Bioconversion of short-chain hydrocarbons to fuels and chemicals

Abstract

An engineered microorganism(s) with novel pathways for the conversion of short-chain hydrocarbons to fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives) is described. Key to this approach is the use of hydrocarbon activation enzymes able to overcome the high stability and low reactivity of hydrocarbon compounds through the cleavage of an inert CH bond. Oxygen-dependent or oxygen-independent activation enzymes can be exploited for this purpose, which when combined with appropriate pathways for the conversion of activated hydrocarbons to key metabolic intermediates, enables the generation of product precursors that can subsequently be converted to desired compounds through established pathways. These novel engineered microorganism(s) provide a route for the production of fuels and chemicals from short chain hydrocarbons such as methane, ethane, propane, butane, and pentane.

Claims

1. A method of producing a product comprising growing a genetically engineered bacteria in a culture broth containing a C1-C5 alkane and a terminal electron acceptor, wherein said genetically engineered bacteria converts the alkane to the product by oxygen-independent activation of the alkane, generating precursor intermediate acetyl-CoA, and producing a product from the acetyl-CoA, and wherein the genetically engineered bacteria selected from the group consisting of E. coli, Bacillus, Streptomyces,Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus comprises a vector expressing enzymes or overexpressing enzymes catalyzing: a) a sequence of reactions for the oxygen-independent activation of the alkane via fumarate addition to a 2-methyl-alkyl-succinate and subsequent conversion of said 2-methyl-alkyl-succinate to an acyl-CoA; b) a sequence of reactions for the generation of the product precursor acetyl-CoA and an acyl-CoA or keto-acid from said acyl-CoA; c) a sequence of reactions for the regeneration of the fumarate through the conversion of said acyl-CoA or keto-acid to the fumarate; and d) a sequence of reactions for the formation of the product from said product precursor acetyl-CoA; wherein the alkane is the sole carbon source in the broth that can be activated; wherein the enzymes comprise an alkyl succinate synthase that catalyzes the addition of the fumarate to the alkane to produce the 2-methyl-alkyl-succinate; wherein one or more of the enzymes are overexpressed; and wherein the method further comprises isolating the product formed in step d).

2. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA comprises: a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; b. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; c. an overexpressed 2-methyl-alkyl-malonyl-CoA mutase that catalyzes the isomerization of said 2-methyl-alkyl-succinyl-CoA to a 2-methyl-alkyl-malonyl-CoA; and d. an overexpressed 2-methyl-alkyl-malonyl-CoA decarboxylase that catalyzes the decarboxylation of said 2-methyl-alkyl-malonyl-CoA to the acyl-CoA.

3. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA and generation of the product precursor acetyl-CoA and the acyl-CoA or the keto-acid comprises: a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; b. an overexpressed 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; c. an overexpressed 2-methyl-alkyl-succinyl-CoA dehydrogenase that catalyzes the conversion of said 2-methyl-alkyl-succinyl-CoA to 2-methyl-alkyl-2-butenoyl-CoA; d. an overexpressed mesaconyl-C1-CoA-C4-CoA transferase that catalyzes the conversion of said 2-methyl-alkyl-2-butenoyl-CoA to 3-methyl-alkyl-2-butenoyl-CoA; e. an overexpressed mesaconyl-C4-CoA hydratase that catalyzes the conversion of said 3-methyl-alkyl-2-butenoyl-CoA to 3-methyl-alkyl-3-hydroxy-succinyl-CoA; and f. an overexpressed citramalyl-CoA lyase that catalyzes the conversion of said 3-methyl-alkyl-3-hydroxy-succinyl-CoA to the product precursor acetyl-CoA and the keto-acid.

4. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA and generation of the product precursor acetyl-CoA and the acyl-CoA comprises: a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; b. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; c. an overexpressed 2-methyl-alkyl-succinyl-CoA dehydrogenase that catalyzes the conversion of said 2-methyl-alkyl-succinyl-CoA to 2-methyl-alkyl-2-butenoyl-CoA; d. an overexpressed mesaconyl-CoA hydratase/-methylmalyl-CoA dehydratase that catalyzes the conversion of said 2-methyl-alkyl-2-butenoyl-CoA to 3-hydroxy-2-methyl-alkyl-succinyl-CoA; e. an overexpressed -methylmalyl-CoA lyase that catalyzes the conversion of said 3-hydroxy-2-methyl-alkyl-succinyl-CoA to glyoxylate and the acyl-CoA; f. an overexpressed glyoxylate carboligase that catalyzes the conversion of said glyoxylate to tartronate semialdehyde; g. an overexpressed tartronate semialdehyde reductase that catalyzes the conversion of said tartronate semialdehyde to D-glycerate; h. an overexpressed glycerate kinase that catalyzes the conversion of said D-glycerate to 3-phospho-D-glycerate; i. glycolytic enzymes that catalyze the conversion of said 3-phospho-D-glycerate to pyruvate, wherein the glycolytic enzymes are selected from the group consisting of phosphoglycerate mutase, enolase, and pyruvate kinase; and j. a pyruvate formate lyase or pyruvate dehydrogenase that catalyze the conversion of said pyruvate to the acetyl-CoA.

5. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the generation of the product precursor acetyl-CoA and the acyl-CoA comprises: a. an overexpressed acyl-CoA dehydrogenase that catalyzes the conversion of said acyl-CoA to a transenoyl-CoA; b. an overexpressed enoyl-CoA hydratase that catalyzes the hydration of said transenoyl-CoA to a 3-hydroxyacyl-CoA; c. an overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the oxidation of said 3-hydroxyacyl-CoA to a -ketoacyl-CoA; and d. an overexpressed thiolase that catalyzes the cleavage of the acetyl-CoA from said -ketoacyl-CoA to produce the acetyl-CoA and an acyl-CoA 2-carbons shorter than said acyl-CoA in step a.

6. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the regeneration of fumarate from the acyl-CoA or the keto-acid comprises: a. an overexpressed propionyl-CoA carboxylase that catalyzes the carboxylation of propionyl-CoA to (S)-methyl-malonyl-CoA; b. an overexpressed methyl-malonyl-CoA epimerase that catalyzes the interconversion of said (S)-methyl-malonyl-CoA to (R)-methyl-malonyl-CoA; c. an overexpressed methyl-malonyl-CoA mutase that catalyzes the isomerization of said (R)-methyl-malonyl-CoA to succinyl-CoA; d. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or succinyl-CoA synthetase that catalyzes the conversion of said succinyl-CoA to succinate; and e. an overexpressed succinate dehydrogenase that catalyzes the conversion of said succinate to fumarate.

7. The method of claim 1, wherein said pathway for the regeneration of the fumarate from the acyl-CoA or the keto-acid comprises: a. an overexpressed malate dehydrogenase for the conversion of said keto-acid to malate, wherein the keto-acid is pyruvate; and b. an overexpressed fumarase for the dehydration of said malate to fumarate.

8. The method of claim 1, wherein said pathway for the regeneration of the fumarate from the acyl-CoA or the keto-acid comprises: a. an overexpressed carboxylic acid omega hydroxylase that catalyzes the conversion of said keto-acid to an omega-hydroxy-2-keto-acid; b. an overexpressed alcohol dehydrogenase that catalyzes the conversion of said omega-hydroxy-2-keto acid to an omega-oxo-2-keto-acid; c. an overexpressed aldehyde dehydrogenase that catalyzes the conversion of said omega-oxo-2-keto-acid to a dicarboxylic 2-keto-acid; d. an overexpressed ketoreductase or malate dehydrogenase that catalyzes the conversion of said dicarboxylic 2-keto-acid to malate; and e. an overexpressed fumarase for the dehydration of said malate to fumarate.

9. The method of claim 1, wherein said alkyl succinate synthase is encoded by Azoarcus sp. HxN1 masB, Azoarcus sp. HxN1 masC, Azoarcus sp. HxN1 masD, Azoarcus sp. HxN1 masE, Azoarcus sp. HxN1 masG, Desulfosarcina sp. BuS5 A39W_RS0101550, Desulfosarcina sp. BuS5 A39W_RS0101545, Desulfosarcina sp. BuS5 A39W_RS0101540, Desulfosarcina sp. BuS5 A39W_RS0101535, Desulfosarcina sp. BuS5 A39W_RS19630, or Desulfosarcina sp. BuS5 A39W_RS0101580.

10. The method of claim 1, wherein said enzymes catalyzing the sequence of reactions for the generation of the product precursor acetyl-CoA comprises: a. an overexpressed acyl-CoA dehydrogenase that catalyzes the conversion of said acyl-CoA to a transenoyl-CoA; b. an overexpressed enoyl-CoA hydratase that catalyzes the hydration of said transenoyl-CoA to a 3-hydroxyacyl-CoA; c. an overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the oxidation of said 3-hydroxyacyl-CoA to a -ketoacyl-CoA; and d. an overexpressed thiolase that catalyzes the cleavage of the acetyl-CoA from said -ketoacyl-CoA to produce the acetyl-CoA and an acyl-CoA 2-carbons shorter than said starting acyl-CoA in step a.

11. The method of claim 1, wherein the sequence of reactions for the formation of the product from the product precursor acetyl-CoA is selected: a. a reverse beta oxidation (BOX-R) cycle comprised of: i. an overexpressed thiolase that catalyzes the non-decarboxylative condensation of an acyl-CoA primer with a 2-carbon donor acetyl-CoA to produce a -ketoacyl-CoA; ii. an overexpressed 3-oxoacyl-[acyl-carrier-protein] reductase or overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the reduction of a -ketoacyl-CoA to a -hydroxyacyl-CoA; iii. an overexpressed 3-hydroxyacyl-[acyl-carrier-protein] dehydratase or an overexpressed enoyl-CoA hydratase or 3-hydroxyacyl-CoA dehydratase that catalyzes the dehydration of a (3R)--hydroxyacyl-CoA to a transenoyl-CoA; iv. an overexpressed enoyl-[acyl-carrier-protein] reductase or acyl-CoA dehydrogenase or trans-enoyl-CoA reductase that catalyzes the reduction of said transenoyl-CoA to an acyl-CoA that is two carbons longer than said acyl-CoA primer; and v. an overexpressed termination pathway that catalyzes the conversion of an intermediate from said BOX-R cycle to said product; or b. a fatty acid biosynthesis (FAS) pathway comprised of: i. an overexpressed acetyl-CoA carboxylase that catalyzes the conversion of acetyl-CoA to malonyl-CoA; ii. an overexpressed malonyl-CoA-[acyl-carrier-protein] (ACP) transacylase that catalyzes the conversion of said malonyl-CoA to malonyl-ACP; iii. an overexpressed -ketoacyl-ACP synthase that catalyzes the decarboxylative condensation of said malonyl-ACP with an acyl-ACP primer to produce a -ketoacyl-ACP; iv. an overexpressed 3-oxoacyl-ACP reductase that catalyzes the reduction of said -ketoacyl-ACP to a -hydroxyacyl-ACP; v. an overexpressed 3-hydroxyacyl-ACP dehydratase that catalyzes the dehydration of a (3R)--hydroxyacyl-ACP to a transenoyl-ACP; vi. an overexpressed enoyl-ACP reductase that catalyzes the reduction of a transenoyl-ACP to an acyl-ACP that is two carbons longer than said acyl-ACP primer; and vii. an overexpressed termination pathway that catalyzes the conversion of an intermediate from said FAS pathway to said product.

12. The method of claim 11, wherein said termination pathway is selected from the group consisting of: a. a CoA cleaving thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase, or an ACP cleaving thioesterase said product is selected from the group consisting of carboxylic acids, (3R)--hydroxy carboxylic acids, -keto carboxylic acids, and ,-unsaturated carboxylic acids; b. an alcohol-forming coenzyme-A thioester reductase, an aldehyde-forming CoA thioester reductase and an alcohol dehydrogenase, an alcohol-forming ACP thioester reductase, or an aldehyde-forming ACP thioester reductase and an alcohol dehydrogenase and said product is selected from the group consisting of primary alcohols, 1,(3R) diols, -keto primary alcohols, and ,-unsaturated primary alcohols; c. an aldehyde-forming CoA or ACP thioester reductase and an aldehyde decarbonylase and said product is selected from the group consisting of linear alkanes, linear alkan-2-ols, linear methyl-ketones, and 1-alkenes; and d. an aldehyde-forming CoA or ACP thioester reductase and a transaminase and said product is selected from the group consisting of primary amines, 3-hydroxy-amines, 3-keto-amines, and ,-unsaturated primary amines.

13. The method of claim 1, wherein the terminal electron acceptor is SO.sub.4.sup.2, NO.sub.3.sup., Fe.sup.3+, O.sub.2, or Mn.sup.4+.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Generalized pathway for the conversion of alkanes to desired products.

(2) FIG. 2. Example of oxygen-independent alkane activation cycle via fumarate addition and conversion to an acyl-CoA, generation of product precursor acetyl-CoA and propionyl-CoA, regeneration of fumarate from propionyl-CoA, and generalized product formation from acetyl-CoA. Enzymes, 1: alkyl succinate synthase, 2a: succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase/2-methyl-alkyl-succinyl-CoA synthetase, 2b: 2-methyl-alkyl-malonyl-CoA mutase, 2c: 2-methyl-alkyl-malonyl-CoA decarboxylase, 2d: -oxidation enzymes, 2e: propionyl-CoA carboxylase, 2f: methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase, 2g: succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase/succinyl-CoA synthetase, 2h: succinate dehydrogenase.

(3) FIG. 3. Example of one specific oxygen-independent alkane activation cycle via fumarate addition and conversion to an acyl-CoA, generation of product precursor acetyl-CoA and pyruvate (a keto acid), regeneration of fumarate from pyruvate, and generalized product formation from acetyl-CoA.

(4) FIG. 4. Example of one specific oxygen-independent alkane activation cycle via fumarate addition and conversion to an acyl-CoA, generation of product precursor acetyl-CoA and propionyl-CoA, regeneration of fumarate from propionyl-CoA, and generalized product formation from acetyl-CoA. Enzymes, 1: methyl succinate synthase, 2: succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase/2-methyl-alkyl-succinyl-CoA synthetase, 3: 2-methyl-alkyl-succinyl-CoA dehydrogenase, 4: mesaconyl-CoA hydratase/-methylmalyl-CoA dehydratase, 5: -methylmalyl-CoA lyase, 6: propionyl-CoA carboxylase, 7: methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase, 8: succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase/succinyl-CoA synthetase, 9: succinate dehydrogenase, 10: glyoxylate carboligase, 11: tartronate semialdehyde reductase, 12: glycerate kinase, 13: glycolytic enzymes (phosphoglycerate mutase, enolase, pyruvate kinase), 14: pyruvate dehydrogenase or pyruvate formate lyase, 15: -oxidation reversal or fatty acid biosynthesis pathways with desired termination pathways.

(5) FIG. 5. Oxygen-dependent alkane activation via terminal addition of a hydroxyl group to a primary alcohol and conversion to acyl-CoA, generation of precursor acetyl-CoA, and generalized product synthesis from acetyl-CoA. Enzymes, 1: alkane monooxygenase or alkane hydroxylase, 2: alcohol dehydrogenase, 3: aldehyde dehydrogenase, 4: acyl-CoA synthetase, 5: acylating aldehyde dehydrogenase.

(6) FIG. 6A-D. Vectors expressing the components of the oxygen-independent activation enzyme alkyl-succinate synthase from Azoarcus sp. HxN1, MasB (A9J4K0), MasC (A9J4K2), MasD (A9J4K4), MasE (A9J4K6), and MasG (A9J4J6) have been proposed as required components for enzyme function.

(7) FIG. 7. Expression of components of the alkyl-succinate synthase from Azoarcus sp. HxN1, part of the oxygen-independent alkane activation/utilization pathway. Soluble cell extract of E. coli cells expressing MasB (A9J4K0), MasC (A9J4K2), MasD (A9J4K4), MasE (A9J4K6), and MasG (A9J4J6). Lane 1: pETDuet-1-P1-MasCDEB-P2-MasG; Lane 2: pETDuet-1-P1-MasCDE-P2-MasG; Lane 3: pETDuet-1-P1-MasCDEB; Lane 4: pETDuet-1-P1-MasCDE; Lane 5: pETDuet-1.

(8) FIG. 8A-C. Vectors expressing the components of the oxygen-independent activation enzyme alkyl-succinate synthase from Desulfosarcina sp. BuS5. Proteins identified through similarity to Azoarcus sp. HxN1 MasBCDEG as well as physical location in Desulfosarcina sp. BuS5 genome. Mas5: WP_027352796.1; Mas8: WP_051374532.1; Mas11: WP_027352800.1.

(9) FIG. 9A-C. Vectors expressing the components of the oxygen-independent activation enzyme alkyl-succinate synthase from Desulfosarcina sp. BuS5. Proteins identified through similarity to Azoarcus sp. HxN1 MasBCDEG as well as physical location in Desulfosarcina sp. BuS5 genome. Mas5: WP_027352796.1; Mas4: WP_027352795.1; Mas3: WP_027352794.1; Mast: WP_027352793.1; Mas8: WP_051374532.1; Mas11: WP 027352800.1.

(10) FIG. 10A-C. Vectors expressing enzymes for the conversion of fumarate activated hydrocarbon intermediates to product precursors. TaBbsEF: T. aromatica succinyl-CoA:(R)-benzylsuccinate CoA-transferase (Q9KJF0, Q9KJE9); RsMcd: R. sphaeroides (2S)-methylsuccinyl-CoA dehydrogenase (ADC44452.1); RsMch: R. sphaeroides mesaconyl-coenzyme A hydratase (Q3IZ78).

(11) FIG. 11. Expression of enzymes involved in the oxygen-independent alkane utilization pathway. Left: Whole cell extract of E. coli cells expressing T. aromatica succinyl-CoA:(R)-benzylsuccinate CoA-transferase TaBbsEF (Q9KJF0, Q9KJE9), Lane 1: pCDFDuet-1-P1-TaBbsEF; Lane 2: pCDFDuet-1. Right: Soluble cell extract of E. coli cells expressing R. sphaeroides (2S)-methylsuccinyl-CoA dehydrogenase RsMcd (ADC44452.1) and R. sphaeroides mesaconyl-coenzyme A hydratase RsMch (Q3IZ78), Lane 1: pCDFDuet-1-P1-P2-RsMch-RsMcd; Lane 2: pCDFDuet-1-P1-P2-RsMch; Lane 3: pCDFDuet-1. RsMcd: R. sphaeroides (2S)-methylsuccinyl-CoA dehydrogenase (ADC44452.1); RsMch: R. sphaeroides mesaconyl-coenzyme A hydratase (Q3IZ78).

(12) FIG. 12. Oxygen-dependent alkane activation of hexane with P. putida alkane hydroxylase AlkBGT. GC-FID chromatogram for products of whole cell biotransformation of BL21(DE3) cells with indicated vectors shown. 1 mL of cell suspension (OD.sub.550 nm=12) incubated with 250 L hexane at 30 C. for 2 hours. Hexanol only observed only in cells expressing AlkBGT.

(13) FIG. 13. Oxygen-dependent alkane activation of pentane with P. putida alkane hydroxylase AlkBGT. GC-FID chromatogram for products of whole cell biotransformation of BL21(DE3) cells with indicated vectors shown. 1 mL of cell suspension (OD.sub.550 nm=12) incubated with 250 L pentane at 30 C. for 2 hours. Pentanol only observed only in cells expressing AlkBGT.

(14) FIG. 14A-B. Vectors expressing the putative alkane monooxygenase from Rhodobacter sphaeroides. RSP2792: monooxygenase alpha subunit (YP_352924.1); RSP2793: monooxygenase reductase component (YP_352923.1); RSP2794: monooxygenase beta subunit (YP_352922.1); RSP2795: regulatory protein of multicomponent monooxygenase (YP_352921.1).

(15) FIG. 15. TABLE G: list of some possible embodiments.

DETAILED DESCRIPTION

(16) The activation of the short chain hydrocarbon substrates, such as methane of LNG components, requires the high stability and low reactivity of hydrocarbon compounds to be overcome through the cleavage of an inert CH bond. Two main biological approaches can be exploited here for this purpose, 1) the oxygen-independent activation through the addition of fumarate to form a 2-methyl-alkyl-succinyl-CoA, or 2) the oxygen-dependent activation through the terminal addition of a hydroxyl group to the alkane to form a primary alcohol.

(17) The first approach leverages a native pathway for anaerobic hydrocarbon activation used by certain microorganisms for the degradation of C.sub.6-C.sub.20 alkanes (Callaghan, 2013). In this pathway, an alkyl-succinate synthase first forms a branched methyl-alkyl-succinate adduct from the addition of fumarate to the alkane, with further metabolism and fumarate regeneration steps resulting in the conversion of hydrocarbon substrates to central, intracellular metabolites (FIG. 2).

(18) In order to meet ATP requirements of this cycle, native hydrocarbon degrading microorganisms utilize the electrons generated during substrate oxidation for anaerobic respiration with nitrate, metal ions, or sulfate, thereby making hydrocarbon degradation thermodynamically favorable (Mbadinga et al., 2011). An example of this type of process is found in strain HxN1, which can grow anaerobically on alkanes such as hexane when coupled to denitrification, with the key genes required for initial alkane activation proposed (Grundmann et al., 2008). Reports also have suggested similar activation with shorter chain hydrocarbons in bacterial consortiums, although the enzymes responsible have not been identified (Duncan et al., 2009; Kniemeyer et al., 2007).

(19) Furthermore, several potential variations of this pathway exist in which various biological reactions can be utilized to convert the 2-methyl-alkyl-succinate adduct formed via fumarate addition into central metabolic intermediates (FIG. 3 and FIG. 4). Regardless of the pathway selected, the end result of the oxygen-independent hydrocarbon activation pathway is the formation of an acyl-CoA molecule that can be used to generate the product precursor acetyl-CoA. While in certain cases acetyl-CoA is directly formed as a result of initial acyl-CoA formation (FIG. 3 and FIG. 4), other initial acyl-CoA intermediates require further oxidation for the generation of acetyl-CoA (FIG. 2). In all cases, the end result is the generation of acetyl-CoA to serve as a product precursor, as well as another acyl-CoA of keto-acid compound that can be used for fumarate regeneration.

(20) Considering the use of fumarate in the alkane activation cycle, this oxygen-independent activation approach also requires pathways for the regeneration of this compound from an acyl-CoA or keto-acid. Details on the pathways leading to the regeneration of fumarate from an acyl-CoA, such as propionyl-CoA, or a keto-acid, such as pyruvate, are shown in FIG. 2, FIG. 3, and FIG. 4. This regeneration enables the continued operation of the hydrocarbon activation cycle via fumarate addition.

(21) In contrast to hydrocarbon activation via fumarate addition, an alternative pathway for the cleavage of an inert CH bond is the use of an oxygen-dependent activation mechanism in which an alkane hydroxylase/monooxygenase adds a terminal alcohol group to the hydrocarbon, which is then further oxidized (FIG. 5). The end result of the oxygen-dependent activation pathway is an acyl-CoA intermediate than can be used for the generation of the product precursor acetyl-CoA. Given the direct use of oxygen in this pathway for activation, no additional pathways are required to regenerate an activation compounds as in the above oxygen-independent approach.

(22) Once the product precursor acetyl-CoA has been formed through the above described approaches, the desired products can then be synthesized through various native or engineered metabolic pathways. These include both the reversal of the -oxidation cycle and the fatty acid biosynthesis pathway, which both proceed from acetyl-CoA as the starting compound. While the type of condensation reaction for chain elongation varies between the 2 pathways (reversal of the -oxidation cycle utilizes a non-decarboxylative condensation while the fatty acid biosynthesis pathway employs a decarboxylative condensation), both proceed through a series of condensation, reduction, dehydration, and reduction reactions that result in an intermediate 2 carbons longer than the starting unit. As such, each of these pathways can be exploited for the synthesis of a wide range of products through the selection of termination pathways that convert cycle intermediates to products such as carboxylic acids and alcohols among others.

(23) Thus, either of these product synthesis pathways, combined with the key pathway modules for hydrocarbon activation and conversion to an acyl-CoA intermediate, generation of product precursor acetyl-CoA, and generation of an addition acyl-CoA or keto-acid for fumarate when required, and the formation of a desired product from acetyl-CoA enables an engineered microorganism capable of the conversion of short-chain hydrocarbons (e.g. methane, ethane, propane, butane, pentane) to fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives).

(24) The following description provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other details, as the specification would be of inordinate length if one were to list every possible combination of genes/vectors/enzymes/hosts that can be made to convert short chain hydrocarbons to desired fuels and chemicals of interest. Some possible combinations are listed in Table G, however, (FIG. 15).

(25) Enzymes of interest can be expressed from vectors such as pETDuet-1 or pCDFDuet-1 (MERCK, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience.

(26) The genes can be amplified by PCR using primers designed with 15-22 base pairs of homology for the appropriate vector cut site. For enzymes that will not require a 6-histadine tag fusion for purification, pCDFDuet-1 can be linearized with NcoI and EcoRI. Enzymes that will be purified by Ni-NTA column will make use of the 6-HIS tag in pCDFDuet-1. The vector can be linearized using only EcoRI in this case.

(27) The PCR product can be inserted into the vector using e.g., the In-Fusion HD EcoDry Cloning System and the vector transformed by heat shock into competent E. coli cells. Transformants can be selected on solid media containing the appropriate antibiotic. Plasmid DNA can be isolated using any suitable method, including QIAprep Spin Miniprep Kit (QIAGEN, Limburg), and the construct confirmed by PCR and sequencing. Confirmed constructs can be transformed by e.g., electroporation into a host strain such as E. coli for expression, but other host species can be used with suitable expression vectors and possible codon optimization for that host species.

(28) Expression of the desired enzymes from the constructed strain can be conducted in liquid culture, e.g., shaking flasks, bioreactors, chemostats, fermentation tanks and the like. Gene expression is typically induced by the addition of a suitable inducer, when the culture reaches an OD.sub.550 nm of approximately 0.5-0.8. Induced cells can be grown for about 4-8 hours, at which point the cells can be pelleted and saved to 20 C. Expression of the desired protein can be confirmed by running samples on SDS-PAGE.

(29) The expressed enzyme can be directly assayed in crude cell lysates, simply by breaking the cells by chemical, enzymatic, heat or mechanical means. Depending on the expression level and activity of the enzyme, however, purification may be required to be able to measure enzyme activity over background levels. Purified enzymes can also allow for the in vitro assembly of the pathway, allowing for its controlled characterization.

(30) N- or C-terminal HIS-tagged proteins can be purified using e.g., a Ni-NTA Spin Kit (Qiagen, Venlo, Limburg) following the manufacturer's protocol, or other methods could be used. The HIS-tag system was chosen for convenience only, and other tags are available for purification uses. Further, the proteins in the final assembled pathway need not be tagged if they are for in vivo use. Tagging was convenient, however, for the enzyme characterization work performed hereunder.

(31) Reaction conditions for enzyme assays can vary greatly with the type of enzyme to be tested. In general, however, enzyme assays follow a similar general protocol. Purified enzyme or crude lysate is added to suitable reaction buffer. Reaction buffers typically contain salts, necessary enzyme cofactors, and are at the proper pH. Buffer compositions often change depending on the enzyme or reaction type. The reaction is initiated by the addition of substrate, and some aspect of the reaction related either to the consumption of a substrate or the production of a product is monitored.

(32) Choice of the appropriate monitoring method depends on the compound to be measured. Spectrophotometric assays are convenient because they allow for the real time determination of enzyme activity by measuring the concentration dependent absorbance of a compound at a certain wavelength. There are not always compounds with a measurable absorbance at convenient wavelengths in the reaction, unfortunately. In these situations, other methods of chemical analysis may be necessary to determine the concentration of the involved compounds.

(33) As an example, cultures for enzymatic assays were conducted in 125 mL Erlenmeyer flasks containing 25 mL LB media inoculated at 3% from an overnight culture. E. coli strains containing constructs expressing genes of interest were grown under appropriate conditions until an optical density of 0.5 was reached, at which point inducer(s) were added and the cells incubated for an additional 4 hrs. Cell harvesting and preparation of crude cell extracts for enzyme assays was conducted as described elsewhere (Dellomonaco et al., 2011). Enzymatic reactions were then monitored on either a Synergy HT plate reader (BioTek Instruments, Inc., Winooski, VT) or a Biomate 5 Spectrophotometer (Thermo Scientific, Waltham, MA) according to established protocols.

(34) Furthermore, whole cell biotransformations represent another method to determine enzyme/pathway functionality. As an example, biotransformations for alkane activation were conducted using E. coli BL21(DE3) with appropriate vectors. 750 L of an overnight LB culture with appropriate antibiotics was used to inoculate 25 mL LB media with 10 M FeSO.sub.4 in a 125 mL Pyrex Erlenmeyer flask. Flasks were incubated at 37 C. and 200 rpm in an NBS Benchtop Incubator Shaker until an optical density of 0.5 was reached, at which point appropriate inducers were added. Cultures were grown for 4 hrs post-induction and then centrifuged (8000 rpm, 22 C., 5 min) and re-suspended to an optical density 12 in 100 mM KP.sub.i buffer (pH 7.0) with 1% glycerol (Koch et al., 2009). 250 L alkane was then added to a 2 mL cell suspension in a 5 mL glass vial and incubated with rotation (60 rpm) at 30 C. for 2 hrs, Following the biotransformations, products (alcohols) were extracted and analyzed as previously described (Kim et al., 2015).

(35) Gas chromatography (GC) is convenient for the quantification of volatile substances, of which fatty acids and alcohols are of particular relevance. Internal standards, typically one or more molecules of similar type not involved in the reaction, are added to the reaction mixture, and the reaction mixture is extracted with an organic solvent, such as hexane. Fatty acid samples, for example, can be dried under a stream of nitrogen and converted to their trimethylsilyl derivatives using N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine in a 1:1 ratio. After 30 minutes incubation, the samples are once again dried and resuspended in hexane to be applied to the gas chromatograph (GC). Aldehyde samples do not need to be derivatized. Samples can be run e.g., on a Varian CP-3800 gas chromatograph (VARIAN ASSOCIATES, Palo Alto, CA) equipped with a flame ionization detector and HP-5 capillary column (AGILENT TECH., CA).

(36) Once pathways have been fully studied in vitro, they can be constructed in vivo with greater confidence. The strain construction for the in vivo pathway operation should allow for the well-defined, controlled expression of the enzymes of the pathway. As before, E. coli or yeast will be a host of choice for the in vivo pathway, but other hosts could be used. The Duet system, for example, allows for the simultaneous expression of up to eight proteins by induction with IPTG in E. coli, and initial experiments will use this host.

(37) Pathway enzymes can also be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of plasmids. A large number of genes that can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with plasmid expression.

(38) DNA constructs for chromosomal integration usually include an antibiotic resistance marker with flanking FRT sites for removal, as described by Datsenko and Wanner, a well characterized promoter, a ribosome binding site, the gene of interest, and a transcriptional terminator. The overall product is a linear DNA fragment with 50 base pairs of homology for the target site on the chromosome flanking each side of the construct.

(39) However, the Flp-FRT recombination method is only one system for adding genes to a chromosome, and other systems are available, such as the RecBCD pathway, the RecF pathway, RecA recombinase, non-homologous end joining (NHEJ), Cre-Lox recombination, TYR recombinases and integrases, SER resolvases/invertases, SER integrases, PhiC31 Integrase, and the like. Chromosomal modifications in E. coli can also achieved by the method of recombineering, as originally described by Datsenko and Wanner.

(40) In a recombineering method, for example, the cells are prepared for electroporation following standard techniques, and the cells transformed with linear DNA that contains flanking 50 base pair targeting homology for the desired modification site. For seamless integration of a DNA construct, a two-step approach can be taken using a cassette that contains both positive and negative selection markers, such as the combination of cat and sacB. In the first round of recombineering, the cat-sacB cassette with targeting homology for the desired modification site is introduced to the cells. The cat gene provides resistance to chloramphenicol, which allows for positive recombinants to be selected for on solid media containing chloramphenicol.

(41) A positive isolate can be subjected to a second round of recombineering introducing the desired DNA construct with targeting homology for sites that correspond to the removal of the cat-sacB cassette. The sacB gene encodes for an enzyme that provides sensitivity to sucrose. Thus, growth on media containing sucrose allows for the selection of recombinants in which the cat-sacB construct was removed. P1 phage lysates can be made from isolates confirmed by PCR and sequencing. The lysates can be used to transduce the modification into desired strains, as described previously.

(42) Engineered strains expressing the designed pathway can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for hydrocarbon utilization can be tested by the use of short-chain alkanes as a substrate in MOPS minimal media, as described by Neidhardt, supplemented with appropriate antibiotics, and inducers.

(43) Analysis of culture media after fermentation provides insight into the performance of the engineered pathway. Quantification of hydrocarbons and longer chain fatty acid and alcohol products can be analyzed by GC. Other metabolites, such as short chain organic acids and alcohols can be analyzed by high pressure liquid chromatograph (HPLC). Once the pathway is fully functional, the cultures can be grown in chemostat, providing continuous uninterrupted production of product if desired.

(44) Various -omics techniques, such as microarray or 2D-PAGE can give information about gene expression or protein expression, respectively. Genome scale modeling allows for the identification of additional modifications to the host strain that might lead to improved performance. Deletion of competing pathways, for example, might increase carbon flux through the engineered pathway for product production.

(45) Standard molecular biology techniques were used for gene cloning, plasmid isolation, and E. coli transformation. Native E. coli genes were amplified from E. coli MG1655 genomic DNA using primers to append 15 bp of homology on each end of the gene insert for recombination into the vector backbone. Genes from other organisms were codon optimized and synthesized by either GeneArt (LIFE TECH., CA or GENSCRIPT, NJ). Plasmids were linearized by the appropriate restriction enzymes and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system (CLONTECH LAB. CA). The mixture was subsequently transformed into Stellar competent cells (CLONTECH LAB.).

(46) Transformants that grew on solid media (LB+Agar) supplemented with the appropriate antibiotic were isolated and screened for the gene insert by PCR. Plasmid was isolated from the verified transformants and the sequence of the gene insert was further confirmed by DNA sequencing (LONE STAR LABS, TX). Plasmids (also referred to as vectors) in each case contain at least one promoter, a ribosome binding site for each gene, the gene(s) of interest, at least one terminator, an origin of replication, and an antibiotic resistance marker. Exemplary plasmids are shown in FIGS. 6A-D, 8A-C, 9A-C, 10A-C and 14.

(47) Genes that encode the enzymes of the engineered pathway were cloned and expressed as described above. The crude protein extracts or purified enzymes were assessed for their ability to catalyze the proposed reactions. Tables -F below describe the characterization of enzymes for required pathway steps depicted in FIG. 2, FIG. 3, FIG. 4, and FIG. 5. In addition to the experimental data provided, several literature examples are listed to provide additional evidence to the ability of known enzymes to possess the required enzymatic activity of several pathway steps. Thus, a great variety of enzymes are exemplified herein.

(48) TABLE B describes the characterization of enzymes involved in hydrocarbon activation/utilization and conversion to acyl-CoA intermediates through the fumarate addition pathway as depicted in FIG. 2.

(49) TABLE-US-00003 TABLE B Characterization of enzymes involved in the pathway for bioconversion of hydrocarbons via fumarate addition as depicted in FIG. 2 Measured specific Enzyme activity (mol/mg Number Enzyme class Enzyme Substrate protein/min) Reference 1 alkylsuccinate synthase See Table C for details 2a succinyl-CoA: 2-methyl-alkyl- T. aromatica Methylsuccinate Methylsuccinyl-CoA Leutwein and succinyl-CoA transferase/2- BSCT and succinyl-CoA formation observed Heider (2001) methyl-alkyl-succinyl-CoA synthetase 2b 2-methyl-alkyl-malonyl-CoA R. Sphaeroides ethylmalonyl-CoA 0.05, K.sub.m: 60 M Erb et al (2008) mutase Ecm (methylsuccinyl-CoA product) 2c 2-methyl-alkyl-malonyl-CoA M. musculus ethylmalonyl-CoA 8800, K.sub.m: 0.96 M Linster et al decarboxylase ECHDC1 (2011) methylmalonyl- K.sub.m: 3.1 M Linster et al CoA (2011) 2e propionyl-CoA carboxylase M. sedula Pcc propionyl-CoA 3.3, K.sub.m: 70 M (75 C.) Hugler et al (2003) S. coelicolor Pcc propionyl-CoA 0.2, K.sub.m: 76 M Arabolaza et al (2010) 2f methylmalonyl-CoA epimerase P. horikoshii Mce (S)-2- 162, K.sub.m: 79 M Bobik and methylmalonyl- Rasche (2004) CoA methylmalonyl-CoA mutase P. freudenreichii (R)-2- 26, K.sub.m: 124 M Chowdhury et al subsp. shermanii methylmalonyl- (1999); Padovani Mcm CoA et al (2006) 2g succinyl-CoA:2-methyl-alkyl- E. coli SucCD Succinate 18.6, K.sub.m: 141 M Nolte at al succinyl-CoA (reversible (2014) transferase/succinyl-CoA reaction) synthetase 2h succinate dehydrogenase E. coli SdhCDAB succinate K.sub.m: 2.5 M Maklashina at al (2001) 2d: -oxidation enzymes Acyl-CoA dehydrogenase E. coli FadE butyryl-CoA 0.008 0.001 This work Enoyl-CoA hydratase E. coli FadB crotonyl-CoA 0.051 0.004 This work 3-hydroxyacyl-CoA E. coli FadB 3-hydroxybutyryl- 0.185 0.001 This work dehydrogenase CoA 3-ketoacyl-CoA thiolase E. coli AtoB acetoacetyl-CoA 17.1 1.2 This work E. coli FadA acetoacetyl-CoA 0.013 0.002 This work R. eutropha BktB acetoacetyl-CoA 27.0 1.1 This work S. collinus FadA acetoacetyl-CoA 115.6 1.0 This work P. putida FadAx acetoacetyl-CoA 30.9 0.3 This work

(50) Additional details on alkylsuccinate synthase enzymes, required for the activation of hydrocarbons via fumarate addition are provided in TABLE C:

(51) TABLE-US-00004 TABLE C Observed alkane activation via fumarate addition (alkylsuccinate synthases) Hydro- carbon Substrate Organism(s) Product Literature Evidence Reference Methane bacterial consortium methylsuccinate Product detected (2.08 M) in Duncan at al. (Alaskan North Slope) oil field sample (2009) bacterial consortium methylsuccinate Product detected in production Bian at al field sample from oil reservoir (2015) Ethane bacterial consortium ethylsuccinate Product detected (1.77 M) in Duncan et al. (Alaskan North Slope) oil field sample (2009) bacterial consortium ethylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Propane bacterial consortium methylethylsuccinate Product detected (2.18 M) in Duncan et al. (Alaskan North Slope) oil field sample (2009) Desulfosarcina sp. BuS5 methylethylsuccinate/n- Terminal and sub-terminal Kniemeyer et propylsuccinate product formation from bacterial al. (2007) cultures bacterial consortium methylethylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Butane bacterial consortiums methylpropylsuccinate Product detected (0.76 M) in Duncan et al. (Alaskan North Slope) field sample oil (2009) Desultosarcina sp. BuS5 methylpropylsuccinate Terminal and sub-terminal Kniemeyer et product formation from bacterial al. (2007) cultures bacterial consortium methylpropylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Pentane Azoarcus sp. H N1 Methylbutylsuccinate Product formation from crude Webner (2012) enzyme assay (~5% relative to hexane) bacterial consortium methylbutylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Hexane Azoarcus sp. H N1 methylpentylsuccinate Product formation from crude Webner (2012) enzyme assay (55 M in crude extract assays) bacterial consortium methylpentylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Heptane Azoarcus sp. H N1 methylhexylsuccinate Product formation from crude Webner (2012) enzyme assay (~120% relative to hexane) bacterial consortium methylhexylsuccinate Product detected in production Bian et al field sample from oil reservoir (2015) Octane Azoarcus sp. H N1 methylheptylsuccinate Product formation from crude Webner (2012) enzyme assay (~50% relative to hexane) bacterial consortium methylheptylsuccinate Product detected in production Bian at al field sample from oil reservoir (2015)

(52) Included in these alkylsuccinate synthase enzymes are those from Azoarcus sp. HxN1 (Grundmann et al., 2008) and Desulfosarcina sp. BuS5 (Kniemeyer et al., 2007) for which the catalytic subunit, and associated subunits for the case of Azoarcus sp. HxN1, have been proposed. Genes encoding the proposed subunits of the Azoarcus sp. HxN1 alkylsuccinate synthase have been cloned into required expression vectors as shown in FIG. 6. Evaluation of the expression of alkylsuccinate synthase subunits in E. coli is shown in FIG. 7.

(53) Furthermore, several candidate alkylsuccinate synthase subunits and associated proteins have been identified through a BLAST search of Azoarcus sp. HxN1 alkylsuccinate synthase subunits against the recently sequenced Desulfosarcina sp. BuS5 genome, a strain that degrades propane and butane via fumarate addition. This search identified several enzymes within close physical proximity to the large catalytic MasD subunit (WP_027352796.1), including 2 enzymes with high similarity to the Azoarcus sp. HxN1 MasE subunit (WP_027352794.1 and WP_027352793.1), a protein with similarity to alkylsuccinate synthase gamma subunits from Desulfatibacillum alkenivorans AK-01 and Smithella sp. SCADC (WP_027352795.1), and enzymes with similarity to the Azoarcus sp. HxN1 MasG activating enzyme (WP_051374532.1) and other radical SAM enzymes (WP_027352800.1). In all, 13 proteins appear to be encoded from an operon within the Desulfosarcina sp. BuS5 genome spanning the locus from 66239-79342 in the associated genome sequence NZ_AXAM01000003.1. Genes encoding potential subunits of the alkylsuccinate synthase (WP_027352793.1, WP_027352794.1, WP_027352795.1, and WP_027352796.1) and activation enzyme (WP_051374532.1 and WP_027352800.1) have been cloned into required expression vectors as shown in FIG. 8 and FIG. 9.

(54) This type of homology search can be further exploited to identify other potential alkylsuccinate synthase enzymes/operons through a BLAST search of these enzymes against other hydrocarbon degrading species. For example, a search of the Desulfosarcina sp. BuS5 MasD subunit (WP_027352796.1) was used to identify potential MasD subunits from species such as Peptococcaceae bacterium SCADC1_2_3 (WP_036734374.1), Desulfoglaeba alkanexedens ALDC (ADJ51097.1), and Peptococcaceae bacterium BRH_c4a (KJS01634.1), among others.

(55) Furthermore, additional enzymes required for conversion of fumarate activated hydrocarbon intermediates to product precursors have been investigated. Enzymes such as T. aromatica succinyl-CoA:(R)-benzylsuccinate CoA-transferase (Q9KJF0, Q9KJE9), R. sphaeroides (2S)-methylsuccinyl-CoA dehydrogenase (ADC44452.1), and R. sphaeroides mesaconyl-coenzyme A hydratase (Q3IZ78) enable the conversion of the fumarate activated hydrocarbons to product precursors, which can then be converted to desired products. Genes encoding the above proteins have been cloned into required expression vectors (FIG. 10A-C) and their expression in E. coli evaluated (FIG. 11).

(56) The above described enzymes for hydrocarbon activation/utilization and conversion to acyl-CoA intermediates through the fumarate addition pathway provide a route for the bioconversion of short-chain hydrocarbons of varying chain length. For the specific case of methane activation/utilization, the pathways depicted in FIG. 3 and FIG. 4 can also be exploited for the conversion of fumarate activated methane into product precursors as well as for the regeneration of fumarate. TABLE D and TABLE E describe the characterization of enzymes involved in these versions of the bioconversion pathway:

(57) TABLE-US-00005 TABLE D Characterization of enzymes involved in the pathway for bioconversion of hydrocarbons via fumarate addition as depicted in FIG. 3 Measured specific activity Enzyme name Enzyme Substrate (mol/mg protein/min) Reference alkylsuccinate synthase See Table B for details succinyl-CoA: 2-methyl-alkyl- T. aromatica Methylsuccinate Methylsuccinyl-CoA Leutwein and succinyl-CoA transferase/2-methyl-alkyl-succinyl- BSCT and succinyl- formation observed Heider (2001) CoA synthetase CoA 2-methyl-alkyl-succinyl-CoA R. sphaeroides (2S)- mesaconyl-CoA formation Erb et al (2009) dehydrogenase Mcd methylsuccinyl- observed CoA mesaconyl-C1-CoA-C4 transferase C. aurantiacus mesaconyl-C1- mesaconyl-C4-CoA Zarzycki et al (2009) Mct CoA formation observed mesaconyl-C4-CoA hydratase C. aurantiacus mesaconyl-C4- (S)-citramalyl-CoA Zarzycki et al (2009) Meh CoA formation observed L-malyl-CoA/citramalyl-CoA lyase C. aurantiacus (S)-citramalyl- Acetyl-CoA and pyruvate Zarzycki et al (2009) Mmc CoA formation observed pyruvic-malic carboxylase E. coli MaeA pryuvate 1.3, K.sub.m: 16 mM Stols and Donnelly (1997) fumarase E. coli FumA L-malate 706, K.sub.m: 700 mM van Vugt- Lussenburg et al (2013)

(58) TABLE-US-00006 TABLE E Characterization of enzymes involved in the pathway for bioconversion of hydrocarbons via fumarate addition as depicted in FIG. 4 Measured specific Enzyme activity (mol/mg Number Enzyme class Enzyme Substrate protein/min) Reference 1 alkylsuccinate synthase See Table C for details 2 succinyl-CoA:2-methyl-alkyl- T. aromatica Methylsuccinate Methylsuccinyl-CoA Leutwein and succinyl-CoA transferase/2-methyl- BSCT and succinyl-CoA formation observed Heider (2001) alkyl-succinyl-CoA synthetase 3 2-methyl-alkyl-succinyl-CoA R. sphaeroides (2S)- mesaconyl-CoA Erb et al (2009) dehydrogenase Mcd methylsuccinyl- formation observed CoA 4 mesaconyl CoA hydratase/ R. sphaeroides methylmalyl 1400, mesaconyl CoA Zarzycki et al methylmalyl-CoA dehydratase Mch CoA (reverse formation observed (2008) reaction) 5 -methylmalyl-CoA lyase R. sphaeroides -methylmalyl- 26, K.sub.m: 10 M Erb et al (2010) Mcl1 CoA 6 propionyl-CoA carboxylase M. sedula Pcc propionyl-CoA 3.3, K.sub.m: 70 M Hugler et al (75 C.) (2003) S. coelicolor propionyl-CoA 0.2, K.sub.m: 76 M Arabolaza et al Pcc (2010) 7 methylmalonyl-CoA epimerase P. horikoshii (S)-2- 162, K.sub.m: 79 M Bobik and Rasche Mce methylmalonyl- (2004) CoA methylmalonyl-CoA mutase P. (R)-2- 26, K.sub.m: 124 M Chowdhury et al freudenreichii methylmalonyl- (1999); Padovani subsp. CoA et al (2006) shermanii Mcm 8 succinyl-CoA:2-methyl-alkyl- E. coli SucCD Succinate 18.6, K.sub.m: 141 M Nolte et al (2014) succinyl-CoA transferase/succinyl- (reversible CoA synthetase reaction) 9 succinate dehydrogenase E. coli succinate K.sub.m: 2.5 M Maklashina et al SdhCDAB (2001) 10 glyoxylate carboligase E. coli Gcl glyoxylate 17.5, K.sub.m: 900 M Kaplun et al (2008) 11 tartronate semialdehyde reductase E. coli GarR tartronate K.sub.m: 280 M Njau et al (2000) semialdehyde 12 glycerate kinase E. coli GlxK D-glycerate K.sub.m: 70 M Ornston and Ornston (1969)

(59) Together, these enzymatic components described above provide a full route for the conversion of short chain hydrocarbons to acyl-CoA (e.g. acetyl-CoA) product precursors, which can subsequently be converted into fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives) through numerous product synthesis pathways, such as beta-oxidation reversal (BOX-R) or fatty acid biosynthesis. BOX-R is not described in great detail herein, since it has been described in the inventor's prior patents and publications.

(60) The use of an oxygen-dependent activation mechanism in which an alkane hydroxylase adds a terminal alcohol group to the hydrocarbon can also be exploited for the bioconversion of short chain hydrocarbons to fuels and chemicals as depicted in FIG. 5. Characterization of required enzymes for hydrocarbon activation and conversion to product precursors is shown in TABLE F:

(61) TABLE-US-00007 TABLE F Characterization of enzymes involved in the pathway for bioconversion of hydrocarbons via hydroxylation as depicted in FIG. 5 Measured specific Enzyme activity (mol/mg Number Enzyme class Enzyme Substrate protein/min) Reference 1 alkane monooxygenase M. capsulatus (Bath) Methane K.sub.m,methane: 3 M Green and or alkane hydroxylase sMMO Dalton (1986) M. capsulatus (Bath) 0.139 0.005 Sirajuddin et pMMO al. (2014) Thauera butanivorans K.sub.m,methane: 1100 M Cooley et al. sBMO (2009) Thauera butanivorans Ethane K.sub.m,methane: 2.2 M Cooley et al. sBMO (2009) P. putida AlkBFG Propane Propanol formation Koch et al. observed (2009) Mycobacterium sp. Propanol formation Koch et al. strain HXN-1500 observed (2009) CYP153A6 Thauera butanivorans K.sub.m,propane: 0.94 M Cooley et al. sBMO (2009) P. putida AlkBFG Butane Butanol formation Koch et al. observed (2009) Mycobacterium sp. Butanol formation Koch et al. strain HXN-1500 observed (2009) CYP153A6 Thauera butonivorans K.sub.m,butane: 0.24 M Cooley et al. sBMO (2009) P. putida AlkBGT Pentane Pentanol formation This work observed, see FIG. 13 P. putida AlkBGT Hexane Hexanol formation This work observed, see FIG. 12 2 alcohol dehydrogenase E. coli FucO butryaldehyde 5.08 0.08 This work 3 Coenzyme A-acylating L. Reuteri PduP propionaldehyde 25 0.2, Sabet-Azad et aldehyde dehydrogenase K.sub.m: 28 mM al. (2013) 4 acyl-CoA synthetase E. coli Acs acetate K.sub.m: 200 M Brown et al. (1977) P. Aeruginosa butyryl- butyrate 0.255, K.sub.m: 10 M Shimizu et al. CoA synthetase (1981) 5 acylating aldehyde E. coli AdhE butyryl-CoA 0.073 0.00 This work dehydrogenase E. coli MhpF butyryl-CoA 0.009 0.003 This work -oxidation enzymes Acyl-CoA dehydrogenase E. coli FadE butyryl-CoA 0.008 0.001 This work Enoyl-CoA hydratase E. coli FadB crotonyl-CoA 0.051 0.004 This work 3-hydroxyacyl-CoA E. coli FadB 3-hydroxybutyryl- 0.185 0.001 This work dehydrogenase CoA 3-ketoacyl-CoA thiolase E. coli AtoB acetoacetyl-CoA 17.1 1.2 This work E. coli FadA acetoacetyl-CoA 0.013 0.002 This work R. eutropha BktB acetoacetyl-CoA 27.0 1.1 This work S. collinus FadA acetoacetyl-CoA 115.6 1.0 This work P. putida FadAx acetoacetyl-CoA 30.9 0.3 This work

(62) In addition to the demonstrated activation of hydrocarbon such as pentane and hexane to the associated alcohols, the enzymes required for the conversion of these alcohols to product precursors have also been characterized. The combination of these enzymatic components provide a full route for the conversion of short chain hydrocarbons to acyl-CoA (e.g. acetyl-CoA) product precursors, which can subsequently be converted into fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives) through numerous product synthesis pathways, such as beta-oxidation reversal or fatty acid biosynthesis.

(63) The enzymes described in TABLE F represent a number of examples of enzymes with required pathway activity, however this list is not representative of all available enzymes. Many additional enzymes have been shown to possess the required activities in literature and as such, can easily be integrated with the pathway framework. Furthermore, sequence similarity can also be used for the identification of additional enzymes. As an example, components of putative alkane monooxygenase system in Rhodobacter sphaeroides were identified through a BLAST using the soluble methane monoxygenase (sMMO) system of Methylococcus capsulatus (Bath). Required hydroxylase alpha (YP_352924.1) and reductase (YP_352923.1) subunits were identified, which are part of a 4 gene operon also including a hydroxylase beta subunit (YP_352922.1) and a regulatory protein (YP_352921.1). Genes encoding potential subunits this putative alkane monooxygenase have been cloned into required expression vectors as shown in FIG. 14A-B.

(64) By exploiting either of these hydrocarbon activation described above, various fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives) can be synthesized from short-chain hydrocarbons through the metabolic pathways described and demonstrated herein.

(65) Each of the following is incorporated by reference herein in its entirety for all purposes:

(66) U.S. 61/440,192, filed Feb. 7, 2011, WO2013036812, US20130316413 Reverse beta oxidation pathway by Clomburg et al.

(67) U.S. 61/531,911, filed Sep. 7, 2011, WO2013036812 US20140273110 Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation by Gonzalez & Clomburg

(68) 61/932,057, filed Jan. 27, 2014, WO2015112988, Type II fatty acid synthesis enzymes in reverse beta-oxidation by Gonzalez & Clomburg

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