c00010	ADK13115.1	300433348	Clostridium ljungdahli
CLJU_c01820	ADK13272.1	300433505	Clostridium ljungdahli
CLJU_c17980	ADK14861.1	300435094	Clostridium ljungdahli
CLJU_c17970	ADK14860.1	300435093	Clostridium ljungdahli
CLJU_c22510	ADK15311.1	300435544	Clostridium ljungdahli
CLJU_c26680	ADK15726.1	300435959	Clostridium ljungdahli
CLJU_c29400	ADK15988.1	300436221	Clostridium ljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins or flavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transfer of electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacter jejuni (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7. The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C. kluyveri, encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J Bacteriol 192: 5115-5123 (2010)). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133 (2008); and Herrmann, J Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin.

Protein	GenBank ID	GI Number	Organism
fqrB	NP_207955.1	15645778	Helicobacter pylori
fqrB	YP_001482096.1	157414840	Campylobacter jejuni
RPA3954	CAE29395.1	39650872	Rhodopseudomonas palustris
Fpr	BAH29712.1	225320633	Hydrogenobacter thermophilus
yumC	NP_391091.2	255767736	Bacillus subtilis
Fpr	P28861.4	399486	Escherichia coli
hcaD	AAC75595.1	1788892	Escherichia coli
LOC100282643	NP_001149023.1	226497434	Zea mays
NfnA	YP_001393861.1	153953096	Clostridium kluyveri
NfnB	YP_001393862.1	153953097	Clostridium kluyveri
CcarbDRAFT_2639	ZP_05392639.1	255525707	Clostridium carboxidivorans P7
CcarbDRAFT_2638	ZP_05392638.1	255525706	Clostridium carboxidivorans P7
CcarbDRAFT_2636	ZP_05392636.1	255525704	Clostridium carboxidivorans P7
CcarbDRAFT_5060	ZP_05395060.1	255528241	Clostridium carboxidivorans P7
CcarbDRAFT_2450	ZP_05392450.1	255525514	Clostridium carboxidivorans P7
CcarbDRAFT_1084	ZP_05391084.1	255524124	Clostridium carboxidivorans P7
RnfC	EDK33306.1	146346770	Clostridium kluyveri
RnfD	EDK33307.1	146346771	Clostridium kluyveri
RnfG	EDK33308.1	146346772	Clostridium kluyveri
RnfE	EDK33309.1	146346773	Clostridium kluyveri
RnfA	EDK33310.1	146346774	Clostridium kluyveri
RnfB	EDK33311.1	146346775	Clostridium kluyveri
CLJU_c11410 (RnfB)	ADK14209.1	300434442	Clostridium ljungdahlii
CLJU_c11400 (RnfA)	ADK14208.1	300434441	Clostridium ljungdahlii
CLJU_c11390 (RnfE)	ADK14207.1	300434440	Clostridium ljungdahlii
CLJU_c11380 (RnfG)	ADK14206.1	300434439	Clostridium ljungdahlii
CLJU_c11370 (RnfD)	ADK14205.1	300434438	Clostridium ljungdahlii
CLJU_c11360 (RnfC)	ADK14204.1	300434437	Clostridium ljungdahlii
MOTH_1518 (NfnA)	YP_430370.1	83590361	Moorella thermoacetica
MOTH_1517(NfnB)	YP_430369.1	83590360	Moorella thermoacetica
CHY_1992 (NfnA)	YP_360811.1	78045020	Carboxydothermus hydrogenoformans
CHY_1993 (NfnB)	YP_360812.1	78044266	Carboxydothermus hydrogenoformans
CLJU_c37220 (NfnAB)	YP_003781850.1	300856866	Clostridium ljungdahlii

FIG. 3, Step I—FDH

Formate dehydrogenase (FDH) catalyzes the reversible transfer of electrons from formate to an acceptor. Enzymes with FDH activity are those described herein and for FIG. 1 Step S.

FIG. 3, Step J—Methanol Dehydrogenase (MeDH or MDH)

NAD+ dependent MeDH enzymes (EC 1.1.1.244) catalyze the conversion of methanol and NAD+ to formaldehyde and NADH. An enzyme with this activity was first characterized in Bacillus methanolicus (Heggeset, et al., Applied and Environmental Microbiology, 78(15):5170-5181 (2012)). This enzyme is zinc and magnesium dependent, and activity of the enzyme is enhanced by the activating enzyme encoded by act (Kloosterman et al, J Biol Chem 277:34785-92 (2002)). The act is a Nudix hydrolase. Several of these candidates have been identified and shown to have activity on methanol. Additional NAD(P)+ dependent enzymes can be identified by sequence homology. MeDH enzymes utilizing different electron acceptors are also known in the art Examples include cytochrome dependent enzymes such as mxaIF of the methylotroph Methylobacterium extorquens (Nunn et al, Nucl Acid Res 16:7722 (1988)). MeDH enzymes of methanotrophs such as Methylococcus capsulatis function in a complex with methane monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)). Methanol can also be oxidized to formaldehyde by alcohol oxidase enzymes such as methanol oxidase (EC 1.1.3.13) of Candida boidinii (Sakai et al, Gene 114: 67-73 (1992)).

Protein	GenBank ID	GI Number	Organism
mdh, MGA3_17392	EIJ77596.1	387585261	Bacillus methanolicus MGA3
mdh2, MGA3_07340	EIJ83020.1	387590701	Bacillus methanolicus MGA3
mdh3, MGA3_10725	EIJ80770.1	387588449	Bacillus methanolicus MGA3
act, MGA3_09170	EIJ83380.1	387591061	Bacillus methanolicus MGA3
mdh, PB1_17533	ZP_10132907.1	387930234	Bacillus methanolicus PB1
mdh1, PB1_14569	ZP_10132325.1	387929648	Bacillus methanolicus PB1
mdh2, PB1_12584	ZP_10131932.1	387929255	Bacillus methanolicus PB1
act, PB1_14394	ZP_10132290.1	387929613	Bacillus methanolicus PB1
BFZC1_05383	ZP_07048751.1	299535429	Lysinibacillus fusiformis
BFZC1_20163	ZP_07051637.1	299538354	Lysinibacillus fusiformis
Bsph_4187	YP_001699778.1	169829620	Lysinibacillus sphaericus
Bsph_1706	YP_001697432.1	169827274	Lysinibacillus sphaericus
mdh2	YP_004681552.1	339322658	Cupriavidus necator N-1
nudF1	YP_004684845.1	339325152	Cupriavidus necator N-1
BthaA_010200007655	ZP_05587334.1	257139072	Burkholderia thailandensis E264
BTH_I1076	YP_441629.1	83721454	Burkholderia thailandensis E264
(MutT/NUDIX NTP
pyrophosphatase)
BalcAV_11743	ZP_10819291.1	402299711	Bacillus alcalophilus ATCC 27647
BalcAV_05251	ZP_10818002.1	402298299	Bacillus alcalophilus ATCC 27647
alcohol dehydrogenase	YP_001447544	156976638	Vibrio harveyi ATCC BAA-1116
P3TCK_27679	ZP_01220157.1	90412151	Photobacterium profundum 3TCK
alcohol dehydrogenase	YP_694908	110799824	Clostridium perfringens ATCC 13124
adhB	NP_717107	24373064	Shewanella oneidensis MR-1
alcohol dehydrogenase	YP_237055	66047214	Pseudomonas syringae pv. syringae
			B728a
alcohol dehydrogenase	YP_359772	78043360	Carboxydothermus hydrogenoformans
			Z-2901
alcohol dehydrogenase	YP_003990729	312112413	Geobacillus sp. Y4.1MC1
PpeoK3_010100018471	ZP_10241531.1	390456003	Paenibacillus peoriae KCTC 3763
OBE_12016	EKC54576	406526935	human gut metagenome
alcohol dehydrogenase	YP_001343716	152978087	Actinobacillus succinogenes 130Z
dhaT	AAC45651	2393887	Clostridium pasteurianum DSM 525
alcohol dehydrogenase	NP_561852	18309918	Clostridium perfringens str. 13
BAZO_10081	ZP_11313277.1	410459529	Bacillus azotoformans LMG 9581
alcohol dehydrogenase	YP_007491369	452211255	Methanosarcina mazei Tuc01
alcohol dehydrogenase	YP_004860127	347752562	Bacillus coagulans 36D1
alcohol dehydrogenase	YP_002138168	197117741	Geobacter bemidjiensis Bem
DesmeDRAFT_1354	ZP_08977641.1	354558386	Desulfitobacterium metallireducens DSM
			15288
alcohol dehydrogenase	YP_001337153	152972007	Klebsiella pneumoniae subsp. pneumoniae
			MGH 78578
alcohol dehydrogenase	YP_001113612	134300116	Desulfotomaculum reducens MI-1
alcohol dehydrogenase	YP_001663549	167040564	Thermoanaerobacter sp. X514
ACINNAV82_2382	ZP_16224338.1	421788018	Acinetobacter baumannii Naval-82
alcohol dehydrogenase	YP_005052855	374301216	Desulfovibrio africanus str. Walvis Bay
alcohol dehydrogenase	AGF87161	451936849	uncultured organism
DesfrDRAFT_3929	ZP_07335453.1	303249216	Desulfovibrio fructosovorans JJ
alcohol dehydrogenase	NP_617528	20091453	Methanosarcina acetivorans C2A
alcohol dehydrogenase	NP_343875.1	15899270	Sulfolobus solfataricus P-2
adh4	YP_006863258	408405275	Nitrososphaera gargensis Ga9.2
Ta0841	NP_394301.1	16081897	Thermoplasma acidophilum
PTO1151	YP_023929.1	48478223	Picrophilus torridus DSM9790
alcohol dehydrogenase	ZP_10129817.1	387927138	Bacillus methanolicus PB-1
cgR_2695	YP_001139613.1	145296792	Corynebacterium glutamicum R
alcohol dehydrogenase	YP_004758576.1	340793113	Corynebacterium variabile
HMPREF1015_01790	ZP_09352758.1	365156443	Bacillus smithii
ADH1	NP_014555.1	6324486	Saccharomyces cerevisiae
NADH-dependent	YP_001126968.1	138896515	Geobacillus themodenitrificans NG80-2
butanol dehydrogenase A
alcohol dehydrogenase	WP_007139094.1	494231392	Flavobacterium frigoris
MeDH	WP_003897664.1	489994607	Mycobacterium smegmatis
ADH1B	NP_000659.2	34577061	Homo sapiens
PMI01_01199	ZP_10750164.1	399072070	Caulobacter sp. AP07
YiaY	YP_026233.1	49176377	Escherichia coli
MCA0299	YP_112833.1	53802410	Methylococcus capsulatis
MCA0782	YP_113284.1	53804880	Methylococcus capsulatis
mxaI	YP_002965443.1	240140963	Methylobacterium extorquens
mxaF	YP_002965446.1	240140966	Methylobacterium extorquens
AOD1	AAA34321.1	170820	Candida boidinii
hypothetical protein	EDA87976.1	142827286	Marine metagenome
GOS_1920437			JCVI_SCAF_1096627185304
alcohol dehydrogenase	CAA80989.1	580823	Geobacillus stearothermophilus

An in vivo assay was developed to determine the activity of MeDHs. This assay relies on the detection of formaldehyde (HCHO), thus measuring the forward activity of the enzyme (oxidation of methanol). To this end, a strain comprising a BDOP and lacking frmA, frmB, frmR was created using Lamba Red recombinase technology (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12): 6640-5 (2000). Plasmids expressing MeDHs were transformed into the strain, then grown to saturation in LB medium+antibiotic at 37° C. with shaking. Transformation of the strain with an empty vector served as a negative control. Cultures were adjusted by O.D. and then diluted 1:10 into M9 medium+0.5% glucose+antibiotic and cultured at 37° C. with shaking for 6-8 hours until late log phase. Methanol was added to 2% v/v and the cultures were further incubated for 30 min. with shaking at 37° C. Cultures were spun down and the supernatant was assayed for formaldehyde produced using DETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor, Mich.) according to manufacturer's instructions. The frmA, frmB, frmR deletions resulted in the native formaldehyde utilization pathway to be deleted, which enables the formation of formaldehyde that can be used to detect MeDH activity in the NNOMO.

The activity of several enzymes was measured using the assay described above. The results of four independent experiments are provided in the below Table.

Results of In Vivo Assays Showing Formaldehyde (HCHO) Production by Various NNOMO Comprising a Plasmid Expressing a MeDH.

Accession number HCHO (μM)
Experiment 1
EIJ77596.1       >50
EIJ83020.1       >20
EIJ80770.1       >50
ZP_10132907.1    >20
ZP_10132325.1    >20
ZP_10131932.1    >50
ZP_07048751.1    >50
YP_001699778.1   >50
YP_004681552.1   >10
ZP_10819291.1    <1
Empty vector     2.33
Experiment 2
EIJ77596.1       >50
NP_00659.2       >50
YP_004758576.1   >20
ZP_09352758.1    >50
ZP_10129817.1    >20
YP_001139613.1   >20
NP_014555.1      >10
WP_007139094.1   >10
NP_343875.1      >1
YP_006863258     >1
NP_394301.1      >1
ZP_10750164.1    >1
YP_023929.1      >1
ZP_08977641.1    <1
ZP_10117398.1    <1
YP_004108045.1   <1
ZP_09753449.1    <1
Empty vector     0.17
Experiment 3
EIJ77596.1       >50
NP_561852        >50
YP_002138168     >50
YP_026233.1      >50
YP_001447544     >50
Metalibrary      >50
YP_359772        >50
ZP_01220157.1    >50
ZP_07335453.1    >20
YP_001337153     >20
YP_694908        >20
NP_717107        >20
AAC45651         >10
ZP_11313277.1    >10
ZP_16224338.1    >10
YP_001113612     >10
YP_004860127     >10
YP_003310546     >10
YP_001343716     >10
NP_717107        >10
YP_002434746     >10
Empty vector     0.11
Experiment 4
EIJ77596.1       >20
ZP_11313277.1    >50
YP_001113612     >50
YP_001447544     >20
AGF87161         >50
EDA87976.1       >20
Empty vector     −0.8

FIG. 3, Step K—Spontaneous or Formaldehyde Activating Enzyme

The conversion of formaldehyde and THF to methylenetetrahydrofolate can occur spontaneously. It is also possible that the rate of this reaction can be enhanced by a formaldehyde activating enzyme. A formaldehyde activating enzyme (Fae) has been identified in Methylobacterium extorquens AM1 which catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin (Vorholt, et al., J. Bacteriol., 182(23), 6645-6650 (2000)). It is possible that a similar enzyme exists or can be engineered to catalyze the condensation of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate. Homologs exist in several organisms including Xanthobacter autotrophicus Py2 and Hyphomicrobium denitrtficans ATCC 51888.

Protein	GenBank ID	GI Number	Organism
MexAM1_META1p1766	Q9FA38.3	17366061	Methylobacterium extorquens AM1
Xaut_0032	YP_001414948.1	154243990	Xanthobacter autotrophicus Py2
Hden_1474	YP_003755607.1	300022996	Hyphomicrobium denitrificans
			ATCC 51888

FIG. 3, Step L—Formaldehyde Dehydrogenase

Oxidation of formaldehyde to formate is catalyzed by formaldehyde dehydrogenase. An NAD+ dependent formaldehyde dehydrogenase enzyme is encoded by fdhA of Pseudomonas putida (Ito et al, J Bacteriol 176: 2483-2491 (1994)). Additional formaldehyde dehydrogenase enzymes include the NAD+ and glutathione independent formaldehyde dehydrogenase from Hyphomicrobium zavarzinii (Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione dependent formaldehyde dehydrogenase of Pichia pastoris (Sunga et al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent formaldehyde dehydrogenase of Methylobacter marinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein	GenBank ID	GI Number	Organism
fdhA	P46154.3	1169603	Pseudomonas putida
faoA	CAC85637.1	19912992	Hyphomicrobium zavarzinii
Fld1	CCA39112.1	328352714	Pichia pastoris
fdh	P47734.2	221222447	Methylobacter marinus

In addition to the formaldehyde dehydrogenase enzymes listed above, alternate enzymes and pathways for converting formaldehyde to formate are known in the art. For example, many organisms employ glutathione-dependent formaldehyde oxidation pathways, in which formaldehyde is converted to formate in three steps via the intermediates S-hydroxymethylglutathione and S-formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of this pathway are S-(hydroxymethyl)glutathione synthase (EC 4.4.1.22), glutathione-dependent formaldehyde dehydrogenase (EC 1.1.1.284) and S-formylglutathione hydrolase (EC 3.1.2.12).

FIG. 3, Step M—Spontaneous or S-(hydroxymethyl)glutathione Synthase

While conversion of formaldehyde to S-hydroxymethylglutathione can occur spontaneously in the presence of glutathione, it has been shown by Goenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002)) that an enzyme from Paracoccus denitrificans can accelerate this spontaneous condensation reaction. The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene encoding it, which was named gfa, is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

Protein	GenBank ID	GI Number	Organism
Gfa	Q51669.3	38257308	Paracoccus denitrificans
Gfa	ABP71667.1	145557054	Rhodobacter sphaeroides ATCC 17025
Gfa	Q92WX6.1	38257348	Sinorhizobium meliloti 1021
Gfa	Q98LU4.2	38257349	Mesorhizobium loti MAFF303099

FIG. 3, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase

Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) belongs to the family of class III alcohol dehydrogenases. Glutathione and formaldehyde combine non-enzymatically to form hydroxymethylglutathione, the true substrate of the GS-FDH catalyzed reaction. The product, S-formylglutathione, is further metabolized to formic acid.

Protein	GenBank ID	GI Number	Organism
frmA	YP_488650.1	388476464	Escherichia coli K-12 MG1655
SFA1	NP_010113.1	6320033	Saccharomyces cerevisiae S288c
flhA	AAC44551.1	1002865	Paracoccus denitrificans
adhI	AAB09774.1	986949	Rhodobacter sphaeroides

FIG. 3, Step O—S-formylglutathione Hydrolase

S-formylglutathione hydrolase is a glutathione thiol esterase found in bacteria, plants and animals. It catalyzes conversion of S-formylglutathione to formate and glutathione. The fghA gene of P. denitrificans is located in the same operon with gfa and flhA, two genes involved in the oxidation of formaldehyde to formate in this organism. In E. coli, FrmB is encoded in an operon with FrmR and FrmA, which are proteins involved in the oxidation of formaldehyde. YeiG of E. coli is a promiscuous serine hydrolase; its highest specific activity is with the substrate S-formylglutathione.

Protein	GenBank ID	GI Number	Organism
frmB	NP_414889.1	16128340	Escherichia coli K-12 MG1655
yeiG	AAC75215.1	1788477	Escherichia coli K-12 MG1655
fghA	AAC44554.1	1002868	Paracoccus denitrificans

FIG. 3, Step P—Carbon Monoxide dehydrogenase (CODH)

CODH is a reversible enzyme that interconverts CO and CO₂ at the expense or gain of electrons. The natural physiological role of the CODH in ACS/CODH complexes is to convert CO₂ to CO for incorporation into acetyl-CoA by acetyl-CoA synthase. Nevertheless, such CODH enzymes are suitable for the extraction of reducing equivalents from CO due to the reversible nature of such enzymes. Expressing such CODH enzymes in the absence of ACS allows them to operate in the direction opposite to their natural physiological role (i.e., CO oxidation).

In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The M. thermoacetica gene (Genbank Accession Number: YP430813) is expressed by itself in an operon and is believed to transfer electrons from CO to an external mediator like ferredoxin in a “Ping-pong” reaction. The reduced mediator then couples to other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al., Science 293:1281-1285 (2001)). Similar ACS-free CODH enzymes can be found in a diverse array of organisms including Geobacter metallireducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, C. ljungdahli and Campylobacter curvus 525.92.

Protein	GenBank ID	GI Number	Organism
CODH (putative)	YP_430813	83590804	Moorella thermoacetica
CODH-II (CooS-II)	YP_358957	78044574	Carboxydothermus hydrogenoformans
CooF	YP_358958	78045112	Carboxydothermus hydrogenoformans
CODH (putative)	ZP_05390164.1	255523193	Clostridium carboxidivorans P7
CcarbDRAFT_0341	ZP_05390341.1	255523371	Clostridium carboxidivorans P7
CcarbDRAFT_1756	ZP_05391756.1	255524806	Clostridium carboxidivorans P7
CcarbDRAFT_2944	ZP_05392944.1	255526020	Clostridium carboxidivorans P7
CODH	YP_384856.1	78223109	Geobacter metallireducens GS-15
Cpha266_0148	YP_910642.1	119355998	Chlorobium phaeobacteroides DSM 266
(cytochrome c)
Cpha266_0149 (CODH)	YP_910643.1	119355999	Chlorobium phaeobacteroides DSM 266
Ccel_0438	YP_002504800.1	220927891	Clostridium cellulolyticum H10
Ddes_0382 (CODH)	YP_002478973.1	220903661	Desulfovibrio desulfuricans subsp.
			desulfuricansstr. ATCC 27774
Ddes_0381 (CooC)	YP_002478972.1	220903660	Desulfovibrio desulfuricans subsp.
			desulfuricans str. ATCC 27774
Pcar_0057 (CODH)	YP_355490.1	7791767	Pelobacter carbinolicus DSM 2380
Pcar_0058 (CooC)	YP_355491.1	7791766	Pelobacter carbinolicus DSM 2380
Pcar_0058 (HypA)	YP_355492.1	7791765	Pelobacter carbinolicus DSM 2380
CooS (CODH)	YP_001407343.1	154175407	Campylobacter curvus 525.92
CLJU_c09110	ADK13979.1	300434212	Clostridium ljungdahli
CLJU_c09100	ADK13978.1	300434211	Clostridium ljungdahli
CLJU_c09090	ADK13977.1	300434210	Clostridium ljungdahli

Example III

Methods for Formaldehyde Fixation

Provided herein are exemplary pathways, which utilize formaldehyde produced from the oxidation of methanol (see, e.g., FIG. 1, step A, or FIG. 3, step J) or from FAPs described in Example I (see, e.g., FIG. 1) in the formation of intermediates of certain central metabolic pathways that can be used for the production of compounds disclosed herein.

One exemplary pathway that can utilize formaldehyde produced from the oxidation of methanol is shown in FIG. 1, which involves condensation of formaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate (h6p) by hexulose-6-phosphate synthase (FIG. 1, step B). The enzyme can use Mg²⁺ or Mn²⁺ for maximal activity, although other metal ions are useful, and even non-metal-ion-dependent mechanisms are contemplated. H6p is converted into fructose-6-phosphate by 6P3HI (FIG. 1, step C).

Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol is shown in FIG. 1 and proceeds through dihydroxyacetone. DHAS is a special transketolase that first transfers a glycoaldehyde group from xylulose-5-phosphate to formaldehyde, resulting in the formation of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), which is an intermediate in glycolysis (FIG. 1). The DHA obtained from DHA synthase can be further phosphorylated to form DHA phosphate and assimilated into glycolysis and several other pathways (FIG. 1).

FIG. 1, Steps B and C—Hexulose-6-phosphate Synthase (Step B) and 6P3HI (Step C)

Both of the hexulose-6-phosphate synthase and 6P3HI enzymes are found in several organisms, including methanotrops and methylotrophs where they have been purified (Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition, these enzymes have been reported in heterotrophs such as Bacillus subtilis also where they are reported to be involved in formaldehyde detoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al; 1999. J Bac 181(23):7154-60. Genes for these two enzymes from the methylotrophic bacterium Mycobacterium gastri MB19 have been fused and E. coli strains harboring the hps-phi construct showed more efficient utilization of formaldehyde (Orita et al., 2007, Appl Microbiol Biotechnol. 76:439-445). In some organisms, these two enzymes naturally exist as a fused version that is bifunctional.

Exemplary candidate genes for hexulose-6-phosphate synthase are:

Protein	GenBank ID	GI number	Organism
Hps	AAR39392.1	40074227	Bacillus methanolicus MGA3
Hps	EIJ81375.1	387589055	Bacillus methanolicus PB1
RmpA	BAA83096.1	5706381	Methylomonas aminofaciens
RmpA	BAA90546.1	6899861	Mycobacterium gastri
YckG	BAA08980.1	1805418	Bacillus subtilis
Hps	YP_544362.1	91774606	Methylobacillus flagellatus
Hps	YP_545763.1	91776007	Methylobacillus flagellatus
Hps	AAG29505.1	11093955	Aminomonas aminovorus
SgbH	YP_004038706.1	313200048	Methylovorus sp. MP688
Hps	YP_003050044.1	253997981	Methylovorus glucosetrophus SIP3-4
Hps	YP_003990382.1	312112066	Geobacillus sp. Y4.1MC1
Hps	gb|AAR91478.1	40795504	Geobacillus sp. M10EXG
Hps	YP_007402409.1	448238351	Geobacillus sp. GHH01

Exemplary gene candidates for 6P3HI are:

Protein	GenBank ID	GI number	Organism
Phi	AAR39393.1	40074228	Bacillus methanolicus MGA3
Phi	EIJ81376.1	387589056	Bacillus methanolicus PB1
Phi	BAA83098.1	5706383	Methylomonas aminofaciens
RmpB	BAA90545.1	6899860	Mycobacterium gastri
Phi	YP_545762.1	91776006	Methylobacillus flagellatus KT
Phi	YP_003051269.1	253999206	Methylovorus glucosetrophus SIP3-4
Phi	YP_003990383.1	312112067	Geobacillus sp. Y4.1MC1
Phi	YP_007402408.1	448238350	Geobacillus sp. GHH01

Candidates for enzymes where both of these functions have been fused into a single open reading frame include the following.

Protein	GenBank ID	GI number	Organism
PH1938	NP_143767.1	14591680	Pyrococcus horikoshii OT3
PF0220	NP_577949.1	18976592	Pyrococcus furiosus
TK0475	YP_182888.1	57640410	Thermococcus kodakaraensis
PAB1222	NP_127388.1	14521911	Pyrococcus abyssi
MCA2738	YP_115138.1	53803128	Methylococcus capsulatas
Metal_3152	EIC30826.1	380884949	Methylomicrobium album BG8

FIG. 1, Step D—Dihydroxyacetone Synthase (DHAS)

The DHAS enzyme in Candida boidinii uses thiamine pyrophosphate and Mg²⁺ as cofactors and is localized in the peroxisome. The enzyme from the methanol-growing carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803, was also found to have DHA synthase and kinase activities (Ro et al., 1997, J Bac 179(19):6041-7). DHA synthase from this organism also has similar cofactor requirements as the enzyme from C. boidinii. The K_ms for formaldehyde and xylulose 5-phosphate were reported to be 1.86 mM and 33.3 microM, respectively. Several other mycobacteria, excluding only Mycobacterium tuberculosis, can use methanol as the sole source of carbon and energy and are reported to use DHAS (Part et al., 2003, JBac 185(1):142-7.

Protein	GenBank ID	GI number	Organism
DAS1	AAC83349.1	3978466	Candida boidinii
HPODL_2613	EFW95760.1	320581540	Ogataea parapolymorpha DL-1
			(Hansenula polymorpha DL-1)
	AAG12171.2	18497328	Mycobacter sp. strain JC1 DSM 3803

Example IV

Pathways to 13BDO and CrotOH

Pathways to product 13BDO and CrotOH that utilize the acetyl-CoA produced by the formate assimilation and FaldFPs described herein are shown in FIG. 10. These pathways can begin with the initiation of fatty acid biosynthesis, in which malonyl-ACP is condensed with acetyl-CoA or acetyl-ACP to form acetoacetyl-ACP (step A). The second step involves reduction of acetoacetyl-ACP to 3-hydroxybutyryl-ACP. Following dehydration to crotonyl-ACP and another reduction, butyryl-ACP is formed. The chain elongation typically continues with further addition of malonyl-ACP until a long-chain acyl chain is formed, which is then hydrolyzed by a thioesterase into a free C16 fatty acid. Bacterial fatty acid synthesis systems (FAS II) utilize discreet proteins for each step, whereas fungal and mammalian fatty acid synthesis systems (FAS I) utilize complex multifunctional proteins. The pathways utilize one or more enzymes of fatty acid biosynthesis to produce the C3 and C4 products 13BDO and CrotOH.

Several pathways are shown in FIG. 10 for converting acetoacetyl-ACP to 13BDO. In some pathways, acetoacetyl-ACP is first converted to acetoacetyl-CoA (step D). Alternatively, acetoacetyl-CoA can also be synthesized from acetyl-CoA and malonyl-CoA by acetoacetyl-CoA synthase (EC 2.3.1.194). Additionally, acetyl-CoA can be convert to malonyl-CoA using an acetyl-CoA carboxylase (step T of FIG. 1). Acetoacetyl-CoA can then be hydrolyzed to acetoacetate by a CoA transferase, hydrolase or synthetase (step E of FIG. 10). Acetoacetate is then reduced to 3-oxobutyraldehyde by a carboxylic acid reductase (step F of FIG. 10). Alternately, acetoacetyl-CoA is converted directly to 3-oxobutyraldehyde by a CoA-dependent aldehyde dehydrogenase (step I of FIG. 10). In yet another embodiment, acetoacetyl-ACP is converted directly to 3-oxobutyraldehyde by an acyl-ACP reductase (step J of FIG. 10). 3-Oxobutyraldehyde is further reduced to 13BDO via a 4-hydroxy-2-butanone or 3-hydroxybutyraldehyde intermediate (steps G and S, or steps R and AA of FIG. 10). Another option is the direct conversion of acetoacetyl-CoA to 4-hydroxy-2-butanone by a bifunctional enzyme with aldehyde dehydrogenase/alcohol dehydrogenase activity (step K of FIG. 10). Pathways to 13BDO can also proceed through a 3-hydroxybutyryl-CoA intermediate. This intermediate is formed by the reduction of acetoacetyl-CoA (step P of FIG. 10) or the transacylation of 3-hydroxybutyryl-ACP (step X of FIG. 10). 3-Hydroxybutyryl-CoA is further converted to 3-hydroxybutyrate (step Y of FIG. 10), 3-hydroxybutyraldehyde (step N of FIG. 10) or 13BDO (step O of FIG. 10). Alternately, the 3-hydroxybutyrate intermediate is formed from acetoacetate (step Q of FIG. 10) or via hydrolysis of 3-hydroxybutyryl-ACP (step L of FIG. 10). The 3-hydroxybutyraldehyde intermediate is also the product of 3-hydroxybutyrl-ACP reductase (step M of FIG. 10).

FIG. 10 also shows pathways from malonyl-ACP to CrotOH. In one embodiment, fatty acid initiation and extension enzymes produce the crotonyl-ACP intermediate (steps A, B, C). Crotonyl-ACP is then transacylated, hydrolyzed or reduced to crotonyl-CoA, crotonate or crotonaldehyde, respectively (steps AE, T, U). Crotonyl-CoA and crotonate are interconverted by a CoA hydrolase, transferase or synthetase (step AF). Crotonate is reduced to crotonaldehyde by a carboxylic acid reductase (step AG). In the final step of all pathways, crotonaldehyde is reduced to CrotOH by an aldehyde reductase in step AH. Numerous alternate pathways enumerated in the table below are also encompassed in the invention. Crotonyl-CoA can be reduced to crotonaldehyde or CrotOH (steps V, W). Alternately, the 3-hydroxybutyryl intermediates of the previously described 13BDO pathways can also be converted to CrotOH precursors. For example, dehydration of 3-hydroxybutyryl-CoA, 3-hydroxybutyrate or 3-hydroxybutyraldehyde yields crotonyl-CoA, crotonate or crotonaldehyde, respectively (step AB, AC, AD).

FIG. 10 still further shows pathways for production of 13BDO and CrotOH which can include the conversion of two acetyl-CoA molecules to acetoacetyl-CoA by an acetyl-CoA:acetyl-CoA acyltransferase. FIG. 10 still further shows pathways that include the conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA by a 4-hydroxybutyryl-CoA dehydratase.

Pathways shown in FIG. 10 comprising more than one enzymatic step can also be catalyzed by a single multifunctional enzyme or enzyme complex. For example, 10B and 10C can together be catalyzed by a multifunctional fatty acid synthase complex. The steps converting an acyl-ACP to an aldehyde and further to an alcohol (10 J and 10 G, 10M and 10 AA, 10U and 10 AH) can be catalyzed by an alcohol-forming acyl-ACP reductase.

Several of the enzyme activities required for the reactions shown in FIG. 10 are listed in the table below.

Label	Function	Step
1.1.1.a	Oxidoreductase (oxo to alcohol)	10B, 10G, 10P, 10Q,
		10R, 10S, 10AA, 10AH
1.1.1.c	Oxidoreductase (acyl-CoA to alcohol)	10K, 10O, 10W
1.2.1.b	Oxidoreductase (acyl-CoA to aldehyde)	10I, 10N, 10V
1.2.1.e	Oxidoreductase (acid to aldehyde)	10F, 10Z, 10AG
1.2.1.f	Oxidoreductase (acyl-ACP to aldehyde)	10J, 10M, 10U
2.3.1.e	Acyl-ACP C-acyltransferase (decarboxylating)	10A
2.3.1.f	CoA-ACP acyltransferase	10D, 10X, 10AE,
2.3.1.g	Fatty-acid synthase	10A, 10B, 10C,
2.8.3.a	CoA transferase	10E, 10Y, 10AF
3.1.2.a	CoA hydrolase	10E, 10Y, 10AF
3.1.2.b	Acyl-ACP thioesterase	10H, 10L, 10T,
4.2.1.a	Hydro-lyase	10C, 10AB, 10AC, 10AD
6.2.1.a	CoA synthetase	10E, 10Y, 10AF

1.1.1.a Oxidoreductase (Oxo to Alcohol)

Several reactions shown in FIG. 10 are catalyzed by alcohol dehydrogenase enzymes. These reactions include Steps B, G, P, Q, R, S, AA and AH. Exemplary alcohol dehydrogenase enzymes are described in further detail below.

The reduction of glutarate semialdehyde to 5-hydroxyvalerate by glutarate semialdehyde reductase entails reduction of an aldehyde to its corresponding alcohol. Enzymes with glutarate semialdehyde reductase activity include the ATEG_00539 gene product of Aspergillus terreus and 4-hydroxybutyrate dehydrogenase of Arabidopsis thaliana, encoded by 4hbd (WO 2010/068953A2). The A. thaliana enzyme was cloned and characterized in yeast (Breitkreuz et al., J. Biol. Chem. 278:41552-41556 (2003)).

PROTEIN	GENBANK ID	GI NUMBER	ORGANISM
ATEG_00539	XP_001210625.1	115491995	Aspergillus terreus NIH2624
4hbd	AAK94781.1	15375068	Arabidopsis thaliana

Additional genes encoding enzymes that catalyze the reduction of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol. 66:5231-5235 (2000)), yqhD and fucO from E. coli (Sulzenbacher et al., 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al., 174:7149-7158 (1992)). YqhD catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor, with a preference for chain lengths longer than C(3) (Sulzenbacher et al., 342:489-502 (2004); Perez et al., J Biol. Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilis E has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by bdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 and Cbei_2421 in C. beijerinckii. Additional aldehyde reductase gene candidates in Saccharomyces cerevisiae include the aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylate reductases GOR1 and YPL113C and glycerol dehydrogenase GCY1 (WO 2011/022651A1; Atsumi et al., Nature 451:86-89 (2008)). The enzyme candidates described previously for catalyzing the reduction of methylglyoxal to acetol or lactaldehyde are also suitable lactaldehyde reductase enzyme candidates.

Protein	GENBANK ID	GI NUMBER	ORGANISM
alrA	BAB12273.1	9967138	Acinetobacter sp. strain M-1
ADH2	NP_014032.1	6323961	Saccharomyces cerevisiae
yqhD	NP_417484.1	16130909	Escherichia coli
fucO	NP_417279.1	16130706	Escherichia coli
bdh I	NP_349892.1	15896543	Clostridium acetobutylicum
bdh II	NP_349891.1	15896542	Clostridium acetobutylicum
adhA	YP_162971.1	56552132	Zymomonas mobilis
bdh	BAF45463.1	124221917	Clostridium saccharoperbutylacetonicum
Cbei_1722	YP_001308850	150016596	Clostridium beijerinckii
Cbei_2181	YP_001309304	150017050	Clostridium beijerinckii
Cbei_2421	YP_001309535	150017281	Clostridium beijerinckii
GRE3	P38715.1	731691	Saccharomyces cerevisiae
ALD2	CAA89806.1	825575	Saccharomyces cerevisiae
ALD3	NP_013892.1	6323821	Saccharomyces cerevisiae
ALD4	NP_015019.1	6324950	Saccharomyces cerevisiae
ALD5	NP_010996.2	330443526	Saccharomyces cerevisiae
ALD6	ABX39192.1	160415767	Saccharomyces cerevisiae
HFD1	Q04458.1	2494079	Saccharomyces cerevisiae
GOR1	NP_014125.1	6324055	Saccharomyces cerevisiae
YPL113C	AAB68248.1	1163100	Saccharomyces cerevisiae
GCY1	CAA99318.1	1420317	Saccharomyces cerevisiae

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al., J Forens Sci, 49:379-387 (2004)) and Clostridium kluyveri (Wolff et al., Protein Expr. Purif: 6:206-212 (1995)). Yet another gene is the alcohol dehydrogenase adhI from Geobacillus thermoglucosidasius (Jeon et al., J Biotechnol 135:127-133 (2008)).

PROTEIN	GENBANK ID	GI NUMBER	ORGANISM
4hbd	YP_726053.1	113867564	Ralstonia eutropha H16
4hbd	L21902.1	146348486	Clostridium kluyveri DSM 555
adhI	AAR91477.1	40795502	Geobacillus therinoglucosidasius

Another exemplary aldehyde reductase is methylmalonate semialdehyde reductase, also known as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., J Mol Biol, 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning et al., Biochem J, 231:481-4 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) and Oryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci. Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosa and Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al., J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci. Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci. Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyrate dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from Pseudomonas aeruginosa (Gokam et al., U.S. Pat. No. 739,676, (2008)) and mmsB from Pseudomonas putida.

PROTEIN	GENBANK ID	GI NUMBER	ORGANISM
P84067	P84067	75345323	Thermus thermophilus
3hidh	P31937.2	12643395	Homo sapiens
3hidh	P32185.1	416872	Oryctolagus cuniculus
mmsB	NP_746775.1	26991350	Pseudomonas putida
mmsB	P28811.1	127211	Pseudomonas aeruginosa
dhat	Q59477.1	2842618	Pseudomonas putida

There exist several exemplary alcohol dehydrogenases that convert a ketone to a hydroxyl functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der Oost et al., Eur. J. Biochem. 268:3062-3068 (2001)).

Protein	Genbank ID	GI Number	Organism
mdh	AAC76268.1	1789632	Escherichia coli
ldhA	NP_415898.1	16129341	Escherichia coli
ldh	YP_725182.1	113866693	Ralstonia eutropha
bdh	AAA58352.1	177198	Homo sapiens
adh	AAA23199.2	60592974	Clostridium beijerinckii NRRL B593
adh	P14941.1	113443	Thermoanaerobacter brockii HTD4
sadh	CAD36475	21615553	Rhodococcus ruber
adhA	AAC25556	3288810	Pyrococcus furiosus

A number of organisms encode genes that catalyze the reduction of 3-oxobutanol to 13BDO, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al. J Mol Cat B Enz, 11:513-521 (2001). One of these enzymes, SADH from Candida parapsilosis, was cloned and characterized in E. coli. A mutated Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia alcohol dehydrogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl. Microbiol Biotechnol. 75:1249-1256 (2007)).

Protein	Genbank ID	GI Number	Organism
sadh	BAA24528.1	2815409	Candida parapsilosis

Exemplary alcohol dehydrogenase enzymes include 3-oxoacyl-CoA reductase and acetoacetyl-CoA reductase. 3-Oxoacyl-CoA reductase enzymes (EC 1.1.1.35) convert 3-oxoacyl-CoA molecules into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta-oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Given the proximity in E. coli of paaH to other genes in the phenylacetate degradation operon (Nogales et al., 153:357-365 (2007)) and the fact thatpaaH mutants cannot grow on phenylacetate (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003)), it is expected that the E. coli paaH gene also encodes a 3-hydroxyacyl-CoA dehydrogenase. Additional 3-oxoacyl-CoA enzymes include the gene products of phaC in Pseudomonas putida (Olivera et al., Proc. Natl. Acad. Sci U.S.A 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens (Di et al., 188:117-125 (2007)). These enzymes catalyze the reversible oxidation of 3-hydroxyadipyl-CoA to 3-oxoadipyl-CoA during the catabolism of phenylacetate or styrene.

AcAcCoAR(EC 1.1.1.36) catalyzes the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. This enzyme participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones et al., Microbiol Rev. 50:484-524 (1986)). Acetoacetyl-CoA reducatse also participates in polyhydroxybutyrate biosynthesis in many organisms, and has also been used in metabolic engineering applications for overproducing PHB and 3-hydroxyisobutyrate (Liu et al., Appl. Microbiol. Biotechnol. 76:811-818 (2007); Qui et al., Appl. Microbiol. Biotechnol. 69:537-542 (2006)). The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J Bacteriol. 171:6800-6807 (1989)). Additional gene candidates include phbB from Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309 (2006)). The Z. ramigera gene is NADPH-dependent and the gene has been expressed in E. coli (Peoples et al., Mol. Microbiol 3:349-357 (1989)). Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al., Eur. J Biochem. 174:177-182 (1988)). Additional genes include phaB in Paracoccus denitrificans, Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al., J Biol. Chem. 207:631-638 (1954)). The enzyme from Paracoccus denitrificans has been functionally expressed and characterized in E. coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)). The enzyme from Candida tropicalis is a component of the peroxisomal fatty acid beta-oxidation multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active on acetoacetyl-CoA. The domain has been functionally expressed in E. coli, a crystal structure is available, and the catalytic mechanism is well-understood (Ylianttila et al., Biochem Biophys Res Commun 324:25-30 (2004); Ylianttila et al., J Mol Biol 358:1286-1295 (2006)).

Protein	Genbank ID	GI Number	Organism
fadB	P21177.2	119811	Escherichia coli
fadJ	P77399.1	3334437	Escherichia coli
paaH	NP_415913.1	16129356	Escherichia coli
Hbd2	EDK34807.1	146348271	Clostridium kluyveri
Hbd1	EDK32512.1	146345976	Clostridium kluyveri
phaC	NP_745425.1	26990000	Pseudomonas putida
paaC	ABF82235.1	106636095	Pseudomonas fluorescens
HSD17B10	O02691.3	3183024	Bos taurus
phbB	P23238.1	130017	Zoogloea ramigera
phaB	YP_353825.1	77464321	Rhodobacter sphaeroides
phaB	BAA08358	675524	Paracoccus denitrificans
Hbd	NP_349314.1	15895965	Clostridium acetobutylicum
Hbd	AAM14586.1	20162442	Clostridium beijerinckii
Msed_1423	YP_001191505	146304189	Metallosphaera sedula
Msed_0399	YP_001190500	146303184	Metallosphaera sedula
Msed_0389	YP_001190490	146303174	Metallosphaera sedula
Msed_1993	YP_001192057	146304741	Metallosphaera sedula
Fox2	Q02207	399508	Candida tropicalis

The reduction of acetoacetyl-ACP to 3-hydroxyacetyl-ACP (step B of FIG. 10) is catalyzed by 3-oxoacyl-ACP reductase (EC 1.1.1.100). The E. coli 3-oxoacyl-ACP reductase is encoded by fabG. Key residues responsible for binding the acyl-ACP substrate to the enzyme have been elucidated (Zhang et al, J Biol Chem 278:52935-43 (2003)). Additional enzymes with this activity have been characterized in Bacillus anthracis (Zaccai et al, Prot Struct Funct Gen 70:562-7 (2008)) and Mycobacterium tuberculosis (Gurvitz, Mol Genet Genomics 282:407-16 (2009)). The beta-ketoacyl reductase (KR) domain of eukaryotic fatty acid synthase also catalyzes this activity (Smith, FASEB J, 8:1248-59 (1994)). While many FabG enzymes preferentially utilize NADH, NADH-dependent FabG enzymes also known in the art and are shown in the table below (Javidpour et al, AEM 80: 597-505 (2014)).

Gene	GenBank ID	GI Number	Organism
FabG	P0AEK2.1	84028081	Escherichia coli
FabG	AAP27717.1	30258498	Bacillus anthracis
FabG1	NP_215999.1	15608621	Mycobacterium tuberculosis
FabG4	YP_003030167.1	253797166	Mycobacterium tuberculosis
FabG	EDM75366.1	149815845	Plesiocystis Pacifica
FabG	WP_018008474.1	516633699	Cupriavidus Taiwanensis
FabG	WP_012242413.1	501199395	Acholeplasma Laidlawii
FabG	EDL65432.1	148851283	Bacillus sp SG-1

1.1.1.c Oxidoreductase (Acyl-CoA to Alcohol)

Bifunctional oxidoreductases convert an acyl-CoA to its corresponding alcohol. Enzymes with this activity can be used Steps K, O and W as depicted in FIG. 10.

Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS. Lett. 281:59-63 (1991))) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002))). The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, et al., J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett, 27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., J Bacteriol, 184:2404-2410 (2002); Strauss et al., Eur J Biochem, 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt et al., Env Microbiol, 9:2067-2078 (2007)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Etythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity.

Protein	GenBank ID	GI Number	Organism
adhE	NP_415757.1	16129202	Escherichia coli
adhE2	AAK09379.1	12958626	Clostridium acetobutylicum
bdh I	NP_349892.1	15896543	Clostridium acetobutylicum
bdh II	NP_349891.1	15896542	Clostridium acetobutylicum
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
mcr	AAS20429.1	42561982	Chloroflexus aurantiacus
Rcas_2929	YP_001433009.1	156742880	Roseiflexus castenholzii
NAP1_02720	ZP_01039179.1	85708113	Erythrobacter sp. NAP1
MGP2080_00535	ZP_01626393.1	119504313	marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced to their corresponding alcohols by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al., Plant Physiol, 122:635-644 (2000)).

Protein	GenBank ID	GI Number	Organism
FAR	AAD38039.1	5020215	Simmondsia chinensis

Another candidate for catalyzing these steps is 3-hydroxy-3-methylglutaryl-CoA reductase (or HMG-CoA reductase). This enzyme naturally reduces the CoA group in 3-hydroxy-3-methylglutaryl-CoA to an alcohol forming mevalonate. The hmgA gene of Sulfolobus solfataricus, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, has been cloned, sequenced, and expressed in E. coli (Bochar et al., J Bacteriol. 179:3632-3638 (1997)). S. cerevisiae also has two HMG-CoA reductases in it (Basson et al., Proc. Natl. Acad. Sci. U.S.A 83:5563-5567 (1986)). The gene has also been isolated from Arabidopsis thaliana and has been shown to complement the HMG-COA reductase activity in S. cerevisiae (Learned et al., Proc. Natl. Acad. Sci. U.S.A 86:2779-2783 (1989)).

Protein	GenBank ID	GI Number	Organism
HMG1	CAA86503.1	587536	Saccharomyces cerevisiae
HMG2	NP_013555	6323483	Saccharomyces cerevisiae
HMG1	CAA70691.1	1694976	Arabidopsis thaliana
hmgA	AAC45370.1	2130564	Sulfolobus solfataricus

1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde)

Acyl-CoA reductases in the 1.2.1 family reduce an acyl-CoA to its corresponding aldehyde. Such a conversion is utilized in Steps I, N and V of FIG. 10. Several acyl-CoA reductase enzymes have been described in the open literature and represent suitable candidates for this step. These are described below.

Acyl-CoA reductases or acylating aldehyde dehydrogenases reduce an acyl-CoA to its corresponding aldehyde. Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase, butyryl-CoA reductase and propionyl-CoA reductase (EC 1.2.1.3). Exemplary fatty acyl-CoA reductases enzymes are encoded by acr1 of Acinetobacter calcoaceticus (Reiser, Journal of Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoA reductase activity are encoded by sucD of Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea including Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol., 191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem., 71:58-68 (2007)). Exemplary propionyl-CoA reductase enzymes include pduP of Salmonella typhimurium LT2 (Leal, Arch. Microbiol. 180:353-361 (2003)) and eutE from E. coli (Skraly, WO Patent No. 2004/024876). The propionyl-CoA reductase of Salmonella typhimurium LT2, which naturally converts propionyl-CoA to propionaldehyde, also catalyzes the reduction of 5-hydroxyvaleryl-CoA to 5-hydroxypentanal (WO 2010/068953A2).

Protein	GenBank ID	GI Number	Organism
acr1	YP_047869.1	50086359	Acinetobacter calcoaceticus
acr1	AAC45217	1684886	Acinetobacter baylyi
acr1	BAB85476.1	18857901	Acinetobacter sp. Strain M-1
MSED_0709	YP_001190808.1	146303492	Metallosphaera sedula
Tneu_0421	ACB39369.1	170934108	Thermoproteus neutrophilus
sucD	P38947.1	172046062	Clostridium kluyveri
sucD	NP_904963.1	34540484	Porphyromonas gingivalis
bphG	BAA03892.1	425213	Pseudomonas sp
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
bld	AAP42563.1	31075383	Clostridium saccharoperbutylacetonicum
pduP	NP_460996	16765381	Salmonella typhimurium LT2
eutE	NP_416950	16130380	Escherichia coli

An additional enzyme that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg, Science 318:1782-1786 (2007); and Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp. (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler, J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg, Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol 188:8551-8559 (2006). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

Protein	GenBank ID	GI Number	Organism
Msed_0709	YP_001190808.1	146303492	Metallosphaera sedula
Mcr	NP_378167.1	15922498	Sulfolobus tokodaii
asd-2	NP_343563.1	15898958	Sulfolobus solfataricus
Saci_2370	YP_256941.1	70608071	Sulfolobus acidocaldarius
Ald	AAT66436	49473535	Clostridium beijerinckii
eutE	AAA80209	687645	Salmonella typhimurium
eutE	P77445	2498347	Escherichia coli

1.2.1.e Oxidoreductase (Acid to Aldehyde)

The conversion of an acid to an aldehyde is thermodynamically unfavorable and typically requires energy-rich cofactors and multiple enzymatic steps. Direct conversion of the acid to aldehyde by a single enzyme is catalyzed by an acid reductase enzyme in the 1.2.1 family. An enzyme in this EC class can be used in Steps F, Z and AG of FIG. 10.

Exemplary acid reductase enzymes include carboxylic acid reductase, alpha-aminoadipate reductase and retinoic acid reductase. Carboxylic acid reductase (CAR), found in Nocardia iowensis, catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). The natural substrate of this enzyme is benzoate and the enzyme exhibits broad acceptance of aromatic substrates including p-toluate (Venkitasubramanian et al., Biocatalysis in Pharmaceutical and Biotechnology Industries. CRC press (2006)). The enzyme from Nocardia iowensis, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)). CAR requires post-translational activation by a phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl. Environ. Microbiol 75:2765-2774 (2009)). Expression of the npt gene, encoding a specific PPTase, product improved activity of the enzyme. An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J Antibiot. 60(6):380-387 (2007)). Co-expression of griC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial.

Gene	GenBank Accession No.	GI No.	Organism
car	AAR91681.1	40796035	Nocardia iowensis
npt	ABI83656.1	114848891	Nocardia iowensis
griC	YP_001825755.1	182438036	Streptomyces griseus
griD	YP_001825756.1	182438037	Streptomyces griseus

Additional car and npt genes can be identified based on sequence homology.

Gene name	GI No.	GenBank Accession No.	Organism
fadD9	121638475	YP_978699.1	Mycobacterium bovis BCG
BCG_2812c	121638674	YP_978898.1	Mycobacterium bovis BCG
nfa20150	54023983	YP_118225.1	Nocardia farcinica IFM 10152
nfa40540	54026024	YP_120266.1	Nocardia farcinica IFM 10152
SGR_6790	182440583	YP_001828302.1	Streptomyces griseus subsp.
			griseus NBRC 13350
SGR_665	182434458	YP_001822177.1	Streptomyces griseus subsp.
			griseus NBRC 13350
MSMEG_2956	YP_887275.1	YP_887275.1	Mycobacterium smegmatis MC2 155
MSMEG_5739	YP_889972.1	118469671	Mycobacterium smegmatis MC2 155
MSMEG_2648	YP_886985.1	118471293	Mycobacterium smegmatis MC2 155
MAP1040c	NP_959974.1	41407138	Mycobacterium avium subsp.
			paratuberculosis K-10
MAP2899c	NP_961833.1	41408997	Mycobacterium avium subsp.
			paratuberculosis K-10
MMAR_2117	YP_001850422.1	183982131	Mycobacterium marinum M
MMAR_2936	YP_001851230.1	183982939	Mycobacterium marinum M
MMAR_1916	YP_001850220.1	183981929	Mycobacterium marinum M
TpauDRAFT_33060	ZP_04027864.1	227980601	Tsukamurella paurometabola DSM 20162
TpauDRAFT_20920	ZP_04026660.1	ZP_04026660.1	Tsukamurella paurometabola DSM
CPCC7001_1320	ZP_05045132.1	254431429	Cyanobium PCC7001
DDBDRAFT_0187729	XP_636931.1	66806417	Dictyostelium discoideum AX4

An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified by sequence comparison homology searching.

Gene	GenBank Accession No.	GI No.	Organism
LYS2	AAA34747.1	171867	Saccharomyces cerevisiae
LYS5	P50113.1	1708896	Saccharomyces cerevisiae
LYS2	AAC02241.1	2853226	Candida albicans
LYS5	AAO26020.1	28136195	Candida albicans
Lys1p	P40976.3	13124791	Schizosaccharomyces pombe
Lys7p	Q10474.1	1723561	Schizosaccharomyces pombe
Lys2	CAA74300.1	3282044	Penicillium chrysogenum

1.2.1.f Oxidoreductase (Acyl-ACP to Aldehyde)

The reduction of an acyl-ACP to its corresponding aldehyde is catalyzed by an acyl-ACP reductase (AAR). Such a transformation is depicted in steps J, M and U of FIG. 10. Suitable enzyme candidates include the orfl 594 gene product of Synechococcus elongatus PCC7942 and homologs thereof (Schirmer et al, Science, 329: 559-62 (2010)). The S. elongates PCC7942 acyl-ACP reductase is coexpressed with an aldehyde decarbonylase in an operon that appears to be conserved in a majority of cyanobacterial organisms. This enzyme, expressed in E. coli together with the aldehyde decarbonylase, conferred the ability to produce alkanes. The P. marinus AAR was also cloned into E. coli and, together with a decarbonylase, demonstrated to produce alkanes (US Application 2011/0207203).

Protein	GenBank ID	GI Number	Organism
orf1594	YP_400611.1	81300403	Synechococcus elongatus PCC7942
PMT9312_0533	YP_397030.1	78778918	Prochlorococcus marinus MIT 9312
syc0051_d	YP_170761.1	56750060	Synechococcus elongatus PCC 6301
Ava_2534	YP_323044.1	75908748	Anabaena variabilis ATCC 29413
alr5284	NP_489324.1	17232776	Nostoc sp. PCC 7120
Aazo_3370	YP_003722151.1	298491974	Nostoc azollae
Cyan7425_0399	YP_002481152.1	220905841	Cyanothece sp. PCC 7425
N9414_21225	ZP_01628095.1	119508943	Nodularia spumigena CCY9414
L8106_07064	ZP_01619574.1	119485189	Lyngbya sp. PCC 8106

The reduction of an acyl-ACP to its corresponding alcohol is catalyzed by an acyl-ACP reductase (alcohol forming). Enzymes with this activity catalyze both the reduction of an acyl-ACP to an aldehyde (Steps J, M, U of FIG. 10), and the reduction of the aldehyde to the alcohol (Step G, AA, AH of FIG. 10). Fatty acyl reductase enzymes that use acyl-ACP substrates to produce alcohols are known in the art. Alcohol forming acyl-ACP reductases include Maqu_2220 of Marinobacter aquaeolei VT8 and Hch_05075 of Hahella chejuensis KCTC2396 (see WO2013/048557). These enzymes convert both acyl-ACP substrates and acyl-CoA substrates to their corresponding alcohols. The M. aquaeolei AAR was previously characterized as an aldehyde reductase (Wahlen et al, AEM 75:2758-2764 (2009)) and US 2010/0203614). Alcohol forming acyl-ACP reductase enzymes are shown in the table below.

Protein	GenBank ID	GI Number	Organism
Maqu_2220	ABM19299	120324984	Marinobacter aquaeolei
Hch_05075	YP_436183	83647748	Hahella chejuensis
MDG893_11561	ZP_01892457.1	149374683	Marinobacter algicola DG893
HP15_810	ADP96574.1	311693701	Marinobacter adhaerens HP15
RED65_09894	ZP_01305629.1	94499091	Oceanobacter sp. RED65

2.3.1.e Acyl-ACP C-Acyltransferase (Decarboxylating)

In step A of FIG. 10, acetoacetyl-ACP is formed from malonyl-ACP and either acetyl-CoA or acetyl-ACP. This reaction is catalyzed by an acyl-ACP C-acyltransferase in EC class 2.3.1. The condensation of malonyl-ACP and acetyl-CoA is catalyzed by beta-ketoacyl-ACP synthase (KAS, EC 2.3.1.180). E. coli has three KAS enzymes encoded by fabB, fabF and fabH. FabH (KAS III), the key enzyme of initiation of fatty acid biosynthesis in E. coli, is selective for the formation of acetoacetyl-ACP. FabB and FabF catalyze the condensation of malonyl-ACP with acyl-ACP substrates and function primarily in fatty acid elongation although they can also react with acetyl-ACP and thereby participate in fatty acid inititation. For example, the Bacillus subtilis KAS enzymes are similar to FabH but are less selective, accepting branched acyl-CoA substrates (Choi et al, J Bacteriol 182:365-70 (2000)).

	Protein	GenBank ID	GI Number	Organism
	fabB	AAC75383.1	1788663	Escherichia coli
	fabF	AAC74179.1	1787337	Escherichia coli
	fabH	AAC74175.1	1787333	Escherichia coli
	FabHA	NP_389015.1	16078198	Bacillus subtilis
	FabHB	NP_388898.1	16078081	Bacillus subtilis

Alternately, acetyl-CoA can first be activated to acetyl-ACP and subsequently condensed to acetoacetyl-ACP by two enzymes, acetyl-CoA:ACP transacylase (EC 2.3.1.38) and acetoacetyl-ACP synthase (EC 2.3.1.41). Acetyl-CoA:ACP transacylase converts acetyl-CoA and an acyl carrier protein to acetyl-ACP, releasing CoA. Enzyme candidates for acetyl-CoA:ACP transacylase are described in section EC 2.3.1.f below. Acetoacetyl-ACP synthase enzymes catalyze the condensation of acetyl-ACP and malonyl-ACP. This activity is catalyzed by FabF and FabB of E. coli, as well as the multifunctional eukaryotic fatty acid synthase enzyme complexes described in EC 2.3.1.g.

2.3.1.f CoA-ACP Acyltransferase

The exchange of an ACP moiety for a CoA is catalyzed by enzymes in EC class 2.3.1. This reaction is shown in steps D, X, and AE of FIG. 10. Activation of acetyl-CoA to acetyl-ACP (step A of FIG. 10) is also catalyzed by a CoA:ACP acyltransferase. Enzymes with CoA-ACP acyltransferase activity include acetyl-CoA:ACP transacylase (EC 2.3.1.38) and malonyl-CoA:ACP transacylase (EC 2.3.1.39).

The FabH (KASIII) enzyme of E. coli functions as an acyl-CoA:ACP transacylase, in addition to its primary activity of forming acetoacetyl-ACP. Butyryl-ACP is accepted as an alternate substrate of FabH (Prescott et al, Adv. Enzymol. Relat. Areas Mol, 36:269-311 (1972)). Acetyl-CoA:ACP transacylase enzymes from Plasmodium falciparum and Streptomyces avermitillis have been heterologously expressed in E. coli (Lobo et al, Biochem 40:11955-64 (2001)). A synthetic KASIII (FabH) from P. falciparum expressed in a fabH-deficient Lactococcus lactis host was able to complement the native fadH activity (Du et al, AEM 76:3959-66 (2010)). The acetyl-CoA:ACP transacylase enzyme from Spinacia oleracea accepts other acyl-ACP molecules as substrates, including butyryl-ACP (Shimakata et al, Methods Enzym 122:53-9 (1986)). The sequence of this enzyme has not been determined to date. Malonyl-CoA:ACP transacylase enzymes include FabD of E. coli and Brassica napsus (Verwoert et al, J Bacteriol, 174:2851-7 (1992); Simon et al, FEBS Left 435:204-6 (1998)). FabD of B. napsus was able to complement fabD-deficient E. coli. The multifunctional eukaryotic fatty acid synthase enzyme complexes (described in EC 2.3.1.g) also catalyze this activity.

Protein	GenBank ID	GI Number	Organism
fabH	AAC74175.1	1787333	Escherichia coli
fadA	NP_824032.1	29829398	Streptomyces avermitillis
fabH	AAC63960.1	3746429	Plasmodium falciparum
Synthetic	ACX34097.1	260178848	Plasmodium falciparum
construct
fabH	CAL98359.1	124493385	Lactococcus lactis
fabD	AAC74176.1	1787334	Escherichia coli
fabD	CAB45522.1	5139348	Brassica napsus

2.3.1.g Fatty Acid Synthase

Steps A, B, and C of FIG. 10 can together be catalyzed fatty acid synthase or fatty-acyl-CoA synthase, multifunctional enzyme complexes composed of multiple copies of one or more subunits. The fatty acid synthase of Saccharomyces cerevisiae is a dodecamer composed of two multifunctional subunits FAS1 and FAS2 that together catalyze all the reactions required for fatty acid synthesis: activation, priming, elongation and termination (Lomakin et al, Cell 129:319-32 (2007)). This enzyme complex catalyzes the formation of long chain fatty acids from acetyl-CoA and malonyl-CoA. The favored product of eukaryotic FAS systems is palmitic acid (C16) Similar fatty acid synthase complexes are found in Candida parapsilosis and Thermomyces lanuginosus (Nguyen et al, PLoS One 22:e8421 (2009); Jenni et al, Science 316:254-61 (2007)). The multifunctional Fas enzymes of Mycobacterium tuberculosis and mammals such as Homo sapiens are also suitable candidates (Fernandes and Kolattukudy, Gene 170:95-99 (1996) and Smith et al, Prog Lipid Res 42:289-317 (2003)).

Protein	GenBank ID	GI Number	Organism
FAS1	CAA82025.1	486321	Saccharomyces cerevisiae
FAS2	CAA97948.1	1370478	Saccharomyces cerevisiae
Fas1	ABO37973.1	133751597	Thermomyces lanuginosus
Fas2	ABO37974.1	133751599	Thermomyces lanuginosus
Fas	AAB03809.1	1036835	Mycobacterium tuberculosis
Fas	NP_004095.4	41872631	Homo sapiens

2.8.3.a CoA Transferase

Enzymes in the 2.8.3 family catalyze the reversible transfer of a CoA moiety from one molecule to another. Such a transformation can be utilized for Steps E, Y and AF of FIG. 10. Several CoA transferase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below.

Many transferases have broad specificity and thus can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate, propionate, vinylacetate, butyrate, among others. For example, an enzyme from Roseburia sp. A2-183 was shown to have butyryl-CoA:acetate:CoA transferase and propionyl-CoA:acetate:CoA transferase activity (Charrier et al., Microbiology 152, 179-185 (2006)). Close homologs can be found in, for example, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Eubacterium rectale ATCC 33656. Another enzyme with propionyl-CoA transferase activity can be found in Clostridium propionicum (Selmer et al., Eur J Biochem 269, 372-380 (2002)). This enzyme can use acetate, (R)-lactate, (S)-lactate, acrylate, and butyrate as the CoA acceptor (Selmer et al., Eur J Biochem 269, 372-380 (2002); Schweiger and Buckel, FEBS Letters, 171(1) 79-84 (1984)). Close homologs can be found in, for example, Clostridium novyi NT, Clostridium beijerinckii NCIMB 8052, and Clostridium botulinum C str. Eklund. YgfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
Ach1	AAX19660.1	60396828	Roseburia sp. A2-183
ROSINTL182_07121	ZP_04743841.2	257413684	Roseburia intestinalis L1-82
ROSEINA2194_03642	ZP_03755203.1	225377982	Roseburia inulinivorans
EUBREC_3075	YP_002938937.1	238925420	Eubacterium rectale ATCC 33656
Pct	CAB77207.1	7242549	Clostridium propionicum
NT01CX_2372	YP_878445.1	118444712	Clostridium novyi NT
Cbei_4543	YP_001311608.1	150019354	Clostridium beijerinckii
CBC_A0889	ZP_02621218.1	168186583	Clostridium botulinum C str. Eklund
ygfH	NP_417395.1	16130821	Escherichia coli
CIT292_04485	ZP_03838384.1	227334728	Citrobacter youngae ATCC 29220
SARI_04582	YP_001573497.1	161506385	Salmonella enterica subsp. arizonae serovar
yinte0001_14430	ZP_04635364.1	238791727	Yersinia intermedia ATCC 29909

An additional candidate enzyme is the two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas, which has been shown to have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et al., supra). Similar enzymes based on homology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403 (2007)). These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
pcaI	AAN69545.1	24985644	Pseudomonas putida
pcaJ	NP_746082.1	26990657	Pseudomonas putida
pcaI	YP_046368.1	50084858	Acinetobacter sp. ADP1
pcaJ	AAC37147.1	141776	Acinetobacter sp. ADP1
pcaI	NP_630776.1	21224997	Streptomyces coelicolor
pcaJ	NP_630775.1	21224996	Streptomyces coelicolor
HPAG1_0676	YP_627417	108563101	Helicobacter pylori
HPAG1_0677	YP_627418	108563102	Helicobacter pylori
ScoA	NP_391778	16080950	Bacillus subtilis
ScoB	NP_391777	16080949	Bacillus subtilis

A CoA transferase that can utilize acetate as the CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr. D Biol Clystallogr. 58:2116-2121 (2002)). This enzyme has also been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra) Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583 (1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
atoA	P76459.1	2492994	Escherichia coli K12
atoD	P76458.1	2492990	Escherichia coli K12
actA	YP_226809.1	62391407	Corynebacterium glutamicum ATCC 13032
cg0592	YP_224801.1	62389399	Corynebacterium glutamicum ATCC 13032
ctfA	NP_149326.1	15004866	Clostridium acetobutylicum
ctfB	NP_149327.1	15004867	Clostridium acetobutylicum
ctfA	AAP42564.1	31075384	Clostridium saccharoperbutylacetonicum
ctfB	AAP42565.1	31075385	Clostridium saccharoperbutylacetonicum

Additional exemplary transferase candidates are catalyzed by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., supra; Sohling et al., Eur. J Biochem. 212:121-127 (1993); Sohling et al., J Bacteriol. 178:871-880 (1996)) Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
cat1	P38946.1	729048	Clostridium kluyveri
cat2	P38942.2	172046066	Clostridium kluyveri
cat3	EDK35586.1	146349050	Clostridium kluyveri
TVAG_395550	XP_001330176	123975034	Trichomonas vaginalis G3
Tb11.02.0290	XP_828352	71754875	Trypanosoma brucei

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). These proteins are identified below.

Protein	GenBank ID	GI Number	Organism
gctA	CAA57199.1	1) 559392	2) Acidaminococcus fermentans
3) gctB	4) CAA57200.1	5) 559393	6) Acidaminococcus fermentans

3.1.2.a CoA Hydrolase

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to their corresponding acids. Such a transformation can be utilized in Steps E, Y and AF of FIG. 10. Several such enzymes have been described in the literature and represent suitable candidates for these steps.

For example, the enzyme encoded by acot12 from Rattus norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB (Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al., J Biol Chem, 2006, 281(16):11028-38). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant. Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).

Protein	GenBank Accession No.	GI Number	Organism
acot12	NP_570103.1	18543355	Rattus norvegicus
tesB	NP_414986	16128437	Escherichia coli
acot8	CAA15502	3191970	Homo sapiens
acot8	NP_570112	51036669	Rattus norvegicus
tesA	NP_415027	16128478	Escherichia coli
ybgC	NP_415264	16128711	Escherichia coli
paaI	NP_415914	16129357	Escherichia coli
ybdB	NP_415129	16128580	Escherichia coli
ACH1	NP_009538	6319456	Saccharomyces cerevisiae

Additional hydrolase enzymes include 3-hydroxyisobutyryl-UoA hydrolase which has been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valine degradation (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., Methods Enzymol 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Similar gene candidates can also be identified by sequence homology, including hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus.

Protein	GenBank No.	GI Number	Organism
hibch	Q5XIE6.2	146324906	Rattus norvegicus
hibch	Q6NVY1.2	146324905	Homo sapiens
hibch	P28817.2	2506374	Saccharomyces cerevisiae
BC_2292	AP09256	29895975	Bacillus cereus

Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcus fermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases may also serve as candidates for this reaction step but would require certain mutations to change their function. GeneBank accession numbers for the gctA and gctB genes are listed above.

3.1.2.b Acyl-ACP Thioesterase

Acyl-ACP thioesterase enzymes convert an acyl-ACP to its corresponding acid. Such a transformation is required in steps H, L, T and AP of FIG. 10. Exemplary enzymes include the FatA and FatB isoforms of Arabidopsis thaliana (Salas et al, Arch Biochem Biophys 403:25-34 (2002)). The activities of these two proteins vary with carbon chain length, with FatA preferring oleyl-ACP and FatB preferring palmitoyl-ACP. See 3.1.2.14. A number of thioesterases with different chain length specificities are listed in WO 2008/113041 and are included in the table below [see p 126 Table 2A of patent]. For example, it has been shown previously that expression of medium chain plant thioesterases like FatB from Umbellularia californica in E. coli results in accumulation of high levels of medium chain fatty acids, primarily laurate (C12:0). Similarly, expression of Cuphea palustris FatB1 thioesterase in E. coli led to accumulation of C8-10:0 acyl-ACPs (Dehesh et al, Plant Physiol 110:203-10 (1996)). Similarly, Carthamus tinctorius thioesterase, when expressed in E. coli leads to >50 fold elevation in C 18:1 chain termination and release as free fatty acid (Knutzon et al, Plant Physiol 100:1751-58 (1992)). Methods for altering the substrate specificity of acyl-ACP thioesterases are also known in the art (for example, EP1605048).

Protein	GenBank ID	GI Number	Organism
fatA	AEE76980.1	332643459	Arabidopsis thaliana
fatB	AEE28300.1	332190179	Arabidopsis thaliana
fatB2	AAC49269.1	1292906	Cuphea hookeriana
fatB1	AAC49179.1	1215718	Cuphea palustris
M96568.1:94 . . . 1251	AAA33019.1	404026	Carthamus tinctorius
fatB1	Q41635.1	8469218	Umbellularia californica
tesA	AAC73596.1	1786702	Escherichia coli

4.2.1.a Hydro-Lyase

Several reactions in FIG. 10 depict dehydration reactions, including steps C, AB, AC and AD. Oleate hydratase enzymes catalyze the reversible hydration of non-activated alkenes to their corresponding alcohols. These enzymes represent additional suitable candidates as suggested in WO2011076691. Oleate hydratases from Elizabethkingia meningoseptica and Streptococcus pyogenes have been characterized (WO 2008/119735). Examples include the following proteins.

Protein	GenBank ID	GI Number	Organism
OhyA	ACT54545.1	254031735	Elizabethkingia meningoseptica
HMPREF0841_1446	ZP_07461147.1	306827879	Streptococcus pyogenes ATCC 10782
P700755_13397	ZP_01252267.1	91215295	Psychroflexus torquis ATCC 700755
RPB_2430	YP_486046.1	86749550	Rhodopseudomonas palustris

3-Hydroxyacyl-ACP dehydratase enzymes are suitable candidates for dehydrating 3-hydroxybutyryl-ACP to crotonyl-ACP (step C of FIG. 10). Enzymes with this activity include FabA and FabZ of E. coli, which posess overlapping broad substrate specificities (Heath, J Biol Chem 271:1833-6 (1996)). Fatty acid synthase complexes, described above, also catalyze this reaction. The FabZ protein from Plasmodium falciparum has been crystallized (Kostrew et al, Protein Sci 14:1570-80 (2005)). Additional candidates are the mitochondrial 3-hydroxyacyl-ACP dehydratase encoded by Htd2p in yeast and TbHTD2 in Homo sapiens and Trypanosoma brucei (Kastanoitis et al, Mol Micro 53:1407-21 (2004); Kaija et al, FEBS Lett 582:729-33 (2008)).

Protein	GenBank ID	GI Number	Organism
fabA	AAC74040.1	1787187	Escherichia coli
fabZ	AAC73291.1	1786377	Escherichia coli
PfFabZ	AAK83685.1	15080870	Plasmodium falciparum
Htd2p	NP_011934.1	6321858	Saccharomyces cerevisiae
HTD2	P86397.1	281312149	Homo sapiens

Several additional hydratase and dehydratase enzymes have been described in the literature and represent suitable candidates for these steps. For example, many dehydratase enzymes catalyze the alpha, beta-elimination of water which involves activation of the alpha-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the beta-position (Buckel et al, J Bacteriol, 117:1248-60 (1974); Martins et al, PNAS 101:15645-9 (2004)). Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 3-dehydroquinate dehydratase (EC 4.2.1.10), cyclohexanone hydratase (EC 4.2.1.-) and 2-keto-4-pentenoate dehydratase (EC 4.2.1.80), citramalate hydrolyase and dimethylmaleate hydratase.

2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci 103:12341-6 (2006)). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing bacterial serine dehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with similar functionality in E. barkeri is dimethylmaleate hydratase, a reversible Fe²⁺-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006); Kollmann-Koch et al., Hoppe Seylers. Z Physiol Chem. 365:847-857 (1984)).

Protein	GenBank ID	GI Number	Organism
hmd	ABC88407.1	86278275	Eubacterium barkeri
BACCAP_02294	ZP_02036683.1	154498305	Bacteroides capillosus
ANACOL_02527	ZP_02443222.1	167771169	Anaerotruncus colihominis
NtherDRAFT_2368	ZP_02852366.1	169192667	Natranaerobius thermophilus
dmdA	ABC88408	86278276	Eubacterium barkeri
dmdB	ABC88409	86278277	Eubacterium barkeri

Fumarate hydratase (EC 4.2.1.2) enzymes naturally catalyze the reversible hydration of fumarate to malate. Although the ability of fumarate hydratase to react with 3-oxobutanol as a substrate has not been described in the literature, a wealth of structural information is available for this enzyme and other researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver, 61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., J Bacteriol, 183:461-467 (2001); Woods et al., 954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates are found in Campylobacter jejuni (Smith et al., Int. J Biochem. Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem, 89:1923-1931 (1981)). Similar enzymes with high sequence homology include fum1 from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett, 270:207-213 (2007)).

Protein	GenBank ID	GI Number	Organism
fumA	NP_416129.1	16129570	Escherichia coli
fumB	NP_418546.1	16131948	Escherichia coli
fumC	NP_416128.1	16129569	Escherichia coli
fumC	O69294	9789756	Campylobacter jejuni
fumC	P84127	75427690	Thermus thermophilus
fumH	P14408	120605	Rattus norvegicus
fum1	P93033	39931311	Arabidopsis thaliana
fumC	Q8NRN8	39931596	Corynebacterium glutamicum
MmcB	YP_001211906	147677691	Pelotomaculum thermopropionicum
MmcC	YP_001211907	147677692	Pelotomaculum thermopropionicum

Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is catalyzed by 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzyme participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene products are encoded by mhpD of E. coli (Ferrandez et al., J Bacteriol. 179:2573-2581 (1997); Pollard et al., Eur J Biochem. 251:98-106 (1998)), todG and cmtF of Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, J Bacteriol. 178:1351-1362 (1996)), cnbE of Comamonas sp. CNB-1 (Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD of Burkholderia xenovorans (Wang et al., FEBS J 272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-1,7-dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks et al., J. Am. Chem. Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Roper et al., Gene 156:47-51 (1995); Izumi et al., J Mol. Biol. 370:899-911 (2007)) and E. coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (91% identity, eval=2e-138) and Salmonella enterica (91% identity, eval=4e-138), among others.

Protein	GenBank Accession No.	GI No.	Organism
mhpD	AAC73453.2	87081722	Escherichia coli
cmtF	AAB62293.1	1263188	Pseudomonas putida
todG	AAA61942.1	485738	Pseudomonas putida
cnbE	YP_001967714.1	190572008	Comamonas sp. CNB-1
mhpD	Q13VU0	123358582	Burkholderia xenovorans
hpcG	CAA57202.1	556840	Escherichia coli C
hpaH	CAA86044.1	757830	Escherichia coli W
hpaH	ABR80130.1	150958100	Klebsiella pneumoniae
Sari_01896	ABX21779.1	160865156	Salmonella enterica

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate range (Drevland et al., J Bacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium tetanomotphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch. Microbiol 168:457-463 (1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms.

Protein	GenBank ID	GI Number	Organism
leuD	Q58673.1	3122345	Methanocaldococcus jannaschii

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe²⁺-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857 (1984)).

	Protein	GenBank ID	GI Number	Organism
	dmdA	ABC88408	86278276	Eubacterium barkeri
	dmdB	ABC88409.1	86278277	Eubacterium barkeri

Oleate hydratases represent additional suitable candidates as suggested in WO2011076691. Examples include the following proteins.

Protein	GenBank ID	GI Number	Organism
OhyA	ACT54545.1	254031735	Elizabethkingia meningoseptica
HMPREF0841_1446	ZP_07461147.1	306827879	Streptococcus pyogenes ATCC 10782
P700755_13397	ZP_01252267.1	91215295	Psychroflexus torquis ATCC 700755
RPB_2430	YP_486046.1	86749550	Rhodopseudomonas palustris

Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydration of a range of 3-hydroxyacyl-CoA substrates (Roberts et al., Arch. Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem. 11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)). The enoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)). This transformation is also catalyzed by the crt gene product of Clostridium acetobutylicum, the crt1 gene product of C. kluyveri, and other clostridial organisms Atsumi et al., Metab Eng 10:305-311 (2008); Boynton et al., J Bacteriol. 178:3015-3024 (1996); Hillmer et al., FEBS Lett. 21:351-354 (1972)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci USA 95:6419-6424 (1998)). The gene product of pimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation (Harrison et al., Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park and Yup, Biotechnol Bioeng 86:681-686 (2004)).

Protein	GenBank No.	GI No.	Organism
ech	NP_745498.1	26990073	Pseudomonas putida
crt	NP_349318.1	15895969	Clostridium acetobutylicum
crt1	YP_001393856	153953091	Clostridium kluyveri
phaA	ABF82233.1	26990002	Pseudomonas putida
phaB	ABF82234.1	26990001	Pseudomonas putida
paaA	NP_745427.1	106636093	Pseudomonas fluorescens
paaB	NP_745426.1	106636094	Pseudomonas fluorescens
maoC	NP_415905.1	16129348	Escherichia coli
paaF	NP_415911.1	16129354	Escherichia coli
paaG	NP_415912.1	16129355	Escherichia coli

Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Yang et al., Biochemistry 30:6788-6795 (1991); Yang, J Bacteriol. 173:7405-7406 (1991); Nakahigashi et al., Nucleic Acids Res. 18:4937 (1990)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

	Protein	GenBank ID	GI Number	Organism
	fadA	YP_026272.1	49176430	Escherichia coli
	fadB	NP_418288.1	16131692	Escherichia coli
	fadI	NP_416844.1	16130275	Escherichia coli
	fadJ	NP_416843.1	16130274	Escherichia coli
	fadR	NP_415705.1	16129150	Escherichia coli

6.2.1.a CoA Synthase (Acid-Thiol Ligase)

The conversion of acyl-CoA substrates to their acid products can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes, several of which are reversible. These reactions include Steps E, Y, and AF of FIG. 10. Several enzymes catalyzing CoA acid-thiol ligase or CoA synthetase activities have been described in the literature and represent suitable candidates for these steps.

For example, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on cyclic compounds phenylacetate and indoleacetate (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al, supra). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra; Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional candidate is succinyl-CoA synthetase, encoded by sucCD of E. coli and LSC1 and LSC2 genes of Saccharomyces cerevisiae. These enzymes catalyze the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP in a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., J. Am. Chem. Soc. 123:5822-5823 (2001)).

Protein	GenBank ID	GI Number	Organism
AF1211	NP_070039.1	11498810	Archaeoglobus fulgidus
AF1983	NP_070807.1	11499565	Archaeoglobus fulgidus
Scs	YP_135572.1	55377722	Haloarcula marismortui
PAE3250	NP_560604.1	18313937	Pyrobaculum aerophilum str. IM2
sucC	NP_415256.1	16128703	Escherichia coli
sucD	AAC73823.1	1786949	Escherichia coli
LSC1	NP_014785	6324716	Saccharomyces cerevisiae
LSC2	NP_011760	6321683	Saccharomyces cerevisiae
paaF	AAC24333.2	22711873	Pseudomonas putida
matB	AAC83455.1	3982573	Rhizobium leguminosarum

Another candidate enzyme for these steps is 6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)). Other candidates are found in Bacillus subtilis (Bower et al., J Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690 (1992)).

Protein	GenBank ID	GI Number	Organism
bioW	NP_390902.2	50812281	Bacillus subtilis
bioW	CAA10043.1	3850837	Pseudomonas mendocina
bioW	P22822.1	115012	Bacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem. J 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J 395:147-155 (2006); Wang et al., 360:453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J Biol Chem 265:7084-7090 (1990)) and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al. J Bacteriol 178(14):4122-4130 (1996)). Acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) naturally catalyze the ATP-dependent conversion of acetoacetate into acetoacetyl-CoA.

Protein	Accession No.	GI No.	Organism
phl	CAJ15517.1	77019264	Penicillium chrysogenum
phlB	ABS19624.1	152002983	Penicillium chrysogenum
paaF	AAC24333.2	22711873	Pseudomonas putida
bioW	NP_390902.2	50812281	Bacillus subtilis
AACS	NP_084486.1	21313520	Mus musculus
AACS	NP_076417.2	31982927	Homo sapiens

Like enzymes in other classes, certain enzymes in the EC class 6.2.1 have been determined to have broad substrate specificity. The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Applied and Environmental Microbiology 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium trifolii could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., J. Am. Chem. Soc. 123:5822-5823 (2001)).

FIG. 1, Step T—Acetyl-CoA Carboxylase

Several pathways shown in FIG. 10, in particular, those utilizing an acetoacetyl-CoA synthase (Step AS of FIG. 10, Step U of FIGS. 1 and 2) can also be combined with an acetyl-CoA carboxylase to form malonyl-CoA. This reaction includes Step T of FIGS. 1 and 2. Exemplary acetyl-CoA carboxylase enzymes are described in further detail below.

Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This enzyme is biotin dependent and is the first reaction of fatty acid biosynthesis initiation in several organisms. Exemplary enzymes are encoded by accABCD of E. coli (Davis et al, J Biol Chem 275:28593-8 (2000)), ACC1 of Saccharomyces cerevisiae and homologs (Sumper et al, Methods Enzym 71:34-7 (1981)).

Protein	GenBank ID	GI Number	Organism
ACC1	CAA96294.1	1302498	Saccharomyces cerevisiae
KLLA0F06072g	XP_455355.1	50310667	Kluyveromyces lactis
ACC1	XP_718624.1	68474502	Candida albicans
YALI0C11407p	XP_501721.1	50548503	Yarrowia lipolytica
ANI_1_1724104	XP_001395476.1	145246454	Aspergillus niger
accA	AAC73296.1	1786382	Escherichia coli
accB	AAC76287.1	1789653	Escherichia coli
accC	AAC76288.1	1789654	Escherichia coli
accD	AAC75376.1	1788655	Escherichia coli

FIG. 10, Step AS—Acetoacetyl-CoA Synthase

The conversion of malonyl-CoA and acetyl-CoA substrates to acetoacetyl-CoA can be catalyzed by a CoA synthetase in the 2.3.1 family of enzymes. These reactions include Steps E, Y, and AF of FIG. 10. Several enzymes catalyzing the CoA synthetase activities have been described in the literature and represent suitable candidates for these steps.

3-Oxoacyl-CoA products such as acetoacetyl-CoA, 3-oxopentanoyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA can be synthesized from acyl-CoA and malonyl-CoA substrates by 3-oxoacyl-CoA synthases (Steps 10AS). As enzymes in this class catalyze an essentially irreversible reaction, they are particularly useful for metabolic engineering applications for overproducing metabolites, fuels or chemicals derived from 3-oxoacyl-CoA intermediates such as acetoacetyl-CoA. Acetoacetyl-CoA synthase, for example, has been heterologously expressed in organisms that biosynthesize butanol (Lan et al, PNAS USA (2012)) and poly-(3-hydroxybutyrate) (Matsumoto et al, Biosci Biotech Biochem, 75:364-366 (2011). An acetoacetyl-CoA synthase (EC 2.3.1.194) enzyme (FhsA) has been characterized in the soil bacterium Streptomyces sp. CL190 where it participates in mevalonate biosynthesis (Okamura et al, PNAS USA 107:11265-70 (2010)). Other acetoacetyl-CoA synthase genes can be identified by sequence homology to fhsA.

Protein	GenBank ID	GI Number	Organism
fhsA	BAJ83474.1	325302227	Streptomyces sp CL190
AB183750.1:11991 . . . 12971	BAD86806.1	57753876	Streptomyces sp. KO-3988
epzT	ADQ43379.1	312190954	Streptomyces cinnamonensis
ppzT	CAX48662.1	238623523	Streptomyces anulatus
O3I_22085	ZP_09840373.1	378817444	Nocardia brasiliensis

FIG. 10, Step AT—Acetyl-CoA:Acetyl-CoA Acyltransferase (Acetoacetyl-CoA Thiolase)

Acetoacetyl-CoA thiolase (also known as acetyl-CoA acetyltransferase) converts two molecules of acetyl-CoA into one molecule each of acetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol Biotechnol 2:531-541 (2000), and ERG10 from S. cerevisiae Hiser et al., J. Biol. Chem. 269:31383-31389 (1994)). These genes/proteins are identified in the Table below.

Gene	GenBank ID	GI Number	Organism
AtoB	NP_416728	16130161	Escherichia coli
ThlA	NP_349476.1	15896127	Clostridium acetobutylicum
ThlB	NP_149242.1	15004782	Clostridium acetobutylicum
ERG10	NP_015297	6325229	Saccharomyces cerevisiae

FIG. 10, Step AU—4-Hydroxybutyryl-CoA Dehydratase

4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA. This enzyme possesses an intrinsic vinylacetyl-CoA A-isomerase activity, shifting the double bond from the 3,4 position to the 2,3 position (Scherf et al., Eur. J BioChem. 215:421-429 (1993); and Scherf et al., Arch. Microbiol 161:239-245 (1994)). 4-Hydroxybutyrul-CoA dehydratase enzymes from C. aminobutyricum and C. kluyveri were purified, characterized, and sequenced at the N-terminus (Scherf et al., Eur. J BioChem. 215:421-429 (1993); and Scherf et al., Arch. Microbiol 161:239-245 (1994)). The C. kluyveri enzyme, encoded by abfD, was cloned, sequenced and expressed in E. coli (Gerhardt et al., Arch. Microbiol 174:189-199 (2000)). The abfD gene product from Porphyromonas gingivalis ATCC 33277 is closely related by sequence homology to the Clostridial gene products. These genes/proteins are identified in the Table below.

Gene	GenBank ID	GI Number	Organism
abfD	YP_001396399.1	153955634	Clostridium kluyveri DSM 555
abfD	P55792	84028213	Clostridium aminobutyricum
abfD	YP_001928843	188994591	Porphyromonas gingivalis ATCC 33277

Example V

Enzymatic Pathways for Producing Butadiene from CrotOH

This example describes enzymatic pathways for converting CrotOH to butadiene. The four pathways are shown in FIG. 11. In one pathway, CrotOH is phosphorylated to 2-butenyl-4-phosphate by a CrotOH kinase (Step A). The 2-butenyl-4-phosphate intermediate is again phosphorylated to 2-butenyl-4-diphosphate (Step B). A BDS enzyme catalyzes the conversion of 2-butenyl-4-diphosphate to butadiene (Step C). Such a BDS can be derived from a phosphate lyase enzyme such as isoprene synthase using methods, such as directed evolution, as described herein. In an alternate pathway, CrotOH is directly converted to 2-butenyl-4-diphosphate by a diphosphokinase (step D). In yet another alternative pathway, CrotOH can be converted to butadiene by a CrotOH dehydratase (step E). In yet another pathway, the 2-butenyl-4-phosphate intermediate is directly converted to butadiene by a BDS (monophosphate) (step F). Enzyme candidates for steps A-F are provided below.

CrotOH Kinase (FIG. 11, Step A)

CrotOH kinase enzymes catalyze the transfer of a phosphate group to the hydroxyl group of CrotOH. The enzymes described below naturally possess such activity or can be engineered to exhibit this activity. Kinases that catalyze transfer of a phosphate group to an alcohol group are members of the EC 2.7.1 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.1 enzyme class.

Enzyme Commission Number Enzyme Name
2.7.1.1                  hexokinase
2.7.1.2                  glucokinase
2.7.1.3                  ketohexokinase
2.7.1.4                  fructokinase
2.7.1.5                  rhamnulokinase
2.7.1.6                  galactokinase
2.7.1.7                  mannokinase
2.7.1.8                  glucosamine kinase
2.7.1.10                 phosphoglucokinase
2.7.1.11                 6-phosphofructokinase
2.7.1.12                 gluconokinase
2.7.1.13                 dehydrogluconokinase
2.7.1.14                 sedoheptulokinase
2.7.1.15                 ribokinase
2.7.1.16                 ribulokinase
2.7.1.17                 xylulokinase
2.7.1.18                 phosphoribokinase
2.7.1.19                 phosphoribulokinase
2.7.1.20                 adenosine kinase
2.7.1.21                 thymidine kinase
2.7.1.22                 ribosylnicotinamide kinase
2.7.1.23                 NAD+ kinase
2.7.1.24                 dephospho-CoA kinase
2.7.1.25                 adenylyl-sulfate kinase
2.7.1.26                 riboflavin kinase
2.7.1.27                 erythritol kinase
2.7.1.28                 triokinase
2.7.1.29                 glycerone kinase
2.7.1.30                 glycerol kinase
2.7.1.31                 glycerate kinase
2.7.1.32                 choline kinase
2.7.1.33                 pantothenate kinase
2.7.1.34                 pantetheine kinase
2.7.1.35                 pyridoxal kinase
2.7.1.36                 mevalonate kinase
2.7.1.39                 homoserine kinase
2.7.1.40                 pyruvate kinase
2.7.1.41                 glucose-1-phosphate
                         phosphodismutase
2.7.1.42                 riboflavin
                         phosphotransferase
2.7.1.43                 glucuronokinase
2.7.1.44                 galacturonokinase
2.7.1.45                 2-dehydro-3-
                         deoxygluconokinase
2.7.1.46                 L-arabinokinase
2.7.1.47                 D-ribulokinase
2.7.1.48                 uridine kinase
2.7.1.49                 hydroxymethylpyrimidine kinase
2.7.1.50                 hydroxyethylthiazole kinase
2.7.1.51                 L-fuculokinase
2.7.1.52                 fucokinase
2.7.1.53                 L-xylulokinase
2.7.1.54                 D-arabinokinase
2.7.1.55                 allose kinase
2.7.1.56                 1-phosphofructokinase
2.7.1.58                 2-dehydro-3-deoxygalactonokinase
2.7.1.59                 N-acetylglucosamine kinase
2.7.1.60                 N-acylmannosamine kinase
2.7.1.61                 acyl-phosphate-hexose
                         phosphotransferase
2.7.1.62                 phosphoramidate-hexose
                         phosphotransferase
2.7.1.63                 polyphosphate-glucose
                         phosphotransferase
2.7.1.64                 inositol 3-kinase
2.7.1.65                 scyllo-inosamine 4-kinase
2.7.1.66                 undecaprenol kinase
2.7.1.67                 1-phosphatidylinositol 4-kinase
2.7.1.68                 1-phosphatidylinositol-4-phosphate
                         5-kinase
2.7.1.69                 protein-Np-phosphohistidine-
                         sugar phosphotransferase
2.7.1.70                 identical to EC 2.7.1.37.
2.7.1.71                 shikimate kinase
2.7.1.72                 streptomycin 6-kinase
2.7.1.73                 inosine kinase
2.7.1.74                 deoxycytidine kinase
2.7.1.76                 deoxyadenosine kinase
2.7.1.77                 nucleoside phosphotransferase
2.7.1.78                 polynucleotide 5′-hydroxyl-kinase
2.7.1.79                 diphosphate-glycerol
                         phosphotransferase
2.7.1.80                 diphosphate-serine
                         phosphotransferase
2.7.1.81                 hydroxylysine kinase
2.7.1.82                 ethanolamine kinase
2.7.1.83                 pseudouridine kinase
2.7.1.84                 alkylglycerone kinase
2.7.1.85                 β-glucoside kinase
2.7.1.86                 NADH kinase
2.7.1.87                 streptomycin 3″-kinase
2.7.1.88                 dihydrostreptomycin-6-phosphate
                         3′a-kinase
2.7.1.89                 thiamine kinase
2.7.1.90                 diphosphate-fructose-6-phosphate
                         1-phosphotransferase
2.7.1.91                 sphinganine kinase
2.7.1.92                 5-dehydro-2-deoxygluconokinase
2.7.1.93                 alkylglycerol kinase
2.7.1.94                 acylglycerol kinase
2.7.1.95                 kanamycin kinase
2.7.1.100                S-methyl-5-thioribose kinase
2.7.1.101                tagatose kinase
2.7.1.102                hamamelose kinase
2.7.1.103                viomycin kinase
2.7.1.105                6-phosphofructo-2-kinase
2.7.1.106                glucose-1,6-bisphosphate
                         synthase
2.7.1.107                diacylglycerol kinase
2.7.1.108                dolichol kinase
2.7.1.113                deoxyguanosine kinase
2.7.1.114                AMP-thymidine kinase
2.7.1.118                ADP-thymidine kinase
2.7.1.119                hygromycin-B 7″-O-kinase
2.7.1.121                phosphoenolpyruvate-glycerone
                         phosphotransferase
2.7.1.122                xylitol kinase
2.7.1.127                inositol-trisphosphate 3-kinase
2.7.1.130                tetraacyldisaccharide 4′-kinase
2.7.1.134                inositol-tetrakisphosphate 1-
                         kinase
2.7.1.136                macrolide 2′-kinase
2.7.1.137                phosphatidylinositol 3-kinase
2.7.1.138                ceramide kinase
2.7.1.140                inositol-tetrakisphosphate 5-
                         kinase
2.7.1.142                glycerol-3-phosphate-glucose
                         phosphotransferase
2.7.1.143                diphosphate-purine nucleoside
                         kinase
2.7.1.144                tagatose-6-phosphate kinase
2.7.1.145                deoxynucleoside kinase
2.7.1.146                ADP-dependent
                         phosphofructokinase
2.7.1.147                ADP-dependent glucokinase
2.7.1.148                4-(cytidine 5′-diphospho)-2-C-
                         methyl-D-erythritol kinase
2.7.1.149                1-phosphatidylinositol-5-
                         phosphate 4-kinase
2.7.1.150                1-phosphatidylinositol-3-
                         phosphate 5-kinase
2.7.1.151                inositol-polyphosphate
                         multikinase
2.7.1.153                phosphatidylinositol-4,5-
                         bisphosphate 3-kinase
2.7.1.154                phosphatidylinositol-4-phosphate
                         3-kinase
2.7.1.156                adenosylcobinamide kinase
2.7.1.157                N-acetylgalactosamine kinase
2.7.1.158                inositol-pentakisphosphate 2-
                         kinase
2.7.1.159                inositol-1,3,4-trisphosphate 5/6-
                         kinase
2.7.1.160                2′-phosphotransferase
2.7.1.161                CTP-dependent riboflavin kinase
2.7.1.162                N-acetylhexosamine 1-kinase
2.7.1.163                hygromycin B 4-O-kinase
2.7.1.164                O-phosphoseryl-tRNASec kinase

Mevalonate kinase (EC 2.7.1.36) phosphorylates the terminal hydroxyl group of mevalonate. Gene candidates for this step include erg12 from S. cerevisiae, mvk from Methanocaldococcus jannaschi, MVK from Homo sapeins, and mvk from Arabidopsis thaliana col. Additional mevalonate kinase candidates include the feedback-resistant mevalonate kinase from the archeon Methanosarcina mazei (Primak et al, AEM, in press (2011)) and the Mvk protein from Streptococcus pneumoniae (Andreassi et al, Protein Sci, 16:983-9 (2007)). Mvk proteins from S. cerevisiae, S. pneumoniae and M. mazei were heterologously expressed and characterized in E. coli (Primak et al, supra). The S. pneumoniae mevalonate kinase was active on several alternate substrates including cylopropylmevalonate, vinylmevalonate and ethynylmevalonate (Kudoh et al, Bioorg Med Chem 18:1124-34 (2010)), and a subsequent study determined that the ligand binding site is selective for compact, electron-rich C(3)-substituents (Lefurgy et al, J Biol Chem 285:20654-63 (2010)).

Protein	GenBank ID	GI Number	Organism
erg12	CAA39359.1	3684	Sachharomyces cerevisiae
mvk	Q58487.1	2497517	Methanocaldococcus jannaschii
mvk	AAH16140.1	16359371	Homo sapiens
mvk	NP_851084.1	30690651	Arabidopsis thaliana
mvk	NP_633786.1	21227864	Methanosarcina mazei
mvk	NP_357932.1	15902382	Streptococcus pneumoniae

Glycerol kinase also phosphorylates the terminal hydroxyl group in glycerol to form glycerol-3-phosphate. This reaction occurs in several species, including Escherichia coli, Saccharomyces cerevisiae, and Thermotoga maritima. The E. coli glycerol kinase has been shown to accept alternate substrates such as dihydroxyacetone and glyceraldehyde (Hayashi et al., J Biol. Chem. 242:1030-1035 (1967)). T, maritime has two glycerol kinases (Nelson et al., Nature 399:323-329 (1999)). Glycerol kinases have been shown to have a wide range of substrate specificity. Crans and Whiteside studied glycerol kinases from four different organisms (Escherichia coli, S. cerevisiae, Bacillus stearothermophilus, and Candida mycoderma) (Crans et al., J. Am. Chem. Soc. 107:7008-7018 (2010); Nelson et al., supra, (1999)). They studied 66 different analogs of glycerol and concluded that the enzyme could accept a range of substituents in place of one terminal hydroxyl group and that the hydrogen atom at C2 could be replaced by a methyl group. Interestingly, the kinetic constants of the enzyme from all four organisms were very similar.

Protein	GenBank ID	GI Number	Organism
glpK	AP_003883.1	89110103	Escherichia coli K12
glpK1	NP_228760.1	15642775	Thermotoga maritime MSB8
glpK2	NP_229230.1	15642775	Thermotoga maritime MSB8
Gut1	NP_011831.1	82795252	Saccharomyces cerevisiae

Homoserine kinase is another possible candidate. This enzyme is also present in a number of organisms including E. coli, Streptomyces sp, and S. cerevisiae. Homoserine kinase from E. coli has been shown to have activity on numerous substrates, including, L-2-amino,1,4-butanediol, aspartate semialdehyde, and 2-amino-5-hydroxyvalerate (Huo et al., Biochemistry 35:16180-16185 (1996); Huo et al., Arch. Biochem. Biophys. 330:373-379 (1996)). This enzyme can act on substrates where the carboxyl group at the alpha position has been replaced by an ester or by a hydroxymethyl group. The gene candidates are:

Protein	GenBank ID	GI Number	Organism
thrB	BAB96580.2	85674277	Escherichia coli K12
SACT1DRAFT_4809	ZP_06280784.1	282871792	Streptomyces sp. ACT-1
Thr1	AAA35154.1	172978	Saccharomyces serevisiae

2-Butenyl-4-Phosphate Kinase (FIG. 11, Step B)

2-Butenyl-4-phosphate kinase enzymes catalyze the transfer of a phosphate group to the phosphate group of 2-butenyl-4-phosphate. The enzymes described below naturally possess such activity or can be engineered to exhibit this activity. Kinases that catalyze transfer of a phosphate group to another phosphate group are members of the EC 2.7.4 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.4 enzyme class.

Enzyme Commission Number Enzyme Name
2.7.4.1                  polyphosphate kinase
2.7.4.2                  phosphomevalonate kinase
2.7.4.3                  adenylate kinase
2.7.4.4                  nucleoside-phosphate kinase
2.7.4.6                  nucleoside-diphosphate kinase
2.7.4.7                  phosphomethylpyrimidine kinase
2.7.4.8                  guanylate kinase
2.7.4.9                  dTMP kinase
2.7.4.10                 nucleoside-triphosphate-adenylate kinase
2.7.4.11                 (deoxy)adenylate kinase
2.7.4.12                 T2-induced deoxynucleotide kinase
2.7.4.13                 (deoxy)nucleoside-phosphate kinase
2.7.4.14                 cytidylate kinase
2.7.4.15                 thiamine-diphosphate kinase
2.7.4.16                 thiamine-phosphate kinase
2.7.4.17                 3-phosphoglyceroyl-phosphate-polyphosphate phosphotransferase
2.7.4.18                 farnesyl-diphosphate kinase
2.7.4.19                 5-methyldeoxycytidine-5′-phosphate kinase
2.7.4.20                 dolichyl-diphosphate-polyphosphate phosphotransferase
2.7.4.21                 inositol-hexakisphosphate kinase
2.7.4.22                 UMP kinase
2.7.4.23                 ribose 1,5-bisphosphate phosphokinase
2.7.4.24                 diphosphoinositol-pentakisphosphate kinase
2.7.4.—                  Farnesyl monophosphate kinase
2.7.4.—                  Geranyl-geranyl monophosphate kinase
2.7.4.—                  Phytyl-phosphate kinase
2.7.4.26                 isopentenyl phosphate kinase

Phosphomevalonate kinase enzymes are of particular interest. Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the analogous transformation to 2-butenyl-4-phosphate kinase. This enzyme is encoded by erg8 in Saccharomyces cerevisiae (Tsay et al., Mol. Cell Biol. 11:620-631 (1991)) and mvaK2 in Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus faecalis (Doun et al., Protein Sci. 14:1134-1139 (2005); Wilding et al., J Bacteriol. 182:4319-4327 (2000)). The Streptococcus pneumoniae and Enterococcus faecalis enzymes were cloned and characterized in E. coli (Pilloff et al., J Biol. Chem. 278:4510-4515 (2003); Doun et al., Protein Sci. 14:1134-1139 (2005)). The S. pneumoniae phosphomevalonate kinase was active on several alternate substrates including cylopropylmevalonate phosphate, vinylmevalonate phosphate and ethynylmevalonate phosphate (Kudoh et al, Bioorg Med Chem 18:1124-34 (2010)).

Protein	GenBank ID	GI Number	Organism
Erg8	AAA34596.1	171479	Saccharomyces cerevisiae
mvaK2	AAG02426.1	9937366	Staphylococcus aureus
mvaK2	AAG02457.1	9937409	Streptococcus pneumoniae
mvaK2	AAG02442.1	9937388	Enterococcus faecalis

Additional exemplary enzymes of particular interest in this class include:

Enzyme	Genbank ID	GI Number	Organism
phosphomevalonate kinase	YP_008718968.1	554649894	Camobacterium sp. WN1359
phosphomevalonate kinase	YP_004889541.1	380032550	Lactobacillus plantarum WCFS1
phosphomevalonate kinase	BAD86802.1	57753872	Streptomyces sp. KO-3988
phosphomevalonate kinase	YP_006806525.1	407642766	Nocardia brasiliensis ATCC 700358
phosphomevalonate kinase	YP_008165221.1	521188403	Corynebacterium terpenotabidum Y-11
isopentenyl phosphate kinase	NP_247007.1	15668214	Methanocaldococcus jarmaschii
isopentenyl phosphate kinase	NP_393581.1	16081271	Thermoplasma acidophilum DSM 1728
isopentenyl phosphate kinase	NP_275190.1	15678076	Methanothermobacter thermautotrophicus
isopentenyl phosphate kinase	YP_003356693.1	282164308	Methanocella paludicola SANAE
isopentenyl phosphate kinase	YP_304959.1	73668944	Methanosarcina barkeri Fusaro
isopentenyl phosphate kinase	YP_007714098.1	478483448	Candidatus Methanomethylophilus alvus Mx1201
isopentenyl phosphate kinase	AAB84554.1	2621082	Methanobacterium thermoautotrophicum

Isopentenyl phosphate kinase, E.C. 2.7.4.26, Genbank ID number 2621082, was cloned from Methanobacterium thermoautotrophicum gil2621082 into a plasmid suitable for expression in E. coli., plasmid pZS*13S obtained from R. Lutz (Expressys, Germany) and are based on the pZ Expression System (Lutz, R. & Bujard, H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25, 1203-1210 (1997)).

E. coli (MG1655 variants) were transformed with the expression plasmid and selected and maintained using antibiotic selection with carbenicillin. Cells were grown in LB media with carbenicillin and IPTG at 37° C. then harvested by centrifugation. Lysis was performed using a chemical lysis procedure, and lysate the cooled to 4° C. Streptactin-tagged isopentenyl phosphate kinase was isolated from the cell lysate using Streptactin-Sepharose purification. Activity measurements on native substrate, isopentenyl phosphate, were performed to verify fidelity of the purified enzyme, using a pyruvate kinase-lactate dehydrogenase coupled assay to couple ADP formation from ATP to NADH oxidation. The same assay procedure was used to demonstrate robust activity on crotyl phosphate. In the absence of enzyme, no conversion of crotyl phosphate to crotyl diphosphate was observed (data not shown).

Additional kinase enzymes include fosfomycin kinase (FomA) which is highly homologous to isopentenyl phosphate kinase and is an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas (Mabangalo et al. Biochemistry 51(4):917-925 (2012)). Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase superfamily. IPK from Thermoplasma acidophilum has been shown to have activity on fosmomycin. A exemplary fosfomycin kinase is that from Streptomyces wedmorensis, Genbank ID BAA32493.1 and GI number 3452580.

Farnesyl monophosphate kinase enzymes catalyze the CTP dependent phosphorylation of farnesyl monophosphate to farnesyl diphosphate. Similarly, geranylgeranyl phosphate kinase catalyzes CTP dependent phosphorylation. Enzymes with these activities were identified in the microsomal fraction of cultured Nicotiana tabacum (Thai et al, PNAS 96:13080-5 (1999)). However, the associated genes have not been identified to date. Additional enzymes include those of the EC 2.7.2.8 class. This class is exemplifed by acetylglutamate kinase, including the exemplary enzymes below:

acetylglutamate kinase	NP_126233.1	14520758	Pyrococcus abyssi GE5
acetylglutamate kinase	NP_579365.1	18978008	Pyrococcus furiosus DSM 3638
acetylglutamate kinase	AAB88966.1	2648231	Archaeoglobus fulgidus DSM4304

Butadiene Synthase (BDS) (FIG. 11, Step C)

BDS catalyzes the conversion of 2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described below naturally possess such activity or can be engineered to exhibit this activity. Carbon-oxygen lyases that operate on phosphates are found in the EC 4.2.3 enzyme class. The table below lists several useful enzymes in EC class 4.2.3.

Enzyme Commission Number Enzyme Name
4.2.3.15                 Myrcene synthase
4.2.3.26                 Linalool synthase
4.2.3.27                 Isoprene synthase
4.2.3.36                 Terpentriene sythase
4.2.3.46                 (E,E)-alpha-Farnesene synthase
4.2.3.47                 Beta-Farnesene synthase
4.2.3.49                 Nerolidol synthase

Particularly useful enzymes include isoprene synthase, myrcene synthase and farnesene synthase. Enzyme candidates are described below, and in the enzymes and classes for FIG. 15, Step F.

Isoprene synthase naturally catalyzes the conversion of dimethylallyl diphosphate to isoprene, but can also catalyze the synthesis of 1,3-butadiene from 2-butenyl-4-diphosphate. Isoprene synthases can be found in several organisms including Populus alba (Sasaki et al., FEBS Letters, 2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et al., Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol., 137(2):700-712 (2005)), and Populus tremula x Populus alba, also called Populus canescens (Miller et al., Planta, 2001, 213 (3), 483-487). The crystal structure of the Populus canescens isoprene synthase was determined (Koksal et al, J Mol Biol 402:363-373 (2010)). Additional isoprene synthase enzymes are described in (Chotani et al., WO/2010/031079, Systems Using Cell Culture for Production of Isoprene; Cervin et al., US Patent Application 20100003716, Isoprene Synthase Variants for Improved Microbial Production of Isoprene).

Protein	GenBank ID	GI Number	Organism
ispS	BAD98243.1	63108310	Populus alba
ispS	AAQ84170.1	35187004	Pueraria montana
ispS	CAC35696.1	13539551	Populus tremula × Populus alba

Isoprene synthase, E.C. 4.2.3.27, Genbank ID number 63108310, was cloned from Populus alba into a plasmid suitable for expression in E. coli., plasmid pZS*13S (Expressys, Germany).

E. coli (MG1655 variants) were transformed with the expression plasmid and selected and maintained using antibiotic selection with carbenicillin. Cells were grown in Terrific Broth with carbenicillin to an OD of 0.8 and then gene expression induced by IPTG addition then harvested by centrifugation. Lysis was performed using microfluidization at 0° C. Streptactin-tagged isoprene synthase was isolated from the cell lysate using Streptactin-Sepharose purification. Purified enzyme was tested for its ability to convert its native substrate, dimethylallyl diphosphate, into isoprene, and for its ability to convert crotyl diphosphate into 1,3-butadiene, by incubating purified enzyme with each substrate in sealed screw-cap vials for a period of time before analysis of product in headspace of vial by GC-MS. Fidelity of purified enzyme was confirmed by detection of isoprene. Activity on crotyl diphosphate was confirmed by detection of butadiene. In the absence of enzyme, no butadiene was formed (data not shown).

Myrcene synthase enzymes catalyze the dephosphorylation of geranyl diphosphate to beta-myrcene (EC 4.2.3.15). Exemplary myrcene synthases are encoded by MST2 of Solanum lycopersicum (van Schie et al, Plant Mol Biol 64:D473-79 (2007)), TPS-Myr of Picea abies (Martin et al, Plant Physiol 135:1908-27 (2004)) g-myr of Abies grandis (Bohlmann et al, J Biol Chem 272:21784-92 (1997)) and TPS10 of Arabidopsis thaliana (Bohlmann et al, Arch Biochem Biophys 375:261-9 (2000)). These enzymes were heterologously expressed in E. coli.

Protein	GenBank ID	GI Number	Organism
MST2	ACN58229.1	224579303	Solanum lycopersicum
TPS-Myr	AAS47690.2	77546864	Picea abies
G-myr	O24474.1	17367921	Abies grandis
TPS10	EC07543.1	330252449	Arabidopsis thaliana

Farnesyl diphosphate is converted to alpha-farnesene and beta-farnesene by alpha-farnesene synthase and beta-farnesene synthase, respectively. Exemplary alpha-farnesene synthase enzymes include TPS03 and TPS02 of Arabidopsis thaliana (Faldt et al, Planta 216:745-51 (2003); Huang et al, Plant Physiol 153:1293-310 (2010)), afs of Cucumis sativus (Mercke et al, Plant Physiol 135:2012-14 (2004), eafar of Malus x domestica (Green et al, Phytochem 68:176-88 (2007)) and TPS-Far of Picea abies (Martin, supra). An exemplary beta-farnesene synthase enzyme is encoded by TPS1 of Zea mays (Schnee et al, Plant Physiol 130:2049-60 (2002)).

Protein	GenBank ID	GI Number	Organism
TPS03	A4FVP2.1	205829248	Arabidopsis thaliana
TPS02	P0CJ43.1	317411866	Arabidopsis thaliana
TPS-Far	AAS47697.1	44804601	Picea abies
afs	AAU05951.1	51537953	Cucumis sativus
eafar	Q84LB2.2	75241161	Malus × domestica
TPS1	Q84ZW8.1	75149279	Zea mays

CrotOH Diphosphokinase (FIG. 11, Step D)

CrotOH diphosphokinase enzymes catalyze the transfer of a diphosphate group to the hydroxyl group of CrotOH. The enzymes described below naturally possess such activity or can be engineered to exhibit this activity. Kinases that catalyze transfer of a diphosphate group are members of the EC 2.7.6 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.6 enzyme class.

Enzyme Commission Number Enzyme Name
2.7.6.1                  ribose-phosphate diphosphokinase
2.7.6.2                  thiamine diphosphokinase
2.7.6.3                  2-amino-4-hydroxy-6-hydroxymeth-
                         yldihydropteridine diphosphokinase
2.7.6.4                  nucleotide diphosphokinase
2.7.6.5                  GTP diphosphokinase

Of particular interest are ribose-phosphate diphosphokinase enzymes which have been identified in Escherichia coli (Hove-Jenson et al., J Biol Chem, 1986, 261(15); 6765-71) and Mycoplasma pneumoniae M129 (McElwain et al, International Journal of Systematic Bacteriology, 1988, 38:417-423) as well as thiamine diphosphokinase enzymes. Exemplary thiamine diphosphokinase enzymes are found in Arabidopsis thaliana (Ajjawi, Plant Mol Biol, 2007, 65(1-2); 151-62).

Protein	GenBank ID	GI Number	Organism
prs	NP_415725.1	16129170	Escherichia coli
prsA	NP_109761.1	13507812	Mycoplasma pneumoniae M129
TPK1	BAH19964.1	222424006	Arabidopsis thaliana col
TPK2	BAH57065.1	227204427	Arabidopsis thaliana col

CrotOH Dehydratase (FIG. 11, Step E)

Converting CrotOH to butadiene using a CrotOH dehydratase can include combining the activities of the enzymatic isomerization of CrotOH to MVC then dehydration of MVC to butadiene. An exemplary bifunctional enzyme with isomerase and dehydratase activities is the linalool dehydratase/isomerase of Castellaniella defragrans. This enzyme catalyzes the isomerization of geraniol to linalool and the dehydration of linalool to myrcene, reactants similar in structure to CrotOH, MVC and butadiene (Brodkorb et al, J Biol Chem 285:30436-42 (2010)). Enzyme accession numbers and homologs are listed in the table below.

Protein	GenBank ID	GI Number	Organism
Ldi	E1XUJ2.1	403399445	Castellaniella defragrans
STEHIDRAFT_68678	EIM80109.1	389738914	Stereum hirsutum FP-91666 SS1
NECHADRAFT_82460	XP_003040778.1	302883759	Nectria haematococca mpVI 77-13-4
AS9A_2751	YP_004493998.1	333920417	Amycolicicoccus subflavus DQS3-9A1

Alternatively, a fusion protein or protein conjugate can be generated using well know methods in the art to generate a bi-functional (dual-functional) enzyme having both the isomerase and dehydratase activities. The fusion protein or protein conjugate can include at least the active domains of the enzymes (or respective genes) of the isomerase and dehydratase reactions. For the first step, the conversion of CrotOH to MVC, enzymatic conversion can be catalyzed by a CrotOH isomerase (classified as EC 5.4.4). A similar isomerization, the conversion of 2-methyl-MVC to 3-methyl-2-buten-1-ol, is catalyzed by cell extracts of Pseudomonas putida MB-1 (Malone et al, AEM 65 (6): 2622-30 (1999)). The extract may be used in vitro, or the protein or gene(s) associated with the isomerase activity can be isolated and used, even though they have not been identified to date.

Alternatively, either or both steps can be done by chemical conversion, or by enzymatic conversion (in vivo or in vitro), or any combination. Enzymes having the desired activity for the conversion of MVC to butadiene are provided elsewhere herein.

BDS (Monophosphate) (FIG. 11, Step F)

BDS (monophosphate) catalyzes the conversion of 2-butenyl-4-phosphate to 1,3-butadiene (step F). BDS enzymes described above for Step C in the EC 4.2.3 enzyme class may possess such activity or can be engineered to exhibit this activity.

Example VI

Pathways for the Production of Butadiene from Malonyl-CoA and Acetyl-CoA Via 3H5PP

This example describes enzymatic pathways for converting malonyl-CoA and acetyl-CoA to butadiene via 3H5PP. The five pathways are shown in FIG. 12. Enzyme candidates for steps A-O are provided below.

Malonyl-CoA:acetyl-CoA acyltransferase (FIG. 12, Step A)

In Step A of the pathway described in FIG. 12, malonyl-CoA and acetyl-CoA are condensed to form 3-oxoglutaryl-CoA by malonyl-CoA:acetyl-CoA acyl transferase, a beta-keothiolase. Although no enzyme with activity on malonyl-CoA has been reported to date, a good candidate for this transformation is beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase that converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., supra, (1998)), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., Arch Microbiol. 88:117-125 (2007)), andpaaJfrom E. coli (Nogales et al., Microbiology, 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PAO1, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida. These enzymes can also be employed for the synthesis of 3-oxoglutaryl-CoA, a compound structurally similar to 3-oxoadipyl-CoA.

Protein	GenBank ID	GI Number	Organism
paaJ	NP_415915.1	16129358	Escherichia coli
pcaF	AAL02407	17736947	Pseudomonas knackmussii (B13)
phaD	AAC24332.1	3253200	Pseudomonas putida
pcaF	AAA85138.1	506695	Pseudomonas putida
pcaF	AAC37148.1	141777	Acinetobacter calcoaceticus
paaE	ABF82237.1	106636097	Pseudomonas fluorescens
bkt	YP_777652.1	115360515	Burkholderia ambifaria AMMD
bkt	AAG06977.1	9949744	Pseudomonas aeruginosa PAO1
pcaF	AAG03617.1	9946065	Pseudomonas aeruginosa PAO1

Another relevant beta-ketothiolase is oxopimeloyl-CoA:glutaryl-CoA acyltransferase (EC 2.3.1.16) that combines glutaryl-CoA and acetyl-CoA to form 3-oxopimeloyl-CoA. An enzyme catalyzing this transformation is found in Ralstonia eutropha (formerly known as Alcaligenes eutrophus), encoded by genes bktB and bktC (Slater et al., J. Bacteriol. 180:1979-1987 (1998); Haywood et al., FEMS Microbiology Letters 52:91-96 (1988)). The sequence of the BktB protein is known; however, the sequence of the BktC protein has not been reported. The pim operon of Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA degradation (Harrison et al., Microbiology 151:727-736 (2005)). A beta-ketothiolase enzyme candidate in S. aciditrophicus was identified by sequence homology to bktB (43% identity, evalue=1e-93).

Protein	GenBank ID	GI Number	Organism
bktB	YP_725948	11386745	Ralstonia eutropha
pimB	CAE29156	39650633	Rhodopseudomonas palustris
syn_02642	YP_462685.1	85860483	Syntrophus aciditrophicus

Beta-ketothiolase enzymes catalyzing the formation of beta-ketovaleryl-CoA from acetyl-CoA and propionyl-CoA can also be able to catalyze the formation of 3-oxoglutaryl-CoA. Zoogloea ramigera possesses two ketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a β-oxidation ketothiolase that is also capable of catalyzing this transformation (Slater et al., J. Bacteriol, 180:1979-1987 (1998)). The sequences of these genes or their translated proteins have not been reported, but several candidates in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to btkB from R. eutropha. These include:

Protein	GenBank ID	GI Number	Organism
phaA	YP_725941.1	113867452	Ralstonia eutropha
h16_A1713	YP_726205.1	113867716	Ralstonia eutropha
pcaF	YP_728366.1	116694155	Ralstonia eutropha
h16_B1369	YP_840888.1	116695312	Ralstonia eutropha
h16_A0170	YP_724690.1	113866201	Ralstonia eutropha
h16_A0462	YP_724980.1	113866491	Ralstonia eutropha
h16_A1528	YP_726028.1	113867539	Ralstonia eutropha
h16_B0381	YP_728545.1	116694334	Ralstonia eutropha
h16_B0662	YP_728824.1	116694613	Ralstonia eutropha
h16_B0759	YP_728921.1	116694710	Ralstonia eutropha
h16_B0668	YP_728830.1	116694619	Ralstonia eutropha
h16_A1720	YP_726212.1	113867723	Ralstonia eutropha
h16_A1887	YP_726356.1	113867867	Ralstonia eutropha
phbA	P07097.4	135759	Zoogloea ramigera
bktB	YP_002005382.1	194289475	Cupriavidus taiwanensis
Rmet_1362	YP_583514.1	94310304	Ralstonia metallidurans
Bphy_0975	YP_001857210.1	186475740	Burkholderia phymatum

Additional candidates include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., supra, (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., supra, (2007); Winzer et al., supra, (2000)), and ERG10 from S. cerevisiae (Hiser et al., supra, (1994)).

Protein	GenBank ID	GI Number	Organism
toB	NP_416728	16130161	Escherichia coli
thlA	NP_349476.1	15896127	Clostridium acetobutylicum
thlB	NP_149242.1	15004782	Clostridium acetobutylicum
ERG10	NP_015297	6325229	Saccharomyces cerevisiae

3-Oxoglutaryl-CoA Reductase (Ketone-Reducing) (FIG. 12, Step B)

This enzyme catalyzes the reduction of the 3-oxo group in 3-oxoglutaryl-CoA to the 3-hydroxy group in Step B of the pathway shown in FIG. 12.

3-Oxoacyl-CoA dehydrogenase enzymes convert 3-oxoacyl-CoA molecules into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta-oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Furthermore, the gene products encoded by phaC in Pseudomonas putida U (Olivera et al., supra, (1998)) and paaC in Pseudomonas fluorescens ST (Di et al., supra, (2007)) catalyze the reversible oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene. In addition, given the proximity in E. coli of paaH to other genes in the phenylacetate degradation operon (Nogales et al., supra, (2007)) and the fact that paaH mutants cannot grow on phenylacetate (Ismail et al., supra, (2003)), it is expected that the E. coli paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.

Protein	GenBank ID	GI Number	Organism
fadB	P21177.2	119811	Escherichia coli
fadJ	P77399.1	3334437	Escherichia coli
paaH	NP_415913.1	16129356	Escherichia coli
phaC	NP_745425.1	26990000	Pseudomonas putida
paaC	ABF82235.1	106636095	Pseudomonas fluorescens

3-Hydroxybutyryl-CoA dehydrogenase, acetoacetyl-CoA reductase, catalyzes the reversible NAD(P)H-dependent conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. This enzyme participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones and Woods, supra, (1986)). Enzyme candidates include hbd from C. acetobutylicum (Boynton et al., J Bacteriol. 178:3015-3024 (1996)), hbd from C. beijerinckii (Colby et al., Appl Environ. Microbiol 58:3297-3302 (1992)), and a number of similar enzymes from Metallosphaera sedula (Berg et al., supra, (2007)). The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., supra, (1989)). Yet other genes demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., supra, (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., supra, (2006)). The former gene is NADPH-dependent, its nucleotide sequence has been determined (Peoples and Sinskey, supra, (1989)) and the gene has been expressed in E. coli. Additional genes include hbd1 (C-terminal domain) and hbd2 (N-terminal domain) in Clostridium kluyveri (Hilimer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus (WAKIL et al., supra, (1954)).

Protein	GenBank ID	GI Number	Organism
hbd	NP_349314.1	15895965	Clostridium acetobutylicum
hbd	AAM14586.1	20162442	Clostridium beijerinckii
Msed_1423	YP_001191505	146304189	Metallosphaera sedula
Msed_0399	YP_001190500	146303184	Metallosphaera sedula
Msed_0389	YP_001190490	146303174	Metallosphaera sedula
Msed_1993	YP_001192057	146304741	Metallosphaera sedula
hbd2	EDK34807.1	146348271	Clostridium kluyveri
hbd1	EDK32512.1	146345976	Clostridium kluyveri
HSD17B10	O02691.3	3183024	Bos taurus
phaB	YP_353825.1	77464321	Rhodobacter sphaeroides
phbB	P23238.1	130017	Zoogloea ramigera

3-Hydroxyglutaryl-CoA Reductase (Aldehyde Forming) (FIG. 12, Step C)

3-hydroxyglutaryl-CoA reductase reduces 3-hydroxyglutaryl-CoA to 3-hydroxy-5-oxopentanoate. Several acyl-CoA dehydrogenases reduce an acyl-CoA to its corresponding aldehyde (EC 1.2.1). Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase (Reiser and Somerville, supra, (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., supra, (2002)), and a CoA- and NADP-dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk, supra, (1996); Sohling and Gottschalk, supra, (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., supra, (2000)). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., supra, (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Protein	GenBank ID	GI Number	Organism
acr1	YP_047869.1	50086359	Acinetobacter calcoaceticus
acr1	AAC45217	1684886	Acinetobacter baylyi
acr1	BAB85476.1	18857901	Acinetobacter sp. Strain M-1
sucD	P38947.1	172046062	Clostridium kluyveri
sucD	NP_904963.1	34540484	Porphyromonas gingivalis
bphG	BAA03892.1	425213	Pseudomonas sp
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
bld	AAP42563.1	31075383	Clostridium saccharoperbutylacetonicum

An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg et al., supra, (2007b); Thauer, supra, (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., supra, (2006); Hugler et al., supra, (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., supra, (2006); Berg et al., supra, (2007b)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., supra, (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius. Yet another acyl-CoA reductase (aldehyde forming) candidate is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ. Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra, (1999)).

Protein	GenBank ID	GI Number	Organism
MSED_0709	YP_001190808.1	146303492	Metallosphaera sedula
mcr	NP_378167.1	15922498	Sulfolobus tokodaii
asd-2	NP_343563.1	15898958	Sulfolobus solfataricus
Saci_2370	YP_256941.1	70608071	Sulfolobus acidocaldarius
Ald	AAT66436	9473535	Clostridium beijerinckii
eutE	AAA80209	687645	Salmonella typhimurium
eutE	P77445	2498347	Escherichia coli

3-Hydroxy-5-oxopentanoate Reductase (FIG. 12, Step D)

This enzyme reduces the terminal aldehyde group in 3-hydroxy-5-oxopentanote to the alcohol group. Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase, 1.1.1.a) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., supra, (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., supra, (2008)), yqhD from E. coli which has preference for molecules longer than C(3) (Sulzenbacher et al., supra, (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al., supra, (1992)). The gene product of yqhD catalyzes the reduction of acetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using NADPH as the cofactor (Perez et al., 283:7346-7353 (2008); Perez et al., J Biol. Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)).

Protein	GenBank ID	GI Number	Organism
alrA	BAB12273.1	9967138	Acinetobacter sp. Strain M-1
ADH2	NP_014032.1	6323961	Saccharomyces cerevisiae
yqhD	NP_417484.1	16130909	Escherichia coli
bdh I	NP_349892.1	15896543	Clostridium acetobutylicum
bdh II	NP_349891.1	15896542	Clostridium acetobutylicum
adhA	YP_162971.1	56552132	Zymomonas mobilis

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al., supra, (2004)), Clostridium kluyveri (Wolff and Kenealy, supra, (1995)) and Arabidopsis thaliana (Breitkreuz et al., supra, (2003)). The A. thaliana enzyme was cloned and characterized in yeast [12882961]. Yet another gene is the alcohol dehydrogenase adhI from Geobacillus thermoglucosidasius (Jeon et al., J Biotechnol 135:127-133 (2008)).

Protein	GenBank ID	GI Number	Organism
4hbd	YP_726053.1	113867564	Ralstonia eutropha H16
4hbd	EDK35022.1	146348486	Clostridium kluyveri
4hbd	Q94B07	75249805	Arabidopsis thaliana
adhI	AAR91477.1	40795502	Geobacillus thermoglucosidasius

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) which catalyzes the reversible oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., J Mol Biol 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning et al., Biochem J 231:481-4 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods Enzymol 324:218-228 (2000)) and Oryctolagus cuniculus (Hawes et al., supra, (2000); Chowdhury et al., Biosci. Biotechnol Biochem. 60:2043-2047 (1996)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart et al., J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., supra, (1996); Chowdhury et al., Biosci. Biotechnol Biochem. 67:438-441 (2003)).

Protein	GenBank ID	GI Number	Organism
P84067	P84067	75345323	Thermus thermophilus
mmsb	P28811.1	127211	Pseudomonas aeruginosa
dhat	Q59477.1	2842618	Pseudomonas putida
3hidh	P31937.2	12643395	Homo sapiens
3hidh	P32185.1	416872	Oryctolagus cuniculus

The conversion of malonic semialdehyde to 3-HP can also be accomplished by two other enzymes: NADH-dependent 3-hydroxypropionate dehydrogenase and NADPH-dependent malonate semialdehyde reductase. An NADH-dependent 3-hydroxypropionate dehydrogenase is thought to participate in beta-alanine biosynthesis pathways from propionate in bacteria and plants (Rathinasabapathi B., Journal of Plant Pathology 159:671-674 (2002); Stadtman, J. Am. Chem. Soc. 77:5765-5766 (1955)). This enzyme has not been associated with a gene in any organism to date. NADPH-dependent malonate semialdehyde reductase catalyzes the reverse reaction in autotrophic CO2-fixing bacteria. Although the enzyme activity has been detected in Metallosphaera sedula, the identity of the gene is not known (Alber et al., supra, (2006)).

3,5-Dihydroxypentanoate Kinase (FIG. 12, Step E)

This enzyme phosphorylates 3,5-dihydroxypentanotae in FIG. 12 (Step E) to form 3-hydroxy-5-phosphonatooxypentanoate (3H5PP). This transformation can be catalyzed by enzymes in the EC class 2.7.1 that enable the ATP-dependent transfer of a phosphate group to an alcohol.

A good candidate for this step is mevalonate kinase (EC 2.7.1.36) that phosphorylates the terminal hydroxyl group of the methyl analog, mevalonate, of 3,5-dihydroxypentanote. Some gene candidates for this step are erg12 from S. cerevisiae, mvk from Methanocaldococcus jannaschi, MVK from Homo sapeins, and mvk from Arabidopsis thaliana col.

Protein	GenBank ID	GI Number	Organism
erg12	CAA39359.1	3684	Sachharomyces cerevisiae
mvk	Q58487.1	2497517	Methanocaldococcus jannaschii
mvk	AAH16140.1	16359371	Homo sapiens
M\mvk	NP_851084.1	30690651	Arabidopsis thaliana

Glycerol kinase also phosphorylates the terminal hydroxyl group in glycerol to form glycerol-3-phosphate. This reaction occurs in several species, including Escherichia coli, Saccharomyces cerevisiae, and Thermotoga maritima. The E. coli glycerol kinase has been shown to accept alternate substrates such as dihydroxyacetone and glyceraldehyde (Hayashi and Lin, supra, (1967)). T, maritime has two glycerol kinases (Nelson et al., supra, (1999)). Glycerol kinases have been shown to have a wide range of substrate specificity. Crans and Whiteside studied glycerol kinases from four different organisms (Escherichia coli, S. cerevisiae, Bacillus stearothermophilus, and Candida mycoderma) (Crans and Whitesides, supra, (2010); Nelson et al., supra, (1999)). They studied 66 different analogs of glycerol and concluded that the enzyme could accept a range of substituents in place of one terminal hydroxyl group and that the hydrogen atom at C2 could be replaced by a methyl group. Interestingly, the kinetic constants of the enzyme from all four organisms were very similar. The gene candidates are:

Protein	GenBank ID	GI Number	Organism
glpK	AP_003883.1	89110103	Escherichia coli K12
glpK1	NP_228760.1	15642775	Thermotoga maritime MSB8
glpK2	NP_229230.1	15642775	Thermotoga maritime MSB8
Gut1	NP_011831.1	82795252	Saccharomyces cerevisiae

Homoserine kinase is another possible candidate that can lead to the phosphorylation of 3,5-dihydroxypentanoate. This enzyme is also present in a number of organisms including E. coli, Streptomyces sp, and S. cerevisiae. Homoserine kinase from E. coli has been shown to have activity on numerous substrates, including, L-2-amino,1,4-butanediol, aspartate semialdehyde, and 2-amino-5-hydroxyvalerate (Huo and Viola, supra, (1996); Huo and Viola, supra, (1996)). This enzyme can act on substrates where the carboxyl group at the alpha position has been replaced by an ester or by a hydroxymethyl group. The gene candidates are:

Protein	GenBank ID	GI Number	Organism
thrB	BAB96580.2	85674277	Escherichia coli K12
SACT1DRAFT_4809	ZP_06280784.1	282871792	Streptomyces sp. ACT-1
Thr1	AAA35154.1	172978	Saccharomyces serevisiae

3H5PP Kinase (FIG. 12, Step F)

Phosphorylation of 3H5PP to 3H5PDP is catalyzed by 3H5PP kinase (FIG. 12, Step F). Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the analogous transformation in the mevalonate pathway. This enzyme is encoded by erg8 in Saccharomyces cerevisiae (Tsay et al., Mol. Cell Biol. 11:620-631 (1991)) and mvaK2 in Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus faecalis (Doun et al., Protein Sci. 14:1134-1139 (2005); Wilding et al., J Bacteriol. 182:4319-4327 (2000)). The Streptococcus pneumoniae and Enterococcus faecalis enzymes were cloned and characterized in E. coli (Pilloff et al., J Biol. Chem. 278:4510-4515 (2003); Doun et al., Protein Sci. 14:1134-1139 (2005)).

Protein	GenBank ID	GI Number	Organism
Erg8	AAA34596.1	171479	Saccharomyces cerevisiae
mvaK2	AAG02426.1	9937366	Staphylococcus aureus
mvaK2	AAG02457.1	9937409	Streptococcus pneumoniae
mvaK2	AAG02442.1	9937388	Enterococcus faecalis

3H5PDP Decarboxylase (FIG. 12, Step G)

Butenyl 4-diphosphate is formed from the ATP-dependent decarboxylation of 3H5PDP by 3H5PDP decarboxylase (FIG. 12, Step G). Although an enzyme with this activity has not been characterized to date a similar reaction is catalyzed by mevalonate diphosphate decarboxylase (EC 4.1.1.33), an enzyme participating in the mevalonate pathway for isoprenoid biosynthesis. This reaction is catalyzed by MVD1 in Saccharomyces cerevisiae, MVD in Homo sapiens and MDD in Staphylococcus aureus and Trypsonoma brucei (Toth et al., J Biol. Chem. 271:7895-7898 (1996); Byres et al., J Mol. Biol. 371:540-553 (2007)).

Protein	GenBank ID	GI Number	Organism
MVD1	P32377.2	1706682	Saccharomyces cerevisiae
MVD	NP_002452.1	4505289	Homo sapiens
MDD	ABQ48418.1	147740120	Staphylococcus aureus
MDD	EAN78728.1	70833224	Trypsonoma brucei

Butenyl 4-Diphosphate Isomerase (FIG. 12, Step H)

Butenyl 4-diphosphate isomerase catalyzes the reversible interconversion of 2-butenyl-4-diphosphate and butenyl-4-diphosphate. The following enzymes can naturally possess this activity or can be engineered to exhibit this activity. Useful genes include those that encode enzymes that interconvert isopenenyl diphosphate and dimethylallyl diphosphate. These include isopentenyl diphosphate isomerase enzymes from Escherichia coli (Rodriguez-Concepcion et al., FEBS Lett, 473(3):328-332), Saccharomyces cerevisiae (Anderson et al., J Biol Chem, 1989, 264(32); 19169-75), and Sulfolobus shibatae (Yamashita et al, Eur J Biochem, 2004, 271(6); 1087-93). The reaction mechanism of isomerization, catalyzed by the Idi protein of E. coli, has been characterized in mechanistic detail (de Ruyck et al., J Biol. Chem. 281:17864-17869 (2006)). Isopentenyl diphosphate isomerase enzymes from Saccharomyces cerevisiae, Bacillus subtilis and Haematococcus pluvialis have been heterologously expressed in E. coli (Laupitz et al., Eur. J Biochem. 271:2658-2669 (2004); Kajiwara et al., Biochem. J 324 (Pt 2):421-426 (1997)).

Protein	GenBank ID	GI Number	Organism
Idi	NP_417365.1	16130791	Escherichia coli
IDI1	NP_015208.1	6325140	Saccharomyces cerevisiae
Idi	BAC82424.1	34327946	Sulfolobus shibatae
Idi	AAC32209.1	3421423	Haematococcus pluvialis
Idi	BAB32625.1	12862826	Bacillus subtilis

BDS (FIG. 12, Step I)

BDS catalyzes the conversion of 2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described herein and for FIG. 11 Step C and FIG. 15 Step F naturally possess such activity or can be engineered to exhibit this activity.

3-Hydroxyglutaryl-CoA Reductase (Alcohol Forming) (FIG. 12, Step J)

This step catalyzes the reduction of the acyl-CoA group in 3-hydroxyglutaryl-CoA to the alcohol group. Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., supra, (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., supra, (2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., supra, (1972); Koo et al., supra, (2005)).

Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH-dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., supra, (2002); Strauss and Fuchs, supra, (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra, (2002)). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms can have similar pathways (Klatt et al., supra, (2007)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity.

Protein	GenBank ID	GI Number	Organism
adhE	NP_415757.1	16129202	Escherichia coli
adhE2	AAK09379.1	12958626	Clostridium acetobutylicum
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
mcr	AAS20429.1	42561982	Chloroflexus aurantiacus
Rcas_2929	YP_001433009.1	156742880	Roseiflexus castenholzii
NAP1_02720	ZP_01039179.1	85708113	Erythrobacter sp. NAP1
MGP2080_00535	ZP_01626393.1	119504313	marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced to their corresponding alcohols by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al., Plant Physiology 122:635-644 (2000)).

Protein	GenBank ID	GI Number	Organism
FAR	AAD38039.1	5020215	Simmondsia chinensis

Another candidate for catalyzing this step is 3-hydroxy-3-methylglutaryl-CoA reductase (or HMG-CoA reductase). This enzyme reduces the CoA group in 3-hydroxy-3-methylglutaryl-CoA to an alcohol forming mevalonate. Gene candidates for this step include:

Protein	GenBank ID	GI Number	Organism
HMG1	CAA86503.1	587536	Saccharomyces cerevisiae
HMG2	NP_013555	6323483	Saccharomyces cerevisiae
HMG1	CAA70691.1	1694976	Arabidopsis thaliana
hmgA	AAC45370.1	2130564	Sulfolobus solfataricus

The hmgA gene of Sulfolobus solfataricus, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, has been cloned, sequenced, and expressed in E. coli (Bochar et al., J Bacteriol. 179:3632-3638 (1997)). S. cerevisiae also has two HMG-CoA reductases in it (Basson et al., Proc. Natl. Acad. Sci. U.S.A 83:5563-5567 (1986)). The gene has also been isolated from Arabidopsis thaliana and has been shown to complement the HMG-COA reductase activity in S. cerevisiae (Learned et al., Proc. Natl. Acad. Sci. U.S.A 86:2779-2783 (1989)).

3-Oxoglutaryl-CoA Reductase (Aldehyde Forming) (FIG. 12, Step K)

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde. Thus they can naturally reduce 3-oxoglutaryl-CoA to 3,5-dioxopentanoate or can be engineered to do so. Exemplary genes that encode such enzymes were discussed in FIG. 12, Step C.

3,5-Dioxopentanoate Reductase (Ketone Reducing) (FIG. 12, Step L)

There exist several exemplary alcohol dehydrogenases that convert a ketone to a hydroxyl functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate for this step is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is a dehydrogenase that operates on a 3-hydroxyacid. Another exemplary alcohol dehydrogenase converts acetone to isopropanol as was shown in C. beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)). Methyl ethyl ketone reductase, or alternatively, 2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der et al., Eur. J. Biochem. 268:3062-3068 (2001)).

Protein	GenBank ID	GI Number	Organism
mdh	AAC76268.1	1789632	Escherichia coli
ldhA	NP_415898. 1	16129341	Escherichia coli
ldh	YP_725182.1	113866693	Ralstonia eutropha
bdh	AAA58352.1	177198	Homo sapiens
adh	AAA23199.2	60592974	Clostridium beijerinckii NRRL B593
adh	P14941.1	113443	Thermoanaerobacter brockii HTD4
adhA	AAC25556	3288810	Pyrococcus furiosus
adh-A	CAD36475	21615553	Rhodococcus ruber

A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 13BDO, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al. U.S. Pat. No. 5,413,922. A mutated Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia alcohol dehydrogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl. Microbiol. Biotechnol. 75(6):1249-1256).

Homoserine dehydrogenase (EC 1.1.1.13) catalyzes the NAD(P)H-dependent reduction of aspartate semialdehyde to homoserine. In many organisms, including E. coli, homoserine dehydrogenase is a bifunctional enzyme that also catalyzes the ATP-dependent conversion of aspartate to aspartyl-4-phosphate (Starnes et al., Biochemistry 11:677-687 (1972)). The functional domains are catalytically independent and connected by a linker region (Sibilli et al., J Biol Chem 256:10228-10230 (1981)) and both domains are subject to allosteric inhibition by threonine. The homoserine dehydrogenase domain of the E. coli enzyme, encoded by thrA, was separated from the aspartate kinase domain, characterized, and found to exhibit high catalytic activity and reduced inhibition by threonine (James et al., Biochemistry 41:3720-3725 (2002)). This can be applied to other bifunctional threonine kinases including, for example, hom1 of Lactobacillus plantarum (Cahyanto et al., Microbiology 152:105-112 (2006)) and Arabidopsis thaliana. The monofunctional homoserine dehydrogenases encoded by hom6 in S. cerevisiae (Jacques et al., Biochim Biophys Acta 1544:28-41 (2001)) and hom2 in Lactobacillus plantarum (Cahyanto et al., supra, (2006)) have been functionally expressed and characterized in E. coli.

Protein	GenBank ID	GI number	Organism
thrA	AAC73113.1	1786183	Escherichia coli K12
akthr2	O81852	75100442	Arabidopsis thaliana
hom6	CAA89671	1015880	Saccharomyces cerevisiae
hom1	CAD64819	28271914	Lactobacillus plantarum
hom2	CAD63186	28270285	Lactobacillus plantarum

3,5-Dioxopentanoate Reductase (Aldehyde Reducing) (FIG. 12, Step M)

Several aldehyde reducing reductases are capable of reducing an aldehyde to its corresponding alcohol. Thus they can naturally reduce 3,5-dioxopentanoate to 5-hydroxy-3-oxopentanoate or can be engineered to do so. Exemplary genes that encode such enzymes were discussed in FIG. 12, Step D.

5-Hydroxy-3-oxopentanoate Reductase (FIG. 12, Step N)

Several ketone reducing reductases are capable of reducing a ketone to its corresponding hydroxyl group. Thus they can naturally reduce 5-hydroxy-3-oxopentanoate to 3,5-dihydroxypentanoate or can be engineered to do so. Exemplary genes that encode such enzymes were discussed in FIG. 12, Step L.

3-Oxo-glutaryl-CoA Reductase (CoA Reducing and Alcohol Forming) (FIG. 12, Step O)

3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming) enzymes catalyze the 2 reduction steps required to form 5-hydroxy-3-oxopentanoate from 3-oxo-glutaryl-CoA. Exemplary 2-step oxidoreductases that convert an acyl-CoA to an alcohol were provided for FIG. 12, Step J. Such enzymes can naturally convert 3-oxo-glutaryl-CoA to 5-hydroxy-3-oxopentanoate or can be engineered to do so.

Example VII

Pathways for Converting Pyruvate to 2-Butanol, and 2-Butanol to 3-Butene-2-ol

This example describes an enzymatic pathway for converting pyruvate to 2-butanol, and further to MVC. The MVC product can be isolated as the product, or further converted to 1,3-butadiene via enzymatic or chemical dehydration. Chemical dehydration of MVC to butadiene is well known in the art (Gustav. Egloff and George Hulla, Chem. Rev., 1945, 36 (1), pp 63-141).

Pathways for converting pyruvate to 2-butanol are well known in the art and are incorporated herein by reference (U.S. Pat. No. 8,206,970, WO 2010/057022). One exemplary pathway for converting pyruvate to 2-butanol is shown in FIG. 14. In this pathway, acetolactate is formed from pyruvate by acetolactate synthase (Step A), acetolactate is subsequently decarbxoylated to acetoin by acetolactate decarboxylase (step B). Reduction of acetoin to 2,3-butanediol and subsequent dehydration (Steps 2C-D) yield 2-butanol. Exemplary enzymes for steps A-D are listed in the table below.

Step	Gene	GenBank ID	GI Number	Organism
14A	budB	AAA25079	149211	Klebsiella pneumonia ATCC 25955
14A	alsS	AAA22222	142470	Bacillus subtilis
14A	budB	AAA25055	149172	Klebsiella terrigena
14B	budA	AAU43774	52352568	Klebsiella oxytoca
14B	alsD	AAA22223	142471	Bacillus subtilis
14B	budA	AAA25054	149171	Klebsiella terrigena
14C	sadH	CAD36475	21615553	Rhodococcus ruber
14C	budC	D86412.1	1468938	Klebsiella pneumonia IAM1063
14C	BC_0668	AAP07682	29894392	Bacillus cereus
14C	butB	AAK04995	12723828	Lactococcus lactis
14D	pddC	AAC98386.1	4063704	Klebsiella pneumoniae
14D	pddB	AAC98385.1	4063703	Klebsiella pneumoniae
14D	pddA	AAC98384.1	4063702	Klebsiella pneumoniae
14D	pduC	AAB84102.1	2587029	Salmonella typhimurium
14D	pduD	AAB84103.1	2587030	Salmonella typhimurium
14D	pduE	AAB84104.1	2587031	Salmonella typhimurium
14D	pddA	BAA08099.1	868006	Klebsiella oxytoca
14D	pddB	BAA08100.1	868007	Klebsiella oxytoca
14D	pddC	BAA08101.1	868008	Klebsiella oxytoca
14D	pduC	CAC82541.1	18857678	Lactobacillus collinoides
14D	pduD	CAC82542.1	18857679	Lactobacillus collinoides
14D	pduE	CAD01091.1	18857680	Lactobacillus collinoides

Enzyme candidates for steps 13A and 13B are disclosed below.

2-Butanol Desaturase (FIG. 13A)

Conversion of 2-butanol to MVC is catalyzed by an enzyme with 2-butanol desaturase activity (Step 1A). An exemplary enzyme is MdpJ from Aquincola tertiaricarbonis L108 (Schaefer et al, AEM 78 (17): 6280-4 (2012); Schuster et al, J. Bacteriol 194:972-81 (2012)). This enzyme is a Rieske non-heme mononuclear iron oxygenase, a class of enzymes which typically reacts with aromatic substrates. The MdpJ gene product is active on aliphatic secondary and tertiary alcohol substrates including 2-butanol, 3-methyl-2-butanol and 3-pentanol. The net reaction of MdpJ is conversion of 2-butanol, oxygen and NADH to MVC, NAD and water. The MdpJ gene is colocalized in an operon with several genes that may encode accessory proteins required for activity, listed in the table below. A similar enzyme is found in M. petroleiphilum PM1 (Schuster et al, supra). The mdpK gene encodes a ferredoxin oxidoreductase that may be required for mdpJ activation (Hristova et al, AEM 73: 7347-57 (2007)). Other enzyme candidates can be identified by sequence similarity and are shown in the table below.

Protein	GenBank ID	GI Number	Organism
mdpJ	AEX20406	369794441	Aquincola tertiaricarbonis L108
mdpK	AEX20407	369794442	Aquincola tertiaricarbonis L108
JQ062962.1:4013 . . . 4777	AEX20409	369794444	Aquincola tertiaricarbonis L108
JQ062962.1:4796 . . . 5074	AEX20408	369794443	Aquincola tertiaricarbonis L108
JQ062962.1:5190 . . . 6062	AEX20410	369794445	Aquincola tertiaricarbonis L108
mdpJ	YP_001023560.1	124263090	Methylibium petroleiphilum PM1
mdpK	YP_001023559.1	124263089	Methylibium petroleiphilum PM1
Mpe_B0553	YP_001023558.1	124263088	Methylibium petroleiphilum PM1
Mpe_B0552	YP_001023557.1	124263087	Methylibium petroleiphilum PM1
Mpe_B0551	YP_001023556.1	124263086	Methylibium petroleiphilum PM1
BN115_3999	YP_006902223.1	410421774	Bordetella bronchiseptica MO149
NC_002928.3:4169127 . . . 4170563	NP_886002.1	33598359	Bordetella parapertussis 12822
NZ_GL982453.1:6380824 . . . 6382248	ZP_17009234	NZ_AFRQ01000000	Achromobacter xylosoxidans AXX-A

MVC Dehydratase (FIG. 13B—Also Applicable to Step G of FIG. 15, Step E of 16, Step G of FIG. 17, and Step F of FIG. 18)

Dehydration of MVC to butadiene is catalyzed by a MVC dehydratase enzyme (Step 13B) or by chemical dehydration. Exemplary dehydratase enzymes suitable for dehydrating MVC include oleate hydratase, acyclic 1,2-hydratase and linalool dehydratase enzymes. Oleate hydratases catalyze the reversible hydration of non-activated alkenes to their corresponding alcohols. Oleate hydratase enzymes disclosed in WO2011/076691 and WO 2008/119735 are incorporated by reference herein. Oleate hydratases from Elizabethkingia meningoseptica and Streptococcus pyogenes are encoded by ohyA and HMPREF0841_1446. Acyclic 1,2-hydratase enzymes (eg. EC 4.2.1.131) catalyze the dehydration of linear secondary alcohols, and are thus suitable candidates for the dehydration of MVC to butadiene. Exemplary 1,2-hydratase enzymes include carotenoid 1,2-hydratase, encoded by crtC of Rubrivivax gelatinosus (Steiger et al, Arch Biochem Biophys 414:51-8 (2003)), and lycopene 1,2-hydratase, encoded by cruF of Synechococcus sp. PCC 7002 and Gemmatimonas aurantiaca (Graham and Bryant, J Bacteriol 191: 2392-300 (2009); Takaichi et al, Microbiol 156: 756-63 (2010)). Dehydration oft-butyl alcohol, t-amyl alcohol and 2-methyl-MVC to isobutene, isoamylene and isoprene, respectively, is catalyzed by an unknown enzyme of Aquincola tertiaricarbonis L108 (Schaefer et al, AEM 78 (17): 6280-4 (2012); Schuster et al, J. Bacteriol 194:972-81 (2012); Schuster et al, J Bacteriol 194: 972-81 (2012)). This dehydratase enzyme is also a suitable enzyme candidate for dehydrating MVC to butadiene. The linalool dehydratase/isomerase of Castellaniella defragrans catalyzes the dehydration of linalool to myrcene, reactants similar in structure to MVC and butadiene (Brodkorb et al, J Biol Chem 285:30436-42 (2010)). Enzyme accession numbers and homologs are listed in the table below.

Protein	GenBank ID	GI Number	Organism
OhyA	ACT54545.1	254031735	Elizabethkingia meningoseptica
HMPREF0841_1446	ZP_07461147.1	306827879	Streptococcus pyogenes ATCC 10782
P700755_13397	ZP_01252267.1	91215295	Psychroflexus torquis ATCC 700755
RPB_2430	YP_486046.1	86749550	Rhodopseudomonas palustris
CrtC	AAO93124.1	29893494	Rubrivivax gelatinosus
CruF	YP_001735274.1	170078636	Synechococcus sp. PCC 7002
Ldi	E1XUJ2.1	403399445	Castellaniella defragrans
STEHIDRAFT_68678	EIM80109.1	389738914	Stereum hirsutum FP-91666 SS1
NECHADRAFT_82460	XP_003040778.1	302883759	Nectria haematococca mpVI 77-13-4
AS9A_2751	YP_004493998.1	333920417	Amycolicicoccus subflavus DQS3-9A1

Example VIII

Pathway for Converting 13BDO to MVC and/or Butadiene

FIG. 15 shows pathways for converting 13BDO to MVC and/or butadiene. Enzymes in FIG. 15 are A. 13BDO kinase, B. 3-hydroxybutyrylphosphate kinase, C. 3-hydroxybutyryldiphosphate lyase, D. 13BDO diphosphokinase, E. 13BDO dehydratase, F. 3-hydroxybutyrylphosphate lyase, G. MVC dehydratase or chemical reaction.

Enzyme candidates for catalyzing steps A, B, C, E and F of FIG. 15 are described below. Enzymes for step G are described above.

13BDO Kinase (FIG. 15, Step A)

Phosphorylation of 13BDO to 3-hydroxybutyrylphosphate is catalyzed by an alcohol kinase enzyme. Alcohol kinase enzymes catalyze the transfer of a phosphate group to a hydroxyl group. Kinases that catalyze transfer of a phosphate group to an alcohol group are members of the EC 2.7.1 enzyme class. The enzymes described herein and for FIG. 11, Step A describe several useful kinase enzymes in the EC 2.7.1 enzyme class.

3-Hydroxybutyrylphosphate Kinase (FIG. 15, Step B)

Alkyl phosphate kinase enzymes catalyze the transfer of a phosphate group to the phosphate group of an alkyl phosphate. The enzymes described herein and for FIG. 11 Step B naturally possess such activity or can be engineered to exhibit this activity. Kinases that catalyze transfer of a phosphate group to another phosphate group are members of the EC 2.7.4 enzyme class.

3-Hydroxybutyryldiphosphate Lyase (FIG. 15, Step C)

Diphosphate lyase enzymes catalyze the conversion of alkyl diphosphates to alkenes. Carbon-oxygen lyases that operate on phosphates are found in the EC 4.2.3 enzyme class. The table below lists several useful enzymes in EC class 4.2.3. Exemplary enzyme candidates were described above (see phosphate lyase section FIG. 11 Step C).

Enzyme Commission No. Enzyme Name
4.2.3.5               Chorismate synthase
4.2.3.15              Myrcene synthase
4.2.3.27              Isoprene synthase
4.2.3.36              Terpentriene sythase
4.2.3.46              (E,E)-alpha-Farnesene synthase
4.2.3.47              Beta-Farnesene synthase

13BDO Dehydratase (FIG. 15, Step D)

Exemplary dehydratase enzymes suitable for dehydrating 13BDO to MVC include oleate hydratases and acyclic 1,2-hydratases. Exemplary enzyme candidates are described above, including the MVC dehydratases, EC class 4.2.1.a Hydro-lyases and enzymes for FIG. 13 Step B (“13B”).

13BDO Diphosphokinase (FIG. 15, Step E)

Diphosphokinase enzymes catalyze the transfer of a diphosphate group to an alcohol group. The enzymes described below naturally possess such activity. Kinases that catalyze transfer of a diphosphate group are members of the EC 2.7.6 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.6 enzyme class.

Enzyme Commission No. Enzyme Name
2.7.6.1               ribose-phosphate diphosphokinase
2.7.6.2               thiamine diphosphokinase
2.7.6.3               2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
                      diphosphokinase
2.7.6.4               nucleotide diphosphokinase
2.7.6.5               GTP diphosphokinase

Of particular interest are ribose-phosphate diphosphokinase enzymes, which have been identified in Escherichia coli (Hove-Jenson et al., J Biol Chem, 1986, 261(15); 6765-71) and Mycoplasma pneumoniae M129 (McElwain et al, International Journal of Systematic Bacteriology, 1988, 38:417-423) as well as thiamine diphosphokinase enzymes. Exemplary thiamine diphosphokinase enzymes are found in Arabidopsis thaliana (Ajjawi, Plant Mol Biol, 2007, 65(1-2); 151-62).

Protein	GenBank ID	GI Number	Organism
prs	NP_415725.1	16129170	Escherichia coli
prsA	NP_109761.1	13507812	Mycoplasma pneumoniae M129
TPK1	BAH19964.1	222424006	Arabidopsis thaliana col
TPK2	BAH57065.1	227204427	Arabidopsis thaliana col

3-Hydroxybutyrylphosphate Lyase (FIG. 15, Step F)

Phosphate lyase enzymes catalyze the conversion of alkyl phosphates to alkenes. The enzymes described below, and in section for FIG. 11 Step C, naturally possess such activity or can be engineered to exhibit this activity. Carbon-oxygen lyases that operate on phosphates are found in the EC 4.2.3 enzyme class. The table below lists several relevant enzymes in EC class 4.2.3.

Enzyme Commission Number Enzyme Name
4.2.3.5                  Chorismate synthase
4.2.3.15                 Myrcene synthase
4.2.3.26                 Linalool synthase
4.2.3.27                 Isoprene synthase
4.2.3.36                 Terpentriene sythase
4.2.3.46                 (E,E)-alpha-Farnesene synthase
4.2.3.47                 Beta-Farnesene synthase
4.2.3.49                 Nerolidol synthase
4.2.3.—                  Methylbutenol synthase

Isoprene synthase enzymes catalyzes the conversion of dimethylallyl diphosphate to isoprene. Isoprene synthases can be found in several organisms including Populus alba (Sasaki et al., FEBS Letters, 2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et al., Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol., 137(2):700-712 (2005)), and Populus tremula x Populus alba, also called Populus canescens (Miller et al., Planta, 2001, 213 (3), 483-487). The crystal structure of the Populus canescens isoprene synthase was determined (Koksal et al, J Mol Biol 402:363-373 (2010)). Additional isoprene synthase enzymes are described in (Chotani et al., WO/2010/031079, Systems Using Cell Culture for Production of Isoprene; Cervin et al., US Patent Application 20100003716, Isoprene Synthase Variants for Improved Microbial Production of Isoprene). Another isoprene synthase-like enzyme from Pinus sabiniana, methylbutenol synthase, catalyzes the formation of 2-methyl-MVC (Grey et al, J Biol Chem 286: 20582-90 (2011)).

Protein	GenBank ID	GI Number	Organism
ispS	BAD98243.1	63108310	Populus alba
ispS	AAQ84170.1	35187004	Pueraria montana
ispS	CAC35696.1	13539551	Populus tremula × Populus alba
Tps-MBO1	AEB53064.1	328834891	Pinus sabiniana

Chorismate synthase (EC 4.2.3.5) participates in the shikimate pathway, catalyzing the dephosphorylation of 5-enolpyruvylshikimate-3-phosphate to chorismate. The enzyme requires reduced flavin mononucleotide (FMN) as a cofactor, although the net reaction of the enzyme does not involve a redox change. In contrast to the enzyme found in plants and bacteria, the chorismate synthase in fungi is also able to reduce FMN at the expense of NADPH (Macheroux et al., Planta 207:325-334 (1999)). Representative monofunctional enzymes are encoded by aroC of E. coli (White et al., Biochem. J 251:313-322 (1988)) and Streptococcus pneumoniae (Maclean and Ali, Structure 11:1499-1511 (2003)). Bifunctional fungal enzymes are found in Neurospora crassa (Kitzing et al., J Biol. Chem. 276:42658-42666 (2001)) and Saccharomyces cerevisiae (Jones et al., Mol. Microbiol. 5:2143-2152 (1991)).

Gene	GenBank Accession No.	GI No.	Organism
aroC	NP_416832.1	16130264	Escherichia coli
aroC	ACH47980.1	197205483	Streptococcus pneumoniae
U25818.1:19 . . . 1317	AAC49056.1	976375	Neurospora crassa
ARO2	CAA42745.1	3387	Saccharomyces cerevisiae

Myrcene synthase enzymes catalyze the dephosphorylation of geranyl diphosphate to beta-myrcene (EC 4.2.3.15). Exemplary myrcene synthases are encoded by MST2 of Solanum lycopersicum (van Schie et al, Plant Mol Biol 64:D473-79 (2007)), TPS-Myr of Picea abies (Martin et al, Plant Physiol 135:1908-27 (2004)) g-myr of Abies grandis (Bohlmann et al, J Biol Chem 272:21784-92 (1997)) and TPS10 of Arabidopsis thaliana (Bohlmann et al, Arch Biochem Biophys 375:261-9 (2000)). These enzymes were heterologously expressed in E. coli.

Protein	GenBank ID	GI Number	Organism
MST2	ACN58229.1	224579303	Solanum lycopersicum
TPS-Myr	AAS47690.2	77546864	Picea abies
G-myr	O24474.1	17367921	Abies grandis
TPS10	EC07543.1	330252449	Arabidopsis thaliana

Farnesyl diphosphate is converted to alpha-farnesene and beta-farnesene by alpha-farnesene synthase and beta-farnesene synthase, respectively. Exemplary alpha-farnesene synthase enzymes include TPS03 and TPS02 of Arabidopsis thaliana (Faldt et al, Planta 216:745-51 (2003); Huang et al, Plant Physiol 153:1293-310 (2010)), afs of Cucumis sativus (Mercke et al, Plant Physiol 135:2012-14 (2004), eafar of Malus x domestica (Green et al, Phytochem 68:176-88 (2007)) and TPS-Far of Picea abies (Martin, supra). An exemplary beta-farnesene synthase enzyme is encoded by TPS1 of Zea mays (Schnee et al, Plant Physiol 130:2049-60 (2002)).

Protein	GenBank ID	GI Number	Organism
TPS03	A4FVP2.1	205829248	Arabidopsis thaliana
TPS02	P0CJ43.1	317411866	Arabidopsis thaliana
TPS-Far	AAS47697.1	44804601	Picea abies
afs	AAU05951.1	51537953	Cucumis sativus
eafar	Q84LB2.2	75241161	Malus × domestica
TPS1	Q84ZW8.1	75149279	Zea mays

Example IX

Pathways for Converting Acrylyl-CoA to 3-Butene-2-01 and/or Butadiene

This example describes pathways for converting acrylyl-CoA to MVC, and further to butadiene. The conversion of acrylyl-CoA to MVC is accomplished in four enzymatic steps. Acrylyl-CoA and acetyl-CoA are first condensed to 3-oxopent-4-enoyl-CoA by 3-oxopent-4-enoyl-CoA thiolase, a beta-ketothiolase (Step 4A). The 3-oxopent-4-enoyl-CoA product is subsequently hydrolyzed to 3-oxopent-4-enoate by a CoA hydrolase, transferase or synthetase (Step 4B). Decarboxylation of the 3-ketoacid intermediate by 3-oxopent-4-enoate decarboxylase (Step 4C) yields 3-buten-2-one, which is further reduced to MVC by an alcohol dehydrogenase or ketone reductase (Step 4D). MVC is further converted to butadiene via chemical dehydration or by a dehydratase enzyme.

Enzymes and gene candidates for catalyzing but-3-en-2-ol and butadiene pathway reactions are described in further detail below. Enzymes for step E are described above.

3-oxopent-4-enoyl-CoA thiolase (FIG. 16, Step A)3-oxo-4-hydroxypentanoyl-CoA thiolase (FIG. 17, Step A)3-oxoadipyl-CoA thiolase (FIG. 18, Step A)

Acrylyl-CoA and acetyl-CoA are condensed to form 3-oxopent-4-enoyl-CoA by a beta-ketothiolase (EC 2.3.1.16). Beta-ketothiolase enzymes are also required for the conversion of lactoyl-CoA and acetyl-CoA to 3-oxo-4-hydroxypentanoyl-CoA (FIG. 5A) and succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA (FIG. 6A). Exemplary beta-ketothiolase enzymes are described below.

Beta-ketovaleryl-CoA thiolase catalyzes the formation of beta-ketovalerate from acetyl-CoA and propionyl-CoA. Zoogloea ramigera possesses two ketothiolases that can form beta-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a beta-oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., U.S. Pat. No. 5,958,745). The sequences of these genes or their translated proteins have not been reported, but several genes in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from R. eutropha.

Protein	GenBank ID	GI Number	Organism
phaA	YP_725941.1	113867452	Ralstonia eutropha
h16_A1713	YP_726205.1	113867716	Ralstonia eutropha
pcaF	YP_728366.1	116694155	Ralstonia eutropha
h16_B1369	YP_840888.1	116695312	Ralstonia eutropha
h16_A0170	YP_724690.1	113866201	Ralstonia eutropha
h16_A0462	YP_724980.1	113866491	Ralstonia eutropha
h16_A1528	YP_726028.1	113867539	Ralstonia eutropha
h16_B0381	YP_728545.1	116694334	Ralstonia eutropha
h16_B0662	YP_728824.1	116694613	Ralstonia eutropha
h16_B0759	YP_728921.1	116694710	Ralstonia eutropha
h16_B0668	YP_728830.1	116694619	Ralstonia eutropha
h16_A1720	YP_726212.1	113867723	Ralstonia eutropha
h16_A1887	YP_726356.1	113867867	Ralstonia eutropha
phbA	P07097.4	135759	Zoogloea ramigera
bktB	YP_002005382.1	194289475	Cupriavidus taiwanensis
Rmet_1362	YP_583514.1	94310304	Ralstonia metallidurans
Bphy_0975	YP_001857210.1	186475740	Burkholderia phymatum

Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat. Biotechnol. 21:796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser et al., J. Biol. Chem. 269:31383-31389 (1994)).

Protein	GenBank ID	GI Number	Organism
atoB	NP_416728	16130161	Escherichia coli
thlA	NP_349476.1	15896127	Clostridium acetobutylicum
thlB	NP_149242.1	15004782	Clostridium acetobutylicum
ERG10	NP_015297	6325229	Saccharomyces cerevisiae

Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J. Bacteriol. 176-6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J. Bacteriol. 169:3168-3174 (1987)). The P. putida enzyme is a homotetramer bearing 45% sequence homology to beta-ketothiolases involved in PHB synthesis in Ralstonia eutropha, fatty acid degradation by human mitochondria and butyrate production by Clostridium acetobutylicum (Harwood et al., supra). A beta-ketoadipyl-CoA thiolase in Pseudomonas knackmussii (formerly sp. B13) has also been characterized (Gobel et al., J. Bacteriol. 184:216-223 (2002); Kaschabek et al., supra).

Protein	GenBank ID	GI Number	Organism
pcaF	NP_743536.1	506695	Pseudomonas putida
pcaF	AAC37148.1	141777	Acinetobacter calcoaceticus
catF	Q8VPF1.1	75404581	Pseudomonas knackmussii

3-oxopent-4-enoyl-CoA Hydrolase, Transferase or Synthase (FIG. 16, Step B)3-oxo-4-hydroxypentanoyl-CoA Hydrolase, Transferase or Synthase (FIG. 17, Step B)3,4-dihydroxypentanoyl-CoA Hydrolase, Transferase or Synthase (FIG. 17, Step F)oxoadipyl-CoA Hydrolase, Transferase or Synthase (FIG. 18, Step 6B)

Acyl-CoA hydrolase, transferase and synthase enzymes convert acyl-CoA moieties to their corresponding acids. Such an enzyme can be utilized to convert, for example, 3-oxopent-4-enoyl-CoA to 3-oxopent-4-enoyl-CoA, 3-oxo-4-hydroxypentanoyl-CoA to 3-oxo-4-hydroxypentanoate, 3,4-dihydroxypentanoyl-CoA to 3,4-dihydroxypentanoate or oxoadipyl-CoA to oxoadipate.

CoA hydrolase or thioesterase enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to their corresponding acids. Several CoA hydrolases with different substrate ranges are suitable for hydrolyzing 3-oxopent-4-enoyl-CoA, 3-oxo-4-hydroxypentanoyl-CoA, 3,4-dihydroxypentanoyl-CoA or oxoadipyl-CoA substrates to their corresponding acids. For example, the enzyme encoded by acot12 from Rattus norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB (Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al., J Biol Chem, 2006, 281(16):11028-38). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant. Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)). Additional enzymes with aryl-CoA hydrolase activity include the palmitoyl-CoA hydrolase of Mycobacterium tuberculosis (Wang et al., Chem. Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of E. coli encoded by entH (Guo et al., Biochemistry 48:1712-1722 (2009)). Additional CoA hydrolase enzymes are described above.

Gene name	GenBank Accession #	GI#	Organism
acot12	NP_570103.1	18543355	Rattus norvegicus
tesB	NP_414986	16128437	Escherichia coli
acot8	CAA15502	3191970	Homo sapiens
acot8	NP_570112	51036669	Rattus norvegicus
tesA	NP_415027	16128478	Escherichia coli
ybgC	NP_415264	16128711	Escherichia coli
paaI	NP_415914	16129357	Escherichia coli
ybdB	NP_415129	16128580	Escherichia coli
ACH1	NP_009538	6319456	Saccharomyces cerevisiae
Rv0098	NP_214612.1	15607240	Mycobacterium tuberculosis
entH	AAC73698.1	1786813	Escherichia coli

CoA hydrolase enzymes active on 3-hydroxyacyl-CoA and 3-oxoacyl-CoA intermediates are well known in the art 3-Hydroxyisobutyryl-CoA hydrolase is active on 3-hydroxyacyl-CoA substrates (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Similar gene candidates can also be identified by sequence homology, including hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus. An exemplary 3-oxoacyl-CoA hydrolase is MKS2 of Solanum lycopersicum (Yu et al, Plant Physiol 154:67-77 (2010)). The native substrate of this enzyme is 3-oxo-myristoyl-CoA, which produces a C14 chain length product.

Gene name	GenBank Accession #	GI#	Organism
fadM	NP_414977.1	16128428	Escherichia coli
hibch	Q5XIE6.2	146324906	Rattus norvegicus
hibch	Q6NVY1.2	146324905	Homo sapiens
hibch	P28817.2	2506374	Saccharomyces cerevisiae
BC_2292	AP09256	29895975	Bacillus cereus
MKS2	ACG69783.1	196122243	Solanum lycopersicum

CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Several transformations require a CoA transferase to acyl-CoA substrates to their corresponding acid derivatives. CoA transferase enzymes are known in the art and described below.

The gene products of cat1, cat2, and cat3 of Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., Proc. Natl. Acad. Sci U.S.A 105:2128-2133 (2008); Sohling et al., J Bacteriol. 178:871-880 (1996)) Similar CoA transferase activities are also present in Trichomonas vaginalis, Trypanosoma brucei, Clostridium aminobutyricum and Polphyromonas gingivalis (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004); van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)).

Protein	GenBank ID	GI Number	Organism
cat1	P38946.1	729048	Clostridium kluyveri
cat2	P38942.2	172046066	Clostridium kluyveri
cat3	EDK35586.1	146349050	Clostridium kluyveri
TVAG_395550	XP_001330176	123975034	Trichomonas vaginalis G3
Tb11.02.0290	XP_828352	71754875	Trypanosoma brucei
cat2	CAB60036.1	6249316	Clostridium aminobutyricum
cat2	NP_906037.1	34541558	Porphyromonas gingivalis W83

A fatty acyl-CoA transferase that utilizes acetyl-CoA as the CoA donor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al., 33:902-908 (1968)). This enzyme has a broad substrate range on substrates of chain length C3-C6 (Sramek et al., Arch Biochem Biophys 171:14-26 (1975)) and has been shown to transfer the CoA moiety to acetate from a variety of branched and linear 3-oxo and acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ. Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced at the transcriptional level by acetoacetate, so modification of regulatory control may be necessary for engineering this enzyme into a pathway (Pauli et al., Eur. J Biochem. 29:553-562 (1972)) Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583 (1990); Wiesenbom et al., Appl Environ Microbiol 155:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Gene	GI #	Accession No.	Organism
atoA	2492994	P76459.1	Escherichia coli
atoD	2492990	P76458.1	Escherichia coli
actA	62391407	YP_226809.1	Corynebacterium glutamicum
cg0592	62389399	YP_224801.1	Corynebacterium glutamicum
ctfA	15004866	NP_149326.1	Clostridium acetobutylicum
ctfB	15004867	NP_149327.1	Clostridium acetobutylicum
ctfA	31075384	AAP42564.1	Clostridium saccharoperbutylacetonicum
ctfB	31075385	AAP42565.1	Clostridium saccharoperbutylacetonicum

Beta-ketoadipyl-CoA transferase, also known as succinyl-CoA:3:oxoacid-CoA transferase, is active on 3-oxoacyl-CoA substrates. This enzyme is encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek et al., J Bacteriol. 184:207-215 (2002)). Similar enzymes are found in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)), Streptomyces coelicolor and Pseudomonas knackmussii (formerly sp. B13) (Gobel et al., J Bacteriol. 184:216-223 (2002); Kaschabek et al., J Bacteriol. 184:207-215 (2002)). Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases have been characterized in Helicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein Expr. Purif 53:396-403 (2007)) and Homo sapiens (Fukao, T., et al., Genomics 68:144-151 (2000); Tanaka, H., et al., Mol Hum Reprod 8:16-23 (2002)). Genbank information related to these genes is summarized below.

Gene	GI #	Accession No.	Organism
pcaI	24985644	AAN69545.1	Pseudomonas putida
pcaJ	26990657	NP_746082.1	Pseudomonas putida
pcaI	50084858	YP_046368.1	Acinetobacter sp. ADP1
pcaJ	141776	AAC37147.1	Acinetobacter sp. ADP1
pcaI	21224997	NP_630776.1	Streptomyces coelicolor
pcaJ	21224996	NP_630775.1	Streptomyces coelicolor
catI	75404583	Q8VPF3	Pseudomonas knackmussii
catJ	75404582	Q8VPF2	Pseudomonas knackmussii
HPAG1_0676	108563101	YP_627417	Helicobacter pylori
HPAG1_0677	108563102	YP_627418	Helicobacter pylori
ScoA	16080950	NP_391778	Bacillus subtilis
ScoB	16080949	NP_391777	Bacillus subtilis
OXCT1	NP_000427	4557817	Homo sapiens
OXCT2	NP_071403	11545841	Homo sapiens

The conversion of acyl-CoA substrates to their acid products can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes. CoA synthases that convert ATP to ADP (ADP-forming) are reversible and react in the direction of acid formation, whereas AMP forming enzymes only catalyze the activation of an acid to an acyl-CoA. For fatty acid formation, deletion or attenuation of AMP forming enzymes will reduce backflux. ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a broad substrate range (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al, supra). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra; Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). An additional candidate is succinyl-CoA synthetase, encoded by sucCD of E. coli and LSC1 and LSC2 genes of Saccharomyces cerevisiae. These enzymes catalyze the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP in a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., J. Am. Chem. Soc. 123:5822-5823 (2001)).

Protein	GenBank ID	GI Number	Organism
AF1211	NP_070039.1	11498810	Archaeoglobus fulgidus
AF1983	NP_070807.1	11499565	Archaeoglobus fulgidus
scs	YP_135572.1	55377722	Haloarcula marismortui
PAE3250	NP_560604.1	18313937	Pyrobaculum aerophilum str. IM2
sucC	NP_415256.1	16128703	Escherichia coli
sucD	AAC73823.1	1786949	Escherichia coli
LSC1	NP_014785	6324716	Saccharomyces cerevisiae
LSC2	NP_011760	6321683	Saccharomyces cerevisiae
paaF	AAC24333.2	22711873	Pseudomonas putida
matB	AAC83455.1	3982573	Rhizobium leguminosarum

3-Oxopent-4-enoate Decarboxylase, 3-oxoadipate Decarboxylase (FIG. 16, Step C, FIG. 18, Step C)

Decarboxylase enzymes suitable for decarboxylating 3-ketoacids such as 3-oxopent-4-enoate (FIG. 4C) and 3-oxoadipate (FIG. 6C) include acetoacetate decarboxylase (EC 4.1.1.4), arylmalonate decarboxylase and 3-oxoacid decarboxylase (EC 4.1.1.-). The 3-oxoacid decarboxylase of Lycopersicon hirsutum f. glabratum, encoded by MKS1, decarboxylates a range of 3-ketoacids to form methylketones (Yu et al, Plant Physiol 154: 67-77 (2010)). This enzyme has been functionally expressed in E. coli, where it was active on the substrate 3-ketomyristic acid. Homologous 3-oxoacid decarboxylase genes in Solanum lycopersicum are listed in the table below. Acetoacetate decarboxylase decarboxylates acetoacetate to acetone. The enzyme from Clostridium acetobutylicum, encoded by adc, has a broad substrate specificity and has been shown to decarboxylate 2-methyl-3-oxobutyrate, 3-oxohexanoate, phenyl acetoacetate and 2-ketocyclohexane-1-carboxylate (Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984); Benner and Rozzell, J. Am. Chem. Soc. 103:993-994 (1981); Autor et al., J Biol. Chem. 245:5214-5222 (1970)). A similar acetoacetate decarboxylase has also been characterized in Clostridium beijerinckii (Ravagnani et al., Mol. Microbiol 37:1172-1185 (2000)). An acetoacetate decarboxylase enzyme from Paenibacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylase 3-oxopentanoate (Matiasek et al., Curr. Microbiol 42:276-281 (2001)). The P. polymyxa genome encodes several acetoacetate decarboxylase enzymes, listed in the table below (Niu et al, J Bacteriol 193: 5862-3 (2011)). Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). Additional gene candidates in other organisms, including Clostridium botulinum and Bacillus amyloliquefaciens, can be identified by sequence homology. Arylmalonate decarboxylase (AMDase) catalyzes the decarboxylation of malonate and a range of alpha-substituted derivatives (phenylmalonic acid, 2-methyl-2-phenylmalonic acid, 2-methyl-2-napthylmalonic acid, 2-thienylmalonic acid). AMDase is unusual in that it does not require biotin or other cofactors for activity. Exemplary AMDase enzymes are found in US Patent Application 2010/0311037. A codon optimized variant of the B. bronchiseptica enzyme was heterologously expressed in E. coli and crystallized. Acetolactate decarboxylase enzyme candidates, described above (FIG. 2B) are also applicable here.

Protein	GenBank ID	GI Number	Organism
MKS1	ADK38535.1	300836815	Lycopersicon hirsutum f. glabratum
MKS1a	ADK38537.1	300836819	Solanum lycopersicum
MKS1b	ADK38538.1	300836821	Solanum lycopersicum
MKS1c	ADK38543.1	300836832	Solanum lycopersicum
MKS1d	ADK38539.1	300836824	Solanum lycopersicum
MKS1e	ADK38540.1	300836826	Solanum lycopersicum
adc	NP_149328.1	15004868	Clostridium acetobutylicum
adc	AAP42566.1	31075386	Clostridium saccharoperbutylacetonicum
adc	YP_001310906.1	150018652	Clostridium beijerinckii
Adc3	YP_005960063.1	386041109	Paenibacillus polymyxa
Adc1	YP_005958789.1	386039835	Paenibacillus polymyxa
CLL_A2135	YP_001886324.1	187933144	Clostridium botulinum
RBAM_030030	YP_001422565.1	154687404	Bacillus amyloliquefaciens
S54007.1:545 . . . 1267	AAC60426.1	298239	Bordetella bronchiseptica KU1201

Alternatively, decarboxylation of 3-ketoacids can occur spontaneously in the absence of a decarboxylase enzyme. 3-Ketoacids are known to be inherently unstable and prone to decarboxylation (Kornberg et al, Fed Proc 6:268 (1947)). In one recent study, high yields of methyl ketones were formed from 3-oxoacids in reaction mixtures lacking decarboxylase enzymes (Goh et al, AEM 78: 70-80 (2012)).

3-Buten-2-one Reductase (FIG. 16, Step D)

4-Oxopentanoate Reductase (FIG. 18, Step D)

3-Oxo-4-hydroxypentanoate Reductase (FIG. 17, Step C)

Reduction of 3-buten-2-one to MVC, 4-oxopentanoate to 4-hydroxypentanoate, or 3-oxo-4-hydroxypentanoate to 3,4-dihydroxypentanoate, is catalyzed by secondary alcohol dehydrogenase or ketone reductase enzymes. Secondary alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der Oost et al., Eur. J. Biochem. 268:3062-3068 (2001)). The cloning of the bdhA gene from Rhizobium (Sinorhizobium) meliloti into E. coli conferred the ability to utilize 3-hydroxybutyrate as a carbon source (Aneja and Charles, J. Bacteriol. 181(3):849-857 (1999)). Additional gene candidates can be found in Pseudomonas fragi (Ito et al., J. Mol. Biol. 355(4) 722-733 (2006)) and Ralstonia pickettii (Takanashi et al., Antonie van Leeuwenoek, 95(3):249-262 (2009)). Recombinant 3-ketoacid reductase enzymes with broad substrate range and high activity have been characterized in US Application 2011/0201072, and are incorporated by reference herein. The mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). Yet another secondary ADH, sadH of Candida parapsilosis, demonstrated activity on 3-oxobutanol (Matsuyama et al. J Mol Cat B Enz, 11:513-521 (2001)). Enzyme candidates for converting acrolein to 2,3-butanediol (Step 2C) and 2-butanone to 2-butanol (Step E) are also applicable here.

Gene	GenBank Accession No.	GI No.	Organism
adh	AAA23199.2	60592974	Clostridium beijerinckii NRRL B593
adh	P14941.1	113443	Thermoanaerobacter brockii HTD4
sadh	CAD36475	21615553	Rhodococcus ruber
adhA	AAC25556	3288810	Pyrococcus furiosus
PRK13394	BAD86668.1	57506672	Pseudomonas fragi
Bdh1	BAE72684.1	84570594	Ralstonia pickettii
Bdh2	BAE72685.1	84570596	Ralstonia pickettii
Bdh3	BAF91602.1	158937170	Ralstonia pickettii
bdh	AAA58352.1	177198	Homo sapiens
sadh	BAA24528.1	2815409	Candida parapsilosis

Allyl alcohol dehydrogenase enzymes are suitable for reducing 3-buten-2-one to MVC. An exemplary allyl alcohol dehydrogenase is the NtRed-1 enzyme from Nicotiana tabacum (Matsushima et al, Bioorg Chem 36: 23-8 (2008)). A similar enzyme has been characterized in Pseudomonas putida MB1 but the enzyme has not been associated with a gene to date (Malone et al, AEM 65: 2622-30 (1999)). Yet another allyl alcohol dehydrogenase is the geraniol dehydrogenase enzymes of Castellaniella defragrans, Carpoglyphus lactis and Ocimum basilicum (Lueddeke et al, AEM 78:2128-36 (2012)).

Gene	GenBank Accession No.	GI No.	Organism
NT-RED1	BAA89423	6692816	Nicotiana tabacum
geoA	CCF55024.1	372099287	Castellaniella defragrans
GEDH1	Q2KNL6.1	122200955	Ocimum basilicum
GEDH	BAG32342.1	188219500	Carpoglyphus lactis

3-Oxo-4-hydroxypentanoyl-CoA Reductase (FIG. 17, Step E)

Reduction of 3-oxo-4-hydroxypentanoyl-CoA to 3,4-dihydroxypentanoyl-CoA (FIG. 5E) is catalyzed by a 3-hydroxyacyl-CoA dehydrogenase (also called 3-oxoacyl-CoA reductase). 3-Hydroxyacyl-CoA dehydrogenase enzymes are often involved in fatty acid beta-oxidation and aromatic degradation pathways. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). Another 3-hydroxyacyl-CoA dehydrogenase from E. coli is paaH (Ismail et al., European Journal of Biochemistry 270:3047-3054 (2003)). Additional 3-oxoacyl-CoA enzymes include the gene products of phaC in Pseudomonas putida (Olivera et al., Proc. Natl. Acad. Sci U.S.A 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens (Di et al., 188:117-125 (2007)). These enzymes catalyze the reversible oxidation of 3-hydroxyadipyl-CoA to 3-oxoadipyl-CoA during the catabolism of phenylacetate or styrene. Other suitable enzyme candidates include AAO72312.1 from E. gracilis (Winkler et al., Plant Physiology 131:753-762 (2003)) and paaC from Pseudomonas putida (Olivera et al., PNAS USA 95:6419-6424 (1998)). Enzymes catalyzing the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd of Clostridium acetobutylicum (Youngleson et al., J Bacteriol. 171:6800-6807 (1989)), phbB from Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)), phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309 (2006)) and paaH1 of Ralstonia eutropha (Machado et al, Met Eng, In Press (2012)). The Z. ramigera enzyme is NADPH-dependent and also accepts 3-oxopropionyl-CoA as a substrate (Ploux et al., Eur. J Biochem. 174:177-182 (1988)). Additional genes include phaB in Paracoccus denitrificans, Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al., J Biol. Chem. 207:631-638 (1954)). The enzyme from Paracoccus denitrificans has been functionally expressed and characterized in E. coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)). The enzyme from Candida tropicalis is a component of the peroxisomal fatty acid beta-oxidation multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active on acetoacetyl-CoA. The domain has been functionally expressed in E. coli, a crystal structure is available, and the catalytic mechanism is well-understood (Ylianttila et al., Biochem Biophys Res Commun 324:25-30 (2004); Ylianttila et al., J Mol Biol 358:1286-1295 (2006)). 3-Hydroxyacyl-CoA dehydrogenases that accept longer acyl-CoA substrates (eg. EC 1.1.1.35) are typically involved in beta-oxidation. An example is HSD17B10 in Bos taurus (Wakil et al., J Biol. Chem. 207:631-638 (1954)). The pig liver enzyme is preferentially active on short and medium chain acyl-CoA substrates whereas the heart enzyme is less selective (He et al, Biochim Biophys Acta 1392:119-26 (1998)). The S. cerevisiae enzyme FOX2 is active in beta-degradation pathways and also has enoyl-CoA hydratase activity (Hiltunen et al, J Biol Chem 267: 6646-6653 (1992)).

Protein	GENBANK ID	GI NUMBER	ORGANISM
fadB	P21177.2	119811	Escherichia coli
fadJ	P77399.1	3334437	Escherichia coli
paaH	NP_415913.1	16129356	Escherichia coli
Hbd2	EDK34807.1	146348271	Clostridium kluyveri
Hbd1	EDK32512.1	146345976	Clostridium kluyveri
phaC	NP_745425.1	26990000	Pseudomonas putida
paaC	ABF82235.1	106636095	Pseudomonas fluorescens
HSD17B10	O02691.3	3183024	Bos taurus
phbB	P23238.1	130017	Zoogloea ramigera
phaB	YP_353825.1	77464321	Rhodobacter sphaeroides
paaH1	CAJ91433.1	113525088	Ralstonia eutropha
phaB	BAA08358	675524	Paracoccus denitrificans
Hbd	NP_349314.1	15895965	Clostridium acetobutylicum
Hbd	AAM14586.1	20162442	Clostridium beijerinckii
Msed_1423	YP_001191505	146304189	Metallosphaera sedula
Msed_0399	YP_001190500	146303184	Metallosphaera sedula
Msed_0389	YP_001190490	146303174	Metallosphaera sedula
Msed_1993	YP_001192057	146304741	Metallosphaera sedula
Fox2	Q02207	399508	Candida tropicalis
HSD17B10	O02691.3	3183024	Bos taurus
HADH	NP_999496.1	47523722	Bos taurus
3HCDH	AAO72312.1	29293591	Euglena gracilis
FOX2	NP_012934.1	6322861	Saccharomyces cerevisiae

Example X

Pathways for Converting Lactoyl-CoA to MVC and/or Butadiene

This example describes pathways for converting lactoyl-CoA to MVC, and further to butadiene. The conversion of lactoyl-CoA to MVC is accomplished in four enzymatic steps. Lactoyl-CoA and acetyl-CoA are first condensed to 3-oxo-4-hydroxypentanoyl-CoA by 3-oxo-4-hydroxypentanoyl-CoA thiolase, a beta-ketothiolase (Step 17A). In one pathway, the 3-oxo-4-hydroxypentanoyl-CoA product is converted to its corresponding acid by a CoA hydrolase, transferase or synthetase (Step 17B). Reduction of the 3-oxo ketone by an alcohol dehydrogenase yields 3,4-dihydroxypentanoate (Step 17C). Alternately, 3,4-dihydroxypentanoate intermediate is formed from 3-oxo-4-hydroxypentanoyl-CoA by a 3-oxo-4-hydroxypentanoyl-CoA reductase and a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase (Steps E and F, respectively). Decarboxylation of 3,4-dihydroxypentanoate yields MVC (Step 17D). MVC is further converted to butadiene via chemical dehydration or by a dehydratase enzyme (Step 17G). In an alternate pathway, 3,4-dihydroxypentanoate is dehydrated to 4-oxopentanoate by a diol dehydratase (Step 17H). 4-Oxopentanoate is reduced to 4-hydroxypentanoate, and then decarboxylated to MVC by an alkene-forming decarboxylase (Steps 17I-17J).

Enzymes and gene candidates for catalyzing but-3-en-2-ol and butadiene pathway reactions are described in further detail below. Enzymes for catalyzing steps A, B, C, E, F, G and H are described above.

3,4-Dihydroxypentanoate Decarboxylase (FIG. 17, Step D)

Olefin-forming decarboxylase enzymes suitable for converting 3,4-dihydroxypentanoate to MVC include mevalonate diphosphate decarboxylase (MDD, EC 4.1.1.33) and similar enzymes. MDD participates in the mevalonate pathway for isoprenoid biosynthesis, where it catalyzes the ATP-dependent decarboxylation of mevalonate diphosphate to isopentenyl diphosphate. The MDD enzyme of S. cerevisiae was heterolgously expressed in E. coli, where it was shown to catalyze the decarboxylation of 3-hydroxyacids to their corresponding alkenes (WO 2010/001078; Gogerty and Bobik, Appl. Environ. Microbiol., 8004-8010, Vol. 76, No. 24 (2010)). Products formed by this enzyme include isobutylene, propylene and ethylene. Two evolved variants of the S. cerevisiae MDD, ScMDD1 (1145F) and ScMMD2 (R74H), achieved 19-fold and 38-fold increases in isobutylene-forming activity compared to the wild-type enzyme (WO 2010/001078), Other exemplary MDD genes are MVD in Homo sapiens and MDD in Staphylococcus aureus and Trypsonoma brucei (Toth et al., J Biol. Chem. 271:7895-7898 (1996); Byres et al., J Mol. Biol. 371:540-553 (2007)).

Protein	GenBank ID	GI Number	Organism
MDD	NP_014441.1	6324371	Saccharomyces cerevisiae
MVD	NP_002452.1	4505289	Homo sapiens
MDD	ABQ48418.1	147740120	Staphylococcus aureus
MDD	EAN78728.1	70833224	Trypsonoma brucei

4-Hydroxypentanoate Decarboxylase (FIG. 17, Step J and FIG. 18, Step E)

An olefin-forming decarboxylase enzyme catalyzes the conversion of 4-hydroxypentanoate to MVC. An exemplary terminal olefin-forming fatty acid decarboxylase is encoded by the oleT gene product of Jeotgalicoccus sp. ATCC8456 (Rude et al, AEM 77(5):1718-27 (2011)). This enzyme is a member of the cytochrome P450 family of enzymes and is similar to P450s that catalyze fatty acid hydroxylation. OleT and homologs are listed in the table below. Additional olefin-forming fatty acid decarboxylase enzymes are described in US 2011/0196180 and WO/2013028792.

Protein	GenBank ID	GI Number	Organism
oleT	ADW41779.1	320526718	Jeotgalicoccus sp. ATCC8456
MCCL_0804	BAH17511.1	222120176	Macrococcus caseolyticus
SPSE _1582	ADX76840.1	323464687	Staphylococcus pseudintermedius
faaH	ADC49546.1	288545663	Bacillus pseudofirmus
cypC2	EGQ19322.1	339614630	Sporosarcina newyorkensis
cypC	BAK15372.1	32743900	Solibacillus silvestris
Bcoam_010100017440	ZP_03227611.1	205374818	Bacillus coahuilensis
SYNPCC7002_A2265	YP_001735499.1	170078861	Synechococcus sp. PCC 7002
Cyan7822_1848	YP_003887108.1	307151724	Cyanothece sp. PCC 7822
PCC7424_1874	YP_002377175	218438846	Cyanothece sp. PCC 7424
LYNGBM3L 11290	ZP_08425909.1	332705833	Lyngbya majuscule 3L
LYNGBM3L_74520	ZP_08432358.1	332712432	Lyngbya majuscule 3L
Hoch_0800	YP_003265309	262194100	Haliangium ochraceum DSM 14365

3,4-Dihydroxypentanoate Dehydratase (FIG. 17, Step H)

A diol dehydratase enzyme with activity on 3,4-dihydroxypentanoate is required to form 4-oxopentanoate in FIG. 5H. Exemplary diol dehydratase enzymes described above for the dehydration of 2,3-butanediol to 2-butanol are also applicable here. Additional diol dehydratase enzymes are listed in the table below.

Enzyme Commission No. Enzyme Name
4.2.1.5               arabinonate dehydratase
4.2.1.6               galactonate dehydratase
4.2.1.7               altronate dehydratase
4.2.1.8               mannonate dehydratase
4.2.1.9               dihydroxy-acid dehydratase
4.2.1.12              phosphogluconate dehydratase
4.2.1.25              L-arabinonate dehydratase
4.2.1.28              propanediol dehydratase
4.2.1.30              glycerol dehydratase
4.2.1.32              L(+)-tartrate dehydratase
4.2.1.39              gluconate dehydratase
4.2.1.40              glucarate dehydratase
4.2.1.41              5-dehydro-4-deoxyglucarate dehydratase
4.2.1.42              galactarate dehydratase
4.2.1.43              2-dehydro-3-deoxy-L-arabinonate dehydratase
4.2.1.44              myo-inosose-2 dehydratase
4.2.1.45              CDP-glucose 4,6-dehydratase
4.2.1.46              dTDP-glucose 4,6-dehydratase
4.2.1.47              GDP-mannose 4,6-dehydratase
4.2.1.76              UDP-glucose 4,6-dehydratase
4.2.1.81              D(−)-tartrate dehydratase
4.2.1.82              xylonate dehydratase
4.2.1.90              L-rhamnonate dehydratase
4.2.1.109             methylthioribulose 1-phosphate dehydratase

Diol dehydratase enzymes include dihydroxy-acid dehydratase (EC 4.2.1.9), propanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30) and myo-inositose dehydratase (EC 4.2.1.44).

Adenosylcobalamin-dependent diol dehydratases contain alpha, beta and gamma subunits, which are all required for enzyme function. Exemplary propanediol dehydratase candidates are found in Klebsiella pneumoniae (Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480 (1976); Tobimatsu et al., Biosci. Biotechnol Biochem. 62:1774-1777 (1998)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J Biol. Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase gene candidates in other organisms are well known in the art (e.g. U.S. Pat. No. 5,686,276).

Protein	GenBank ID	GI Number	Organism
pddC	AAC98386.1	4063704	Klebsiella pneumoniae
pddB	AAC98385.1	4063703	Klebsiella pneumoniae
pddA	AAC98384.1	4063702	Klebsiella pneumoniae
pduC	AAB84102.1	2587029	Salmonella typhimurium
pduD	AAB84103.1	2587030	Salmonella typhimurium
pduE	AAB84104.1	2587031	Salmonella typhimurium
pddA	BAA08099.1	868006	Klebsiella oxytoca
pddB	BAA08100.1	868007	Klebsiella oxytoca
pddC	BAA08101.1	868008	Klebsiella oxytoca
pduC	CAC82541.1	18857678	Lactobacillus collinoides
pduD	CAC82542.1	18857679	Lactobacillus collinoides
pduE	CAD01091.1	18857680	Lactobacillus collinoides

Enzymes in the glycerol dehydratase family (EC 4.2.1.30) are also diol dehydratases. Exemplary gene candidates are encoded by gldABC and dhaB123 in Klebsiella pneumoniae (World Patent WO 2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480 (1976)), dhaBCE in Clostridium pasteuranum (Macis et al., FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacter freundii (Seyfried et al., J Bacteriol. 178:5793-5796 (1996)). Variants of the B12-dependent diol dehydratase from K. pneumoniae with 80- to 336-fold enhanced activity were recently engineered by introducing mutations in two residues of the beta subunit (Qi et al., J. Biotechnol. 144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivation kinetics were developed by DuPont using error-prone PCR (WO 2004/056963).

Protein	GenBank ID	GI Number	Organism
gldA	AAB96343.1	1778022	Klebsiella pneumonia
gldB	AAB96344.1	1778023	Klebsiella pneumonia
gldC	AAB96345.1	1778024	Klebsiella pneumoniae
dhaB1	ABR78884.1	150956854	Klebsiella pneumoniae
dhaB2	ABR78883.1	150956853	Klebsiella pneumoniae
dhaB3	ABR78882.1	150956852	Klebsiella pneumoniae
dhaB	AAC27922.1	3360389	Clostridium pasteuranum
dhaC	AAC27923.1	3360390	Clostridium pasteuranum
dhaE	AAC27924.1	3360391	Clostridium pasteuranum
dhaB	P45514.1	1169287	Citrobacter freundii
dhaC	AAB48851.1	1229154	Citrobacter freundii
dhaE	AAB48852.1	1229155	Citrobacter freundii

If a B12-dependent diol dehydratase is utilized, heterologous expression of the corresponding reactivating factor is recommended. B12-dependent diol dehydratases are subject to mechanism-based suicide activation by substrates and some downstream products. Inactivation, caused by a tight association with inactive cobalamin, can be partially overcome by diol dehydratase reactivating factors in an ATP-dependent process. Regeneration of the B12 cofactor requires an additional ATP. Diol dehydratase regenerating factors are two-subunit proteins. Exemplary candidates are found in Klebsiella oxytoca (Mori et al., J Biol. Chem. 272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633-6639 (1997); Chen et al., J Bacteriol. 176:5474-5482 (1994)), Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol Lett. 209:69-74 (2002)), Klebsiella pneumonia (World Patent WO 2008/137403).

Protein	GenBank ID	GI Number	Organism
ddrA	AAC15871	3115376	Klebsiella oxytoca
ddrB	AAC15872	3115377	Klebsiella oxytoca
pduG	AAB84105	16420573	Salmonella typhimurium
pduH	AAD39008	16420574	Salmonella typhimurium
pduG	YP_002236779	206579698	Klebsiella pneumonia
pduH	YP_002236778	206579863	Klebsiella pneumonia
pduG	CAD01092	29335724	Lactobacillus collinoides
pduH	AJ297723	29335725	Lactobacillus collinoides

B12-independent diol dehydratase enzymes are glycyl radicals that utilize S-adenosylmethionine (SAM) as a cofactor and function under strictly anaerobic conditions. The glycerol dehydrogenase and corresponding activating factor of Clostridium butyricum, encoded by dhaB1 and dhaB2, have been well-characterized (O'Brien et al., Biochemistry 43:4635-4645 (2004); Raynaud et al., Proc. Natl. Acad. Sci U.S.A 100:5010-5015 (2003)). This enzyme was recently employed in a 1,3-propanediol overproducing strain of E. coli and was able to achieve very high titers of product (Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additional B12-independent diol dehydratase enzyme and activating factor from Roseburia inulinivorans was shown to catalyze the conversion of 2,3-butanediol to 2-butanone (US 2009/09155870). A B12-independent, oxygen sensitive and membrane bound diol dehydratase from Clostridium glycolycum catalyzes the dehydration of 1,2-ethanediol to acetaldehyde; however the gene has not been identified to date (Hartmanis et al, Arch Biochem Biophys, 245:144-152 (1986)).

Protein	GenBank ID	GI Number	Organism
dhaB1	AAM54728.1	27461255	Clostridium butyricum
dhaB2	AAM54729.1	27461256	Clostridium butyricum
rdhtA	ABC25539.1	83596382	Roseburia inulinivorans
rdhtB	ABC25540.1	83596383	Roseburia inulinivorans

Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independent enzyme participating in branched-chain amino acid biosynthesis. In its native role, it converts 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate, a precursor of isoleucine. In valine biosynthesis the enzyme catalyzes the dehydration of 2,3-dihydroxy-isovalerate to 2-oxoisovalerate. The DHAD from Sulfolobus solfataricus has a broad substrate range and activity of a recombinant enzyme expressed in E. coli was demonstrated on a variety of aldonic acids (KIM et al., J. Biochem. 139:591-596 (2006)). The S. solfataricus enzyme is tolerant of oxygen unlike many diol dehydratase enzymes. The E. coli enzyme, encoded by ilvD, is sensitive to oxygen, which inactivates its iron-sulfur cluster (Flint et al., J. Biol. Chem. 268:14732-14742 (1993)). Similar enzymes have been characterized in Neurospora crassa (Altmiller et al., Arch. Biochem. Biophys. 138:160-170 (1970)), Salmonella typhimurium (Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)) and Corynebacterium glutamicum (Holatko et al, J Biotechnol 139:203-10 (2009)). Other groups have shown that the overexpression of one or more Aft proteins or homologs thereof improves DHAD activity (US Patent Application 2011/0183393. In Saccharomyces cerevisiae, the Aft1 and Aft2 proteins are transcriptional activators that regulate numerous proteins related to the acquisition, compartmentalization, and utilization of iron.

Protein	GenBank ID	GI Number	Organism
ilvD	NP_344419.1	15899814	Sulfolobus solfataricus
ilvD	AAT48208.1	48994964	Escherichia coli
ilvD	NP_462795.1	16767180	Salmonella typhimurium
ilvD	XP_958280.1	85090149	Neurospora crassa
ilvD	CAB57218.1	6010023	Corynebacterium glutamicum
Aft1	P22149.2	1168370	Saccharomyces cerevisiae
Aft2	Q08957.1	74583775	Saccharomyces cerevisiae

Example X1

Pathways for Converting Succinyl-CoA to MVC and/or Butadiene

This example describes pathways for converting succinyl-CoA to MVC, and further to butadiene. The conversion of succinyl-CoA to MVC is accomplished in five enzymatic steps. Succinyl-CoA and acetyl-CoA are first condensed to 3-oxoadipyl-CoA by 3-oxoadipyl-CoA thiolase, a beta-ketothiolase (Step 6A). The 3-oxoadipyl-CoA product is converted to its corresponding acid by a CoA hydrolase, transferase or synthetase (Step 6B). Decarboxylation of the 3-oxoacid to 4-oxopentanoate (Step 6C), and subsequent reduction by a 4-oxopentanoate reductase yields 4-hydroxypentanoate (Step 6D). Oxidative decarboxylation of 4-hydroxypentanoate yields MVC (Step 6E). MVC is further converted to butadiene via chemical dehydration or by a dehydratase enzyme (Step 5G).

Enzymes and gene candidates for catalyzing but-3-en-2-ol and butadiene pathway reactions are described herein. Enzymes for steps A-F are described above.

Example XII

Identification of MVC Regulatory Elements

Organisms that metabolize MVC or its methylated analog, 2-methyl-MVC, can be examined for regulatory elements responsive to MVC or MVC pathway intermediates. For example, the genome of Pseudomonas putida MB-1 encodes an alcohol dehydrogenase and aldehyde dehydrogenase that is induced by 3-methyl-2-buten-3-ol (Malone et al, AEM 65: 2622-30 (1999)). The promoter of these genes can be used in several capacities, such as, being linked to expression of a fluorescent protein or other indicator that can be used to identify when MVC is produced and in some aspect the quantity of MVC produced by an organism of the invention.

Example XIII

Chemical dehydration of 1,3-BDO to Butadiene 13BDO (also referred to as 13BDO) can be a biosynthetic pathway intermediate to the product butadiene as described herein, or 13BDO can be the biosynthetic product After biosynthetic production of 13BDO is achieved, access to butadiene can be accomplished by 13BDO isolation, optional purification, and subsequent chemical (or enzymatic) dehydration to butadiene. Provided is a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces 13BDO according to any of the methods described herein; and (b) isolating the 13BDO from the fermentation broth; and (c) converting the isolated 13BDO produced by culturing the non-naturally occurring microbial organism to butadiene. Optionally, and preferably, after step (b) and before step (c) the isolated 13BDO is purified by a process comprising one, two, three or four additional purification steps that include one, two or more distillation steps, a salt reduction or removal step, and/or a water reduction or removal step.

In the embodiment where 1,3-BDO is the biosynthetic product, 1,3-BDO can be converted to butadiene by dehydration—two waters are removed. In one embodiment 1,3-BDO is first dehydrated to CrotOH that is then further dehydrated to butadiene.

Following the dehydration step, the resulting butadiene is isolated and purified by a suitable method including those described herein. Un-reacted 13BDO and other byproducts can be recycled to the dehydration step or purged from the process.

Example XIV

Chemical Dehydration of CrotOH to Butadiene

CrotOH can be a biosynthetic pathway intermediate to the product butadiene as described herein, or CrotOH can be the biosynthetic product. After biosynthetic production of CrotOH is achieved, access to butadiene can be accomplished by CrotOH isolation, optional purification, and subsequent chemical (or enzymatic) dehydration to butadiene. Provided is a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces CrotOH according to any of the methods described herein; and (b) isolating the CrotOH from the fermentation broth; and (c) converting the isolated CrotOH produced by culturing the non-naturally occurring microbial organism to butadiene. Converting the alcohol to butadiene can be performed by dehydration enzymatically or chemically, with or without a catalyst. Optionally, after step (b) and before step (c) the isolated CrotOH is purified by a process comprising one, two, three or four additional purification steps that include one, two or more distillation steps, a salt reduction or removal step, and/or a water reduction or removal step. Following fermentation the CrotOH is isolated from the fermentation broth prior to enzymatic or catalytic dehydration to butadiene. The isolation comprises a distillation step. The normal boiling point of CrotOH is about 122 degrees C., which does not suggest an easy separation from fermentation broth. The preferred isolation process described herein exploits a CrotOH-water azeotrope to facilitate isolation. Its azeotrope with water occurs at approximately 90 to 95 degrees C. It is widely recognized that an azeotrope typically causes complications and challenges for a separations process. Further the presence of impurities and byproducts in the fermentation broth point away from a simple, short isolation process. A simple, short isolation process would be even more avoided for use with a biomass feedstock that contains more and varied impurities and byproducts than a purified sugar feedstock, e.g. dextrose. Despite these complications, the present inventors recognized the presence of the azeotrope and that its presence in the fermentation broth facilitates and simplifies the isolation process. Exploiting this property to provide a simple isolation process is unique for the fermentation production of CrotOH because of the presence of water. Since the azeotrope has a higher relative volatility than water (normal boiling point of water is 100 degrees C.), the azeotropic mixture can be removed directly from the aqueous fermentation broth as the overheads from a distillation column. Water (non-azeotrope), feedstock impurities, microbial biomass, and fermentation byproducts that have lower relative volatilities will be left behind in the distillation column bottoms. Accordingly, the distillation step will be at a temperature that vaporizes the azeotrope and minimizes vaporization of the other materials in the fermentation broth, typically about 90 to 95 degrees C., and in one embodiment can be about 94.2 degrees C.

The isolated CrotOH, for example as an azeotropic mixture with water, can be dehydrated to butadiene in Step (c). In one such embodiment, the CrotOH, e.g. as a CrotOH-water azeotrope, is subjected to a one-step catalytic dehydration to butadiene without any additional drying or purification. Optionally, if a higher purity of CrotOH is preferred for the catalytic dehydration the CrotOH can be dried, for example by passing the azeotropic mixture through a molecular sieve or via azeotropic distillation using a third component such as an organic solvent, e.g., benzene. The dried CrotOH can optionally undergo further refining and purification as needed to obtain a desired purity for catalytic dehydration to butadiene. Alternatively, a purification step can precede a drying step, or can occur at the same time, or where multiple drying and/or purification steps are used they can occur in any order.

The dehydration of alcohols to olefins, specifically butadiene, is known in the art and can include various thermal processes, both catalyzed and non-catalyzed. In some embodiments, a catalyzed thermal dehydration employs a metal oxide catalyst or silica. For example, CrotOH can be dehydrated over bismuth molybdate (Adams, C. R. J. Catal. 10:355-361, 1968) to produce 1,3-butadiene. Also see Winfield, Catalytic Dehydration and Hydration, Chapter 2, in Catalysis Volume VII: Oxidation, Hydration, Dehydration and Cracking Catalysis, 1960, ed. Paul H. Emmett, Reinhold Publishing Corporation, New York N.Y. USA.

Dehydration can be achieved via activation of the alcohol group and subsequent elimination by standard elimination mechanisms such as E1 or E2 elimination. Activation can be achieved by way of conversion of the alcohol group to a halogen such as iodide, chloride, or bromide. Activation can also be accomplished by way of a sulfonyl, phosphate or other activating functionality that convert the alcohol into a good leaving group. In some embodiments, the activating group is a sulfate or sulfate ester selected from a tosylate, a mesylate, a nosylate, a brosylate, and a triflate. In some embodiments, the leaving group is a phosphate or phosphate ester. In some such embodiments, the dehydrating agent is phosphorus pentoxide.

Dehydration reactions can be carried out in both gas and liquid phases with both heterogeneous and homogeneous catalyst systems in many different reactor configurations. Typically, the catalysts used are stable to the water that is generated by the reaction. The water is usually removed from the reaction zone with the product. The resulting alkene(s) either exit the reactor in the gas or liquid phase (e.g., depending upon the reactor conditions) and are captured by a downstream purification process or are further converted in the reactor to other compounds (such as butadiene or isoprene) as described herein. The water generated by the dehydration reaction exits the reactor with unreacted alcohol and alkene product(s) and is separated by distillation or phase separation. Because water is generated in large quantities in the dehydration step, the dehydration catalysts used are generally tolerant to water and a process for removing the water from substrate and product may be part of any process that contains a dehydration step. For this reason, it is possible to use wet MVC as a substrate for a dehydration reaction and remove this water with the water generated by the dehydration reaction (e.g., using a zeolite catalyst as described U.S. Pat. Nos. 4,698,452 and 4,873,392). Additionally, neutral alumina and zeolites will dehydrate alcohols to alkenes but generally at higher temperatures and pressures than the acidic versions of these catalysts. Dehydration of alcohols, including CrotOH, to butadiene is described in Gustav. Egloff and George. Hulla, Chem. Rev., 1945, 36 (1), pp 63-141.

In a typical process for converting CrotOH into butadiene, CrotOH is passed, either neat or in a solvent and either in presence or absence of steam, over a solid inorganic, organic or metal-containing dehydration catalyst heated to temperatures in the range 40-400° C. inside of the reaction vessel or tube, leading to elimination of water and release of butadiene as a gas, which is condensed (butadiene bp=−4.4° C.) and collected in a reservoir for further processing, storage, or use. Typical catalysts can include bismuth molybdate, phosphate-phosphoric acid, cerium oxide, kaolin-iron oxide, kaolin-phosphoric acid, silica-alumina, and alumina. Typical process throughputs are in the range of 0.1-20,000 kg/h. Typical solvents are toluene, heptane, octane, ethylbenzene, and xylene.

Following the dehydration step, the resulting butadiene is isolated and purified by a suitable method including those described herein. Un-reacted CrotOH and other byproducts can be recycled to the dehydration step or purged from the process.

Accordingly, the route to butadiene via CrotOH isolation has a significant advantage versus the route via 13BDO in part because it requires fewer separation steps and only one versus two dehydrations. More separation steps are required for 13BDO since it is more miscible in water and its normal boiling point is about 205 degrees C. Due to the unique physical properties of CrotOH, the isolation route as described herein allows its fermentation production with low-quality, impure biomass feedstock. Isolating CrotOH from salts and other impurities is not as difficult as for 13BDO since the crotyl-alcohol azeotrope can be distilled directly from the broth leaving a bulk of the impurities behind in the distillation bottoms.

Example XV

Chemical dehydration of MVC to Butadiene 3-Buten-2-ol (also referred to as methyl vinyl carbinol; MVC) can be a biosynthetic pathway intermediate to the product butadiene as described herein, or MVC can be the biosynthetic product After biosynthetic production of MVC is achieved, access to butadiene can be accomplished by MVC isolation, optional purification, and subsequent chemical (or enzymatic) dehydration to butadiene. Provided is a process for the production of butadiene that includes (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces MVC according to any of the methods described herein; and (b) isolating the MVC from the fermentation broth; and (c) converting the isolated MVC produced by culturing the non-naturally occurring microbial organism to butadiene. Converting MVC to butadiene can be performed by dehydration enzymatically or chemically, with or without a catalyst. Optionally, after step (b) and before step (c) the isolated MVC is purified by a process comprising one, two, three or four additional purification steps that include one, two or more distillation steps, a salt reduction or removal step, and/or a water reduction or removal step.

Following fermentation as described herein, MVC can be isolated from the fermentation broth prior to catalytic dehydration to butadiene. MVC has a boiling point approximating that of water. The azeotrope of MVC and water occurs at about 87 degrees C. It is widely recognized that an azeotrope typically causes complications and challenges for a separations process. Further the presence of impurities and byproducts in the fermentation broth point away from a simple, short isolation process. A simple, short isolation process would be even more avoided for use with a biomass feedstock that contains more and varied impurities and byproducts than a purified sugar feedstock, e.g. dextrose. Despite these complications, the present inventors recognized the presence of the MVC-water azeotrope and that its presence in the fermentation broth facilitates and simplifies the isolation process. Exploiting this property to provide a simple isolation process is unique for the fermentation production of MVC because of the presence of water. Since the azeotrope has a higher relative volatility than water (normal boiling point of water is 100 degrees C.), the azeotropic mixture can be removed directly from the aqueous fermentation broth as the overheads from a distillation column. Water (non-azeotrope), feedstock impurities, microbial biomass, and fermentation byproducts that have lower relative volatilities will be left behind in the distillation column bottoms.

The isolated MVC, for example as an azeotropic mixture with water, can be dehydrated to butadiene in step (c). In one such embodiment, the MVC, e.g. as a MVC-water azeotrope, is subjected to a one-step catalytic dehydration to butadiene without any additional drying or purification. Optionally, if a higher purity of MVC is preferred for the catalytic dehydration the MVC can be dried, for example by passing the azeotropic mixture through a molecular sieve or via azeotropic distillation using a third component such as an organic solvent, e.g., benzene. The dried MVC can optionally undergo further refining and purification as needed to obtain a desired purity for catalytic dehydration to butadiene. Alternatively, a purification step can precede a drying step, or can occur at the same time, or where multiple drying and/or purification steps are used they can occur in any order.

The dehydration of alcohols to olefins, specifically butadiene, are known in the art and can include various thermal processes, both catalyzed and non-catalyzed. In some embodiments, a catalyzed thermal dehydration employs a metal oxide catalyst or silica. Step (c) of the process, dehydration, can be performed enzymatically or by chemically in the presence of a catalyst. For example, see Winfield, Catalytic Dehydration and Hydration, Chapter 2, in Catalysis Volume VII: Oxidation, Hydration, Dehydration and Cracking Catalysis, 1960, ed. Paul H. Emmett, Reinhold Publishing Corporation, New York, N.Y. USA.

Dehydration can be achieved via activation of the alcohol group and subsequent elimination by standard elimination mechanisms such as E1 or E2 elimination. Activation can be achieved by way of conversion of the alcohol group to a halogen such as iodide, chloride, or bromide. Activation can also be accomplished by way of a sulfonyl, phosphate or other activating functionality that convert the alcohol into a good leaving group. In some embodiments, the activating group is a sulfate or sulfate ester selected from a tosylate, a mesylate, a nosylate, a brosylate, and a triflate. In some embodiments, the leaving group is a phosphate or phosphate ester. In some such embodiments, the dehydrating agent is phosphorus pentoxide.

Dehydration reactions can be carried out in both gas and liquid phases with both heterogeneous and homogeneous catalyst systems in many different reactor configurations. Typically, the catalysts used are stable to the water that is generated by the reaction. The water is usually removed from the reaction zone with the product. The resulting alkene(s) either exit the reactor in the gas or liquid phase (e.g., depending upon the reactor conditions) and are captured by a downstream purification process or are further converted in the reactor to other compounds (such as butadiene or isoprene) as described herein. The water generated by the dehydration reaction exits the reactor with unreacted alcohol and alkene product(s) and is separated by distillation or phase separation. Because water is generated in large quantities in the dehydration step, the dehydration catalysts used are generally tolerant to water and a process for removing the water from substrate and product may be part of any process that contains a dehydration step. For this reason, it is possible to use wet MVC as a substrate for a dehydration reaction and remove this water with the water generated by the dehydration reaction (e.g., using a zeolite catalyst as described U.S. Pat. Nos. 4,698,452 and 4,873,392). Additionally, neutral alumina and zeolites will dehydrate alcohols to alkenes but generally at higher temperatures and pressures than the acidic versions of these catalysts. Dehydration of MVC to butadiene is well known in the art (Gustay. Egloff and George. Hulla, Chem. Rev., 1945, 36 (1), pp 63-141). See also U.S. Pat. No. 2,400,409 entitled “Methods for dehydration of alcohols.”

Following the dehydration step, the resulting butadiene is isolated and purified by a suitable method including those described herein. Un-reacted MVC and other byproducts can be recycled to the dehydration step or purged from the process.

Accordingly, the route to butadiene via MVC isolation has a significant advantage versus the route via 13BDO in part because it requires fewer separation steps and only one versus two dehydrations. More separation steps are required for 13BDO since it is more miscible in water and its normal boiling point is about 205 degrees C. Due to the unique physical properties of MVC, the isolation route as described herein allows its fermentation production with low-quality, impure biomass feedstock. Isolating MVC from salts and other impurities is not as difficult as for 13BDO since the MVC-water azeotrope can be distilled directly from the broth leaving a bulk of the impurities behind in the distillation bottoms.

Example XVI

Pathways for Producing 3-buten-1-ol and Butadiene from Crotonyl-CoA

This example describes pathways for converting crotonyl-CoA to 3-buten-1-ol and butadiene. The pathways are shown in FIG. 19. Relevant enzymes include: crotonyl-CoA delta-isomerase, vinylacetyl-CoA reductase, 3-buten-1-al reductase, and 3-buten-1-ol dehydratase. Step D can also be catalyzed via chemical dehydration. The conversion of crotonyl-CoA to 3-buten-1-ol can be accomplished in three enzymatic steps. Crotonyl-CoA can be first converted to vinylacetyl-CoA by a crotonyl-CoA delta-isomerase (Step A of FIG. 19). The vinylacetyl-CoA can be subsequently reduced to 3-buten-1-al by a vinylacetyl-CoA reductase (Step B of FIG. 19). 3-buten-1-al can be further reduced to 3-buten-1-ol by a 3-buten-1-al reductase (Step C of FIG. 19). Further dehydration of the 3-buten-1-ol product to butadiene can be performed by an enzyme, that is, 3-buten-1-ol dehydratase, or chemical catalyst (Step D of FIG. 19).

Crotonyl-CoA Delta-Isomerase (FIG. 19, Step A)

Crotonyl-CoA delta-isomerase shifts the double bond of crotonyl-CoA from the 2- to the 3-position, forming vinylacetyl-CoA. Exemplary enzymes that catalyze this transformation or similar transformations include 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta-isomerase (EC 5.3.3.3), fatty acid oxidation complexes and delta-3, delta-2-enoyl-CoA isomerase (EC 5.3.3.8). 4-Hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta-isomerase enzymes catalyze the reversible conversion of crotonyl-CoA to vinylacetyl-CoA (also called but CoA), and subsequent dehydration to 4-hydroxybutyryl-CoA. These enzymes are bifunctional, catalyzing both the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA, and also the isomerization of vinylacetyl-CoA and crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta-isomerase enzymes from C. aminobutyrium and C. kluyveri were purified, characterized, and sequenced at the N-terminus (Scherf et al., Arch. Microbiol 161:239-245 (1994); Scherf and Buckel, Eur. J Biochem. 215:421-429 (1993)). The C. kluyveri enzyme, encoded by abfD, was cloned, sequenced and expressed in E. coli (Gerhardt et al., Arch. Microbiol 174:189-199 (2000)). The abfD gene product from Porphyromonas gingivalis ATCC 33277 is closely related by sequence homology to the Clostridial gene products. 4-Hydroxybutyryl-CoA dehydratase/isomerase activity was also detected in Metallosphaera sedula, and is likely associated with the Msed_1220 gene (Berg et al, Science 318(5857):1782-6 (2007). Delta isomerization reactions are also catalyzed by the fatty acid oxidation complex. In E. coli, the fadJ and fadB gene products convert cis-3-enoyl-CoA molecules to trans-2-enoyl-CoA molecules under aerobic and anaerobic conditions, respectively (Campbell et al, Mol Micro 47(3):793-805 (2003)). A monofunctional delta-isomerase isolated from Cucumis sativus peroxisomes catalyzes the reversible conversion of both cis- and trans-3-enoyl-CoA into trans-2-enoyl-CoA (Engeland et al, Eur J Biochem, 196 (3):699-705 (1991). The gene associated with this enzyme has not been identified to date. A number of multifunctional proteins (MFP) from Cucumis sativus also catalyze this activity, including the gene product of MFP-a (Preisig-Muller et al, J Biol Chem 269:20475-81 (1994)).

Protein	GenBank ID	GI Number	Organism
abfD	P55792	84028213	Clostridium aminobutyricum
abfD	YP_001396399.1	153955634	Clostridium kluyveri
abfD	YP_001928843	188994591	Porphyromonas gingivalis
Msed_1220	ABP95381.1	145702239	Metallosphaera sedula
fadJ	AAC75401.1	1788682	Escherichia coli
fadB	AAC76849.1	1790281	Escherichia coli
MFP-a	Q39659.1	34922495	Cucumis sativus

3,2-Trans-enoyl-CoA isomerase enzymes (EC 5.3.3.8) interconvert trans-2-enoyl-CoA and trans-3-enoyl-CoA substrates. Enzymes in this class are found in Saccharomyces cerevisiae and mammals such as Rattus norvegicus and Homo sapiens. A crystal structure of the S. cerevisiae enzyme is available (Mursula et al, J Mol Biol 309: 845-53 (2001)). 3,2-Trans-enoyl-CoA isomerase isozymes found in rat liver mitochondria are active on short and longer chain enoyl-CoA substrates including hex-2-enoyl-CoA (Palosaari et al, J Biol Chem 266:10750-3 (1991); Yu et al, Biochim Biophys Acta 1760:1874-83 (2006)). Substrate specificities are described in Zhang et al, J Biol Chem 277: 9127-32 (2002). Two other well-characterized enzyme candidates are the human mitochondrial 3,2-trans-enoyl-CoA isomerase (Partanen et al, J Mol Biol 342:1197-208 (2004)) and the peroxisomal mammalian enzyme PEC1 found in humans and mice (Geisbrecht et al, J Biol Chem 274:21797-803 (1999)).

Protein	GenBank ID	GI Number	Organism
ECU	Q05871.1	60392229	Saccharomyces cerevisiae
ECU	P42126.1	1169204	Homo sapiens
ECI1	NP_059002.2	162287040	Rattus norvegicus
ECI2	Q5XIC0.1	81883743	Rattus norvegicus
PEC1	NP_001159482.1	260275230	Homo sapiens

Vinylacetyl-CoA Reductase (FIG. 19, Step B)

An acyl-CoA reductase with vinylacetyl-CoA reductase activity catalyzes the reduction of vinylacetyl-CoA to 3-buten-1-al. Alternately, a bifunctional acyl-CoA reductase/aldehyde reductase can catalyze the conversion of vinylacetyl-CoA directly to 3-buten-1-ol. Exemplary monofunctional and bifunctional acyl-CoA reductase enzyme candidates described above and in Example IV. Additional enzyme candidates are described below.

Acyl-CoA dehydrogenases that reduce an acyl-CoA to its corresponding aldehyde are shown in the table below. Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase, butyryl-CoA reductase and propionyl-CoA reductase (EC 1.2.1.3).

Enzyme Commission Number Enzyme Name
1.2.1.10                 Acetaldehyde dehydrogenase (acetylating)
1.2.1.42                 (Fatty) acyl-CoA reductase
1.2.1.44                 Cinnamoyl-CoA reductase
1.2.1.50                 Long chain fatty acyl-CoA reductase
1.2.1.57                 Butanal dehydrogenase
1.2.1.75                 Malonate semialdehyde dehydrogenase
1.2.1.76                 Succinate semialdehyde dehydrogenase
1.2.1.81                 Sulfoacetaldehyde dehydrogenase
1.2.1.—                  Propanal dehydrogenase
1.2.1.—                  Hexanal dehydrogenase

Exemplary fatty acyl-CoA reductases enzymes are encoded by acr1 of Acinetobacter calcoaceticus (Reiser, Journal of Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoA reductase activity are encoded by sucD of Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea including Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol, 191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem., 71:58-68 (2007)). Exemplary propionyl-CoA reductase enzymes include pduP of Salmonella typhimurium LT2 (Leal, Arch. Microbiol. 180:353-361 (2003)) and eutE from E. coli (Skraly, WO Patent No. 2004/024876). The propionyl-CoA reductase of Salmonella typhimurium LT2, which naturally converts propionyl-CoA to propionaldehyde, also catalyzes the reduction of 5-hydroxyvaleryl-CoA to 5-hydroxypentanal (WO 2010/068953A2). The propionaldehyde dehydrogenase of Lactobacillus reuteri, PduP, has a broad substrate range that includes buyraldehyde, valeraldehyde and 3-hydroxypropionaldehyde (Luo et al, Appl Microbiol Biotech, 89: 697-703 (2011).

Protein	GenBank ID	GI Number	Organism
acr1	YP_047869.1	50086359	Acinetobacter calcoaceticus
acr1	AAC45217	1684886	Acinetobacter baylyi
acr1	BAB85476.1	18857901	Acinetobacter sp. Strain M-1
MSED_0709	YP_001190808.1	146303492	Metallosphaera sedula
Tneu_0421	ACB39369.1	170934108	Thermoproteus neutrophilus
sucD	P38947.1	172046062	Clostridium kluyveri
sucD	NP_904963.1	34540484	Porphyromonas gingivalis
bphG	BAA03892.1	425213	Pseudomonas sp
adhE	AAV66076.1	55818563	Leuconostoc mesenteroides
bld	AAP42563.1	31075383	Clostridium saccharoperbutylacetonicum
pduP	NP_460996	16765381	Salmonella typhimurium LT2
eutE	NP_416950	16130380	Escherichia coli

The NAD(P)⁺ dependent oxidation of acetaldehyde to acetyl-CoA is catalyzed by acylating acetaldehyde dehydrogenase (EC 1.2.1.10). Acylating acetaldehyde dehydrogenase enzymes of E. coli are encoded by adhE and mhpF (Ferrandez et al, J Bacteriol 179:2573-81 (1997)). The Pseudomonas sp. CF600 enzyme, encoded by dmpF, participates in meta-cleavage pathways and forms a complex with 4-hydroxy-2-oxovalerate aldolase (Shingler et al, J Bacteriol 174:711-24 (1992)). Solventogenic organisms such as Clostridium acetobutylicum encode bifunctional enzymes with alcohol dehydrogenase and acetaldehyde dehydrogenase activities. The bifunctional C. acetobutylicum enzymes are encoded by bdh I and adhE2 (Walter, et al., J. Bacteriol. 174:7149-7158 (1992); Fontaine et al., J. Bacteriol. 184:821-830 (2002)). Yet another candidate for acylating acetaldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This gene is very similar to the eutE acetaldehyde dehydrogenase genes of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

Protein	GenBank ID	GI Number	Organism
adhE	NP_415757.1	16129202	Escherichia coli
mhpF	NP_414885.1	16128336	Escherichia coli
dmpF	CAA43226.1	45683	Pseudomonas sp. CF600
adhE2	AAK09379.1	12958626	Clostridium acetobutylicum
bdh I	NP_349892.1	15896543	Clostridium acetobutylicum
Ald	AAT66436	49473535	Clostridium beijerinckii
eutE	NP_416950	16130380	Escherichia coli
eutE	AAA80209	687645	Salmonella typhimurium

3-Buten-1-al Reductase (FIG. 19, Step C)

Reduction of 3-buten-1-al to 3-buten-1-ol is catalyzed by an aldehyde reductase or alcohol dehydrogenase (EC 1.1.1-). Exemplary alcohol dehydrogenase enzyme candidates suitable for catalyzing this reaction are described above and in Example IV.

3-buten-1-ol Dehydratase (FIG. 19, Step D)

Dehydration of 3-buten-1-ol to butadiene is catalyzed by a hydro-lyase (EC 4.2.1.a). Exemplary hydro-lyase enzyme candidates suitable for catalyzing this reaction are described above and in Example IV.

Example XVII

Chemical Dehydration of 3-buten-1-ol to Butadiene

Alcohols can be converted to olefins by reaction with a suitable dehydration catalyst under appropriate conditions. Typical dehydration catalysts that convert alcohols such as butanols and pentanols into olefins include various acid treated and untreated alumina (e.g., γ-alumina) and silica catalysts and clays including zeolites (e.g., β-type zeolites, ZSM-5 or Y-type zeolites, fluoride-treated β-zeolite catalysts, fluoride-treated clay catalysts, etc.), sulfonic acid resins (e.g., sulfonated styrenic resins such as Amberlyst® 15), strong acids such as phosphoric acid and sulfuric acid, Lewis acids such boron trifluoride and aluminum trichloride, and many different types of metal salts including metal oxides (e.g., zirconium oxide or titanium dioxide) and metal chlorides (e.g., Latshaw B E, Dehydration of Isobutanol to Isobutylene in a Slurry Reactor, Department of Energy Topical Report, February 1994).

Dehydration reactions can be carried out in both gas and liquid phases with both heterogeneous and homogeneous catalyst systems in many different reactor configurations. Typically, the catalysts used are stable to the water that is generated by the reaction. The water is usually removed from the reaction zone with the product. The resulting alkene(s) either exit the reactor in the gas or liquid phase (e.g., depending upon the reactor conditions) and are captured by a downstream purification process or are further converted in the reactor to other compounds (such as butadiene or isoprene) as described herein. The water generated by the dehydration reaction exits the reactor with unreacted alcohol and alkene product(s) and is separated by distillation or phase separation. Because water is generated in large quantities in the dehydration step, the dehydration catalysts used are generally tolerant to water and a process for removing the water from substrate and product may be part of any process that contains a dehydration step. For this reason, it is possible to use wet (i.e., up to about 95% or 98% water by weight) alcohol as a substrate for a dehydration reaction and remove this water with the water generated by the dehydration reaction (e.g., using a zeolite catalyst as described U.S. Pat. Nos. 4,698,452 and 4,873,392). Additionally, neutral alumina and zeolites will dehydrate alcohols to alkenes but generally at higher temperatures and pressures than the acidic versions of these catalysts.

Dehydration of 3-buten-1-ol to butadiene is well known in the art (Gustav. Egloff and George. Hulla, Chem. Rev., 1945, 36 (1), pp 63-141).

Example XVIII

Co-Utilization of Sugar 2 and Sugar 1

This example describes the utilization of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1, using E. coli strains bearing different xR mutants.

E. coli strains with a mutation that can utilize Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 were obtained by screening for Sugar 2 utilization under selective pressure in the presence of Sugar 1. Strains capable of co-utilizing Sugar 2 in the presence of Sugar 1 were obtained by selection in a continuous culture mode (chemostat) under sugar limited-conditions. The initial sugar ratio in the medium was 10:1 (Sugar 1:Sugar 2). The chemostat was operated at a dilution rate of 0.2/h during approximately 400 hours. A sample of the population was plated on selective M9-Sugar 2 agar. Several clones were tested for co-consumption of Sugar 1 and Sugar 2. The positive strains able to utilize Sugar 2 in the presence of Sugar 1 were sequenced and revealed a mutation at position 121 of XR with a serine substitution of the original arginine. Variants of XR as described herein can be assayed for desired activity in vivo by the methods described herein and other methods well known in the art.

E. coli strain MG1655 having the xR mutation (arginine to serine) and wild-type MG1655 were used to test the Sugar 2 use in the presence of a catabolite-repressing concentration of Sugar 1. In FIG. 20, the data of Sugar 2 use in the presence of a catabolite-repressing concentration of Sugar 1 by MG1655 having the xR mutation are shown in squares, while those of wild-type MG1655 are shown in diamonds. Compared to wild-type MG1655, MG1655 having the xR mutation provided improved use of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1. In FIG. 25, the data of Sugar 2 use in the presence of a catabolite-repressing concentration of Sugar 3 by MG1655 are shown in diamonds. Compared to wild-type MG1655, the MG1655 with the xR mutation R121S improved Sugar 2 utilization in the presence of catabolite-repressing concentration of Sugar 3. These results shows that the arginine to serine mutation at position 121 of XR allows Sugar 2 to escape from Sugar 1 and Sugar 3 catabolite repression.

In addition, the E. coli strain variant of MG1655 having the xR mutation (arginine to serine) and the variant without the xR mutation were used to test the Sugar 2 use in the presence of a catabolite-repressing concentration of Sugar 1. In FIG. 21, the data of Sugar 2 use in the presence of a catabolite-repressing concentration of Sugar 1 by the E. coli strain variant of MG1655 having the xR mutation are shown in “Xs,” while those of the variant without the xR mutation are shown in triangles. Compared to the E. coli strain variant of MG1655 without xR, the E. coli strain variant of MG1655 having the xR mutation provided immediate and complete use of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1. The E. coli strain variant of MG1655 mentioned herein differs from MG1655 by containing, amongst other things, heterologous sucrose operon genes cscA, cscB and cscK, that allow sucrose use.

Further, strains having the xR mutation (arginine to serine) were tested on biomass sugar. Biomass source and pretreatment determines the sugar content, type and amounts. In one example, sugars are about 50% of the biomass content by weight, with Sugar 1 predominating, generally at about 50% of the sugar mass. Of the remaining monosaccharides, Sugar 2 is typically second most abundant, followed by Sugar 3 and by galactose. Of disaccharides (DP2), isomaltose (alpha 1-6 Sugar 1-Sugar 1) is abundant, followed by other unidentified DP2 (hex-hex) sugars, by xylobiose (beta 1-4 xyl-xyl), and by cellobiose (alpha 1-4 Sugar 1-Sugar 1). Salts and organic acids, including pyruvate, formate, succinate, acetate and lactate, are also present. The table below shows an exemplary biomass sugar composition. Evaluations showed that Sugar 2 use was immediate and complete using the strains having the xR mutation of the invention on biomass sugar. Sugars were analyzed and quantitiated by known art methods.

	Sugar 3	Cellobiose	DP2 (Hex-Hex)	Galactose	Sugar 1	iso-Mal	Xylobiose	Sugar 2
AVERAGE	6.1	6.2	36.6	3.5	365.7	5.6	12.3	180.3

In order to identify additional mutants that allow the co-utilization of Sugar 2 and Sugar 1, an NNK library was generated at position 121 of XR by site-specific mutagenesis. An NNK library was generated using well known methods in the art based on the E. coli XR-encoding gene xR.

The NNK library was screened for different mutations at position 121 that allowed Sugar 2 to escape from Sugar 1 catabolite repression. Table 1 lists the mutations at position 121 and their performance in Sugar 2 consumption relative to the wild-type variant (Arginine).

TABLE 1
Amino Acid Substitutions Relieving Catabolite Repression
Amino acid    Faster Sugar 2 consumption than WT (Arg)
Cysteine      Yes
Serine        Yes (original mutation)
Threonine     Yes
Glycine       Yes
Histidine     Yes
Valine        Yes
Methioine     Yes
Tyrosine      Yes
Isoleucine    Yes
Alanine       Yes
Leucine       Yes
Proline       Yes
Phenylalanine Yes
Tryptophane   Possibly (depending on time course)

E. coli strains bearing different mutations at position 121 were grown in a media containing 0.6% Sugar 1 and 0.4% Sugar 2. The growth of the E. coli strains was recorded by measuring the optical density at 600 nm wavelength (“OD600”) of the cells at different time points. FIG. 22 records the growth of 11 different xR mutants, compared to wild-type xR, in the media containing 0.6% Sugar 1 and 0.4% Sugar 2. FIG. 22 shows that the mutants that have OD600 measurements between those of the Arg and Ser mutants resulted in intermediate growth improvement.

In addition, the residual Sugar 1 and Sugar 2 concentrations in the fermentation broths were measured at different time points using Megazyme kits (Megazyme International Ireland, Ireland) according to manufacturer instructions. All strains successfully and similarly used Sugar 1 (data not shown). FIG. 23 records the utilization rate of Sugar 2 in the presence of a catabolite-repressing concentration of Sugar 1 for 15 different xR mutants compared to wild-type xR. The results indicate that several mutants that have rates between those of the Arg and Ser mutants resulted in intermediate Sugar 2 utilization rates. Further, FIG. 24 records and ranks the amounts of residual Sugar 2 at a single time point following 40 minutes of fermentation for 15 different xR mutants compared to wild-type xR in the presence of catabolite-repressing concentrations of Sugar 1.

Example XIX

Co-Utilization of Sugar 2, Sugar 3, and Sugar 1

This example describes the co-utilization of Sugar 2, Sugar 3, and Sugar 1, using the xR mutation (arginine to serine) and constitutive expression of araE.

Constitutive expression of araE was achieved by placing araE under a constitutive promoter. Three different promoters of increasing transcriptional strength, p100, p107, and p115, were each used to express araE. The resulting E. coli strains were tested for Sugar 3 use. The culture conditions, growth conditions and measurements were performed similarly to what was described in Example XVIII. The constitutive expression of araE resulted in increased Sugar 3 use. AraE was from an heterologous source (Corynebacterium glutamicum), and in this example overexpression of the native E. coli AraE was not performed. The C. glutamicum AraE is a 479 amino acid protein of sequence of GenBank ID: BAH60837.1 and its encoding gene sequence is identified as GI:238231325.

The xR mutation (arginine to serine) and constitutive expression of araE were combined to test the use of Sugar 1, Sugar 2 and Sugar 3, on both pure sugar mixtures and biomass. It was observed that the combination of the xR mutation and constitutive expression of araE provided co-utilization of Sugar 2, Sugar 3, and Sugar 1. Despite the increased Sugar 3 use, Sugar 2 was used in the presence of catabolite-repressing concentrations of Sugar 3.

Example XX

Improvement of Sugar 2 Use by xylFGH Overexpression

This example describes the improvement of Sugar 2 use by overexpression of xylFGH.

Overexpression of xylFGH was achieved by constitutively expressing xylFGH. The resulting E. coli strains were tested for Sugar 2 use. The culture conditions, growth conditions and measurements were performed as described in Example XVIII. It was observed was that overexpression of xylFGH resulted in a dramatic improvement in Sugar 2 use (in wild-type xR background). In addition, the improvement in Sugar 2 use was only apparent in native xylFGH context (p100-xylFGH) but not in the refactored xylFGH (p100-xylF-p100-xylGH. This suggests that the region between xylF and xylGH plays an important role, possibly in regulating, RNA stabilizing, or fine tuning the levels of the 3 subunits.

Example XXI

In Vivo Labeling Assay for Conversion of Methanol to CO₂

This example describes a functional methanol pathway in a microbial organism.

Strains with functional reductive TCA branch and pyruvate formate lyase deletion were grown aerobically in LB medium overnight, followed by inoculation of M9 high-seed media containing IPTG and aerobic growth for 4 hrs. These strains had MeDH/ACT pairs in the presence and absence of formaldehyde dehydrogenase or FDH. ACT is an activator protein (a Nudix hydrolase). At this time, strains were pelleted, resuspended in fresh M9 medium high-seed media containing 2% ¹³CH₃OH, and sealed in anaerobic vials. Head space was replaced with nitrogen and strains grown for 40 hours at 37° C. Following growth, headspace was analyzed for ¹³CO₂. Media was examined for residual methanol as well as BDO and byproducts. All constructs expressing MeDH(MeDH) mutants and MeDH/ACT pairs grew to slightly lower ODs than strains containing empty vector controls. This is likely due to the high expression of these constructs (Data not shown). One construct (2315/2317) displayed significant accumulation of labeled CO₂ relative to controls in the presence of FalDH, FDH or no coexpressed protein. This shows a functional MeOH pathway in E. coli and that the endogenous glutathione-dependent formaldehyde detoxification genes (frmAB) are sufficient to carry flux generated by the current MeDH/ACT constructs.

2315 is internal laboratory designation for the MeDH from Bacillus methanolicus MGA3 (GenBank Accession number: E1177596.1; GI number: 387585261), and 2317 is internal laboratory designation for the activator protein from the same organism (locus tag: MGA3_09170; GenBank Accession number:EIJ83380; GI number: 387591061).

Sequence analysis of the NADH-dependent MeDH from Bacillus methanolicus places the enzyme in the alcohol dehydrogenase family III. It does not contain any tryptophan residues, resulting in a low extinction coefficient (18,500 M⁻¹, cm⁻¹) and should be detected on SDS gels by Coomassie staining.

The enzyme has been characterized as a multisubunit complex built from 43 kDa subunits containing one Zn and 1-2 Mg atoms per subunit. Electron microscopy and sedimentation studies determined it to be a decamer, in which two rings with five-fold symmetry are stacked on top of each other (Vonck et al., J. Biol. Chem. 266:3949-3954, 1991). It is described to contain a tightly but not covalently bound cofactor and requires exogenous NAD⁺ as e⁻-acceptor to measure activity in vitro. A strong increase (10-40-fold) of in vitro activity was observed in the presence of an activator protein (ACT), which is a homodimer (21 kDa subunits) and contains one Zn and one Mg atom per subunit.

The mechanism of the activation was investigated by Kloosterman et al., J Biol. Chem. 277:34785-34792, 2002, showing that ACT is a Nudix hydrolase and Hektor et al., J Biol. Chem. 277:46966-46973, 2002, demonstrating that mutation of residue S97 to G or Tin MeDH changes activation characteristics along with the affinity for the cofactor. While mutation of residues G15 and D88 had no significant impact, a role of residue G13 for stability as well as of residues G95, D100, and K103 for the activity is suggested. Both papers together propose a hypothesis in which ACT cleaves MeDH-bound NAD⁺. MeDH retains AMP bound and enters an activated cycle with increased turnover.

The stoichiometric ratio between ACT and MeDH is not well defined in the literature. Kloosterman et al., supra determine the ratio of dimeric Act to decameric MeDH for full in vitro activation to be 10:1. In contrast, Arfman et al. J Biol. Chem. 266:3955-3960, 1991 determined a ratio of 3:1 in vitro for maximum and a 1:6 ratio for significant activation, but observe a high sensitivity to dilution. Based on expression of both proteins in Bacillus, the authors estimate the ratio in vivo to be around 1:17.5.

However, our in vitro experiments with purified activator protein (2317A) and MeDH(2315A) showed the ratio of ACT to MeDH to be 10:1. This in vitro test was done with 5 M methanol, 2 mM NAD and 10 μM MeDH2315A at pH 7.4.

Example XXII

Attenuation or Disruption of Endogenous Enzymes

This example provides endogenous enzyme targets for attenuation or disruption that can be used for enhancing carbon flux through acetyl-CoA.

DHA Kinase

Methylotrophic yeasts typically utilize a cytosolic DHA kinase to catalyze the ATP-dependent activation of DHA to DHAP. DHAP together with G3P is combined to form fructose-1,6-bisphosphate (FBP) by FBP aldolase. FBP is then hydrolyzed to F6P by fructose bisphosphatase. The net conversion of DHA and G3P to F6P by this route is energetically costly (1 ATP) in comparison to the F6P aldolase route, described above and shown in FIG. 1. DHA kinase also competes with F6P aldolase for the DHA substrate. Attenuation of endogenous DHA kinase activity will thus improve the energetics of formaldehyde assimilation pathways, and also increase the intracellular availability of DHA for DHA synthase. DHA kinases of Saccharomyces cerevisiae, encoded by DAK1 and DAK2, enable the organism to maintain low intracellular levels of DHA (Molin et al, J Biol Chem 278:1415-23 (2003)). In methylotrophic yeasts DHA kinase is essential for growth on methanol (Luers et al, Yeast 14:759-71 (1998)). The DHA kinase enzymes of Hansenula polymorpha and Pichia pastoris are encoded by DAK (van der Klei et al, Curr Genet 34:1-11 (1998); Luers et al, supra). DAK enzymes in other organisms can be identified by sequence similarity to known enzymes.

Protein	GenBank ID	GI Number	Organism
DAK1	NP_013641.1	6323570	Saccharomyces cerevisiae
DAK2	NP_116602.1	14318466	Saccharomyces cerevisiae
DAK	AAC27705.1	3171001	Hansenula polymorpha
DAK	AAC39490.1	3287486	Pichia pastoris
DAK2	XP_505199.1	50555582	Yarrowia lipolytica

Methanol Oxidase

Attenuation of redox-inefficient endogenous methanol oxidizing enzymes, combined with increased expression of a cytosolic NADH-dependent MeDH, will enable redox-efficient oxidation of methanol to formaldehyde in the cytosol. Methanol oxidase, also called alcohol oxidase (EC 1.1.3.13), catalyzes the oxygen-dependent oxidation of methanol to formaldehyde and hydrogen peroxide. In eukaryotic organisms, alcohol oxidase is localized in the peroxisome. Exemplary methanol oxidase enzymes are encoded by AOD of Candida boidinii (Sakai and Tani, Gene 114:67-73 (1992)); and AOX of H. polymorphs, P. methanolica and P. pastoris (Ledeboer et al, Nucl Ac Res 13:3063-82 (1985); Koutz et al, Yeast 5:167-77 (1989); Nakagawa et al, Yeast 15:1223-1230 (1999)).

Protein	GenBank ID	GI Number	Organism
AOX2	AAF02495.1	6049184	Pichia methanolica
AOX1	AAF02494.1	6049182	Pichia methanolica
AOX1	AAB57849.1	2104961	Pichia pastoris
AOX2	AAB57850.1	2104963	Pichia pastoris
AOX	P04841.1	113652	Hansenula polymorpha
AOD1	Q00922.1	231528	Candida boidinii
AOX1	AAQ99151.1	37694459	Ogataea pini

PQQ-Dependent MeDH

PQQ-dependent MeDH from M. extorquens (mxaIF) uses cytochrome as an electron carrier (Nunn et al, Nucl Acid Res 16:7722 (1988)). MeDH enzymes of methanotrophs such as Methylococcus capsulatis function in a complex with methane monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)). Note that of accessory proteins, cytochrome CL and PQQ biosynthesis enzymes are needed for active MeDH. Attenuation of one or more of these required accessory proteins, or retargeting the enzyme to a different cellular compartment, would also have the effect of attenuating PQQ-dependent MeDH activity.

7) Protein	8) GenBank ID	9) GI Number	10) Organism
11) MCA0299	12) YP_112833.1	13) 53802410	14) Methylococcus capsulatis
15) MCA0782	16) YP_113284.1	17) 53804880	18) Methylococcus capsulatis
19) mxaI	20) YP_002965443.1	21) 240140963	22) Methylobacterium extorquens
23) mxaF	24) YP_002965446.1	25) 240140966	26) Methylobacterium extorquens

DHA Synthase and Other Competing Formaldehyde Assimilation and Dissimilation Pathways

Carbon-efficient formaldehyde assimilation can be improved by attenuation of competing formaldehyde assimilation and dissimilation pathways. Exemplary competing assimilation pathways in eukaryotic organisms include the peroxisomal dissimilation of formaldehyde by DHA synthase, and the DHA kinase pathway for converting DHA to F6P, both described herein. Exemplary competing endogenous dissimilation pathways include one or more of the enzymes shown in FIG. 1.

Methylotrophic yeasts normally target selected methanol assimilation and dissimilation enzymes to peroxisomes during growth on methanol, including methanol oxidase, DHA synthase and S-(hydroxymethyl)-glutathione synthase (see review by Yurimoto et al, supra). The peroxisomal targeting mechanism comprises an interaction between the peroxisomal targeting sequence and its corresponding peroxisomal receptor (Lametschwandtner et al, J Biol Chem 273:33635-43 (1998)). Peroxisomal methanol pathway enzymes in methylotrophic organisms contain a PTS1 targeting sequence which binds to a peroxisomal receptor, such as Pex5p in Candida boidinii (Horiguchi et al, J Bacteriol 183:6372-83 (2001)). Disruption of the PTS1 targeting sequence, the Pex5p receptor and/or genes involved in peroxisomal biogenesis would enable cytosolic expression of DHA synthase, S-(hydroxymethyl)-glutathione synthase or other methanol-inducible peroxisomal enzymes. PTS1 targeting sequences of methylotrophic yeast are known in the art (Horiguchi et al, supra). Identification of peroxisomal targeting sequences of unknown enzymes can be predicted using bioinformatic methods (eg. Neuberger et al, J Mol Biol 328:581-92 (2003))).

Example XXIII

Methanol Assimilation Via MeDH and the Ribulose Monophosphate Pathway

This example shows that co-expression of an active MeDH(MeDH) and the enzymes of the Ribulose Monophosphate (RuMP) pathway can effectively assimilate methanol derived carbon.

An experimental system was designed to test the ability of a MeDH in conjunction with the enzymes H6P synthase (HPS) and 6P3HI (PHI) of the RuMP pathway to assimilate methanol carbon into the glycolytic pathway and the TCA cycle. Escherichia coli strain ECh-7150 (ΔlacIA, ΔpflB, ΔptsI, ΔPpckA(pckA), ΔPglk(glk), glk::glfB, ΔhycE, ΔfrmR, ΔfrmA, ΔfrmB) was constructed to remove the glutathione-dependent formaldehyde detoxification capability encoded by the FrmA and FrmB enzyme. This strain was then transformed with plasmid pZA23S variants that either contained or lacked gene 2616A encoding a fusion of the HPS and PHI enzymes. These two transformed strains were then each transformed with pZS*13S variants that contained gene 2315L (encoding an active MeDH), or gene 2315 RIP2 (encoding a catalytically inactive MeDH), or no gene insertion. Genes 2315 and 2616 are internal nomenclatures for NAD-dependent MeDH from Bacillus methanolicus MGA3 and 2616 is a fused phs-hpi constructs as described in Orita et al. (2007) Appl Microbiol Biotechnol 76:439-45.

The six resulting strains were aerobically cultured in quadruplicate, in 5 ml minimal medium containing 1% arabinose and 0.6 M 13C-methanol as well as 100 ug/ml carbenicillin and 25 μg/ml kanamycin to maintain selection of the plasmids, and 1 mM IPTG to induce expression of the MeDH and HPS-PHI fusion enzymes. After 18 hours incubation at 37° C., the cell density was measured spectrophotometrically at 600 nM wavelength and a clarified sample of each culture medium was submitted for analysis to detect evidence of incorporation of the labeled methanol carbon into TCA-cycle derived metabolites. The label can be further enriched by deleting the gene araD that competes with ribulose-5-phosphate.

¹³C carbon derived from labeled methanol provided in the experiment was found to be significantly enriched in the metabolites pyruvate, lactate, succinate, fumarate, malate, glutamate and citrate, but only in the strain expressing both catalytically active MeDH 2315L and the HPS-PHI fusion 2616A together (data not shown). Moreover, this strain grew significantly better than the strain expressing catalytically active MeDH but lacking expression of the HPS-PHI fusion (data not shown), suggesting that the HPS-PHI enzyme is capable of reducing growth inhibitory levels of formaldehyde that cannot be detoxified by other means in this strain background. These results show that co-expression of an active MeDH and the enzymes of the RuMP pathway can effectively assimilate methanol derived carbon and channel it into TCA-cycle derived products.

Example XXIV

Pathway for Producing 2,4-Pentadienoate from Propionyl-CoA

This example describes a pathway for converting propionyl-CoA to 2,4-pentadienoate, shown in FIG. 27. Enzymes include: 3-oxopentanoyl-CoA thiolase or synthase, 3-oxopentanoyl-CoA reductase, 3-hydroxypentanoyl-CoA dehydratase, pent-2-enoyl-CoA isomerase, pent-3-enoyl-CoA dehydrogenase, one or more of 2,4-pentadienoyl-CoA hydrolase, transferase or synthetase and pent-2-enoyl-CoA dehydrogenase.

Propionyl-CoA is formed as a metabolic intermediate in numerous biological pathways including the 3-hydroxypropionate/4-hydroxybutyrate and 3-hydroxypropionate cycles of CO2 fixation, conversion of succinate or pyruvate to propionate, glyoxylate assimilation and amino acid degradation. In the pathways of FIG. 27, propionyl-CoA is further converted to 2,4-pentadienoate. In the first step of the pathway, propionyl-CoA and acetyl-CoA are condensed to 3-oxopentanoyl-CoA by 3-oxopentanoyl-CoA thiolase. Alternately, propionyl-CoA and malonyl-CoA are condensed by an enzyme with 3-oxopentanoyl-CoA synthase activity. Alternately, the 3-oxopentanoyl-CoA intermediate can be formed in two steps by first converting propionyl-CoA and malonyl-ACP to 3-oxopentanoyl-ACP, then converting the ACP to the CoA. 3-Oxopentanoyl-CoA is then reduced to 3-hydroxypentanoyl-CoA, and subsequently dehydrated to pent-2-enoyl-CoA by a 3-oxoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase, respectively (steps B, C). A delta-isomerase shifts the double bond from the 2- to the 3-position, forming pent-3-enoyl-CoA, the substrate for pent-3-enoyl-CoA dehydrogenase (steps D and E). Together the enzymes catalyzing steps B, C, D and E participate in the reverse direction in 5-aminovalerate utilizing organisms such as Clostridium aminovalericum. Alternately the pent-2-enoyl-CoA intermediate is oxidized to 2,4-pentadienoyl-CoA by a pent-2-enoyl-CoA dehydrogenase. In the final step of the pathway, 2,4-pentadienoyl-CoA is converted to its corresponding acid by a CoA hydrolase, transferse or synthetase (step F). 2,4-Pentadiene can be isolated as a product, or 2,4-pentadienoate or 2,4-pentadienoyl-CoA can be further converted to butadiene as depicted in FIG. 27. Enzymes and gene candidates for converting propionyl-CoA to 2,4-pentadienoate are described in further detail in Example XXV.

Example XXV

Enzyme Candidates for the Reactions Shown in FIGS. 26 and 27

Label	Function	Step
1.1.1.a	Oxidoreductase (oxo to alcohol)	26B, 26I, 26N, 26P, 27B,
1.3.1.a	Oxidoreducatse (alkane to alkene)	27E
2.3.1.b	Beta-ketothiolase	26A, 26M, 27A
2.8.3.a	Coenzyme-A transferase	26F, 26O, 26G, 26T, 26E,
		26H, 27F
3.1.2.a	Thiolester hydrolase	26F, 26O, 26G, 26T, 26E,
	(CoA specific)	26H, 27F
4.1.1.a	Decarboxylase	26U, 26V, 26Y, 26X, 27X
4.2.1.a	Hydro-lyase	26S, 26K, 26L, 26R, 26D,
		26C, 26J, 26Q, 26W, 27C
5.3.3.a	Delta-isomerase	27D
6.2.1.a	CoA synthetase	26F, 26O, 26G, 26T, 26E,
	(Adic-thiol ligase)	26H, 27F

1.1.1.a Oxidoreductase (Oxo to Alcohol)

Several reactions shown in FIGS. 26 and 27 can be catalyzed by alcohol dehydrogenase enzymes. These reactions include Steps B, I, N and P of FIG. 26, Step B of FIG. 27. Exemplary genes encoding enzymes that catalyze the reduction of an aldehyde to alcohol are described herein and above with regard to oxidoreductases (oxo to alcohol) EC class 1.1.1.a in relation to FIG. 10. For example, alcohol dehydrogenase enzymes that reduce 3-oxoacyl-CoA substrates to their corresponding 3-hydroxyacyl-CoA product are also relevant to the pathways depicted in FIG. 27 (step B) and include exemplary enzymes 3-oxoacyl-CoA reductase and acetoacetyl-CoA reductase as described for FIG. 10 above.

1.3.1.a Oxidoreducatse (Alkane to Alkene)

Step E of FIG. 27 entail oxidation of pent-3-enoyl-CoA to 2,4-pentadienoyl-CoA. Exemplary enzyme candidates are described below.

The oxidation of pent-3-enoyl-CoA or pent-2-enoyl-CoA to 2,4-pentadienoyl-CoA is catalyzed by 2,4-pentadienoyl-CoA forming dehydrogenase enzymes. 2,4-Dienoyl-CoA reductase enzymes (EC 1.3.1.34) are suitable candidates for these transformations. Generally, bacterial 2,4-dienoyl-CoA reductases yield 2-enoyl-CoA products, whereas eukaryotic 2,4-dienoyl-CoA reductases yield 3-enoyl-CoA products (Dommes and Kunau, J Biol Chem, 259:1781-1788 (1984)). The fadH gene product of E. coli is an NADPH-dependent 2,4-dienoyl-CoA reductase, which participates in the beta-oxidation of unsaturated fatty acids (Tu et al, Biochem, 47:1167-1175 (2008). A series of mutant DCR enzymes were constructed and shown to yield both 2-enoyl-CoA and 3-enoyl-CoA products (Tu et al, supra). Eukaryotic DCR enzymes have been characterized in humans and the mouse (Koivuranta et al, Biochem J, 304:787-792 (1994); Geisbrecht et al, J Biol Chem 274:25814-20 (1999); Miinalainen et al, PLoS genet 5: E1000543 (2009)). The 2,4-pentadienoyl-CoA reductase of Clostridium aminovalericum was shown to catalyze the oxidation of 3-pent-3-enoyl-CoA to 2,4-pentadienoyl-CoA. This enzyme has been purified, characterized and crystallized (Eikmanns, Acta Cryst, D50: 913-914 (1994) and Eikmanns and Buckel, Eur J Biochem 198:263-266 (1991)). The electron carrier of this enzyme is not known; however, it is not NAD(P)H. The sequence of the enzyme has not been published to date.

	Protein	GenBank ID	GI Number	Organism
	fadH	NP_417552.1	16130976	Escherichia coli
	Decr1	Q16698.1	3913456	Homo sapiens
	Pdcr	Q9WV68.1	90109767	Mus musculus
	Decr	NP_080448.1	13385680	Mus musculus

2-Enoate reductase enzymes in the EC classes 1.3.* are known to catalyze the reversible reduction of a wide variety of α,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J Biol Chem 276:5779-5787 (2001)). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been characterized (Seedorf et al., PNAS 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and Moorella thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel et al., 135:51-57 (1983)). It has been reported based on these sequence results that the C. tyrobutyricum enr is very similar to the FadH dienoyl CoA reductase of E. coli (Rohdich et al., supra). The M. thermoaceticum enr gene was expressed in a catalytically active form in E. coli (Rohdich et al., supra). This enzyme exhibits activity on a broad range of alpha, beta-unsaturated carbonyl compounds.

Protein	GenBank ID	GI Number	Organism
enr	ACA54153.1	169405742	Clostridium botulinum A3 str
enr	CAA71086.1	2765041	Clostridium tyrobutyricum
enr	CAA76083.1	3402834	Clostridium kluyveri
enr	YP_430895.1	83590886	Moorella thermoacetica

Another candidate 2-enoate reductase is maleylacetate reductase (MAR, EC 1.3.1.32), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. MAR enzymes naturally participate in aromatic degradation pathways (Kaschabek et al., J Bacteriol. 175:6075-6081 (1993); Kaschabek et al., J Bacteriol. 177:320-325 (1995); Camara et al., J Bacteriol. (2009); Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)). The enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek et al., 175:6075-6081 (1993); Kaschabek et al., 177:320-325 (1995)), and the coding gene was cloned and sequenced (Kasberg et al., J Bacteriol. 179:3801-3803 (1997)). Additional MAR gene candidates include clcE gene from Pseudomonas sp. strain B13 (Kasberg et al., J Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., 180:3503-3508 (1998)), the macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha (Seibert et al., J Bacteriol. 175:6745-6754 (1993)) and NCgl1112 in Corynebacterium glutamicum (Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)). A MAR in Pseudomonas reinekei MTJ, encoded by ccaD, was recently identified (Camara et al., J Bacteriol. (2009)).

Gene	GI #	Accession No.	Organism
clcE	3913241	O30847.1	Pseudomonas sp. strain B13
macA	7387876	O84992.1	Rhodococcus opacus
macA	5916089	AAD55886	Cupriavidus necator
tfdFII	1747424	AC44727.1	Ralstonia eutropha JMP134
NCgl1112	19552383	NP_600385	Corynebacterium glutamicum
ccaD	ABO61029.1	134133940	Pseudomonas reinekei MT1

An exemplary enoate reductase that favors the alkene-forming oxidative direction is succinate dehydrogenase (EC classes 1.3.99 or 1.3.5), also known as succinate-ubiquinone oxidoreductase and complex II. SDH is a membrane-bound enzyme complex that converts succinate to fumarate and transfers electrons to ubiquinone. The enzyme is composed of two catalytic subunits, encoded by sdhAB, and two membrane subunits encoded by sdhCD. Although the E. coli SDH is reversible, the enzyme is 50-fold more proficient in oxidizing succinate than reducing fumarate (Maldashina et al J Biol. Chem. 281:11357-11365 (2006)).

	Protein	GenBank ID	GI Number	Organism
	sdhA	AAC73817.1	1786942	Escherichia coli
	sdhB	AAC73818.1	1786943	Escherichia coli
	sdhC	AAC73815.1	1786940	Escherichia coli
	sdhD	AAC73816.1	1786941	Escherichia coli

An exemplary acyl-CoA dehydrogenase or enoyl-CoA reductase is the gene product of bcd from Clostridium acetobutylicum (Atsumi et al., 10:305-311 (2008); Boynton et al., J Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA (EC 1.3.99.2). This enzyme participates in the acetyl-CoA fermentation pathway to butyrate in Clostridial species (Jones et al., Microbiol Rev. 50:484-524 (1986)). Activity of butyryl-CoA reductase can be enhanced by expressing bcd in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase (EC 1.3.1.44) from E. gracilis (Hoffmeister et al., J Biol. Chem 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme (Hoffineister et al, supra). A close homolog of the protein from the prokaryote Treponema denticola, encoded by TDE0597, has also been cloned and expressed in E. coli (Tucci et al., FEBS Left, 581:1561-1566 (2007)). Six genes in Syntrophus aciditrophicus were identified by sequence homology to the C. acetobutylicum bcd gene product. The S. aciditrophicus genes syn_02637 and syn_02636 bear high sequence homology to the etfAB genes of C. acetobutylicum, and are predicted to encode the alpha and beta subunits of an electron transfer flavoprotein.

Protein	GenBank ID	GI Number	Organism
bcd	NP_349317.1	15895968	Clostridium acetobutylicum
etfA	NP_349315.1	15895966	Clostridium acetobutylicum
etfB	NP_349316.1	15895967	Clostridium acetobutylicum
TER	Q5EU90.1	62287512	Euglena gracilis
TDE0597	NP_971211.1	42526113	Treponema denticola
syn_02587	ABC76101	85721158	Syntrophus aciditrophicus
syn_02586	ABC76100	85721157	Syntrophus aciditrophicus
syn_01146	ABC76260	85721317	Syntrophus aciditrophicus
syn_00480	ABC77899	85722956	Syntrophus aciditrophicus
syn_02128	ABC76949	85722006	Syntrophus aciditrophicus
syn_01699	ABC78863	85723920	Syntrophus aciditrophicus
syn_02637	ABC78522.1	85723579	Syntrophus aciditrophicus
syn_02636	ABC78523.1	85723580	Syntrophus aciditrophicus

Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic capability to degrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes in the pim operon, pimC and pimD, bear sequence homology to C. acetobutylicum bcd and are predicted to encode a flavin-containing pimeloyl-CoA dehydrogenase (Harrison et al., 151:727-736 (2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison and Harwood, Microbiology 151:727-736 (2005)).

Protein	GenBank ID	GI Number	Organism
pimC	CAE29155	39650632	Rhodopseudomonas palustris
pimD	CAE29154	39650631	Rhodopseudomonas palustris
pimC	BAC53083	27356102	Bradyrhizobium japonicum
pimD	BAC53082	27356101	Bradyrhizobium japonicum

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase (EC 1.3.1.52 and EC 1.3.99.12), an enzyme catalyzing the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme participates in branched-chain fatty acid synthesis in the nematode Ascarius suum and is capable of reducing a variety of linear and branched chain substrates including 2-methylvaleryl-CoA, 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA (Duran et al., 268:22391-22396 (1993)). Two isoforms of the enzyme, encoded by genes acad1 and acad, have been characterized.

	Protein	GenBank ID	GI Number	Organism
	acad1	AAC48316.1	2407655	Ascarius suum
	acad	AAA16096.1	347404	Ascarius suum

2.3.1.b Beta-ketothiolase

Beta-ketothiolase enzymes in the EC class 2.3.1 catalyze the condensation of two acyl-CoA substrates. Step A of FIGS. 26 and 27, and Step M of FIG. 26 include the condensation of either 3-hydroxypropionyl-CoA, acrylyl-CoA or propionyl-CoA with malonyl-CoA or acetyl-CoA. Several beta-ketothiolase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below.

Exemplary beta-ketothiolases with acetoacetyl-CoA thiolase activity include the gene products of atoB from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol Biotechnol 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser et al., J. Biol. Chem. 269:31383-31389 (1994)).

Protein	GenBank ID	GI Number	Organism
atoB	NP_416728	16130161	Escherichia coli
thlA	NP_349476.1	15896127	Clostridium acetobutylicum
thlB	NP_149242.1	15004782	Clostridium acetobutylicum
ERG10	NP_015297	6325229	Saccharomyces cerevisiae

Beta-ketothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl-CoA and propionyl-CoA are also suitable candidates. Zoogloea ramigera possesses two ketothiolases that can form 3-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a beta-oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., U.S. Pat. No. 5,958,745 (1999)). The sequences of these genes or their translated proteins have not been reported, but several candidates in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from R. eutropha. These include:

Protein	GenBank ID	GI Number	Organism
phaA	YP_725941.1	113867452	Ralstonia eutropha
h16_A1713	YP_726205.1	113867716	Ralstonia eutropha
pcaF	YP_728366.1	116694155	Ralstonia eutropha
hl6 B1369	YP_840888.1	116695312	Ralstonia eutropha
h16_A0170	YP_724690.1	113866201	Ralstonia eutropha
h16_A0462	YP_724980.1	113866491	Ralstonia eutropha
h16_A1528	YP_726028.1	113867539	Ralstonia eutropha
h16_B0381	YP_728545.1	116694334	Ralstonia eutropha
h16_B0662	YP_728824.1	116694613	Ralstonia eutropha
h16_B0759	YP_728921.1	116694710	Ralstonia eutropha
h16_B0668	YP_728830.1	116694619	Ralstonia eutropha
h16_A1720	YP_726212.1	113867723	Ralstonia eutropha
h16_A1887	YP_726356.1	113867867	Ralstonia eutropha
phbA	P07097.4	135759	Zoogloea ramigera
bktB	YP_002005382.1	194289475	Cupriavidus taiwanensis
Rmet_1362	YP_583514.1	94310304	Ralstonia metallidurans
Bphy_0975	YP_001857210.1	186475740	Burkholderia phymatum

Another suitable candidate is 3-oxoadipyl-CoA thiolase (EC 2.3.1.174), which converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di et al., Arch. Microbiol 188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PAO1, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida.

Gene name	GI#	GenBank Accession #	Organism
paaJ	16129358	NP_415915.1	Escherichia coli
pcaF	17736947	AAL02407	Pseudomonas knackmussii (B13)
phaD	3253200	AAC24332.1	Pseudomonas putida
pcaF	506695	AAA85138.1	Pseudomonas putida
pcaF	141777	AAC37148.1	Acinetobacter calcoaceticus
paaE	106636097	ABF82237.1	Pseudomonas fluorescens
bkt	115360515	YP_777652.1	Burkholderia ambifaria AMMD
bkt	9949744	AAG06977.1	Pseudomonas aeruginosa PAO1
pcaF	9946065	AAG03617.1	Pseudomonas aeruginosa PAO1

2.8.3.a Coenzyme-A Transferase

Enzymes in the 2.8.3 family catalyze the reversible transfer of a CoA moiety from one molecule to another. Such a transformation can be included by steps F, O, G, T, H, and E of FIG. 26 and step F of FIG. 27. Several CoA transferase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described above for the EC 2.8.3.a Co-A transferase class described for FIG. 10.

3.1.2.a Thiolester Hydrolase (CoA Specific).

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to their corresponding acids. Such a transformation is required by steps F, O, G, T, H, and E of FIG. 26 and step F of FIG. 27. Several such enzymes have been described in the literature and represent suitable candidates for these steps. Suitable enzymes include those described for the EC 3.1.2.a CoA hydrolase above.

4.1.1.a Decarboxylase

The decarboxylation reactions of 2,4-pentadienoate to butadiene (step X of FIGS. 26 and 27) are catalyzed by enoic acid decarboxylase enzymes. Decarboxylase enzymes in the EC class 4.1.1 can also be used to catalyze steps U, Y, and V of FIG. 26. Candidate decarboxylase enzymes are described herein.

Exemplary enzymes are sorbic acid decarboxylase, aconitate decarboxylase, 4-oxalocrotonate decarboxylase and cinnamate decarboxylase. Sothic acid decarboxylase converts sorbic acid to 1,3-pentadiene. Sothic acid decarboxylation by Aspergillus niger requires three genes: padA1, ohbA1, and sdrA (Plumridge et al. Fung. Genet. Bio, 47:683-692 (2010). PadA1 is annotated as a phenylacrylic acid decarboxylase, ohbA1 is a putative 4-hydroxybenzoic acid decarboxylase, and sdrA is a sorbic acid decarboxylase regulator. Additional species have also been shown to decarboxylate sorbic acid including several fungal and yeast species (Kinderlerler and Hatton, Food Addit Contam., 7(5):657-69 (1990); Casas et al., Int J Food Micro., 94(1):93-96 (2004); Pinches and Apps, Int. J. Food Microbiol. 116: 182-185 (2007)). For example, Aspergillus oryzae and Neosartorya fischeri have been shown to decarboxylate sorbic acid and have close homologs to padA1, ohbA1, and sdrA.

Gene name	GenBankID	GI Number	Organism
padA1	XP_001390532.1	145235767	Aspergillus niger
ohbA1	XP_001390534.1	145235771	Aspergillus niger
sdrA	XP_001390533.1	145235769	Aspergillus niger
padA1	XP_001818651.1	169768362	Aspergillus oryzae
ohbA1	XP_001818650.1	169768360	Aspergillus oryzae
sdrA	XP_001818649.1	169768358	Aspergillus oryzae
padA1	XP_001261423.1	119482790	Neosartorya fischeri
ohbA1	XP_001261424.1	119482792	Neosartorya fischeri
sdrA	XP_001261422.1	119482788	Neosartorya fischeri

Aconitate decarboxylase (EC 4.1.1.6) catalyzes the final step in itaconate biosynthesis in a strain of Candida and also in the filamentous fungus Aspergillus terreus (Bonnarme et al. J Bacteriol. 177:3573-3578 (1995); Willke and Vorlop, Appl Microbiol. Biotechnol 56:289-295 (2001)). A cis-aconitate decarboxylase (CAD) (EC 4.1.16) has been purified and characterized from Aspergillus terreus (Dwiarti et al., J. Biosci. Bioeng. 94(1): 29-33 (2002)). Recently, the gene has been cloned and functionally characterized (Kanamasa et al., Appl. Microbiol Biotechnol 80:223-229 (2008)) and (WO/2009/014437). Several close homologs of CAD are listed below (EP 2017344A1; WO 2009/014437 A1). The gene and protein sequence of CAD were reported previously (EP 2017344 A1; WO 2009/014437 A1), along with several close homologs listed in the table below.

Gene name	GenBankID	GI Number	Organism
CAD	XP_001209273	115385453	Aspergillus terreus
	XP_001217495	115402837	Aspergillus terreus
	XP_001209946	115386810	Aspergillus terreus
	BAE66063	83775944	Aspergillus oryzae
	XP_001393934	145242722	Aspergillus niger
	XP_391316	46139251	Gibberella zeae
	XP_001389415	145230213	Aspergillus niger
	XP_001383451	126133853	Pichia stipitis
	YP_891060	118473159	Mycobacterium smegmatis
	NP_961187	41408351	Mycobacterium avium subsp. pratuberculosis
	YP_880968	118466464	Mycobacterium avium
	ZP_01648681	119882410	Salinispora arenicola

An additional class of decarboxylases has been characterized that catalyze the conversion of cinnamate (phenylacrylate) and substituted cinnamate derivatives to the corresponding styrene derivatives. These enzymes are common in a variety of organisms and specific genes encoding these enzymes that have been cloned and expressed in E. coli are: pad 1 from Saccharomyces cerevisae (Clausen et al., Gene 142:107-112 (1994)), pdc from Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069 (2001); Qi et al., Metab Eng 9:268-276 (2007); Rodriguez et al., J. Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella oxytoca (Uchiyama et al., Biosci. Biotechnol. Biochem. 72:116-123 (2008); Hashidoko et al., Biosci. Biotech. Biochem. 58:217-218 (1994)), Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), and padC from Bacillus subtilis and Bacillus pumilus (Shingler et al., 174:711-724 (1992)). A ferulic acid decarboxylase from Pseudomonas fluorescens also has been purified and characterized (Huang et al., J. Bacteriol. 176:5912-5918 (1994)). Importantly, this class of enzymes have been shown to be stable and do not require either exogenous or internally bound co-factors, thus making these enzymes ideally suitable for biotransformations (Sariaslani, Annu. Rev. Microbiol. 61:51-69 (2007)).

Protein	GenBank ID	GI Number	Organism
pad1	AAB64980.1	1165293	Saccharomyces cerevisae
pdc	AAC45282.1	1762616	Lactobacillus plantarum
pad	BAF65031.1	149941608	Klebsiella oxytoca
padC	NP_391320.1	16080493	Bacillus subtilis
pad	YP_804027.1	116492292	Pedicoccus pentosaceus
pad	CAC18719.1	11691810	Bacillus pumilus

4-Oxalocronate decarboxylase catalyzes the decarboxylation of 4-oxalocrotonate to 2-oxopentanoate. This enzyme has been isolated from numerous organisms and characterized. The decarboxylase typically functions in a complex with vinylpyruvate hydratase. Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al., 174:711-724 (1992)), xylII and xylIII from Pseudomonas putida (Kato et al., Arch. Microbiol 168:457-463 (1997); Stanley et al., Biochemistry 39:3514 (2000); Lian et al., J. Am. Chem. Soc. 116:10403-10411 (1994)) and Reut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al., J Bacteriol, 158:79-83 (1984)). The genes encoding the enzyme from Pseudomonas sp. (strain 600) have been cloned and expressed in E. coli (Shingler et al., J. Bacteriol. 174:711-724 (1992)). The 4-oxalocrotonate decarboxylase encoded by xylI in Pseudomonas putida functions in a complex with vinylpyruvate hydratase. A recombinant form of this enzyme devoid of the hydratase activity and retaining wild type decarboxylase activity has been characterized (Stanley et al., Biochem. 39:718-26 (2000)). A similar enzyme is found in Ralstonia pickettii (formerly Pseudomonas pickettii) (Kukor et al., J Bacteriol. 173:4587-94 (1991)).

Gene	GenBank	GI Number	Organism
dmpH	CAA43228.1	45685	Pseudomonas sp. CF600
dmpE	CAA43225.1	45682	Pseudomonas sp. CF600
xylII	YP_709328.1	111116444	Pseudomonas putida
xylIII	YP_709353.1	111116469	Pseudomonas putida
Reut_B5691	YP_299880.1	73539513	Ralstonia eutropha JMP134
Reut_B5692	YP_299881.1	73539514	Ralstonia eutropha JMP134
xylI	P49155.1	1351446	Pseudomonas putida
tbuI	YP_002983475.1	241665116	Ralstonia pickettii
nbaG	BAC65309.1	28971626	Pseudomonas fluorescens KU-7

The decarboxylation of 2-keto-acids such as 2-oxoadipate is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li et al., Biochemistry. 38:10004-10012 (1999); ter Schure et al., Appl. Environ. Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al., 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., 269:3256-3263 (2002)).

Protein	GenBank ID	GI Number	Organism
pdc	P06672.1	118391	Zymomonas mobilis
pdc1	P06169	30923172	Saccharomyces cerevisiae
pdc	Q8L388	20385191	Acetobacter pasteurians
pdc1	Q12629	52788279	Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al., 42:1820-1830 (2003); Hasson et al., 37:9918-9930 (1998)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Chembiochem. 4:721-726 (2003); Lingen et al., Protein Eng 15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

Protein	GenBank ID	GI Number	Organism
mdlC	P20906.2	3915757	Pseudomonas putida
mdlC	Q9HUR2.1	81539678	Pseudomonas aeruginosa
dpgB	ABN80423.1	126202187	Pseudomonas stutzeri
ilvB-1	YP_260581.1	70730840	Pseudomonas fluorescens

A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., 102:10670-10675 (2005)) has been cloned and functionally expressed. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka et al., Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO.) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

Protein	GenBank ID	GI Number	Organism
kgd	O50463.4	160395583	Mycobacterium tuberculosis
kgd	NP_767092.1	27375563	Bradyrhizobium japonicum USDA110
kgd	NP_105204.1	13473636	Mesorhizobium loti

A fourth candidate enzyme for catalyzing this reaction is branched chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku et al., J Biol Chem. 263:18386-18396 (1988); Smit et al., Appl Environ Microbiol 71:303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol 71:303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria. Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the E1 subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J. Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:12400-12403 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). These enzymes are composed of two alpha and two beta subunits.

Protein	GenBank ID	GI Number	Organism
kdcA	AAS49166.1	44921617	Lactococcus lactis
BCKDHB	NP_898871.1	34101272	Homo sapiens
BCKDHA	NP_000700.1	11386135	Homo sapiens
BCKDHB	P21839	115502434	Bos taurus
BCKDHA	P11178	129030	Bos taunts

A decarboxylase enzyme suitable for decarboxylating 3-ketoacids is acetoacetate decarboxylase (EC 4.1.1.4). The enzyme from Clostridium acetobutylicum, encoded by adc, has a broad substrate specificity and has been shown to decarboxylate numerous alternate substrates including 2-ketocyclohexane carboxylate, 3-oxopentanoate, 2-oxo-3-phenylpropionic acid, 2-methyl-3-oxobutyrate and benzoyl-acetate (Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984); Benner and Rozzell, J. Am. Chem. Soc. 103:993-994 (1981); Autor et at, J Biol. Chem. 245: 5214-5222 (1970)). An acetoacetate decarboxylase has also been characterized in Clostridium beijerinckii (Ravagnani et al., Mol. Microbiol 37:1172-1185 (2000)). The acetoacetate decarboxylase from Bacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylate 3-oxopentanoate (Matiasek et al., Curr. Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence of B. polymyxa is not yet available. Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)). Additional gene candidates in other organisms, including Clostridium botulinum and Bacillus amyloliquefaciens FZB42, can be identified by sequence homology.

Protein	GenBank ID	GI Number	Organism
adc	NP_149328.1	15004868	Clostridium acetobutylicum
adc	AAP42566.1	31075386	Clostridium saccharoperbutylacetonicum
adc	YP_001310906.1	150018652	Clostridium beijerinckii
CLL_A2135	YP_001886324.1	187933144	Clostridium botulinum
RBAM_030030	YP_001422565.1	154687404	Bacillus amyloliquefaciens

Numerous characterized enzymes decarboxylate amino acids and similar compounds, including aspartate decarboxylase, lysine decarboxylase and ornithine decarboxylase. Aspartate decarboxylase (EC 4.1.1.11) decarboxylates aspartate to form beta-alanine. This enzyme participates in pantothenate biosynthesis and is encoded by gene panD in Escherichia coli (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999); Ramjee et al., Biochem. J 323 (Pt 3):661-669 (1997); Merkel et al., FEMS Microbiol Lett. 143:247-252 (1996); Schmitzberger et al., EMBO J 22:6193-6204 (2003)). The enzymes from Mycobacterium tuberculosis (Chopra et al., Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutanicum (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999)) have been expressed and characterized in E. coli.

Protein	GenBank ID	GI Number	Organism
panD	P0A790	67470411	Escherichia coli K12
panD	Q9X4N0	18203593	Corynebacterium glutanicum
panD	P65660.1	54041701	Mycobacterium tuberculosis

Lysine decarboxylase (EC 4.1.1.18) catalyzes the decarboxylation of lysine to cadaverine. Two isozymes of this enzyme are encoded in the E. coli genome by genes cadA and ldcC. CadA is involved in acid resistance and is subject to positive regulation by the cadC gene product (Lemonnier et al., Microbiology 144 (Pt 3):751-760 (1998)). CadC accepts hydroxylysine and S-aminoethylcysteine as alternate substrates, and 2-aminopimelate and 6-aminocaproate act as competitive inhibitors to this enzyme (Sabo et al., Biochemistry 13:662-670 (1974)). The constitutively expressed ldc gene product is less active than CadA (Lemonnier and Lane, Microbiology 144 (Pt 3):751-760 (1998)). A lysine decarboxylase analogous to CadA was recently identified in Vibrio parahaemolyticus (Tanaka et al., J Appl Microbiol 104:1283-1293 (2008)). The lysine decarboxylase from Selenomonas ruminantium, encoded by ldc, bears sequence similarity to eukaryotic ornithine decarboxylases, and accepts both L-lysine and L-ornithine as substrates (Takatsuka et al., Biosci. Biotechnol Biochem. 63:1843-1846 (1999)). Active site residues were identified and engineered to alter the substrate specificity of the enzyme (Takatsuka et al., J Bacteriol. 182:6732-6741 (2000)). Several ornithine decarboxylase enzymes (EC 4.1.1.17) also exhibit activity on lysine and other similar compounds. Such enzymes are found in Nicotiana glutinosa (Lee et al., Biochem. J 360:657-665 (2001)), Lactobacillus sp. 30a (Guirard et al., J Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). The enzymes from Lactobacillus sp. 30a (Momany et al., J Mol. Biol. 252:643-655 (1995)) and V. vulnificus have been crystallized. The V. vulnificus enzyme efficiently catalyzes lysine decarboxylation and the residues involved in substrate specificity have been elucidated (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). A similar enzyme has been characterized in Trichomonas vaginalis but the gene encoding this enzyme is not known (Yarlett et al., Biochem. J 293 (Pt 2):487-493 (1993)).

Protein	GenBank ID	GI Number	Organism
cadA	AAA23536.1	145458	Escherichia coli
ldcC	AAC73297.1	1786384	Escherichia coli
ldc	O50657.1	13124043	Selenomonas ruminantium
cadA	AB124819.1	44886078	Vibrio parahaemolyticus
AF323910.1:1 . . . 1299	AAG45222.1	12007488	Nicotiana glutinosa
odc1	P43099.2	1169251	Lactobacillus sp. 30a
W2_1235	NP_763142.1	27367615	Vibrio vulnificus

Glutaryl-CoA dehydrogenase (GCD, EC 1.3.99.7 and EC 4.1.1.70) is a bifunctional enzyme that catalyzes the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA. Bifunctional GCD enzymes are homotetramers that utilize electron transfer flavoprotein as an electron acceptor (Hartel et al., Arch. Microbiol 159:174-181 (1993)). Such enzymes were first characterized in cell extracts of Pseudomonas strains KB740 and K172 during growth on aromatic compounds (Hartel et al., Arch. Microbiol 159:174-181 (1993)), but the associated genes in these organisms is unknown. Genes encoding glutaryl-CoA dehydrogenase (gcdH) and its cognate transcriptional regulator (gcdR) were identified in Azoarcus sp. CIB (Blazquez et al., Environ. Microbiol 10:474-482 (2008)). An Azoarcus strain deficient in gcdH activity was used to identify a heterologous gcdH gene from Pseudomonas putida (Blazquez et al., Environ. Microbiol 10:474-482 (2008)). The cognate transcriptional regulator in Pseudomonas putida has not been identified but the locus PP 0157 has a high sequence homology (>69% identity) to the Azoarcus enzyme. Additional GCD enzymes are found in Pseudomonas fluorescens and Paracoccus denitrificans (Husain et al., J Bacteriol. 163:709-715 (1985)). The human GCD has been extensively studied, overexpressed in E. coli (Dwyer et al., Biochemistry 39:11488-11499 (2000)), crystallized, and the catalytic mechanism involving a conserved glutamate residue in the active site has been described (Fu et al., Biochemistry 43:9674-9684 (2004)). A GCD in Syntrophus aciditrophicus operates in the CO₂-assimilating direction during growth on crotonate (Mouttaki et al., 73:930-938 (2007))). Two GCD genes in S. aciditrophicus were identified by protein sequence homology to the Azoarcus GcdH: syn_00480 (31%) and syn_01146 (31%). No significant homology was found to the Azoarcus GcdR regulatory protein.

Protein	GenBank ID	GI Number	Organism
gcdH	ABM69268.1	123187384	Azoarcus sp. CIB
gcdR	ABM69269.1	123187385	Azoarcus sp. CIB
gcdH	AAN65791.1	24981507	Pseudomonas putida KT2440
PP_0157 (gcdR)	AAN65790.1	24981506	Pseudomonas putida KT2440
gcdH	YP_257269.1	70733629	Pseudomonas fluorescens Pf-5
gcvA (gcdR)	YP_257268.1	70733628	Pseudomonas fluorescens Pf-5
gcd	YP_918172.1	119387117	Paracoccus denitrificans
gcdR	YP_918173.1	119387118	Paracoccus denitrificans
gcd	AAH02579.1	12803505	Homo sapiens
syn_00480	ABC77899	85722956	Syntrophus aciditrophicus
syn_01146	ABC76260	85721317	Syntrophus aciditrophicus

Alternatively, the carboxylation of crotonyl-CoA to glutaconyl-CoA and subsequent reduction to glutaryl-CoA can be catalyzed by separate enzymes: glutaconyl-CoA decarboxylase and glutaconyl-CoA reductase. Glutaconyl-CoA decarboxylase enzymes, characterized in glutamate-fermenting anaerobic bacteria, are sodium-ion translocating decarboxylases that utilize biotin as a cofactor and are composed of four subunits (alpha, beta, gamma, and delta) (Boiangiu et al., J Mol. Microbiol Biotechnol 10:105-119 (2005); Buckel, Biochim. Biophys. Acta 1505:15-27 (2001)). Such enzymes have been characterized in Fusobacterium nucleatum (Beatrix et al., Arch. Microbiol 154:362-369 (1990)) and Acidaminococcus fermentans (Braune et al., Mol. Microbiol 31:473-487 (1999)). Analogs to the F. nucleatum glutaconyl-CoA decarboxylase alpha, beta and delta subunits are found in S. aciditrophicus. A gene annotated as an enoyl-CoA dehydrogenase, syn_00480, another GCD, is located in a predicted operon between a biotin-carboxyl carrier (syn_00479) and a glutaconyl-CoA decarboxylase alpha subunit (syn_00481). The protein sequences for exemplary gene products can be found using the following GenBank accession numbers shown below. Enoyl-CoA reductase enzymes are described above (see EC 1.3.1).

Protein	GenBank ID	GI Number	Organism
gcdA	CAA49210	49182	Acidaminococcus fermentans
gcdC	AAC69172	3777506	Acidaminococcus fermentans
gcdD	AAC69171	3777505	Acidaminococcus fermentans
gcdB	AAC69173	3777507	Acidaminococcus fermentans
FN0200	AAL94406	19713641	Fusobacterium nucleatum
FN0201	AAL94407	19713642	Fusobacterium nucleatum
FN0204	AAL94410	19713645	Fusobacterium nucleatum
syn_00479	YP_462066	85859864	Syntrophus aciditrophicus
syn_00481	YP_462068	85859866	Syntrophus aciditrophicus
syn_01431	YP_460282	85858080	Syntrophus aciditrophicus
syn_00480	ABC77899	85722956	Syntrophus aciditrophicus

4.2.1.a Hydro-Lyase

The hydration of a double bond can be catalyzed by hydratase enzymes in the 4.2.1 family of enzymes. The removal of water to form a double bond can also be catalyzed by dehydratase enzymes in the 4.2.1 family of enzymes. Hydratase enzymes are sometimes reversible and also catalyze dehydration. Dehydratase enzymes are sometimes reversible and also catalyze hydration. The addition or removal of water from a given substrate is included by steps S, K, L, R, D, C, J, Q, and Win FIG. 26, and by step C in FIG. 27. Several hydratase and dehydratase enzymes have been described in the literature and represent suitable candidates for these steps. Useful enzymes include those described above for the EC 4.2.1.a Hydro-lyase class used in FIG. 10.

5.3.3.a Delta-Isomerase

Several characterized enzymes shift the double bond of enoyl-CoA substrates from the 2- to the 3-position. Exemplary enzymes include 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta-isomerase (EC 5.3.3.3), delta-3, delta-2-enoyl-CoA isomerase (EC 5.3.3.8) and fatty acid oxidation complexes. 4-Hydroxybutyrul-CoA dehydratase enzymes catalyze the reversible conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA. These enzymes are bifunctional, catalyzing both the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA, and also the isomerization of vinylacetyl-CoA and crotonyl-CoA. 4-Hydroxybutyrul-CoA dehydratase enzymes from C. aminobutyrium and C. kluyveri were purified, characterized, and sequenced at the N-terminus (Scherf et al., Arch. Microbiol 161:239-245 (1994); Scherf and Buckel, Eur. J Biochem. 215:421-429 (1993)). The C. kluyveri enzyme, encoded by abfD, was cloned, sequenced and expressed in E. coli (Gerhardt et al., Arch. Microbiol 174:189-199 (2000)). The abfD gene product from Porphyromonas gingivalis ATCC 33277 is closely related by sequence homology to the Clostridial gene products. 4-Hydroxybutyryl-CoA dehydratase/isomerase activity was also detected in Metallosphaera sedula, and is likely associated with the Msed_1220 gene (Berg et al, Science 318(5857):1782-6 (2007). Delta isomerization reactions are also catalyzed by the fatty acid oxidation complex. In E. coli, the fadJ and fadB gene products convert cis-3-enoyl-CoA molecules to trans-2-enoyl-CoA molecules under aerobic and anaerobic conditions, respectively (Campbell et al, Mol Micro 47(3):793-805 (2003)). A monofunctional delta-isomerase isolated from Cucumis sativus peroxisomes catalyzes the reversible conversion of both cis- and trans-3-enoyl-CoA into trans-2-enoyl-CoA (Engeland et al, Eur J Biochem, 196 (3):699-705 (1991). The gene associated with this enzyme has not been identified to date. A number of multifunctional proteins (MFP) from Cucumis sativus also catalyze this activity, including the gene product of MFP-a (Preisig-Muller et al, J Biol Chem 269:20475-81 (1994)).

Gene	GenBank	GI Number	Organism
abfD	P55792	84028213	Clostridium aminobutyricum
abfD	YP_001396399.1	153955634	Clostridium kluyveri
abfD	YP_001928843	188994591	Porphyromonas gingivalis
Msed_1220	ABP95381.1	145702239	Metallosphaera sedula
fadJ	AAC75401.1	1788682	Escherichia coli
fadB	AAC76849.1	1790281	Escherichia coli
MFP-a	Q39659.1	34922495	Cucumis sativus

6.2.1.a CoA Synthetase (Acid-Thiol Ligase)

The conversion of acyl-CoA substrates to their acid products can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes, several of which are reversible. Several reactions shown in FIGS. 26 and 27 are catalyzed by acid-thiol ligase enzymes. These reactions include Steps F, O, G, T, H, and E of FIG. 26 and Step F of FIG. 27. Several enzymes catalyzing CoA acid-thiol ligase or CoA synthetase activities have been described in the literature and represent suitable candidates for these steps. Suitable enzymes are described above in the EC 6.2.1.a CoA synthase (Acid-thiol ligase) as used for FIG. 10.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims

1-13. (canceled)

14. A non-naturally occurring microbial organism having a butadiene pathway and comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, wherein said butadiene pathway comprises a pathway selected from: (1) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (2) 10A, 10D, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (3) 10A, 10D, 10K, 10S, 15A, 15B, 15C, and 15G; (4) 10A, 10H, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (5) 10A, 10J, 10G, 10S, 15A, 15B, 15C, and 15G; (6) 10A, 10J, 10R, 10AA, 15A, 15B, 15C, and 15G; (7) 10A, 10H, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (8) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (9) 10A, 10D, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (10) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (11) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (12) 10A, 10D, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (13) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (14) 10A, 10B, 10M, 10AA, 15A, 15B, 15C, and 15G; (15) 10A, 10B, 10L, 10Z, 10AA, 15A, 15B, 15C, and 15G; (16) 10A, 10B, 10X, 10N, 10AA, 15A, 15B, 15C, and 15G; (17) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (18) 10A, 10D, 10P, 10O, 15A, 15B, 15C, and 15G; (19) 10A, 10B, 10X, 10O, 15A, 15B, 15C, and 15G; (20) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (21) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (22) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (23) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (24) 10A, 10B, 10C, 10AE, 10AB, 100, 15A, 15B, 15C, and 15G; (25) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (26) 10AU, 10AB, 10N, 10AA, 15A, 15B, 15C, and 15G; (27) 10AU, 10AB, 10O, 15A, 15B, 15C, and 15G; (28) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (29) 1T, 10AS, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (30) 1T, 10AS, 10K, 10S, 15A, 15B, 15C, and 15G; (31) 1T, 10AS, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (32) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (33) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (34) 1T, 10AS, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (35) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (36) 1T, 10AS, 10P, 10O, 15A, 15B, 15C, and 15G; (37) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (38) 10AT, 10E, 10F, 10G, 10S, 15A, 15B, 15C, and 15G; (39) 10AT, 10I, 10G, 10S, 15A, 15B, 15C, and 15G; (40) 10AT, 10K, 10S, 15A, 15B, 15C, and 15G; (41) 10AT, 10I, 10R, 10AA, 15A, 15B, 15C, and 15G; (42) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (43) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15B, 15C, and 15G; (44) 10AT, 10P, 10N, 10AA, 15A, 15B, 15C, and 15G; (45) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15B, 15C, and 15G; (46) 10AT, 10P, 10O, 15A, 15B, 15C, and 15G; (47) 10AT, 10E, 10F, 10R, 10AA, 15A, 15B, 15C, and 15G; (48) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (49) 10A, 10D, 10I, 10G, 10S, 15D, and 15G; (50) 10A, 10D, 10K, 10S, 15D, and 15G; (51) 10A, 10H, 10F, 10G, 10S, 15D, and 15G; (52) 10A, 10J, 10G, 10S, 15D, and 15G; (53) 10A, 10J, 10R, 10AA, 15D, and 15G; (54) 10A, 10H, 10F, 10R, 10AA, 15D, and 15G; (55) 10A, 10H, 10Q, 10Z, 10AA, 15D, and 15G; (56) 10A, 10D, 10I, 10R, 10AA, 15D, and 15G; (57) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (58) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (59) 10A, 10D, 10P, 10N, 10AA, 15D, and 15G; (60) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (61) 10A, 10B, 10M, 10AA, 15D, and 15G; (62) 10A, 10B, 10L, 10Z, 10AA, 15D, and 15G; (63) 10A, 10B, 10X, 10N, 10AA, 15D, and 15G; (64) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15D, and 15G; (65) 10A, 10D, 10P, 10O, 15D, and 15G; (66) 10A, 10B, 10X, 10O, 15D, and 15G; (67) 10A, 10D, 10E, 10F, 10R, 10AA, 15D, and 15G; (68) 10A, 10D, 10E, 10F, 10G, 10S, 15D, and 15G; (69) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (70) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15D, and 15G; (71) 10A, 10B, 10C, 10AE, 10AB, 10O, 15D, and 15G; (72) 10AU, 10AB, 10Y, 10Z, 10AA, 15D, and 15G; (73) 10AU, 10AB, 10N, 10AA, 15D, and 15G; (74) 10AU, 10AB, 10O, 15D, and 15G; (75) 1T, 10AS, 10E, 10F, 10G, 10S, 15D, and 15G; (76) 1T, 10AS, 10I, 10G, 10S, 15D, and 15G; (77) 1T, 10AS, 10K, 10S, 15D, and 15G; (78) 1T, 10AS, 10I, 10R, 10AA, 15D, and 15G; (79) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (80) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (81) 1T, 10AS, 10P, 10N, 10AA, 15D, and 15G; (82) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (83) 1T, 10AS, 10P, 10O, 15D, and 15G; (84) 1T, 10AS, 10E, 10F, 10R, 10AA, 15D, and 15G; (85) 10AT, 10E, 10F, 10G, 10S, 15D, and 15G; (86) 10AT, 10I, 10G, 10S, 15D, and 15G; (87) 10AT, 10K, 10S, 15D, and 15G; (88) 10AT, 10I, 10R, 10AA, 15D, and 15G; (89) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (90) 10AT, 10E, 10Q, 10Z, 10AA, 15D, and 15G; (91) 10AT, 10P, 10N, 10AA, 15D, and 15G; (92) 10AT, 10P, 10Y, 10Z, 10AA, 15D, and 15G; (93) 10AT, 10P, 10O, 15D, and 15G; (94) 10AT, 10E, 10F, 10R, 10AA, 15D, and 15G; (95) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (96) 10A, 10D, 10I, 10G, 10S, 15E, 15C, and 15G; (97) 10A, 10D, 10K, 10S, 15E, 15C, and 15G; (98) 10A, 10H, 10F, 10G, 10S, 15E, 15C, and 15G; (99) 10A, 10J, 10G, 10S, 15E, 15C, and 15G; (100) 10A, 10J, 10R, 10AA, 15E, 15C, and 15G; (101) 10A, 10H, 10F, 10R, 10AA, 15E, 15C, and 15G; (102) 10A, 10H, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (103) 10A, 10D, 10I, 10R, 10AA, 15E, 15C, and 15G; (104) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (105) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (106) 10A, 10D, 10P, 10N, 10AA, 15E, 15C, and 15G; (107) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (108) 10A, 10B, 10M, 10AA, 15E, 15C, and 15G; (109) 10A, 10B, 10L, 10Z, 10AA, 15E, 15C, and 15G; (110) 10A, 10B, 10X, 10N, 10AA, 15E, 15C, and 15G; (111) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (112) 10A, 10D, 10P, 10O, 15E, 15C, and 15G; (113) 10A, 10B, 10X, 10O, 15E, 15C, and 15G; (114) 10A, 10D, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (115) 10A, 10D, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (116) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (117) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15E, 15C, and 15G; (118) 10A, 10B, 10C, 10AE, 10AB, 10O, 15E, 15C, and 15G; (119) 10AU, 10AB, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (120) 10AU, 10AB, 10N, 10AA, 15E, 15C, and 15G; (121) 10AU, 10AB, 10O, 15E, 15C, and 15G; (122) 1T, 10AS, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (123) 1T, 10AS, 10I, 10G, 10S, 15E, 15C, and 15G; (124) 1T, 10AS, 10K, 10S, 15E, 15C, and 15G; (125) 1T, 10AS, 10I, 10R, 10AA, 15E, 15C, and 15G; (126) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (127) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (128) 1T, 10AS, 10P, 10N, 10AA, 15E, 15C, and 15G; (129) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (130) 1T, 10AS, 10P, 10O, 15E, 15C, and 15G; (131) 1T, 10AS, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (132) 10AT, 10E, 10F, 10G, 10S, 15E, 15C, and 15G; (133) 10AT, 10I, 10G, 10S, 15E, 15C, and 15G; (134) 10AT, 10K, 10S, 15E, 15C, and 15G; (135) 10AT, 10I, 10R, 10AA, 15E, 15C, and 15G; (136) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (137) 10AT, 10E, 10Q, 10Z, 10AA, 15E, 15C, and 15G; (138) 10AT, 10P, 10N, 10AA, 15E, 15C, and 15G; (139) 10AT, 10P, 10Y, 10Z, 10AA, 15E, 15C, and 15G; (140) 10AT, 10P, 10O, 15E, 15C, and 15G; (141) 10AT, 10E, 10F, 10R, 10AA, 15E, 15C, and 15G; (142) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (143) 10A, 10D, 10I, 10G, 10S, 15A, 15F, and 15G; (144) 10A, 10D, 10K, 10S, 15A, 15F, and 15G; (145) 10A, 10H, 10F, 10G, 10S, 15A, 15F, and 15G; (146) 10A, 10J, 10G, 10S, 15A, 15F, and 15G; (147) 10A, 10J, 10R, 10AA, 15A, 15F, and 15G; (148) 10A, 10H, 10F, 10R, 10AA, 15A, 15F, and 15G; (149) 10A, 10H, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (150) 10A, 10D, 10I, 10R, 10AA, 15A, 15F, and 15G; (151) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (152) 10A, 10D, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (153) 10A, 10D, 10P, 10N, 10AA, 15A, 15F, and 15G; (154) 10A, 10D, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (155) 10A, 10B, 10M, 10AA, 15A, 15F, and 15G; (156) 10A, 10B, 10L, 10Z, 10AA, 15A, 15F, and 15G; (157) 10A, 10B, 10X, 10N, 10AA, 15A, 15F, and 15G; (158) 10A, 10B, 10X, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (159) 10A, 10D, 10P, 10O, 15A, 15F, and 15G; (160) 10A, 10B, 10X, 10O, 15A, 15F, and 15G; (161) 10A, 10D, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (162) 10A, 10D, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (163) 10A, 10B, 10C, 10AE, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (164) 10A, 10B, 10C, 10AE, 10AB, 10N, 10AA, 15A, 15F, and 15G; (165) 10A, 10B, 10C, 10AE, 10AB, 10O, 15A, 15F, and 15G; (166) 10AU, 10AB, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (167) 10AU, 10AB, 10N, 10AA, 15A, 15F, and 15G; (168) 10AU, 10AB, 10O, 15A, 15F, and 15G; (169) 1T, 10AS, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (170) 1T, 10AS, 10I, 10G, 10S, 15A, 15F, and 15G; (171) 1T, 10AS, 10K, 105, 15A, 15F, and 15G; (172) 1T, 10AS, 10I, 10R, 10AA, 15A, 15F, and 15G; (173) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (174) 1T, 10AS, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (175) 1T, 10AS, 10P, 10N, 10AA, 15A, 15F, and 15G; (176) 1T, 10AS, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (177) 1T, 10AS, 10P, 10O, 15A, 15F, and 15G; (178) 1T, 10AS, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (179) 10AT, 10E, 10F, 10G, 10S, 15A, 15F, and 15G; (180) 10AT, 10I, 10G, 10S, 15A, 15F, and 15G; (181) 10AT, 10K, 10S, 15A, 15F, and 15G; (182) 10AT, 10I, 10R, 10AA, 15A, 15F, and 15G; (183) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (184) 10AT, 10E, 10Q, 10Z, 10AA, 15A, 15F, and 15G; (185) 10AT, 10P, 10N, 10AA, 15A, 15F, and 15G; (186) 10AT, 10P, 10Y, 10Z, 10AA, 15A, 15F, and 15G; (187) 10AT, 10P, 10O, 15A, 15F, and 15G; (188) 10AT, 10E, 10F, 10R, 10AA, 15A, 15F, and 15G; (189) 14A, 14B, 14C, 14D, 14E, 13A, and 13B; (190) 15A, 15B, 15C, and 15G; (191) 15D, and 15G; (192) 15E, 15C, and 15G; (193) 15A, 15F, and 15G; (194) 16A, 16B, 16C, 16D, and 16E; (195) 17A, 17B, 17C, 17D, and 17G; (196) 17A, 17E, 17F, 17D, and 17G; (197) 17A, 17B, 17C, 17H, 17I, 17J, and 17G; (198) 18A, 18B, 18C, 18D, 18E, and 18F; (199) 13A, and 13B; (200) 17A, 17E, 17F, 17H, 17I, 17J, and 17G; (201) 10A, 10B, 10C, 10AE, 19A, 19B, 19C, and 19D; (202) 10A, 10B, 10X, 10AB, 19A, 19B, 19C, and 19D; (203) 10A, 10D, 10P, 10AB, 19A, 19B, 19C, and 19D; (204) 1T, 10AS, 10P, 10AB, 19A, 19B, 19C, and 19D; (205) 10AT, 10P, 10AB, 19A, 19B, 19C, and 19D; (206) 10P, 10AB, 19A, 19B, 19C, and 19D; (207) 10AU, 19A, 19B, 19C, and 19D; and (208) 19A, 19B, 19C, and 19D, (209) 11A and 11F; (210) 10A, 10J, 10R, 10AD, 10AH, 11A, and 11F; (211) 10A, 10H, 10F, 10R, 10AD, 10AH, 11A, and 11F; (212) 10A, 10H, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (213) 10A, 10H, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (214) 10A, 10D, 10I, 10R, 10AD, 10AH, 11A, and 11F; (215) 10A, 10D, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (216) 10A, 10D, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (217) 10A, 10D, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (218) 10A, 10D, 10P, 10N, 10AD, 10AH, 11A, and 11F; (219) 10A, 10D, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (220) 10A, 10D, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (221) 10A, 10D, 10P, 10AB, 10V, 10AH, 11A, and 11F; (222) 10A, 10D, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (223) 10A, 10B, 10M, 10AD, 10AH, 11A, and 11F; (224) 10A, 10B, 10L, 10Z, 10AD, 10AH, 11A, and 11F; (225) 10A, 10B, 10L, 10AC, 10AG, 10AH, 11A, and 11F; (226) 10A, 10B, 10X, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (227) 10A, 10B, 10X, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (228) 10A, 10B, 10X, 10AB, 10V, 10AH, 11A, and 11F; (229) 10A, 10B, 10X, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (230) 10A, 10B, 10C, 10U, 10AH, 11A, and 11F; (231) 10A, 10B, 10C, 10T, 10AG, 10AH, 11A, and 11F; (232) 10A, 10B, 10C, 10AE, LOAF, 10AG, 10AH, 11A, and 11F; (233) 10A, 10D, 10P, 10AB, 10W, 11A, and 11F; (234) 10A, 10B, 10X, 10AB, 10W, 11A, and 11F; (235) 10A, 10B, 10C, 10AE, 10W, 11A, and 11F; (236) 10A, 10B, 10C, 10AE, 10V, 10AH, 11A, and 11F; (237) 10I, 10R, 10AD, 10AH, 11A, and 11F; (238) 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (239) 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (240) 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (241) 10P, 10N, 10AD, 10AH, 11A, and 11F; (242) 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (243) 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (244) 10P, 10AB, 10V, 10AH, 11A, and 11F; (245) 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (246) 10P, 10AB, 10W, 11A, and 11F; (247) 1T, 10AS, 10I, 10R, 10AD, 10AH, 11A, and 11F; (248) 1T, 10AS, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (249) 1T, 10AS, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (250) 1T, 10AS, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (251) 1T, 10AS, 10P, 10N, 10AD, 10AH, 11A, and 11F; (252) 1T, 10AS, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (253) 1T, 10AS, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (254) 1T, 10AS, 10P, 10AB, 10V, 10AH, 11A, and 11F; (255) 1T, 10AS, 10P, 10AB, 10AF, 10AG, 10AH, 11A, and 11F; (256) 1T, 10AS, 10P, 10AB, 10W, 11A, and 11F; (257) 10AT, 10I, 10R, 10AD, 10AH, 11A, and 11F; (258) 10AT, 10E, 10F, 10R, 10AD, 10AH, 11A, and 11F; (259) 10AT, 10E, 10Q, 10Z, 10AD, 10AH, 11A, and 11F; (260) 10AT, 10E, 10Q, 10AC, 10AG, 10AH, 11A, and 11F; (261) 10AT, 10P, 10N, 10AD, 10AH, 11A, and 11F; (262) 10AT, 10P, 10Y, 10Z, 10AD, 10AH, 11A, and 11F; (263) 10AT, 10P, 10Y, 10AC, 10AG, 10AH, 11A, and 11F; (264) 10AT, 10P, 10AB, 10V, 10AH, 11A, and 11F; (265) 10AT, 10P, 10AB, LOAF, 10AG, 10AH, 11A, and 11F; (266) 10AT, 10P, 10AB, 10W, 11A, and 11F; (267) 10AU, LOAF, 10AG, 10AH, 11A, and 11F; (268) 10AU, 10W, 11A, and 11F; (269) 10AU, 10V, 10AH, 11A, and 11F; (270) 10A, 10B, 10X, 10N, 10AD, 10AH, 11A, and 11F; and (271) 10A, 10B, 10X, 10N, 10AD, 10AH, and 11E, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10E is an acetoacetyl-CoA hydrolase, transferase or synthetase, wherein 10F is an acetoacetate reductase (acid reducing), wherein 10G is a 3-oxobutyraldehyde reductase (aldehyde reducing), wherein 10H is an acetoacetyl-ACP thioesterase, wherein 10I is an AcAcCoAR(CoA-dependent, aldehyde forming), wherein 10J is an acetoacetyl-ACP reductase (aldehyde forming), wherein 10K is an AcAcCoAR(alcohol forming), wherein 10L is a 3-hydroxybutyryl-ACP thioesterase, wherein 10M is a 3-hydroxybutyryl-ACP reductase (aldehyde forming), wherein 10N is a 3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 10O is a 3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 10P is an AcAcCoAR(ketone reducing), wherein 10Q is an acetoacetate reductase (ketone reducing), wherein 10R is a 3-oxobutyraldehyde reductase (ketone reducing), wherein 10S is a 4-hydroxy-2-butanone reductase, wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10Y is a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein 10Z is a 3-hydroxybutyrate reductase, wherein 10AA is a 3-hydroxybutyraldehyde reductase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 11E is a CrotOH dehydratase, wherein 11F is a BDS (monophosphate), wherein 13A is a 2-butanol desaturase, wherein 13B is a MVC dehydratase, wherein 14A is an acetolactate synthase, wherein 14B is an acetolactate decarboxylase, wherein 14C is a butanediol dehydrogenase, wherein 14D is a butanediol dehydratase, wherein 14E is a butanol dehydrogenase, wherein 15A is a 13BDO kinase, wherein 15B is a 3-hydroxybutyrylphosphate kinase, 15C is a 3-hydroxybutyryldiphosphate lyase, wherein 15D is a 13BDO diphosphokinase, wherein 15E is a 13BDO dehydratase, wherein 15F is a 3-hydroxybutyrylphosphate lyase, wherein 15G is a MVC dehydratase, wherein 16A is a 3-oxopent-4-enoyl-CoA thiolase, wherein 16B is a 3-oxopent-4-enoyl-CoA hydrolase, synthetase or transferase, wherein 16C is a 3-oxopent-4-enoate decarboxylase or spontaneous, wherein 16D is a 3-buten-2-one reductase, wherein 16E is a MVC dehydratase, wherein 17A is a 3-oxo-4-hydroxypentanoyl-CoA thiolase, wherein 17B is a 3-oxo-4-hydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17C is a 3-oxo-4-hydroxypentanoate reductase, wherein 17D is a 3,4-dihydroxypentanoate decarboxylase, wherein 17E is a 3-oxo-4-hydroxypentanoyl-CoA reductase, wherein 17F is a 3,4-dihydroxypentanoyl-CoA transferase, synthetase or hydrolase, wherein 17G is a MVC dehydratase, wherein 17H is a 3,4-dihydroxypentanoate dehydratase, wherein 171 is a 4-oxopentanoate reductase, wherein 17J is a 4-hyd4-oxoperoxypentanoate decarboxylase, wherein 18A is a 3-oxoadipyl-CoA thiolase, wherein 18B is a 3-oxoadipyl-CoA transferase, synthetase or hydrolase, wherein 18C is a 3-oxoadipate decarboxylase or spontaneous, wherein 18D is a 4-oxopentanoate reductase, wherein 18E is a 4-hydroxypentanoate decarboxylase, wherein 18F is a MVC dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase, wherein 19D is a 3-buten-1-ol dehydratase.

15. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve exogenous nucleic acids each encoding a butadiene pathway enzyme.

16. The non-naturally occurring microbial organism of claim 15, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(271).

17-24. (canceled)

25. The non-naturally occurring microbial organism of claim 14, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

26. The non-naturally occurring microbial organism of claim 14, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

27. The non-naturally occurring microbial organism of claim 14, wherein said microbial organism is a species of bacteria, yeast, or fungus.

28-31. (canceled)

32. A method for producing butadiene comprising culturing the non-naturally occurring microbial organism of claim 14 under conditions and for a sufficient period of time to produce butadiene.

33. The method of claim 32, wherein said method further comprises separating the butadiene from other components in the culture.

34. The method of claim 33, wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

35-166. (canceled)

167. A non-naturally occurring microbial organism having a 3-buten-1-ol pathway and comprising at least one exogenous nucleic acid encoding a 3-buten-1-ol pathway enzyme expressed in a sufficient amount to produce 3-buten-1-ol, wherein said 3-buten-1-ol pathway comprises a pathway selected from: (1) 10A, 10B, 10C, 10AE, 19A, 19B, and 19C; (2) 10A, 10B, 10X, 10AB, 19A, 19B, and 19C; (3) 10A, 10D, 10P, 10AB, 19A, 19B, and 19C; (4) 1T, 10AS, 10P, 10AB, 19A, 19B, and 19C; (5) 10AT, 10P, 10AB, 19A, 19B, and 19C; (6) 10P, 10AB, 19A, 19B, and 19C; (7) 10AU, 19A, 19B, and 19C; and (8) 19A, 19B, and 19C, wherein 1T is an acetyl-CoA carboxylase, wherein 10A is a 3-ketoacyl-ACP synthase, wherein 10B is an acetoacetyl-ACP reductase, wherein 10C is a 3-hydroxybutyryl-ACP dehydratase, wherein 10D is an acetoacetyl-CoA:ACP transferase, wherein 10P is an AcAcCoAR(ketone reducing), wherein 10X is a 3-hydroxybutyryl-CoA:ACP transferase, wherein 10AB is a 3-hydroxybutyryl-CoA dehydratase, wherein 10AE is a crotonyl-CoA:ACP transferase, wherein 10AS is an acetoacetyl-CoA synthase, wherein 10AT is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 10AU is a 4-hydroxybutyryl-CoA dehydratase, wherein 19A is a crotonyl-CoA delta-isomerase, wherein 19B is a vinylacetyl-CoA reductase, wherein 19C is a 3-buten-1-al reductase.

168. The non-naturally occurring microbial organism of claim 167, wherein said microbial organism comprises one, two, three, four, five, six, or seven exogenous nucleic acids each encoding a 3-buten-1-ol pathway enzyme.

169. The non-naturally occurring microbial organism of claim 168, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(8).

170-177. (canceled)

178. The non-naturally occurring microbial organism of claim 167, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

179. The non-naturally occurring microbial organism of claim 167, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

180. The non-naturally occurring microbial organism of claim 167, wherein said microbial organism is a species of bacteria, yeast, or fungus.

181-184. (canceled)

185. A method for producing 3-buten-1-ol, comprising culturing the non-naturally occurring microbial organism of claim 167 under conditions and for a sufficient period of time to produce 3-buten-1-ol.

186. A method for producing butadiene, comprising culturing the non-naturally occurring microbial organism of claim 167 under conditions and for a sufficient to produce 3-buten-1-ol, and chemically dehydrating said 3-buten-1-ol to produce butadiene.

187-209. (canceled)

210. An isolated nucleic acid molecule selected from: (a) a nucleic acid molecule encoding an amino acid sequence of XR, wherein said amino acid sequence comprises an amino acid substitution at position 121 as set forth in Table 1;(b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes an amino acid substitution at position 121 as set forth in Table 1, and(c) a nucleic acid molecule that is complementary to (a) or (b).

211. A non-naturally occurring microbial organism having an enzymatic pathway for producing a product wherein said organism comprises the nucleic acid of claim 210 or deregulated AraE.

212-213. (canceled)

214. A method of making a product comprising culturing the non-naturally occurring microbial organism of claim 211 under conditions and for a sufficient period of time to produce the product, wherein the culturing comprises the co-utilization of Sugar 1 and Sugar 2, Sugar 1 and Sugar 3, Sugar 2 and Sugar 3, or Sugar 1, Sugar 2 and Sugar 3.

215-226. (canceled)