METHOD FOR IMPROVING THE MICROBIAL PRODUCTION OF FAT TALCOHOLS OF A DEFINITE LENGTH IN THE PRESENCE OF METHANOL

Description

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to biosynthetic processes, and more specifically to organisms having specific length fatty alcohol, fatty aldehyde or fatty acid biosynthetic capacity or having isopropanol biosynthetic capacity.

[0002] Primary alcohols are a product class of compounds having a variety of industrial applications which include a variety of biofuels and specialty chemicals. Primary alcohols also can be used to make a large number of additional industrial products including polymers and surfactants. For example, higher primary alcohols, also known as fatty alcohols (C 4 -C 24 ) and their ethoxylates are used as surfactants in many consumer detergents, cleaning products and personal care products worldwide such as laundry powders and liquids, dishwashing liquid and hard surface cleaners. They are also used in the manufacture of a variety of industrial chemicals and in lubricating oil additives. Specific length fatty alcohols, such as octanol and hexanol, have useful organoleptic properties and have long been employed as fragrance and flavor materials. Smaller chain length C 4 -C 8 alcohols (e.g., butanol) are used as chemical intermediates for production of derivatives such as acrylates used in paints, coatings, and adhesives applications.

[0003] Fatty alcohols are currently produced from, for example, hydrogenation of fatty acids, hydroformylation of terminal olefins, partial oxidation of n-paraffins and the Al- catalyzed polymerization of ethylene. Unfortunately, it is not commercially viable to produce fatty alcohols directly from the oxidation of petroleum-based linear hydrocarbons (n-paraffins). This impracticality is because the oxidation of n-paraffins produces primarily secondary alcohols, tertiary alcohols or ketones, or a mixture of these compounds, but does not produce high yields of fatty alcohols. Additionally, currently known methods for producing fatty alcohols suffer from the disadvantage that they are restricted to feedstock which is relatively expensive, notably ethylene, which is produced via the thermal cracking of petroleum. In addition, current methods require several steps, and several catalyst types.

[0004] Fatty alcohol production by microorganisms involves fatty acid synthesis followed by acyl-reduction steps. The universal fatty acid biosynthesis pathway found in most cells has been investigated for production of fatty alcohols and other fatty acid derivatives. There is currently a great deal of improvement that can be achieved to provide more efficient biosynthesis pathways for fatty alcohol production with significantly higher theoretical product and energy yields.

[0005] Isopropanol (IPA) is a colorless, flammable liquid that mixes completely with most solvents, including water. The largest use for IPA is as a solvent, including its well known yet small use as "rubbing alcohol," which is a mixture of IPA and water. As a solvent, IPA is found in many everyday products such as paints, lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, and pharmaceuticals. Low-grade IPA is also used in motor oils. The second largest use is as a chemical intermediate for the production of isopropylamines, isopropylethers, and isopropyl esters. Isopropanol can potentially be dehydrated to form propylene, a polymer precursor with an annual market of more than 2 million metric tons.

[0006] Current global production capacity of IPA is approximately 6 B lb / yr, with approximately 74% of global IPA capacity concentrated in the US, Europe, and Japan. Isopropanol is manufactured by two petrochemical routes. The predominant process entails the hydration of propylene either with or without sulfuric acid catalysis. Secondarily, IPA is produced via hydrogenation of acetone, which is a by-product formed in the production of phenol and propylene oxide. High-priced propylene is currently driving costs up and margins down throughout the chemical industry motivating the need for an expanded range of low cost feedstocks.

[0007] Thus, there exists a need for alternative means for effectively producing commercial quantities of fatty alcohols, isopropanol and related compounds. The present invention satisfies this need and provides related advantages as well.SUMMARY OF INVENTION

[0008] The invention is defined in the claims and provides non-naturally occurring microbial organisms as defined in the claims containing a fatty alcohol, fatty aldehyde, or fatty acid pathway.

[0009] The invention is concerned with a non-naturally occurring microbial organism producing a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, the organism having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway, wherein said formaldehyde fixation pathway comprises: (1) a) a 3-hexulose-6phosphate synthase and b) a 6-phospho-3-hexuloisomerase; wherein said formate assimilation pathway comprises: (2) a) a formyltetrahydrofolate synthetase, b) a methenyltetrahydrofolate cyclohydrolase, c) a methylenetetrahydrofolate dehydrogenase, d) a glycine cleavage system, e) a serine hydroxymethyltransferase, and f) a serine deaminase; wherein said methanol metabolic pathway comprises: (3) a methanol dehydrogenase; the microbial organism further comprising: A) a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE) cycle and / or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway, wherein said MI-FAE cycle comprises one or more thiolase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said MD-FAE cycle comprises one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said termination pathway is selected from: (4A) an acyl-CoA reductase (aldehyde forming); (5A) a) an acyl-CoA reductase (aldehyde forming) and b) an alcohol dehydrogenase; or (6A) an acyl-CoA reductase (alcohol forming), wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the MI-FAE cycle, the MD-FAE cycle or the termination pathway is encoded by at least one exogenous nucleic acid; or B) a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway, wherein said FAACPE cycle comprises one or more β-ketoacyl-ACP synthase, one or more β-ketoacyl-ACP reductase, one or more β-hydroxyacyl-ACP reductase, and one or more enoyl ACP-reductase, wherein said termination pathway is selected from: (4B) a fatty acyl-ACP reductase; (5B) a) a thioesterase and b) a carboxylic acid reductase (CAR); or (6B) a) a thioesterase, b) a CAR, and c) a fatty aldehyde reductase, wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the FAACPE cycle or the termination pathway is encoded by at least one exogenous nucleic acid, wherein the organism is a species of bacteria, wherein the species of bacteria is E. coli.

[0010] The present invention is also concerned with a method for producing a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions for a sufficient period of time to produce said compound of Formula (I). In a preferred embodiments, R 3 can be H.

[0011] In the non-naturally occurring microbial organism of the invention, each of said formaldehyde fixation pathway, said formate assimilation pathway, and said methanol metabolic pathway can comprise at least one exogenous nucleic acid encoding at least one metabolic pathway enzyme.

[0012] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MI-FAE cycle and an acyl-CoA reductase (aldehyde forming).

[0013] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MI-FAE cycle, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase.

[0014] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MI-FAE cycle and an acyl-CoA reductase (alcohol forming).

[0015] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MD-FAE cycle and an acyl-CoA reductase (aldehyde forming).

[0016] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MD-FAE cycle, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase.

[0017] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an MD-FAE cycle and an acyl-CoA reductase (alcohol forming).

[0018] In the non-naturally occurring microbial organism of the invention, the microbial organism can comprise an FAACPE cycle and a fatty acyl-ACP reductase.

[0019] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an FAACPE cycle, a thioesterase, and a CAR.

[0020] In the non-naturally occurring microbial organism of the invention, the microbial organism can have an FAACPE cycle, a thioesterase, a CAR, and a fatty aldehyde reductase.

[0021] For production of a fatty alcohol, fatty aldehyde, or fatty acid, in some embodiments, the non-naturally occurring microbial organism of the invention has: a formaldehyde fixation pathway, a formate assimilation pathway, and a methanol metabolic pathway; and a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE) cycle and / or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway, as depicted in Figures 1, 2, 7, 8 and 10. Alternatively, in some embodiments, the non-naturally occurring microbial organism of the invention has: a formaldehyde fixation pathway, a formate assimilation pathway, and a methanol metabolic pathway; and a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway, as depicted in Figures 1, 10 and 12.

[0022] In one aspect, the formaldehyde fixation pathway, formate assimilation pathway, and a methanol metabolic pathway present in the microbial organisms of the invention enhances the availability of substrates and / or pathway intermediates, such as acetyl-CoA and malonyl-CoA, and / or reducing equivalents, which can be utilized for fatty alcohol, fatty aldehyde, or fatty acid, production through one or more fatty alcohol, fatty aldehyde, or fatty acid pathways of the invention. For example, in some embodiments, a non-naturally occurring microbial organism of the invention that includes a methanol metabolic pathway can enhance the availability of reducing equivalents in the presence of methanol and / or convert methanol to formaldehyde, a substrate for the formaldehyde fixation pathway. Likewise, a non-naturally occurring microbial organism of the invention having a formate assimilation pathway can reutilize formate to generate substrates and pathway intermediates such as formaldehyde, pyruvate and / or acetyl-CoA. Such substrates, intermediates and reducing equivalents can be used to increase the yield of a fatty alcohol, a fatty aldehyde, or a fatty acidproduced by the microbial organism.

[0023] The microbial organisms of the invention advantageously enhance the production of substrates and / or pathway intermediates for the production of a chain length specific fatty alcohol, fatty aldehyde, fatty acid. In order to be able to produce a chain length specific compound, the enzymes of the MI-FAE cycle, the MD-FAE cycle, the FAACPE cycle and / or the termination pathway are selective for a particular substrate. Accordingly, the substrate of each of the enzymes of the MI-FAE cycle, the MD-FAE cycle and / or the termination pathway can be independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein said one or more enzymes of the MI-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said fatty alcohol, fatty aldehyde or fatty acid of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said fatty alcohol, fatty aldehyde or fatty acid of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said fatty alcohol, fatty aldehyde or fatty acid of Formula (I). Alternatively, the substrate of each of the enzymes of the FAACPE cycle and / or the termination pathway can be independently selected from a compound of Formula (II) or malonyl-ACP: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the one or more enzymes of the FAACPE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and wherein the one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

[0024] The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde, or a fatty acid by culturing a non-naturally occurring microbial organism containing a fatty alcohol, fatty aldehyde, or fatty acid pathway as described herein under conditions and for a sufficient period of time to produce a fatty alcohol, fatty aldehyde, or fatty acid.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures concerned with subject-matter not encompassed by the claims are for reference. Figure 1 shows exemplary metabolic pathways enabling the conversion of CO2, formate, formaldehyde (Fald), methanol (MeOH), glycerol, xylose (XYL) and glucose (GLC) to acetyl-CoA (ACCOA) and exemplary endogenous enzyme targets for optional attenuation or disruption. The exemplary pathways and endogenous enzyme targets can be combined with the cycles and pathways depicted herein that utilize ACCOA, such as those depicted in Figures 1, 11 and 12. The enzyme targets are indicated by arrows having "X" markings. The endogenous enzyme targets include DHA kinase, methanol oxidase (AOX), PQQ-dependent methanol dehydrogenase (PQQ) and / or DHA synthase. The enzymatic transformations shown are carried out by the following enzymes: A) methanol dehydrogenase, B) 3-hexulose-6-phosphate synthase, C) 6-phospho-3-hexuloisomerase, D) dihydroxyacetone synthase, E) formate reductase, F) formate ligase, formate transferase, or formate synthetase, G) fonnyl-CoA reductase, H) formyltetiahydrofolate synthetase, I) methenyltetrahydrofolate cyclohydrolase, J) methylenetetrahydrofolate dehydrogenase, 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) fructose-6-phosphate phosphoketolase, U) xylulose-5-phosphate phosphoketolase, V) phosphotiansacetylase, W) acetate kinase, X) acetyl-coa transferase, synthetase, or ligase, Y) lower glycolysis including glyceraldehyde-3-phosphate dehydrogenase, Z) fructose-6-phosphate aldolase. See abbreviation list below for compound names. Figure 2 shows an exemplary MI-FAE cycle and / or MD-FAE cycle in combination with termination pathways for production of fatty alcohols, aldehydes, or acids from the acyl-CoA intermediate of the MI-FAE cycle or MD-FAE cycle. Enzymes are: A. Thiolase; B. 3-Oxoacyl-CoA reductase; C. 3-Hydroxyacyl-CoA dehydratase; D. Enoyl-CoA reductase; E. Acyl-CoA reductase (aldehyde forming); F. Alcohol dehydrogenase; G. Acyl-CoA reductase (alcohol forming); H. acyl-CoA hydrolase, transferase or synthase; J. Acyl-ACP reductase; K. Acyl-CoA:ACP acyltransferase; L. Thioesterase; N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase; O. Elongase; and P. acyl-ACP reductase (alcohol forming). Figure 3 shows exemplary pathways for production of cytosolic acetyl-CoA from pyruvate or threonine. Enzymes are: A. pyruvate oxidase (acetate-forming); B. acetyl-CoA synthetase, ligase or transferase; C. acetate kinase; D. phosphotransacetylase; E. pyruvate decarboxylase; F. acetaldehyde dehydrogenase; G. pyruvate oxidase (acetyl-phosphate forming); H. pyruvate dehydrogenase, pyruvate:ferredoxin oxidoreductase, pyruvate:NAD(P)H oxidoreductase or pyruvate formate lyase; I. acetaldehyde dehydrogenase (acylating); and J. threonine aldolase. Figure 4 shows exemplary pathways for production of acetyl-CoA from phosphoenolpyruvate (PEP). Enzymes are: A. PEP carboxylase or PEP carboxykinase; B. oxaloacetate decarboxylase; C. malonate semialdehyde dehydrogenase (acetylating); D. acetyl-CoA carboxylase or malonyl-CoA decarboxylase; F. oxaloacetate dehydrogenase or oxaloacetate oxidoreductase; G. malonate semialdehyde dehydrogenase (acylating); H. pyruvate carboxylase; J. malonate semialdehyde dehydrogenase; K. malonyl-CoA synthetase or transferase; L. malic enzyme; M. malate dehydrogenase or oxidoreductase; and N. pyruvate kinase or PEP phosphatase. Figure 5 shows exemplary pathways for production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA using citrate and malate transporters. Enzymes are: A. citrate synthase; B. citrate transporter; C. citrate / malate transporter; D. ATP citrate lyase; E. citrate lyase; F. acetyl-CoA synthetase or transferase; H. cytosolic malate dehydrogenase; I. malate transporter; J. mitochondrial malate dehydrogenase; K. acetate kinase; and L. phosphotransacetylase. Figure 6 shows exemplary pathways for production of cytosolic acetyl-CoA from mitochondrial acetyl-CoA using citrate and oxaloacetate transporters. Enzymes are: A. citrate synthase; B. citrate transporter; C. citrate / oxaloacetate transporter; D. ATP citrate lyase; E. citrate lyase; F. acetyl-CoA synthetase or transferase; G) oxaloacetate transporter; K) acetate kinase; and L) phosphotransacetylase. Figure 7 shows an exemplary MI-FAE cycle and / or MD-FAE cycle for elongating the linear alkyl of R 1 . Enzymes are: A. Thiolase; B. 3-Ketoacyl-CoA reductase; C. 3-Hydroxyacyl-CoA dehydratase; D. Enoyl-CoA reductase; and E. Elongase. Figure 8 shows an exemplary termination cycle for generating a fatty alcohol, fatty aldehyde or fatty acid from any of the MI-FAE cycle intermediates or MD-FAE cycle intermediates of Figure 7. Enzymes are: E. MI-FAE / MD-FAE intermediate-CoA reductase (aldehyde forming); F. Alcohol dehydrogenase; G. MI-F AE / MD-F AE intermediate-CoA reductase (alcohol forming); H. MI-FAE / MD-FAE intermediate-CoA hydrolase, transferase or synthase; J. MI-FAE / MD-FAE intermediate-ACP reductase; K. MI-FAE / MD-FAE intermediate-CoA:ACP acyltransferase; L. Thioesterase; N. Aldehyde dehydrogenase (acid forming) or carboxylic acid reductase; and P. acyl-ACP reductase (alcohol forming). R1 is C1-24 linear alkyl; R 3 is H, OH, or oxo (=O) and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four. Figure 9 shows exemplary compounds that can be produced from the four MI-FAE or MD-FAE cycle intermediates using the cycles depicted in Figure 6 and the termination pathways depicted in Figure 7. R is C 1-24 linear alkyl. Figure 10 shows exemplary metabolic pathways that provide the extraction of reducing equivalents from methanol, hydrogen, or carbon monoxide. Enzymes are: A) methanol methyltransferase, B) methylenetetrahydrofolate reductase, C) methylenetetrahydrofolate dehydrogenase, D) methenyltetrahydrofolate cyclohydrolase, E) formyltetrahydrofolate deformylase, F) formyltetrahydrofolate synthetase, G) formate hydrogen lyase, H) hydrogenase, I) formate dehydrogenase, J) methanol dehydrogenase, K) spontaneous or formaldehyde activating enzyme, L) formaldehyde dehydrogenase, M) spontaneous or S-(hydroxymethyl)glutathione synthase, N) Glutathione-Dependent Formaldehyde Dehydrogenase, O) S-formylglutathione hydrolase, P) carbon monoxide dehydrogenase. See abbreviation list below for compound names. Figure 11 shows exemplary metabolic pathways enabling the conversion of acetyl-CoA to isopropanol. Enzymes are: T) acetyl-CoA carboxylase, U) acetoacetyl-CoA synthase, V) acetyl-CoA:acetyl-CoA acyltransferase, W) acetoacetyl-CoA hydrolase, acetoacetyl-CoA transferase, acetoacetyl-CoA ligase, phosphotransacetoacetylase / acetoacetate kinase, X) acetoacetate decarboxylase, Y) acetone reductase (or isopropanol dehydrogenase). See abbreviation list below for compound names. Figure 12 shows an exemplary β-ketoacyl-ACP pathway, a FAACPE cycle in combination with termination pathways for production of fatty alcohols, aldehydes, or acids from the acyl-ACP intermediate of the FAACPE cycle. Enzymes are: A) Acetyl-CoA carboxylase, B) Malonyl-CoA ACP transacylase, C) Acetoacetyl-ACP synthase, D) β-Ketoacyl-ACP synthase, E) β-Ketoacyl-ACP reductase, F) β- Hydroxyacyl-ACP reductase, G) Enoyl ACP-reductase, H) β-Ketoacyl-ACP synthase, I) Thioesterase, J) Fatty acyl-ACP reductase, K) Acyl-CoA synthase, L) Acyl-CoA reductase, M) Fatty aldehyde reductase, N) Fatty alcohol forming acyl-CoA reductase (FAR), O) Carboxylic acid reductase (CAR), and P) acyl-ACP reductase (alcohol forming). Figure 13 depicts the production of 1,3-butanediol (Figure 13A) or ethanol (Figure 13B) in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example XIII. Figure 14 depicts the production of pyruvic acid (Figure 14A), succinic acid (Figure 14B), acetic acid (Figure 14C) or glucose (Figure 14D) in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example XIII. Figure 15 depicts the production of 1,3-butanediol in S. cerevisiae transformed with plasmids comprising genes encoding various MI-FAE cycle and termination pathway enzymes, either with or without pflAV or PDH bypass, as provided in Example XIII. Figure 16 depicts the estimated specific activity of five thiolases for acetyl-CoA condensation activity in E. coli as provided in Example XIV. Figures 17A and 17B depict the estimated specific activity of two thiolases (1491 and 560) cloned in dual promoter yeast vectors with 1495 (a 3-hydroxybutyryl-CoA dehydrogenase) for acetyl-CoA condensation activity in E. coli as provided in Example XIV. Figure 18 depicts the time course of fluorescence detection of oxidation of NADH, which is used to measure the metabolism of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase, as provided in Example XIV. Acetoacetyl-CoA is metabolized to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase. The reaction requires oxidation of NADH, which can be monitored by fluorescence at an excitation wavelength at 340 nm and an emission at 460 nm. The oxidized form, NAD+, does not fluoresce. 1495, the Hbd from Clostridium beijerinckii, was assayed in the dual promoter yeast vectors that contained either 1491 (vector id = pY3Hd17) or 560 (vector id = pY3Hd16). Figure 19 depicts levels of NAD(P)H oxidation in the presence of 1 or 5 ug / ml NADH or 1 or 5 ug / ml NADPH, and shows that the Hbd prefers NADH over NADPH, as provided in Example XIV. Figure 20 depicts the activity data for crude lysates of an aldehyde reductase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde and requires NAD(P)H oxidation, which can be used to monitor enzyme activity, as provided in Example XIV. The Ald from Lactobacillus brevis (Gene ID 707) was cloned in a dual vector that contained the alcohol dehydrogenase from Clostridium saccharoperbutylacetonicum (Gene ID 28). These two enzymes were cloned in another dual promoter yeast vector containing a Leu marker. A 707 lysate from E. coli was used as a standard. Figure 21 depicts the evaluation of ADH (Gene 28) in the dual promoter vector with ALD (Gene 707) with butyraldehyde, a surrogate substrate for 3-hydroxybutyraldehyde. 1,3-BDO is formed by an alcohol dehydrogenase (Adh), which reduces 3-hydroxybutyraldehyde in the presence of NAD(P)H, and the oxidation of NAD(P)H is used to monitor the reaction. Figure 22 depictes exemplary pathways for production of propionyl-CoA. Enzymes are: A) PEP carboxykinase, B) PEP carboxylase, C) Pyruvate kinase, D) Pyruvate carboxylase, E) Malate dehydrogenase, F) Fumarase, G) Fumarate reductase, H) Succinyl-CoA synthetase, I) Succinyl-CoA:3-ketoacid-CoA transferase, J) Methylmalonyl-CoA mutase, K) Methyl-malonyl-CoA epimerase, L) Methylmalonyl-CoA decarboxylase. See abbreviation list below for compound names. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention is defined in the claims and is directed to metabolic and biosynthetic processes and microbial organisms capable of producing fatty alcohols, fatty aldehydes, or fatty acids. The invention disclosed herein is based, at least in part, on non-naturally occurring microbial organisms capable of synthesizing fatty alcohols, fatty aldehydes, or fatty acids using a formaldehyde fixation pathway, a formate assimilation pathway and a methoanol metabolic pathway with a malonyl-CoA-independent fatty acid elongation (MI-FAE) cycle and / or malonyl-CoA dependent fatty acid elongation cycle (MD-FAE) cycle in combination with a termination pathway, or in some embodiments a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway.

[0027] 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; 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; ACTP = acetyl-phosphate; ACCOA = acetyl-CoA; AACOA = acetoacetyl-CoA; MALCOA = malonyl-CoA; FTHF = formyltetrahydrofolate; THF = tetrahydrofolate; E4P = erythrose-4-phosphate: Xu5P = xyulose-5-phosphate; Ru5P = ribulose-5-phosphate; S7P = sedoheptulose-7-phosphate: RSP = ribose-5-phosphate; TCA = tricarboxylic acid; PEP = Phosphoenolpyruvate; OAA = Oxaloacetate; MAL = malate; FUM = Fumarate; SUCC = Succinate; SUCCOA = Succinyl-CoA; (R)-MMCOA = R-Methylmalonyl-CoA; (S)-MMCOA= S-Methylmalonyl-CoA; PPCOA = Propionyl-CoA.

[0028] 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.

[0029] 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 fatty alcohol, fatty aldehyde or fatty alcohol biosynthetic pathway.

[0030] 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.

[0031] 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.

[0032] 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. In the present invention, the non-naturally occurring microbial organism is Escherichia coli.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] "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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 a fatty alcohol, fatty aldehyde or fatty acid product 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 a fatty alcohol, fatty aldehyde or fatty acid product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.

[0041] The term "fatty alcohol," as used herein, is intended to mean an aliphatic compound that contains one or more hydroxyl groups and contains a chain of 4 or more carbon atoms. The fatty alcohol possesses the group - CH 2 OH that can be oxidized so as to form a corresponding aldehyde or acid having the same number of carbon atoms. A fatty alcohol can also be a saturated fatty alcohol, an unsaturated fatty alcohol, a 1,3-diol, or a 3-oxo-alkan-1-ol. Exemplary fatty alcohols include a compound of Formula (III)-(VI): wherein R 1 is a C 1-24 linear alkyl.

[0042] The term "fatty aldehyde," as used herein, is intended to mean an aliphatic compound that contains an aldehyde (CHO) group and contains a chain of 4 or more carbon atoms. The fatty aldehyde can be reduced to form the corresponding alcohol or oxidized to form the carboxylic acid having the same number of carbon atoms. A fatty aldehyde can also be a saturated fatty aldehyde, an unsaturated fatty aldehyde, a 3-hydroxyaldehyde or 3-oxoaldehyde. Exemplary fatty aldehydes include a compound of Formula (VII)-(X): wherein R 1 is a C 1-24 linear alkyl.

[0043] The term "fatty acid," as used herein, is intended to mean an aliphatic compound that contains a carboxylic acid group and contains a chain of 4 or more carbon atoms. The fatty acid can be reduced to form the corresponding alcohol or aldehyde having the same number of carbon atoms. A fatty acid can also be a saturated fatty acid, an unsaturated fatty acid, a 3-hydroxyacid or a 3-oxoacids. Exemplary fatty acids include a compound of Formula (XI)-(XIV): wherein R 1 is a C 1-24 linear alkyl.

[0044] The term "alkyl" refers to a linear saturated monovalent hydrocarbon. The alkyl can be a linear saturated monovalent hydrocarbon that has 1 to 24 (C 1-24 ), 1 to 17 (C 1-17 ), or 9 to 13 (C 9-13 ) carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl. For example, C 9-13 alkyl refers to a linear saturated monovalent hydrocarbon of 9 to 13 carbon atoms.

[0045] As used herein, the phrase "enhance carbon flux" is intended to mean to intensify, increase, or further improve the extent or flow of metabolic carbon through or to a desired pathway, pathway product, intermediate, or compound. The intensity, increase or improvement can be relative to a predetermined baseline of a pathway product, intermediate or compound. For example, an increased yield of acetyl-CoA can be achieved per mole of methanol with a phosphoketolase enzyme described herein (see, e.g., Figure 1) than in the absence of a phosphoketolase enzyme. Similarly, an increased yield of acetyl-CoA can be achieved per mole of methanol with the formale assimilation enzymes (see, e.g., Figure 1) than in the absence of the enzymes. Since an increased yield of acetyl-CoA can be achieved, a higher yield of acetyl-CoA derived products, such as fatty alcohols, fatty acids, fatty aldehydes or isopropanol of the invention, can also be achieved.

[0046] Provided herein are methanol metabolic pathways and a methanol oxidation pathway to improve that availability of reducing equivaments and / or substrants for production of a compound of the invention. Because 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 fatty alcohols, fatty acids, fatty aldehydes or isopropanol, and their intermediates, it is a desireable substrate for the non-naturally occurring microbial organisms of the invention. Employing one or more methanol metabolic enzymes as described herein, for example as shown in Figures 1 and 10, methanol can enter central metabolism in most production hosts by employing methanol dehydrogenase (Figure 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 Figure 1, which involves condensation of formaldehyde and D-ribulose-5-phosphate to form hexulose-6-phosphate (H6P) by hexulose-6-phosphate synthase (Figure 1, step B). The enzyme can use Mg 2+< or Mn 2+< 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 6-phospho-3-hexuloisomerase (Figure 1, step C). Another exemplary pathway that involves the detoxification and assimilation of formaldehyde produced from the oxidation of methanol proceeds through dihydroxyacetone. Dihydroxyacetone synthase (Figure 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).

[0047] By combining the pathways for methanol oxidation (Figure 1, step A) and formaldehyde fixation (Figure 1, Steps B and C or Step D), molar yields of 0.333 mol acetyl-CoA / mol methanol can be achieved for production of a fatty alcohol, a fatty acid, a fatty aldehyde, isopropanol, and their intermediates. The following maximum theoretical yield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid (e.g., a C12), a fatty aldehyde (e.g., a C12) are thus made possible by combining the steps for methanol oxidation, formaldehyde fixation, and product synthesis. 18 CH 4 O + 9 O 2 → C 12 H 26 O + 6 CO 2 + 23 H 2 O(Fatty Alcohol on MeOH)18 CH 4 O + 10 O 2 → C 12 H 24 O 2 + 6 CO 2 + 24 H 2 O(Fatty Acid on MeOH)18 CH 4 O + 9.5 O 2 → C 12 H 24 O + 6 CO 2 + 24 H 2 O(Fatty Aldehyde on MeOH)

[0048] 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 Figure 1. For example, the CO 2 generated by conversion of pyruvate to acetyl-CoA (Figure 1, step R) can be converted to formate via formate dehydrogenase (Figure 1, step S). Alternatively, pyruvate formate lyase, which forms formate directly instead of CO 2 , can be used to convert pyruvate to acetyl-CoA (Figure 1, step Q). Formate can be converted to formaldehyde by using: 1) formate reductase (Figure 1, step E), 2) a formyl-CoA synthetase, transferase, or ligase along with formyl-CoA reductase (Figure 1, steps F-G), or 3) formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, and formaldehyde-forming enzyme (Figure 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 Figure 1, steps H-I-J-L-M-N or Figure 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,

[0049] By combining the pathways for methanol oxidation (Figure 1, step A), formaldehyde fixation (Figure 1, Steps B and C or Step D), and formate reutilization, molar yields as high as 0.500 mol acetyl-CoA / mol methanol can be achieved for production of a fatty alcohol, a fatty acid, a fatty aldehyde, isopropanol, and their intermediates. Thus, for example, the following maximum theoretical yield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid (e.g., a C12), and a fatty aldehyde (e.g., a C12), are thus made possible by combining the steps for methanol oxidation, formaldehyde fixation, formate reutilization, and product synthesis. 12 CH 4 O → C 12 H 26 O + 11 H 2 O(Fatty Alcohol on MeOH)12 CH 4 O + O 2 → C 12 H 24 O 2 + 12 H 2 O(Fatty Acid on MeOH)12 CH 4 O + 0.5 O 2 → C 12 H 24 O + 12 H 2 O(Fatty Aldehyde on MeOH)

[0050] 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, the following maximum theoretical yield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid (e.g., a C12), and a fatty aldehyde (e.g., a C12), on glucose are made possible by combining the steps for formaldehyde fixation, formate reutilization, and product synthesis. 3 C 6 H 12 O 6 → C 12 H 26 O + 5 H 2 O + 6 CO 2 (Fatty Alcohol on glucose)3 C 6 H 12 O 6 → 1.0588 C 12 H 24 O 2 + 5.2941 H 2 O + 5.2941 CO 2 (Fatty Acid on glucose)3 CH 12 O 6 → 1.0286 C 12 H 24 O + 5.6571 H 2 O + 5.6571 CO 2 (Fatty Aldehyde on glucose)

[0051] Similarly, the maximum theoretical yield of a fatty alcohol, a fatty acid, a fatty aldehyde, or isopropanol from glycerol can be increased by enabling fixation of formaldehyde from generation and utilization of formate. The following maximum theoretical yield stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid (e.g., a C12), and a fatty aldehyde (e.g., a C12), on glycerol are thus made possible by combining the steps for formaldehyde fixation, formate reutilization, and product synthesis. 6 C 3 H 3 O 3 → 1.1667 C 12 H 26 O + 8.8333 H 2 O + 4 CO 2 (Fatty Alcohol on glycerol)6 C 3 H 3 O 3 → 1.2353 C 12 H 24 O 2 + 9.1765 H 2 O + 3.1765 CO 2 (Fatty Acid on glycerol)6 C 3 H 3 O 3 → 1.2000 C 12 H 24 O + 9.6000 H 2 O + 3.6000 CO 2 (Fatty Aldehyde on glycerol)

[0052] 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 Figure 10. Reducing equivalents can also be extracted from hydrogen and carbon monoxide by employing hydrogenase and carbon monoxide dehydrogenase enzymes, respectively, as shown in Figure 10. 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, H 2 , 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.

[0053] The reducing equivalents produced by the metabolism of methanol, hydrogen, and carbon monoxide can be used to power several fatty alcohol, fatty acid, fatty aldehyde, and isopropanol production pathways. For example, the maximum theoretical yield of a fatty alcohol, a fatty acid, a fatty aldehyde, or isopropanol from glucose and glycerol 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 fatty alcohol, a fatty acid, and a fatty aldehyde, on glucose and glycerol are made possible. 2 C 6 H 12 O 6 + 12 H 2 → C 12 H 26 O + 11 H 2 O(Fatty Alcohol on glucose + external redox)2 C 6 H 12 O 6 + 10 H 2 → C 12 H 24 O 2 + 10 H 2 O(Fatty Acid on glucose + external redox)2 C 6 H 12 O 6 + 11 H 2 → C 12 H 24 O + 11 H 2 O(Fatty Aldehyde on glucose + external redox)4 C3H 8 O 3 + 8 H 2 → C 12 H 26 O + 11 H 2 O(Fatty Alcohol on glycerol + external redox)4 C 3 H 3 O 3 + 6 H 2 → C 12 H 24 O 2 + 10 H 2 O(Fatty Acid on glycerol + external redox)4 C 3 H 3 O 3 + 7 H 2 → C 12 H 24 O + 11 H 2 O(Fatty Aldehyde on glycerol + external redox)

[0054] In most instances, achieving such maximum yield stoichiometries may require some oxidation of reducing equivalents (e.g., H 2 + ½ O 2 → H 2 O, CO + V 2 O 2 → CO 2 , CH 4 O + 1.5 O 2 → CO 2 + 2 H 2 O, CH 12 O 6 + 6 O 2 → 6 CO 2 + 6 H 2 O) 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 a fatty alcohol, a fatty acid, a fatty aldehyde, isopropanol, and their intermediates, directly from CO 2 .

[0055] 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 compound 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.

[0056] In some embodiments, the microorganisms of the invention can utilize a heterologous MI-FAE cycle and / or a MD-FAE cycle coupled with an acyl-CoA termination pathway to form fatty alcohols, fatty aldehydes, or fatty acids. The MI-FAE cycle can include a thiolase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase. The MD-FAE cycle can include an elongase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase and an enoyl-CoA reductase. Each passage through the MI-FAE cycle and / or the MD-FAE cycle results in the formation of an acyl-CoA elongated by a single two carbon unit compared to the acyl-CoA substrate entering the elongation cycle. Products can be even or odd chain length, depending on the initial substrate entering the acyl-CoA elongation pathway, i.e. two acety-CoA substrates, malonyl-CoA or one acetyl-CoA substrate combined with a propionyl-CoA substrate. Elongation of the two acetyl-CoA substrates or malonyl-CoA produces an even chain length product, whereas elongation with the propionyl-CoA substrate produces an odd chain length product. A termination pathway catalyzes the conversion of a MI-FAE intermediate and / or a MD-FAE intermediate, such as the acyl-CoA, to its corresponding fatty alcohol, fatty aldehyde, or fatty acid product. MI-FAE cycle, MD-FAE cycle and termination pathway enzymes can be expressed in one or more compartments of the microorganism. For example, in one embodiment, all MI-FAE cycle and termination pathway enzymes are expressed in the cytosol. In another embodiment, all MD-FAE cycle and termination pathway enzymes are expressed in the cytosol. Additionally, the microorganisms of the invention can be engineered to optionally secret the desired product into the culture media or fermentation broth for further manipulation or isolation.

[0057] In some embodiments, the microorganisms of the invention can utilize a heterologous FAACPE cycle coupled with an acyl-ACP termination pathway to form fatty alcohols, fatty aldehydes, or fatty acids. The FAACPE cycle includes a β-ketoacyl-ACP synthase, a β-ketoacyl-ACP reductase, a β-hydroxyacyl-ACP reductase, and a enoyl ACP-reductase. Each passage through the FAACPE cycle results in the formation of an acyl-ACP elongated by a single two carbon unit compared to the acyl-ACP substrate entering the elongation cycle. Products can be even or odd chain length, depending on the initial substrate entering the FAACPE pathway, i.e. acetoacetyl-ACP or 3-oxovaleryl-ACP. Elongation of the acetoacetyl-ACP substrates produces an even chain length product, whereas elongation with the 3-oxovaleryl-ACP substrate produces an odd chain length product. A termination pathway catalyzes the conversion of a FAACPE intermediate, such as the acyl-ACP, to its corresponding fatty alcohol, fatty aldehyde, or fatty acid product. FAACPE cycle and termination pathway enzymes can be expressed in one or more compartments of the microorganism. For example, in one embodiment, all FAACPE cycle and termination pathway enzymes are expressed in the cytosol. Additionally, the microorganisms of the invention can be engineered to optionally secret the desired product into the culture media or fermentation broth for further manipulation or isolation.

[0058] Products of the invention include fatty alcohols, fatty aldehydes, or fatty acids derived from intermediates of the MI-FAE cycle, MD-FAE cycle, and / or FAACPE cycle. For example, alcohol products can include saturated fatty alcohols, unsaturated fatty alcohols, 1,3-diols, and 3-oxo-alkan-1-ols. Aldehyde products can include saturated fatty aldehydes, unsaturated fatty aldehydes, 3-hydroxyaldehydes and 3-oxoaldehydes. Acid products can include saturated fatty acids, unsaturated fatty acids, 3-hydroxyacids and 3-oxoacids. These products can further be converted to derivatives such as fatty esters, either by chemical or enzymatic means. Methods for converting fatty alcohols to esters are well known in the art.

[0059] The invention also encompasses fatty alcohol, fatty aldehyde, and fatty acid chain-length control strategies in conjunction with host strain engineering strategies, such that the non-naturally occurring microorganism of the invention efficiently directs carbon and reducing equivalents toward fermentation products of a specific chain length.

[0060] Recombinant microorganisms of the invention can produce commercial quantities of a fatty alcohol, fatty aldehyde, or fatty acid ranging in chain length from four carbon atoms (C 4 ) to twenty-four carbon atoms (C 24 ) or more carbon atoms. The microorganism of the invention can produce a desired product that is at least 50%, 60%, 70%, 75%, 85%, 90%, 95% or more selective for a particular chain length. The carbon chain-length of the product can be controlled by one or more enzymes of the MI-FAE cycle (steps A / B / C / D of Figures 7) and / or one or more enzymes of the MD-FAE cycle (steps E / B / C / D of Figure 7) in combination with one or more termination pathway enzymes (steps E-N of Figure 8). Chain length can be capped during the elongation cycle by one or more MI-FAE cycle enzymes (thiolase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and / or enoyl-CoA reductase) exhibiting selectivity for MI-FAE cycle substrates having a number of carbon atoms that are no greater than the desired product size. Alternatively, or in addition, chain length can be capped during the elongation cycle by one or more MD-FAE cycle enzymes (elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and / or enoyl-CoA reductase). Chain length can be further constrained by one or more enzymes catalyzing the conversion of the MI-FAE cycle intermediate to the fatty alcohol, fatty aldehyde or fatty acid product such that the one or more termination enzymes only reacts with substrates having a number of carbon atoms that are no less than the desired fatty alcohol, fatty aldehyde or fatty acid product.

[0061] The termination pathway enzymes catalyzing conversion of a MI-FAE-CoA intermediate or MD-FAE-CoA intermediate to a fatty alcohol can include enzyme combinations of a fatty acyl-CoA reductase (alcohol or aldehyde forming), a fatty aldehyde reductase, an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolase and / or a carboxylic acid reductase (see, e.g., pathways G; E / F; K / J / F; HIN / F; or K / L / N / F of Figure 8). Termination pathway enzymes for converting a MI-FAE-CoA intermediate or MD-FAE-CoA intermediate to a fatty acid can include enzyme combinations of a thioesterase, a CoA hydrolase, an acyl-CoA:ACP acyltransferase, an aldehyde dehydrogenase and / or an acyl-ACP reductase (see, e.g., pathways H; K / L; EN; K / J / N of Figure 8). For production of a fatty aldehyde, the termination pathway enzymes can include enzyme combinations of a fatty acyl-CoA reductase (aldehyde forming), an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolase and / or a carboxylic acid reductase (see, e.g., pathways E; K / J; H / N; or K / L / N of Figure 8).

[0062] The carbon chain-length of the product can also be controlled by one or more enzymes of the FAACPE cycle (steps H / E / F / G of Figures 12) in combination with one or more termination pathway enzymes (steps I-O of Figure 12). Chain length can be capped during the elongation cycle by one or more FAACPE cycle enzymes (β-ketoacyl-ACP synthase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP reductase, and / or enoyl ACP-reductase) exhibiting selectivity for FAACPE cycle substrates having a number of carbon atoms that are no greater than the desired product size. Chain length can be further constrained by one or more enzymes catalyzing the conversion of the FAACPE cycle intermediate to the fatty alcohol, fatty aldehyde or fatty acid product such that the one or more termination enzymes only reacts with substrates having a number of carbon atoms that are no less than the desired fatty alcohol, fatty aldehyde or fatty acid product.

[0063] The termination pathway enzymes catalyzing conversion of a FAACPE cycle intermediate to a fatty alcohol can include enzyme combinations of a thioesterase, a fatty acyl-ACP reductase, an acyl-CoA synthase, an acyl-CoA reductase, a fatty aldehyde reductase, a fatty alcohol forming acyl-CoA reductase (FAR), and / or a carboxylic acid reductase (CAR) (see, e.g., pathways J / M; I / K / L / M; I / O / M; and I / K / N of Figure 12). Termination pathway enzyme for converting a FAACPE intermediate to a fatty acid can include a thioesterase (see, e.g., pathways I of Figure 12). For production of a fatty aldehyde, the termination pathway enzymes can include combinations of a thioesterase, a fatty acyl-ACP reductase, an acyl-CoA synthase, an acyl-CoA reductase, a fatty aldehyde reductase, and / or a carboxylic acid reductase (CAR), (see, e.g., pathways J; I / K / L; and I / O of Figure 12).

[0064] The non-naturally occurring microbial organisms of the invention can also efficiently direct cellular resources, including carbon, energy and reducing equivalents, to the production of fatty alcohols, fatty aldehydes and fatty acids, thereby resulting in improved yield, productivity and / or titer relative to a naturally occurring organism. In one embodiment, the microorganism is modified to increase cytosolic acetyl-CoA levels. In another embodiment, the microorganism is modified to efficiently direct cytosolic acyl-CoA into fatty alcohols, fatty aldehydes or fatty acids rather than other byproducts or cellular processes. Enzymes or pathways that lead to the formation of byproducts can be attenuated or deleted. Exemplary byproducts include, but are not limited to, ethanol, glycerol, lactate, acetate, esters and carbon dioxide. Additional byproducts can include fatty-acyl-CoA derivatives such as alcohols, alkenes, alkanes, esters, acids and aldehydes. Accordingly, a byproduct can include any fermentation product diverting carbon and / or reducing equivalents from the product of interest.

[0065] In another embodiment, the availability of reducing equivalents or redox ratio is increased. In yet another embodiment, the cofactor requirements of the microorganism are balanced such that the same reduced cofactors generated during carbon assimilation and central metabolism are utilized by MI-FAE cycle, MD-FAE cycle and / or termination pathway enzymes. In yet another embodiment, the fatty alcohol, fatty aldehyde or fatty acid producing organism expresses a transporter which exports the fatty alcohol, fatty aldehyde or fatty acid from the cell.

[0066] Microbial organisms capable of fatty alcohol production are exemplified herein with reference to the Saccharomyces cerevisaie genetic background. However, with the complete genome sequence available now for thousands of species (with more than half of these available on public databases such as the NCBI), the identification of an alternate species homolog for one or more genes, including for example, orthologs, paralogs and nonorthologous gene displacements, and the interchange of genetic alterations between eukaryotic organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling production of fatty alcohols described herein with reference to a particular organism such as Saccharomyces cerevisiae can be readily applied to other microorganisms, such as Escherichia coli. Given the teachings and guidance provided herein, those skilled in the art understand that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[0067] In some aspects of the invention, production of fatty alcohols, fatty aldehydes and fatty acids through the MI-FAE cycle and termination pathways disclosed herein are particularly useful because the cycle and pathways result in higher product and ATP yields than through naturally occurring biosynthetic pathways such as the well-known malonyl-CoA dependent fatty acid synthesis pathway, or in some aspects the malonyl-ACP dependent fatty acid sysnthesis pathway. For example, using acetyl-CoA as a C 2 extension unit (e.g. step A, Figure 2) instead of malonyl-acyl carrier protein (malonyl-ACP) saves one ATP molecule per unit flux of acetyl-CoA entering the MI-FAE cycle. The MI-FAE cycle results in acyl-CoA instead of acyl-ACP, and can preclude the need of the ATP-consuming acyl-CoA synthase reactions for the production of octanol and other fatty alcohols, fatty aldehydes or fatty acids if acetyl-CoA is used as the extender unit. The fatty alcohol, fatty aldehyde and fatty acid producing organisms of the invention can additionally allow the use of biosynthetic processes to convert low cost renewable feedstock for the manufacture of chemical products.

[0068] Chain length control can be achieved using a combination of highly active enzymes with suitable substrate ranges appropriate for biosynthesis of the desired fatty alcohol, fatty aldehyde, or fatty acid. Chain length of the product can be controlled using one or more enzymes of MI-FAE cycle, MD-FAE cycle, FAACPE cycle or termination pathway. As described herein, chain length can be capped during the MI-FAE cycle by one or more MI-FAE cycle enzymes (thiolase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and / or enoyl-CoA reductase), in the case of the MD-FAE cycle, one or more MD-FAE cycle enzymes (elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and / or enoyl-CoA reductase), and in the case of the FAACPE cycle, one or more enzymes (β-ketoacyl-ACP synthase, β-ketoacylcl-ACP reductase, (3-hydroxyacyl-ACP reductase and / or enoyl ACP-reductase), exhibiting selectivity for MI-FAE cycle, MD-FAE cycle and / or FAACPE cycle substrates having a number of carbon atoms that are no greater than the desired product size. Since enzymes are reversible, any of the elongation pathway enzymes can serve in this capacity. Selecting enzymes with broad substrate ranges but defined chain-length boundaries enables the use of a single enzyme to catalyze multiple cycles of elongation, while conferring product specificity. To further hone specificity and prevent the accumulation of shorter byproducts, selectivity is further constrained by product-forming termination enzymes, such that one or more enzymes are selective for acyl-CoA, acyl-ACP or other termination pathway substrates having a number of carbon atoms that are no less than the desired chain length. The deletion or attenuation of endogenous pathway enzymes that produce different chain length products can further hone product specificity.

[0069] Using the approaches outlined herein, one skilled in the art can select enzymes from the literature with characterized substrate ranges that selectively produce a fatty alcohol, fatty aldehyde or fatty acid product of a specific chain length. To selectively produce fatty alcohols, fatty aldehydes or fatty acids of a desired length, one can utilize combinations of known enzymes in the literature with different selectivity ranges as described above. For example, a non-naturally occurring microbial organism that produces C 16 fatty alcohol can express enzymes such as the Rattus norvegicus Acaala thiolase and the enoyl-CoA reducatse of Mycobacterium smegmatis, which only accept substrates up to length C 16 . Coupling one or both chain elongation enzymes with a C 16 -C 18 fatty acyl-CoA reductase (alcohol or aldehyde forming) such as FAR of Simmondsia chinensis further increases product specificity by reducing the synthesis of shorter alcohol products. As another example, a non-naturally occurring microbial organism of the invention can selectively produce alcohols of length C 14 by combining the 3-hydroxyacyl-CoA dehydratase of Arabidopsis thaliana with the acyl-CoA reductase Acr1 of Acinetobacter sp. Strain M-1. To produce 3-oxoacids of length C 14 , one can, for example, combine the rat thiolase with the 3-oxoacyl-CoA hydrolase of Solanum lycopersicum. As still a further example, to produce C 18 fatty acids, one can combine the Salmonella enterica fadE enoyl-CoA reductase with the tesB thioesterase of E. coli. In yet another example, selective production of C 6 alcohols are formed by combining the paaH1 thiolase from Ralstonia eutropha with the Leifsonia sp. S749 alcohol dehydrogenase lsadh.

[0070] Exemplary MI-FAE cycle, MD-FAE cycle and termination pathway enzymes are described in detail in Example IV. The biosynthetic enzymes described herein exhibit varying degrees of substrate specificity. Exemplary substrate ranges of enzymes characterized in the literature are shown in the table below and described in further detail in Example IV. Pathway step Chain length Gene Organism 2AC4AtoBEscherichia coli2AC6PhaDPseudomonas putida2AC6-C8BktBRalstonia eutropha2AC10-C16AcaalaRattus norvegicus2BC4HbdClostridium acetobutylicum2BC4-C6paaH1Ralstonia eutropha2BC4-C10HADHSus scrofa2B / CC4-C18FadBEscherichia coli2B / CC4-C18Fox2Candida tropicalis2B / CC4-C18Fox2Saccharomyces cerevisiae2CC4-C6crtClostridium acetobutylicum2CC4-C7pimFRhodopseudomonas palustris2CC4-C14MFP2Arabidopsis thaliana2DC4-C6ECR1Euglena gracilis2DC6-C8ECR3Euglena gracilis2DC8-10ECR2Euglena gracilis2DC8-C16ECRRattus norvegicus2DC10-C16ECRMycobacterium smegmatis2DC2-C18fadESalmonella enterica2EC2-C4bphGPseudomonas sp2EC4BldClostridium saccharoperbutylacetonicum2EC12-C20ACRAcinetobacter calcoaceticus2EC14-C18Acr1Acinetobacter sp. Strain M-12EC16-C18Rv1543, Rv3391Mycobacterium tuberculosis2EC18FAR1, FAR2Mus musculus2EC12-C20orf1594Synechococcus elongatus PCC79422EC6-C18Maqu 2220Marinobacter aquaeolei2FC6-C7lsadhLeifsonia sp. S7492FC2-C8yqhDEscherichia coli2FC3-C10AdhPseudomonas putida2FC2-C14alrAAcinetobacter sp. strain M-12FC2-C30ADH1Geobacillus thermodenitrificans2FC3-C8ADH6Saccharomyces cerevisiae s288c2GC2adhEEscherichia coli2GC2-C8adhe2Clostridium acetobutylicum2GC14-C16At3g11980Arabidopsis thaliana2GC16At3g44560Arabidopsis thaliana2GC16-C18FARSimmondsia chinensis2HC4Cat2Clostridium kluyveri2HC4-C6Acot12Rattus norveyicus2HC14MKS2Solanum lycopersicum2LC8-C10fatB2Cuphea hookeriana2LC12fatBUmbellularia california2LC14-C16fatB3Cuphea hookeriana2LC18tesAEscherichia coli2NC12-C18CarNocardia iowensis2NC12-C16CarMycobacterium sp. (strain JLS)2OC4-C8ELO1Trypanosoma brucei2OC10-C12ELO2Trypanosoma brucei2OC14-C16ELO3Trypanosoma brucei2OC14-C16ELO1Saccharomyces cerevisiae2OC18-C20ELO2Saccharomyces cerevisiae2OC22-C24ELO3Saccharomyces cerevisiae

[0071] Taking into account the differences in chain-length specificities of each enzyme in the MI-FAE cycle, MD-FAE cycle or FAACPE cycle, one skilled in the art can select one or more enzymes for catalyzing each elongation cycle reaction step (e.g., steps A-D or steps E / B / C / D of Figure 6, or H / E / F / G of Figure 12). For example, for the thiolase step of the MI-FAE cycle, some thiolase enzymes such as bktB of Ralstonia eutropha catalyze the elongation of short- and medium-chain acyl-CoA intermediates (C 6 -C 8 ), whereas others such as Acaa1a of R. norvegicus are active on longer-chain substrates (C 10 -C 16 ). Thus, a microbial organism producing a fatty alcohol, fatty aldehyde or fatty acid can comprise one, two, three, four or more variants of a thiolase, elongase, 3-oxoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and / or enoyl-CoA reductase.

[0072] Chain length specificity of enzymes can be assayed by methods well known in the art (eg. Wrensford et al, Anal Biochem 192:49-54 (1991)). The substrate ranges of fatty alcohol, fatty aldehyde, or fatty acid producing enzymes can be further extended or narrowed by methods well known in the art. Variants of biologically-occurring enzymes can be generated, for example, by rational and directed evolution, mutagenesis and enzyme shuffling as described herein. As one example, a rational engineering approach for altering chain length specificity was taken by Denic and Weissman (Denic and Weissman, Cell 130:663-77 (2008)). Denic and Weissman mapped the region of the yeast elongase protein ELOp responsible for chain length, and introduced mutations to vary the length of fatty acid products. In this instance, the geometry of the hydrophobic substrate pocket set an upper boundary on chain length. A similar approach can be useful for altering the chain length specificities of enzymes of the MI-FAE cycle, MD-FAE cycle and / or termination pathways.

[0073] Enzyme mutagenesis, expression in a host, and screening for fatty alcohol production is another useful approach for generating enzyme variants with improved properties for the desired application. For example, US patent application 2012 / 0009640 lists hundreds of variants of Marinobacter algicola and Marinobacter aquaeolei FAR enzymes with improved activity over the wild type enzyme, and varying product profiles.

[0074] Enzyme mutagenesis (random or directed) in conjunction with a selection platform is another useful approach. For example, Machado and coworkers developed a selection platform aimed at increasing the activity of acyl-CoA elongation cycle enzymes on longer chain length substrates (Machado et al., Met Eng 14(5):504-511 (2012)). Machado et al. identified the chain-length limiting step of their pathway (a 3-hydroxyacyl-CoA dehydrogenase) and evolved it for improved activity on C 6 -C 8 substrates using an anaerobic growth rescue platform. Additional variants of enzymes useful for producing fatty alcohols are listed in the table below Enzyme Protein / GenBankID / GI number Organism Variant(s) Reference 3-Ketoacyl-CoA thiolaseAcaa2Rattus norvegicusH352A, H352E, H352K, H352YZeng et al., Prot. Expr. Purif. 35: 320-326 (2004)NP_569117.1GI:184268663-Hydroxyacyl-CoA dehydrogenaseHadhRattus norvegicusS137A, S137C, S137TLiu et al., Prot. Expr. Purif. 37:344-351 (2004).NP_476534.1GI:17105336Enoyl-CoA hydrataseEch1Rattus norvegicusE144A, E144A / Q162L, E164A, Q162A, Q162L, Q162MKiema et al., Biochem. 38:2991-2999 (1999)NP_072116.1GI:12018256Enoyl-CoA reductaseInhAMycobacterium tuberculosisK165A, K165Q, Y158FPoletto,S. et al., Prot. Expr. Purif. 34: 118-125 (2004).AAY54545.1GI:66737267Acyl-CoA reductaseLuxCPhoto bacterium phosphoreumC171S, C279S, C286SLee, C. et al., Biochim. Biophys. Acta. 1338: 215-222 (1997).AAT00788.1GI:46561111Alcohol dehydrogenaseYADH-1Saccharomyces cerevisiaeD223G, D49N, E68Q, G204A, G224I, H47R, H51E, L203ALeskovac et al., FEMS Yeast Res. 2(4):481-94 (2002).P00330.4GI:1168350Fatty alcohol forming acyl-CoA reductase (FAR)AdhEEscherichia coliA267T / E568K, A267TMembrillo et al., J. Biol. Chem. 275(43): 333869-75 (2000).NP_415757.1GI: 16129202

[0075] 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 or S. cerevisiae 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 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.

[0076] 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.

[0077] 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 III 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.

[0078] 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.

[0079] 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.

[0080] Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having fatty alcohol, fatty aldehyde or fatty acid 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.

[0081] 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.

[0082] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x _dropoff 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

[0083] In some embodiments, the invention provides a non-naturally occurring microbial organism having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway as depicted in Figures 1 and 10, and a MI-FAE cycle or a MD-FAE cycle in combination with a termination pathway as depicted in Figures 2, 7 and 8, wherein said formaldehyde fixation pathway comprises: (1) 1B and 1C; (2) 1D; or (3) 1D and 1Z, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein said formate assimilation pathway comprises a pathway selected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase, 1F is a formate ligase, a formate transferase, or a formate synthetase, wherein 1G is a fonnyl-CoA reductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 11 is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a methylenetetrahydrofolate dehydrogenase, 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 1O is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase, wherein said methanol metabolic pathway comprises a pathway selected from: (12) 10J; (13) 10A, (14) 10A and 10B; (15) 10A, 10B and 10C; (16) 10J, 10K and 10C; (17) 10J, 10M, and 10N; (18) 10J and 10L; (19) 10J, 10L and 10G; (20) 10J, 10L, and 10I; (21) 10A, 10B, 10C, 10D, and 10E; (22) 10A, 10B, 10C, 10D, and 10F; (23) 10J, 10K, 10C, 10D, and 10E; (24) 10J, 10K, 10C, 10D, and 10F; (25) 10J, 10M, 10N, and 10O; (26) 10A, 10B, 10C, 10D, 10E, and 10G; (27) 10A, 10B, 10C, 10D, 10F, and 10G; (28) 10J, 10K, 10C, 10D, 10E, and 10G; (29) 10J, 10K, 10C, 10D, 10F, and 10G; (30) 10J, 10M, 10N, 10O, and 10G; (31) 10A, 10B, 10C, 10D, 10E, and 10I; (32) 10A, 10B, 10C, 10D, 10F, and 10I; (33) 10J, 10K, 10C, 10D, 10E, and 10I; (34) 10J, 10K, 10C, 10D, 10F, and 10I; and (35) 10J, 10M, 10N, 10O, and 101, wherein 10A is a methanol methyltransferase, wherein 10B is a methylenetetrahydrofolate reductase, wherein 10C is a methylenetetrahydrofolate dehydrogenase, wherein 10D is a methenyltetrahydrofolate cyclohydrolase, wherein 10E is a formyltetrahydrofolate deformylase, wherein 10F is a formyltetrahydrofolate synthetase, wherein 10G is a formate hydrogen lyase, wherein 10I is a formate dehydrogenase, wherein 10J is a methanol dehydrogenase, wherein 10K is a formaldehyde activating enzyme or spontaneous, wherein 10L is a formaldehyde dehydrogenase, wherein 10M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 10N is a glutathione-dependent formaldehyde dehydrogenase, wherein 10O is a S-formylglutathione hydrolase, wherein the MI-FAE cycle includes one or more thiolase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the MD-FAE cycle includes one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3 -hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the termination pathway includes a pathway selected from: (36) 2H; (37) 2K and 2L; (38) 2E and 2N; (39) 2K, 2J, and 2N; (40) 2E; (41) 2K and 2J; (42) 2H and 2N; (43) 2K, 2L, and 2N; (44) 2E and 2F; (45) 2K, 2J, and 2F; (46) 2H, 2N, and 2F; (47) 2K, 2L, 2N, and 2F; (48) 2G; (49) 2P, wherein 2E is an acyl-CoA reductase (aldehyde forming), wherein 2F is an alcohol dehydrogenase, wherein 2G is an acyl-CoA reductase (alcohol forming), wherein 2H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein 2J is an acyl-ACP reductase, wherein 2K is an acyl-CoA:ACP acyltransferase, wherein 2L is a thioesterase, wherein 2N is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase, wherein 2P is an acyl-ACP reductase (alcohol forming) wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO,; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the MI-FAE cycle, the MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the MI-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

[0084] In some embodiments, the invention provides a non-naturally occurring microbial organism having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway as depicted in Figures 1 and 10, and a FAACPE cycle in combination with a termination pathway as depicted in Figure 12, wherein said formaldehyde fixation pathway comprises: (1) 1B and 1C; (2) 1D; (3) 1D and 1Z, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein said formate assimilation pathway comprises a pathway selected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase, 1F is a formate ligase, a formate transferase, or a formate synthetase, wherein 1G is a fonnyl-CoA reductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a methylenetetrahydrofolate dehydrogenase, 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 1O is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase, wherein said methanol metabolic pathway comprises a pathway selected from: (12) 10J; (13) 10A, (14) 10A and 10B; (15) 10A, 10B and 10C; (16) 10J, 10K and 10C; (17) 10J, 10M, and 10N; (18) 10J and 10L; (19) 10J, 10L and 10G; (20) 10J, 10L, and 10I; (21) 10A, 10B, 10C, 10D, and 10E; (22) 10A, 10B, 10C, 10D, and 10F; (23) 10J, 10K, 10C, 10D, and 10E; (24) 10J, 10K, 10C, 10D, and 10F; (25) 10J, 10M, 10N, and 10O; (26) 10A, 10B, 10C, 10D, 10E, and 10G; (27) 10A, 10B, 10C, 10D, 10F, and 10G; (28) 10J, 10K, 10C, 10D, 10E, and 10G; (29) 10J, 10K, 10C, 10D, 10F, and 10G; (30) 10J, 10M, 10N, 10O, and 10G; (31) 10A, 10B, 10C, 10D, 10E, and 101; (32) 10A, 10B, 10C, 10D, 10F, and 101; (33) 10J, 10K, 10C, 10D, 10E, and 101; (34) 10J, 10K, 10C, 10D, 10F, and 10I; and (35) 10J, 10M, 10N, 10O, and 10I, wherein 10A is a methanol methyltransferase, wherein 10B is a methylenetetrahydrofolate reductase, wherein 10C is a methylenetetrahydrofolate dehydrogenase, wherein 10D is a methenyltetrahydrofolate cyclohydrolase, wherein 10E is a formyltetrahydrofolate deformylase, wherein 10F is a formyltetrahydrofolate synthetase, wherein 10G is a formate hydrogen lyase, wherein 10I is a formate dehydrogenase, wherein 10J is a methanol dehydrogenase, wherein 10K is a formaldehyde activating enzyme or spontaneous, wherein 10L is a formaldehyde dehydrogenase, wherein 10M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 10N is a glutathione-dependent formaldehyde dehydrogenase, wherein 10O is a S-formylglutathione hydrolase, wherein said FAACPE cycle comprises one or more β-ketoacyl-ACP synthase, one or more β-ketoacyl-ACP reductase, one or more β-hydroxyacyl-ACP reductase, and one or more enoyl ACP-reductase, wherein said termination pathway comprises a pathway selected from: (36) 12I; (37) 12J; (38) 12I, 12K, and 12L; (39) 12I and 120; (40) 12J and 12M; (41) 12I, 12K, 12L, and 12M; (42) 121, 120, and 12M; (43) 12I, 12K and 12N; (44) 12P, wherein 12I is a thioesterase, wherein 12J is a fatty acyl-ACP reductase, wherein 12K is an acyl-CoA synthase, wherein 12L is an acyl-CoA reductase, wherein 12M is a fatty aldehyde reductase, wherein 12N is a fatty alcohol forming acyl-CoA reductase (FAR), wherein 12O is a carboxylic acid reductase (CAR), wherein 12P is an acyl-ACP reductase (alcohol forming), wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the FAACPE cycle or the termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the FAACPE cycle and the termination pathway are independently selected from a compound of Formula (II) or malonyl-ACP: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the FAACPE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

[0085] In some aspects of the invention, non-naturally occurring microbial organism of the invention can produce a compound of Formula (I) wherein R 1 is C 1-17 linear alkyl. In another aspect of the invention, the R 1 of the compound of Formula (I) is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl..

[0086] In some aspects of the invention, the microbial organism includes two, three, or four exogenous nucleic acids each encoding an enzyme of the MI-FAE cycle, the MD-FAE cycle, or the FAACPE cycle. In some aspects of the invention, the microbial organism includes two, three, or four exogenous nucleic acids each encoding an enzyme of the termination pathway. In some aspects of the invention, the microbial organism includes one, two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a formaldehyde fixation pathway enzyme, a formate assimilation pathway enzyme, or a methanol metabolic pathway enzyme. In some aspects of the invention, the microbial organism includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(49) for a microbial organism having a MI-FAE cycle or a MD-FAE cycle in combination with a termination pathway as depicted in Figures 1, 2, 7, 8 and 10. In some aspects of the invention, the microbial organism includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(44) for a microbial organism having a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway as depicted in Figures 1, 10 and 12.

[0087] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle, FAACPE cycle or termination pathway is expressed in a sufficient amount to produce a fatty alcohol selected from the Formulas (III)-(VI): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0088] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle, FAACPE cycle or termination pathway is expressed in a sufficient amount to produce a fatty aldehyde selected from the Formula (VII)-(X): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0089] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein the one or more enzymes of the MI-FAE cycle, MD-FAE cycle, FAACPE cycle or termination pathway is expressed in a sufficient amount to produce a fatty acid selected from the Formula (XI)-(XIV): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0090] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein one or more enzymes of the MI-FAE cycle and / or MD-FAE cycle are each selective for a compound of Formula (II) wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11, linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0091] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein one or more enzymes of the FAACPE cycle are each selective for a compound of Formula (II) wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, Cis linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0092] In some embodiments, the invention provides a non naturally occurring microbial organism, wherein one or more enzymes of the termination pathway are each selective for a compound of Formula (II) wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl, C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, Cis linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0093] In some embodiments, a non-naturally occurring microbial organism of the invention having a formate assimilation pathway further includes wherein the formate assimilation pathway comprises: (1) 1Q; (2) 1R, and 1S; (3) 1Y and 1Q; (4) 1Y, 1R and 1S, wherein 1Q is a pyruvate formate lyase, wherein IRis a pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, or a pyruvate:NADP+ oxidoreductase, wherein 1S is a formate dehydrogenase, wherein 1Y is a glyceraldehydes-3-phosphate dehydrogenase or an enzyme of lower glycolysis. In addition to a glyceraldehyde-3-phosphate dehydrogenase, lower glycolysis includes a phosphoglycerate kinase, a phosphoglyceromutase, an enolase, a pyruvate kinase or a PTS-dependant substrate import. Accordingly, in some embodiments, the formate assimilation pathway comprising 1Y includes an enzyme selected from a phosphoglycerate kinase, a phosphoglyceromutase, an enolase, a pyruvate kinase and a PTS-dependant substrate import.

[0094] In some embodiments, a non-naturally occurring micoribial organism of the invention includes a methanol oxidation pathway. Such a pathway can include at least one exogenous nucleic acid encoding a methanol oxidation pathway enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol. An exemplary methanol oxidation pathway enzyme is a methanol dehydrogenase. Accordingly, in some embodiments, a non-naturally occurring micoribial organism of the invention includes at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to produce formaldehyde in the presence of methanol.

[0095] In some embodiments, the exogenous nucleic acid encoding an methanol dehydrogenase 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 methanol dehydrogenase 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 methanol dehydrogenase is selected from those provided herein, e.g., as exemplified in Example II (see Figure 1, Step A, or Figure 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 Figure 1, Step A, or Figure 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.

[0096] In certain embodiments, the exogenous nucleic acid encoding an methanol dehydrogenase is expressed in a sufficient amount to produce at least 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 30X, 40X, 50X, 100X or more formaldehyde in culture medium or intracellularly. In other embodiments, the exogenous nucleic acid encoding an methanol dehydrogenase is capable of producing an amount of formaldehyde at least 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 30X, 40X, 50X, 100X, or a range thereof, in culture medium or intracellularly. In some embodiments, the range is from 1X to 100X. In other embodiments, the range is from 2X to 100X. In other embodiments, the range is from 5X to 100X. In other embodiments, the range is from 10X to 100X. In other embodiments, the range is from 50X to 100X. In some embodiments, the amount of formaldehyde production is at least 20X. In other embodiments, the amount of formaldehyde production is at least 50X. 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 methanol dehydrogenase is selected from those provided herein, e.g., as exemplified in Example II (see Figure 1, Step A, or Figure 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 Figure 1, Step A, or Figure 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.

[0097] In some embodiments, the at least one exogenous nucleic acid included in the non-naturally occurring microbial organism of the invention is a heterologous nucleic acid. Accordingly, in some embodiments, the at least one exogenous nucleic acid encoding a formaldehyde fixation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a formate assimilation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a MI-FAE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a MD-FAE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a FAACPE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a termination pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding an acetoacetyl-ACP pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a 3-oxovalery-ACP pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding an isopropanol pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a methanol oxidation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a hydrogenase or a carbon monoxide dehydrogenase is a heterologous nucleic acid.

[0098] In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium.

[0099] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde or fatty acid 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, H6P to F6P, Fald to DHA and G3P, DHA and G3P to F6P, F6P to ACTP and E4P, ACTP to ACCOA, ACTP to acetate, acetate to ACCOA, Xu5P to ACTP and G3P, G3P to PYR, PYR to formate and ACCOA, PYR to CO2 and ACCOA, CO2 to formate, formate to Fald, formate to Formyl-CoA, Fonnyl-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, two acetyl-CoA molecules to a 3-ketoacyl-CoA, acetyl-CoA plus propionyl-CoA to a ketoacyl-CoA, malonyl-CoA to 3-ketoacyl-CoA, a 3-ketoacyl-CoA to a 3-hydroxyacyl-CoA, a 3-hydroxyacyl-CoA to an enoyl-CoA, an enoyl-CoA to an acyl-CoA, an acyl-CoA plus an acetyl-CoA to a 3-ketoacyl-CoA, an acyl-CoA plus malonyl-CoA to a 3-ketoacyl-CoA, an acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, an acyl-CoA to a fatty alcohol, an acyl-CoA to an acyl-ACP, an acyl-ACP to a fatty acid, an acyl-CoA to a fatty acid, an acyl-ACP to a fatty aldehyde, a fatty acid to a fatty aldehyde, a fatty aldehyde to a fatty acid, formaldehyde to S-hydroxymethylglutathione, S-hydroxymethylglutathione to S-formylglutathione to formate, formaldehyde to formate, MeOH to methyl-THF, methyl-THF to methylene-THF, formaldehyde to methylene-THF, methylene-THF to methenyl-THF, methenyl-THF to formyl-THF, formyl-THF to formate, formaldehyde to formate, ACCOA to MALCOA, ACCOA to AACOA, MALCOA to AACOA, AACOA to acetoacetate, acetoacetate to acetone, acetone to isopropanol, malonyl-CoA to malonyl-ACP, malonyl-ACP and acetyl-CoA to acetoacetyl-ACP, malonyl-ACP and acetyl-ACP to acetoacetyl-ACP, malonyl-ACP and propionyl-CoA to 3-oxovalery-ACP, malonyl-ACP and an acyl-ACP to a β-ketoacyl-ACP, a β-ketoacyl-ACP to a β-hydroxyacyl-ACP, a β-hydroxyacyl-ACP to a trans-2-enoyl-ACP, a trans-2-enoyl-ACP to an acyl-ACP, an acyl-ACP to a fatty acid, an acyl-ACP to a fatty aldehyde, a fatty acid to a fatty aldehyde, a fatty acid to an acyl-CoA, an acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, a fatty aldehyde to a fatty alcohol, PEP to OAA, OAA to MAL, MAL to FUM, FUM to SUCC, SUCCOA to (R)-MMCOA, (R)-MMCOA to (S)-MMCOA, MMCOA to PPCOA, PEP to PYR, pyruvate to acetate, acetate to acetyl-CoA, pyruvate to acetyl-CoA, pyruvate to acetaldehyde, threonin to acetaldehyde, acetaldehyde to acetate, acetaldehyde to acetyl-CoA, pyruvate to acetyl-phosphate, acetate to acetyl-phosphate, acetyl-phosphate to acetyl-CoA, phosphoenolpyruvate (PEP) to pyruvate, pyruvate to malate, malate to oxaloacetate, pyruvate to oxaloacetate, PEP to oxaloacetate, oxaloacetate to malonate semialdehyde, oxaloacetate to malonyl-CoA, malonate semialdehyde to malonate, malonate to malonyl-CoA, malonate semialdehyde to malonyl-CoA, malonyl-CoA to acetyl-CoA, malonate semialdehyde to acetyl-CoA, oxaloacetate plus acetyl-CoA to citrate, citrate to oxaloacetate plus acetyl-CoA, citrate to oxaloacetate plus acetate, and oxaloacetate to malate. 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 fatty alcohol, fatty aldehyde, fatty acid, or isopropanol pathway, such as that shown in Figure 1-12 and 22.

[0100] While generally described herein as a microbial organism that contains a fatty alcohol, fatty aldehyde, or fatty acid pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a fatty alcohol, fatty aldehyde, or fatty acid pathway enzyme or protein expressed in a sufficient amount to produce an intermediate of a fatty alcohol, fatty aldehyde, or fatty acid pathway. For example, as disclosed herein, a fatty alcohol, fatty aldehyde, or fatty acid pathway is exemplified in Figures 1-12 and 22. Therefore, in addition to a microbial organism containing a fatty alcohol, fatty aldehyde, or fatty acid pathway that produces fatty alcohol, fatty aldehyde, or fatty acid, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a fatty alcohol, fatty aldehyde, or fatty acid or pathway enzyme, where the microbial organism produces a fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate, for example, pyruvate, formate, formaldehyde, H6P, DHA, G3P, F6P, ACTP, E4P, formyl-CoA, FTHF, methenyl-THF, methylene-THF, glycine, serine, methyl-THF, CO2, a 3-ketoacyl-CoA, a 3-hydroxyacyl-CoA, an enoyl-CoA, a β-ketoacyl-ACP, a β-hydroxyacyl-ACP, a trans-2-enoyl-ACP, an acyl-CoA, an acyl-ACP, acetoacetate, acetone, acetate, acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonate semialdehyde, malonate, malonyl-ACP, propionyl-CoA, malonyl-CoA, acetyl-CoA, or citrate.

[0101] It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-12 and 22, 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 fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate can be utilized to produce the intermediate as a desired product.

[0102] 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, fatty alcohol, fatty aldehyde, acid and / or isopropanol pathways to enhance the yield and / or production of a product such as any of the fatty alcohol, fatty aldehyde, fatty acids and / or isopropanol described herein.

[0103] Metabolic modifications for enabling redox- and carbon-efficient cytosolic methanol utilization in a eukaryotic or prokaryotic organism are exemplified in further detail below.

[0104] 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 methanol oxidation pathways and / or one or more endogenous or exogenous formaldehyde assimilation pathway enzymes in the cytosol. An exemplary pathway for methanol oxidation includes NADH dependent methanol dehydrogenase as shown in Figure 1. Exemplary pathways for converting cytosolic formaldehyde into glycolytic intermediates also are shown in Figure 1. Such pathways include methanol oxidation via expression of an cytosolic NADH dependent methanol dehydrogenase, formaldehyde fixation via expression of cytosolic DHA synthase, both methanol oxidation via expression of an cytosolic NADH dependent methanol dehydrogenase 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 formaldehyde fixation pathways. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and / or when utilization of ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation attenuation of DHA synthase.

[0105] For example, in the carbon-efficient DHA pathway of formaldehyde assimilation shown in Fig 1, step D, formaldehyde is converted to dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (GAP) by DHA synthase (Fig 1D). DHA and G3P are then converted to fructose-6-phosphate in one step by F6P aldolase (Fig 1C) 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, RSP epimerase and Ru5P epimerase) regenerates xylulose-5-phosphate, the DHA synthase substrate. Conversion of F6P to acetyl-phosphate and E4P (Fig 11), or Xu5P to G3P and acetyl-phosphate (Fig 1T and 1U) by one or more phosphoketolase enzymes results in the carbon-efficient generation of cytosolic acetyl-CoA. Exemplary enzymes catalyzing each step of the carbon efficient DHA pathway are described elsewhere herein.

[0106] An alternate carbon efficient pathway for formaldehyde assimilation proceeding through ribulose-5-phosphate (Ru5P) is shown in Fig 1, 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 (Fig 1B). 6-Phospho-3-hexuloisomerase converts H6P to F6P (Fig 1C). Regeneration of Ru5P from F6P proceeds by pentose phosphate pathway enzymes. Carbon-efficient phosphoketolase enzymes catalyze the conversion of F6P and / or Xu5P to acetyl-phosphate and pentose phosphate intermediates. Exemplary enzymes catalyzing each step of the carbon efficient RuMP pathway are described elsewhere herein.

[0107] 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 6-phospho-3-hexuloisomerase. 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 methanol dehydrogenase, formaldehyde fixation via expression of cytosolic 3-Hu6P synthase and 6-phospho-3-hexuloisomerase, both methanol oxidation via expression of an cytosolic NADH dependent dehydrogenase and formaldehyde fixation via expression of cytosolic 3-Hu6P synthase and 6-phospho-3-hexuloisomerase alone or together with the metabolic modifications exemplified below that attenuate less beneficial methanol oxidation and / or formaldehyde fixation pathways. Such attenuating metabolic modifications include, for example, attenuation of alcohol oxidase, attenuation of DHA kinase and / or when utilization of ribulose-5-phosphate (Ru5P) pathway for formaldehyde fixation attenuation of DHA synthase.

[0108] 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 Figure 1. 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 methanol dehydrogenase, 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 formaldehyde fixation pathways. 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).

[0109] 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.

[0110] 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.

[0111] 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 fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid biosynthesis. Thus, a non-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 fatty alcohol, fatty aldehyde, or fatty acid.

[0112] Depending on the fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathways. For example, fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid can be included, such as a thiolase, a 3-oxoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA redutase, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase, for production of a fatty alcohol.

[0113] 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 fatty alcohol, fatty aldehyde, or fatty acid 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 or eight up to all nucleic acids encoding the enzymes or proteins constituting a fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid pathway precursors such as acetyl-CoA, malonyl-ACP, malonyl-CoA or propionyl-CoA.

[0114] 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.

[0115] 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 fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid biosynthetic capability. For example, a non-naturally occurring microbial organism having a fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of a thiolase and an acyl-CoA reductase (alcohol forming), or alternatively a 2-oxoacyl-CoA reductase and an acyl-CoA hydrolase, or alternatively a enoyl-CoA reductase and an acyl-CoA reductase (aldehyde forming), or alternatively a methanol methyltransferase and an acetone reductase, or alternatively a 3-hexulose-6-phosphate synthase and an enoyl ACP-reductase, 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 thiolase, an enoyl-CoA reductase and a aldehyde dehydrogenase (acid forming), or alternatively a 3-hydroxyacyl-coA dehydratase, an acyl-CoA:ACP acyltransferase and a thioesterase, or alternatively a 3-oxoacyl-CoA reductase, an acyl-CoA hydrolase and a carboxylic acid reductase, or alternatively a dihydroxyacetone synthase, a S-formylglutathione hydrolase and an acetoacetyl-CoA ligase, or alternatively a 6-phospho-3-hexuloisomerase, a β-hydroxyacyl-ACP reductase and a fatty alcohol forming acyl-CoA reductase, 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.

[0116] Sources of encoding nucleic acids for a fatty alcohol, fatty aldehyde, or fatty acid 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, 255956237 Penicillium chrysogenum Wisconsin 54-1255, Acetobacter pasteurians, Acidaminococcus fermentans,Acinetobacter baumαrrnii Naval-82, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1, Actinobacillus succinogenes, Actinobacillus succinogenes 130Z, Aedes aegypti, Allochromatium vinosum DSM 180, Aminomonas aminovorus, Anabaena variabilis ATCC 29413, Anaerobiospirillum succiniciproducens, Aquifex aeolicus, Arabidopsis thaliana, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Arthrobacter globiformis, Ascaris suum, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624, AspergillusSynechococcus elongatus PCC 6301, Azotobacter vinelandii DJ, B. subtilis 168, Bacillus alcalophilus ATCC 27647, Bacillus anthracis, Bacillus azotoformans LMG 9581, Bacillus cereus, Bacillus cereus ATCC 14579, Bacillus coagulans 36D1, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus selenitireducens MLS10, Bacillus sp. SG-1, Bacillus sphaericus, Bacillus subtilis, Bacteroides fragilis, Bifidobacterium bifidum, Bifidobacterium longum NCC2705, Bombyx mori, Bos taurus, Bradyrhizobium japonicum, Bradyrhizobium japonicum USDA110, Brassica juncea, Brassica napsus, Burkholderia ambifaria AMMD, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia phymatum, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderiales bacterium Joshi_001, butyrate-producing bacterium L2-50, Caenorhabditis elegans, Campylobacter curvus 525.92, Campylobacter jejuni, Candida albicans, Candida boidinii, Candida methylica, Candida parapsilosis, Candida tropicalis, Candida tropicalis MYA-3404, Candida tropicalis MYA-3404, Candida tropicalis" Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenoformans Z-2901, Carthamus tinctorius, Caulobacter sp. AP07, Chlamydomonas reinhardtii, Chlorobium limicola, Chlorobium phaeobacteroides DSM266, Chlorobium tepidum, Chloroflexus aurantiacus, Cinnamonum camphorum, Citrobacter koseri ATCC BAA-895, Citrus junos, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium aminobutyricum, Clostridium beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium carboxidivorans P7, Clostridium cellulolyticum H10, Clostridium cellulovorans 743B, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahlii DSM 13528, Clostridium pasteurianum, Clostridium pasteurzanum DSM 525, Clostridium perfringens. Clostridium perfringens ATCC 13124, Clostridium perfringens str. 13, Clostridium phytofermentans ISDg, Clostridium saccharoperbutylacetonicum, Clostridium symbiosum, Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp., Corynebacterium sp. U-96, Corynebacterium ulcerans, Corynebacterium variabile, Cryptosporidium parvum Iowa II, Cuphea hookenana, Cuphea palustris, Cuprzavidus necator, Cuprzavidus necator N-1, Cupriavidus taiwanensis, Cyunobium PCC7001, Cyanothece sp. PCC 7425, Danio rerio, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacterium metallireducens DSM 15288, Desulfococcus oleovorans Hxd3, Desulfotomaculum reducerrs MI-1, Desulfovibrio africanus, Desulfovibrio africanus str. Walvis Bay, Desulfovibrio alaskensis, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Desulfovibriofructosovorans JJ, Desulfovibrio vulgaris str. 'Miyazaki F', Desulfovibrio vulgaris str. Hilderrborough, Dictyostelium discoideum AX4, E. coli, Erythrobacter sp. NAP1, Escherichia coli DH1, Escherichia coli K-12, Escherichia coli K-12 MG1655, Escherichia coli K-12 MG1655 niger CBS 513.88, Escherichia coli LW1655F+, Escherichia coli MG1655, Escherichia coli str. K-12 substr. MG1655, Euglena gracilis, Flavobacterium frigoris, Fusobacterium nucleatum, Geobacillus sp. GHH01, Geobacillus sp. M10EXG, Geobacillus sp. Y4.1MC1, Geobacillus themodenitrificans NG80-2, Geobacillus thermodenitrificans, Geobacter bemidjiensis Bern, Geobacter metallireducens GS-15, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Haemophilus influenza, Haloarcula marismortui, Haloarcula mαrismortui ATCC 43049, Halomonas sp. HTNK1, Helianthus annuus, Helicobacter pylori, Helicobacter pylori 26695, Homo sapiens, human gut metagenome, Hydrogenobacter thermophilus, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Kineococcus radiotolerans, Klebsiella pneumonia, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Kluyveromyces lactis, Kluyveromyces lactis NRRL Y-1140, Lactobacillus acidophilus, Lactobacillus brevis ATCC 367, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis subsp. lactis, Leifsonia sp. S749, Leuconostoc mesenteroides, Listeria monocytogenes, Lyngbya sp. PCC 8106, Lysinibacillusfusiformis, Lysinibacillus sphaericus, Mannheimia succiniciproducens, marine gamma proteobacterium HTCC2080, Marinobacter aquaeolei, Megathyrsus maximus, Mesorhizobium loti, Mesorhizobium loti MAFF303099, Metallosphaera sedula, Metallosphaera sedula, Metarhizium acridum CQMa 102, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanosarcina mazei Tuc01, Methanosarcina thermophila, Methanothermobacter thermautotrophicus, Methylobacillusflagellates, Methylobacillusflagellatus KT, Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens AM1, Methylococcus capsulatis, Methylomicrobium album BG8, Methylomonas aminofaciens, Methylovorus glucosetrophus SIP 3-4, Methylovorus sp. MP688, Moorella thermoacetica, Moorella thermoacetica ATCC 39073, Mus musculus, Mycobacter sp. strain JC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri, Mycobacterium kansasii ATCC 12478, Mycobacterium mannum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium smegmatis str. MC2 155, Mycobacterium sp. strain JLS, Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Neurospora crassa OR74A, Nicotiana tabacum, Nitrosopumilus salaria BD31, Nitrososphaera gargensis Ga9.2, Nocardia brasiliensis, Nocardia farcinica IFM 10152, Nocardia iowensis, Nocardia iowensis (sp. NRRL 5646), Nodularia spumigerra CCY9414, Nostoc azollae, Nostoc sp. PCC 7120, Ogataea parapolymorpha DL-1 (Hansenulapolymorpha DL-1), Oxalobacter formigenes, Paenibacilluspeoriae KCTC 3763, Paracoccus denitrificans, Pelobacter carbinolicus DSM2380, Penicillium chrysogenum, Perkinsus marinus ATCC 50983, Photobacterium leiognathi PL741, Photobacterium phosphoreum, Photobacterium profundum 3TCK, Phtomonas sp., Pichia pastoris, Pichia pastoris GS115, Picrophilus torridus DSM9790, Plasmodium falciparum, Porphyromonas gingivalis, Porphyromonas gingivalis W83, Prochlorococcus marinus MIT 9312, Propionibacterium acnes, Propionibacterium fredenreichii sp. shermanii, Propionibacterium freudenreichii, Propionibacterium freundenreichii subsp. Shermanii, Propionigenium modestum, Pseudomonas aeruginosa, Pseudomonas aeruginosa PA01, Pseudomonas fluorescens, Pseudomonas fluorescens Pf0-1, Pseudomonas knackmussii, Pseudomonas knackmussii (Bl3), Pseudomonas mendocina, Pseudomonas putida, Pseudomonas putida GB-1, Pseudomonas putida GB-1Trypanosoma brucei, Pseudomonas sp, Pseudomonas sp. CF600, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas syringae pv. syringae B728a, Pyrobaculum aerophilum str. IM2, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha H16, Ralstonia metallidurans, Rattus norvegicus, Rhizobium leguminosarum, Rhizopus oryzae, Rhodobacter capsulatus, Rhodobacter sphαeroides, Rhodobacter sphaeroides ATCC 17025, Rhodococcus erythropolis SK121, Rhodococcus opacus B4, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Roseiflexus castenholzii, Saccahromyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enteric, Salmonella enterica, Salmonella enterica LT2, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Salmonella enterica Typhimurium, Salmonella typhimurium, Salmonella typhimurium LT2, Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386, Shewanella oneidensis MR-1, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Simmondsia chinensis, Sinorhizobium meliloti 1021, Solanum lycopersicum, Sordaria macrospora, Staphylococcus aureus, Staphylococcus aureus MW2, Streptococcus mutans, Streptococcus mutans UA159, Streptococcus pneumoniae, Streptococcus sanguinis, Streptomyces anulatus, Streptomyces avermitilis MA-4680, Streptomyces avermitillis, Streptomyces cinnamonensis, Streptomyces coelicolor, Streptomyces glaucescens, Streptomyces griseus subsp. griseus NBRC 13350, Streptomyces luridus, Streptomyces sp CL190, Streptomyces sp CL190, Streptomyces sp. KO-3988, Streptomyces viridochromogenes, Streptomyces wedmorensis, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus solfataricus P-2, Sulfolobus tokodaii, Sulfurihydrogenibium subterraneum, Sulfurimonas denitrificans, Sus scrofa, Synechococcus elongatus PCC 6301, Synechococcus elongatus PCC7942, Synechococcus sp. PCC 7002, Synechocystis str. PCC 6803, Syntrophobacter fumαroxidans, Syntrophus aciditrophicus, Thauera aromatic, Thermoanaerobacter ethanolicus JW 200, Thermoanaerobacter pseudethanolicus ATCC 33223, Thermoanaerobacter sp. X514, Thermoanaerobacter tengcongensis MB4, Thermoanaerobobacter brockii, Thermococcus kodakaraensis, Thermococcus litoralis, Thermomyces lanuginosus, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritime, Thiocapsa roseopersicina, Treponema denticola, Trichomonas vaginalis G3, Triticum aestivum, Trypanosoma brucei, Trypanosoma cruzi strain CL Brener, Tsukamurella paurometabola DSM 20162, Umbellularia californica, uncultured organism, Veillonella parvula, Vibrio hαrveyi ATCC BAA-1116, Xanthobacter autotrophicus Py2, Xenopus tropicalis, Yarrowia lipolytica, Yersinia frederiksenii, Zea mays, Zoogloea ramigera, Zymomonas mobilis, Zymomonas mobilis subsp. mobilis ZM4, Clostridium beijerinickii, Deinococcus radiodurans R1,Aquifex aeolicus VF5, Methanocaldococcusjanaschii, Yersinia pestis, Bifidobacterium animalis lactis, Bifidobacterium dentium ATCC 27678, Bifidobacterium pseudolongum subsp. globosum, Bifidobacterium breve, Lactobacillus paraplantarum, Corynebacterium glutamicum ATCC 13032, 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 fatty alcohol, fatty aldehyde, fatty acid or isopropanol 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 fatty alcohol, fatty aldehyde, or fatty acid 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.

[0117] In some instances, such as when an alternative fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathway exists in an unrelated species, fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid. A nucleic acid molecule encoding a fatty alcohol, fatty aldehyde, or fatty acid pathway enzyme 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.

[0118] 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, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X 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, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in 1X SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene 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).

[0119] A nucleic acid molecule encoding a fatty alcohol, fatty aldehyde, fatty acid or isopropanol 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 fatty alcohol, fatty aldehyde, or fatty acid 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.

[0120] 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.

[0121] Methods for constructing and testing the expression levels of a non-naturally occurring fatty alcohol, fatty aldehyde, or fatty acid-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).

[0122] Exogenous nucleic acid sequences involved in a pathway for production of fatty alcohol, fatty aldehyde, or fatty acid 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 Nterminal 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 (Hoffmeister et al., J. Biol. Chem. 280:43294338 (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.

[0123] An expression vector or vectors can be constructed to include one or more fatty alcohol, fatty aldehyde, or fatty acid 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.

[0124] In some embodiments, the invention provides a method for producing a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and - - - - - - represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, comprising culturing a non-naturally occurring microbial organism of the invention under conditions and for a sufficient period of time to produce the compound of Formula (I).

[0125] In some aspects of the invention, the microbial organism used in a method of the invention includes a non-naturally occurring having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway as depicted in Figures 1 and 10, and a MI-FAE cycle or a MD-FAE cycle in combination with a termination pathway as depicted in Figures 2, 7 and 8, wherein said formaldehyde fixation pathway comprises: (1) 1B and 1C; (2) ID; or (3) 1D and 1Z, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein said formate assimilation pathway comprises a pathway selected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (11) 1H, 1I, 1J, 1O, and 1P, 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 formyltetrahydrofolate synthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a methylenetetrahydrofolate dehydrogenase, 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 1O is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase, wherein said methanol metabolic pathway comprises a pathway selected from: (12) 10J; (13) 10A, (14) 10A and 10B; (15) 10A, 10B and 10C; (16) 10J, 10K and 10C; (17) 10J, 10M, and 10N; (18) 10J and 10L; (19) 10J, 10L and 10G; (20) 10J, 10L, and 101; (21) 10A, 10B, 10C, 10D, and 10E; (22) 10A, 10B, 10C, 10D, and 10F; (23) 10J, 10K, 10C, 10D, and 10E; (24) 10J, 10K, 10C, 10D, and 10F; (25) 10J, 10M, 10N, and 10O; (26) 10A, 10B, 10C, 10D, 10E, and 10G; (27) 10A, 10B, 10C, 10D, 10F, and 10G; (28) 10J, 10K, 10C, 10D, 10E, and 10G; (29) 10J, 10K, 10C, 10D, 10F, and 10G; (30) 10J, 10M, 10N, 10O, and 10G; (31) 10A, 10B, 10C, 10D, 10E, and 10I; (32) 10A, 10B, 10C, 10D, 10F, and 10I; (33) 10J, 10K, 10C, 10D, 10E, and 10I; (34) 10J, 10K, 10C, 10D, 10F, and 10I; and (35) 10J, 10M, 10N, 10O, and 10I, wherein 10A is a methanol methyltransferase, wherein 10B is a methylenetetrahydrofolate reductase, wherein 10C is a methylenetetrahydrofolate dehydrogenase, wherein 10D is a methenyltetrahydrofolate cyclohydrolase, wherein 10E is a formyltetrahydrofolate deformylase, wherein 10F is a formyltetrahydrofolate synthetase, wherein 10G is a formate hydrogen lyase, wherein 10I is a formate dehydrogenase, wherein 10J is a methanol dehydrogenase, wherein 10K is a formaldehyde activating enzyme or spontaneous, wherein 10L is a formaldehyde dehydrogenase, wherein 10M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 10N is a glutathione-dependent formaldehyde dehydrogenase, wherein 10O is a S-formylglutathione hydrolase, wherein the MI-FAE cycle includes one or more thiolase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the MD-FAE cycle includes one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein the termination pathway includes a pathway selected from: (36) 2H; (37) 2K and 2L; (38) 2E and 2N; (39) 2K, 2J, and 2N; (40) 2E; (41) 2K and 2J; (42) 2H and 2N; (43) 2K, 2L, and 2N; (44) 2E and 2F; (45) 2K, 2J, and 2F; (46) 2H, 2N, and 2F; (47) 2K, 2L, 2N, and 2F; (48) 2G; and (49) 2P, wherein 2E is an acyl-CoA reductase (aldehyde forming), wherein 2F is an alcohol dehydrogenase, wherein 2G is an acyl-CoA reductase (alcohol forming), wherein 2H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein 2J is an acyl-ACP reductase, wherein 2K is an acyl-CoA:ACP acyltransferase, wherein 2L is a thioesterase, wherein 2N is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase, wherein 2P is an acyl-ACP reductase (alcohol forming) wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the MI-FAE cycle, MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the MI-FAE cycle, the MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the MI-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

[0126] In some aspects of the invention, the microbial organism used in a method of the invention includes a non-naturally occurring having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway as depicted in Figures 1 and 10, and a FAACPE cycle in combination with a termination pathway as depicted in Figure 12, wherein said formaldehyde fixation pathway comprises: (1) 1B and 1C; (2) 1D; (3) 1D and 1Z, wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein said formate assimilation pathway comprises a pathway selected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase, 1F is a formate ligase, a formate transferase, or a formate synthetase, wherein 1G is a fonnyl-CoA reductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a methylenetetrahydrofolate dehydrogenase, 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 1O is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase, wherein said methanol metabolic pathway comprises a pathway selected from: (12) 10J; (13) 10A, (14) 10A and 10B; (15) 10A, 10B and 10C; (16) 10J, 10K and 10C; (17) 10J, 10M, and 10N; (18) 10J and 10L; (19) 10J, 10L and 10G; (20) 10J, 10L, and 10I; (21) 10A, 10B, 10C, 10D, and 10E; (22) 10A, 10B, 10C, 10D, and 10F; (23) 10J, 10K, 10C, 10D, and 10E; (24) 10J, 10K, 10C, 10D, and 10F; (25) 10J, 10M, 10N, and 10O; (26) 10A, 10B, 10C, 10D, 10E, and 10G; (27) 10A, 10B, 10C, 10D, 10F, and 10G; (28) 10J, 10K, 10C, 10D, 10E, and 10G; (29) 10J, 10K, 10C, 10D, 10F, and 10G; (30) 10J, 10M, 10N, 10O, and 10G; (31) 10A, 10B, 10C, 10D, 10E, and 10I; (32) 10A, 10B, 10C, 10D, 10F, and 10I; (33) 10J, 10K, 10C, 10D, 10E, and 10I; (34) 10J, 10K, 10C, 10D, 10F, and 10I; and (35) 10J, 10M, 10N, 10O, and 10I, wherein 10A is a methanol methyltransferase, wherein 10B is a methylenetetrahydrofolate reductase, wherein 10C is a methylenetetrahydrofolate dehydrogenase, wherein 10D is a methenyltetrahydrofolate cyclohydrolase, wherein 10E is a formyltetrahydrofolate deformylase, wherein 10F is a formyltetrahydrofolate synthetase, wherein 10G is a formate hydrogen lyase, wherein 10I is a formate dehydrogenase, wherein 10J is a methanol dehydrogenase, wherein 10K is a formaldehyde activating enzyme or spontaneous, wherein 10L is a formaldehyde dehydrogenase, wherein 10M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 10N is a glutathione-dependent formaldehyde dehydrogenase, wherein 10O is a S-formylglutathione hydrolase, wherein said FAACPE cycle comprises one or more β-ketoacyl-ACP synthase, one or more β-ketoacyl-ACP reductase, one or more β-hydroxyacyl-ACP reductase, and one or more enoyl ACP-reductase, wherein said termination pathway comprises a pathway selected from: (36) 12I; (37) 12J; (38) 12I, 12K, and 12L; (39) 12I and 120; (40) 12J and 12M; (41) 121, 12K, 12L, and 12M; (42) 121, 120, and 12M; (43) 121, 12K and 12N, and (44) 12P, wherein 12I is a thioesterase, wherein 12J is a fatty acyl-ACP reductase, wherein 12K is an acyl-CoA synthase, wherein 12L is an acyl-CoA reductase, wherein 12M is a fatty aldehyde reductase, wherein 12N is a fatty alcohol forming acyl-CoA reductase (FAR), wherein 120 is a carboxylic acid reductase (CAR), wherein 12P is an acyl-ACP reductase (alcohol forming),wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the FAACPE cycle or the termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I): wherein R 1 is C 1-24 linear alkyl; R 2 is CH 2 OH, or CHO; R 3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four, wherein the substrate of each of said enzymes of the FAACPE cycle and the termination pathway are independently selected from a compound of Formula (II) or malonyl-ACP: wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (=O); R 4 is S-CoA, ACP, OH or H; andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R 3 is attached is four; wherein said one or more enzymes of the FAACPE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

[0127] In some embodiments, the microbial organism has a formaldehyde fixation pathway, a formate assimilation pathway, a methanol metabolic pathway and a MI-FAE cycle in combination with a termination pathway. In some embodiments, the microbial organism has a formaldehyde fixation pathway, a methanol metabolic pathway and a MD-FAE cycle in combination with a termination pathway.

[0128] In some embodiments, the invention provides a method for producing a compound of Formula (I) wherein R 1 is C 1-17 linear alkyl. In another aspect of the invention, the R 1 of the compound of Formula (I) is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl..

[0129] In some aspects of the invention, the microbial organism used in a method of the invention includes two, three, or four exogenous nucleic acids each encoding an enzyme of the MI-FAE cycle, the MD-FAE cycle, or the FAACPE cycle. In some aspects of the invention, the microbial organism includes two, three, or four exogenous nucleic acids each encoding an enzyme of the termination pathway. In some aspects of the invention, the microbial organism includes one, two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a formaldehyde fixation pathway enzyme, a formate assimilation pathway enzyme, or a methanol metabolic pathway enzyme. In some aspects of the invention, the microbial organism includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(49) for a microbial organism having a MI-FAE cycle or a MD-FAE cycle in combination with a termination pathway as depicted in Figures 1, 2, 7, 8 and 10. In some aspects of the invention, the microbial organism includes exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(44) for a microbial organism having a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway as depicted in Figures 1, 10 and 12.

[0130] In some embodiments, the invention provides a method for producing a fatty alcohol selected from the Formulas (III)-(VI): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0131] In some embodiments, the invention provides a method for producing a fatty aldehyde selected from the Formulas (VII)-(X): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0132] In some embodiments, the invention provides a method for producing a fatty acid selected from the Formulas (XI)-(XIV): wherein R 1 is C 1-24 linear alkyl, or alternatively R 1 is C 1-17 linear alkyl, or alternatively R 1 is C 9-13 linear alkyl. In some aspects of the invention, R 1 is C 1 linear alkyl, C 2 linear alkyl, C 3 linear alkyl, C 4 linear alkyl, C 5 linear alkyl, C 6 linear alkyl, C 7 linear alkyl, C 8 linear alkyl, C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or C 13 linear alkyl, C 14 linear alkyl, C 15 linear alkyl, C 16 linear alkyl, C 17 linear alkyl, C 18 linear alkyl, C 19 linear alkyl, C 20 linear alkyl, C 21 linear alkyl, C 22 linear alkyl, C 23 linear alkyl, or C 24 linear alkyl.

[0133] In some aspects of the invention, the microbial organism used in a method of the invention having a formate assimilation pathway further includes wherein the formate assimilation pathway comprises: (1) 1Q; (2) 1R, and 1S; (3) 1Y and 1Q; or (4) 1Y, 1R, and 1S, 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 formate dehydrogenase wherein 1Y is a glyceraldehydes-3-phosphate dehydrogenase or an enzyme of lower glycolysis. In addition to a glyceraldehyde-3-phosphate dehydrogenase, lower glycolysis includes a phosphoglycerate kinase, a phosphoglyceromutase, an enolase, a pyruvate kinase or a PTS-dependant substrate import. Accordingly, in some embodiments, the formate assimilation pathway comprising 1Y includes an enzyme selected from a phosphoglycerate kinase, a phosphoglyceromutase, an enolase, a pyruvate kinase and a PTS-dependant substrate import.

[0134] In some aspects of the invention, the microbial organism used in a method of the invention includes a methanol oxidation pathway. Such a pathway can include at least one exogenous nucleic acid encoding a methanol oxidation pathway enzyme expressed in a sufficient amount to produce formaldehyde in the presence of methanol. An exemplary methanol oxidation pathway enzyme is a methanol dehydrogenase. Accordingly, in some aspects, the microbial organism used in the method of the invention includes a non-naturally occurring having at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to produce formaldehyde in the presence of methanol.

[0135] In some aspects of the invention, the microbial organism used in a method of the invention includes a non-naturally occurring having at least one exogenous nucleic acid that is a heterologous nucleic acid. Accordingly, in some embodiments, the at least one exogenous nucleic acid encoding a formaldehyde fixation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a formate assimilation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a MI-FAE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a MD-FAE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a FAACPE cycle enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a termination pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding an acetoacetyl-ACP pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a 3-oxovalery-ACP pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding an isopropanol pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a methanol oxidation pathway enzyme described herein is a heterologous nucleic acid. In some embodiments, the at least one exogenous nucleic acid encoding a hydrogenase or a carbon monoxide dehydrogenase is a heterologous nucleic acid.

[0136] In other aspects, the invention further provides methods for producing elevated or enhanced yields of biosynthetic products such as a fatty alcohol, fatty aldehyde, and / or fatty acid.

[0137] The methods for producing enhanced yields of a fatty alcohol, fatty aldehyde, and / or fatty acid described herein include using a non-naturally occurring microbial organisms having one or more of the various pathway configurations employing a methanol dehydrogenase for methanol oxidation, a formaldehyde fixation pathway, and / or a phosphoketolase 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, fatty alcohol, fatty aldehyde, and / or fatty acid 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 formaldehyde fixation pathways and / or increase the production of reducing equivalents.

[0138] 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 alcohol, fatty aldehyde, fatty acid and / or isopropanol pathway to enhance the yield and / or production of a product such as any of the fatty alcohol, fatty aldehyde, and / or fatty acids described herein.

[0139] 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 methanol dehydrogenase (MeDH), one or more formaldehyde assimilation pathways and / or one or more phosphoketolases.

[0140] 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 Figure 1. Such pathways include methanol oxidation via expression of a cytosolic NADH dependent methanol dehydrogenase, formaldehyde fixation via expression of cytosolic DHA synthase, both methanol oxidation via expression of an cytosolic NADH dependent methanol dehydrogenase 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 formaldehyde fixation pathways. 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).

[0141] 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 methanol dehydrogenase, 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 formaldehyde fixation pathways. 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).

[0142] 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 methanol dehydrogenase, 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 formaldehyde fixation pathways. 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).

[0143] In some embodiments, the method for producing a fatty alcohol, fatty aldehyde, or fatty acid 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 methanol dehydrogenase, 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 methanol dehydrogenase. 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 methanol dehydrogenase, or alternatively DHA kinase, methanol oxidase, and PQQ-dependent methanol dehydrogenase, 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 methanol dehydrogenase and DHA synthase.

[0144] Suitable purification and / or assays to test for the production of fatty alcohol, fatty aldehyde, or fatty acid 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.

[0145] The fatty alcohol, fatty aldehyde, or fatty acid 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.

[0146] 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 fatty alcohol, fatty aldehyde, or fatty acid producers can be cultured for the biosynthetic production of fatty alcohol, fatty aldehyde, or fatty acid. Accordingly, in some embodiments, the invention provides culture medium having the fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate described herein. In some aspects, the culture medium can also be separated from the non-naturally occurring microbial organisms of the invention that produced the fatty alcohol, fatty aldehyde, or fatty acid 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.

[0147] For the production of fatty alcohol, fatty aldehyde, or fatty acid, 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 August 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 fatty alcohol, fatty aldehyde, or fatty acid yields.

[0148] 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.

[0149] 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, 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 certain embodiments, methanol is used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In a specific embodiment, the methanol is the only (sole) carbon source. 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 carbohydrate 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 yet other embodiments, the carbon source is a sugar-containing biomass and crude glycerol without treatment. In some embodiments, the carbon source is a sugar-containing biomass, methanol and a carbohydrate. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods provided herein 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 succinate and other pathway intermediates.

[0150] 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.

[0151] 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.

[0152] In one embodiment, the carbon source is methanol or formate. In certain embodiments, methanol is used as a carbon source in a formaldehyde fixation pathway provided herein. In one embodiment, the carbon source is methanol or formate. In other embodiments, formate is used as a carbon source in a formaldehyde fixation pathway provided herein. In specific embodiments, methanol is used as a carbon source in a methanol oxidation pathway provided herein, either alone or in combination with the fatty alcohol, fatty aldehyde, or fatty acid pathways provided herein. In one embodiment, the carbon source is methanol. In another embodiment, the carbon source is formate.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] In addition to renewable feedstocks such as those exemplified above, the fatty alcohol, fatty aldehyde, or fatty acid producing 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 fatty alcohol, fatty aldehyde, or fatty acid producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

[0158] 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 H 2 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 H 2 and CO, syngas can also include CO 2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO 2 .

[0159] 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, fatty alcohol, fatty aldehyde, fatty acid and any of the intermediate metabolites in the fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and / or secretes fatty alcohol, fatty aldehyde, or fatty acid when grown on a carbohydrate or other carbon source and produces and / or secretes any of the intermediate metabolites shown in the fatty alcohol, fatty aldehyde, or fatty acid pathway when grown on a carbohydrate or other carbon source. The fatty alcohol, fatty aldehyde, or fatty acid producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, a 3-ketoacyl-CoA, a 3-hydroxyacyl-CoA, an enoyl-CoA, an acyl-CoA, an acyl-ACP, acetate, acetaldehyde, acetyl-phosphate, oxaloacetate, matate, malonate semialdehyde, malonate, malonyl-CoA, acetyl-CoA, or citrate.

[0160] 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 fatty alcohol, fatty aldehyde, or fatty acid pathway enzyme or protein in sufficient amounts to produce fatty alcohol, fatty aldehyde, or fatty acid. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce fatty alcohol, fatty aldehyde, or fatty acid. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of fatty alcohol, fatty aldehyde, or fatty acid resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration of fatty alcohol, fatty aldehyde, or fatty acid 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.

[0161] 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 August 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 fatty alcohol, fatty aldehyde, or fatty acid producers can synthesize fatty alcohol, fatty aldehyde, or fatty acid 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, fatty alcohol, fatty aldehyde, or fatty acid producing microbial organisms can produce fatty alcohol, fatty aldehyde, or fatty acid intracellularly and / or secrete the product into the culture medium.

[0162] 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, N 2 / CO 2 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 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.

[0163] 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.

[0164] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of fatty alcohol, fatty aldehyde, or fatty acid 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-camitine 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 10mM, no more than about 50mM, no more than about 100mM or no more than about 500mM.

[0165] 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 fatty alcohol, fatty aldehyde, or fatty acid or any fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid or fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate, or for side products generated in reactions diverging away from a fatty alcohol, fatty aldehyde, or fatty acid pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

[0166] 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.

[0167] 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 CO 2 , which can possess a larger amount of carbon-14 than its petroleum-derived counterpart.

[0168] The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 10 12< 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 ( 14< N). 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".

[0169] 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.

[0170] 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 April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

[0171] The biobased content of a compound is estimated by the ratio of carbon-14 ( 14< C) to carbon-12 ( 12< C). Specifically, the Fraction Modem (Fm) is computed from the expression: Fm = (S-B) / (M-B), where B, S and M represent the 14< C / 12< C ratios of the blank, the sample and the modem reference, respectively. Fraction Modem is a measurement of the deviation of the 14< C / 12< C ratio of a sample from "Modem." Modem 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 δ 13< C VPDS =-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 ofNBS Oxalic Acid I (SRM 4990b) normalized to δ 13< C VPDB =-19 permil. This is equivalent to an absolute (AD 1950) 14< C / 12< C ratio of 1.176 ± 0.010 x 10 -12< (Karlen et al., Arkiv Geofysik, 4:465471 (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 C 12< over C 13< over C 14< , and these corrections are reflected as a Fm corrected for δ 13< .

[0172] 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 mil. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modem 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 modem carbon source. As described herein, such a "modem" source includes biobased sources.

[0173] As described in ASTM D6866, the percent modem 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.

[0174] 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.

[0175] 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 (PPT) 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).

[0176] Accordingly, in some embodiments, the present invention provides fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid 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 CO 2 . In some embodiments, the present invention provides fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid 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.

[0177] Further, the present invention relates to the biologically produced fatty alcohol, fatty aldehyde, or fatty acid or fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the fatty alcohol, fatty aldehyde, or fatty acid or a fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO 2 that occurs in the environment. For example, in some aspects the invention provides bioderived fatty alcohol, fatty aldehyde, or fatty acid or a bioderived fatty alcohol, fatty aldehyde, or fatty acid intermediate having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO 2 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 CO 2 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived fatty alcohol, fatty aldehyde, or fatty acid or a bioderived fatty alcohol, fatty aldehyde, or fatty acid 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 fatty alcohol, fatty aldehyde, or fatty acid, 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 biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials or acrylates having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO 2 that occurs in the environment, wherein the biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials or acrylates are generated directly from or in combination with bioderived fatty alcohol, fatty aldehyde, or fatty acid or a bioderived fatty alcohol, fatty aldehyde, or fatty acid pathway intermediate as disclosed herein.

[0178] Fatty alcohol, fatty aldehyde or fatty acid is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials and acrylates. Accordingly, in some embodiments, the invention provides biobased biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials and acrylates comprising one or more bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

[0179] 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.

[0180] In some embodiments, the invention provides a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate comprising bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, wherein the bioderived fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate includes all or part of the fatty alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate used in the production of a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. For example, the final biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate can contain the bioderived fatty alcohol, fatty aldehyde or fatty acid, fatty alcohol, fatty aldehyde or fatty acid pathway intermediate, or a portion thereof that is the result of the manufacturing of the biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. Such manufacturing can include chemically reacting the bioderived fatty alcohol, fatty aldehyde or fatty acid, or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) with itself or another compound in a reaction that produces the final biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. Thus, in some aspects, the invention provides a biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fiagrance, flavor material or acrylate 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 fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway intermediate as disclosed herein. In some aspects, when the product is a biobased polymer that includes or is obtained from a bioderived fatty alcohol, fatty aldehyde or fatty acid, or or fatty alcohol, fatty aldehyde or fatty acid pathway intermediate described herein, the biobased polymer can be molded using methods well known in the art. Accordingly, in some embodiments, provided herein is a molded product comprising the biobased polymer described herein.

[0181] In some embodiments, the invention provides a solvent, a paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or a polymer comprising bioderived isopropanol or bioderived isopropanol pathway intermediate, wherein the bioderived isopropanol or bioderived isopropanol pathway intermediate includes all or part of the isopropanol or isopropanol pathway intermediate used in the production of a solvent, a paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or a polymer. For example, the final solvent, paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or polymer can contain the bioderived isopropanol, isopropanol pathway intermediate, or a portion thereof that is the result of the manufacturing of a solvent, paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or a polymer. Such manufacturing can include chemically reacting the bioderived isopropanol or bioderived isopropanol pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final solvent, paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or polymer. Thus, in some aspects, the invention provides a biobased solvent, paint, lacquer, thinner, ink, adhesive, cleaner, disinfectant, cosmetic, toiletry, de-icer, pharmaceutical, motor oil, isopropylamine, isopropylether, isopropyl ester, propylene or polymer 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 isopropanol or bioderived isopropanol pathway intermediate as disclosed herein.

[0182] 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.

[0183] As described herein, one exemplary growth condition for achieving biosynthesis of fatty alcohol, fatty aldehyde, or fatty acid 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 N 2 / CO 2 mixture or other suitable non-oxygen gas or gases.

[0184] The culture conditions described herein can be scaled up and grown continuously for manufacturing of fatty alcohol, fatty aldehyde, or fatty acid. 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 fatty alcohol, fatty aldehyde, or fatty acid. Generally, and as with non-continuous culture procedures, the continuous and / or near-continuous production of fatty alcohol, fatty aldehyde, or fatty acid will include culturing a non-naturally occurring fatty alcohol, fatty aldehyde, or fatty acid 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.

[0185] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of fatty alcohol, fatty aldehyde, or fatty acid 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.

[0186] In addition to the above fermentation procedures using the fatty alcohol, fatty aldehyde, or fatty acid producers of the invention for continuous production of substantial quantities of fatty alcohol, fatty aldehyde, or fatty acid, the fatty alcohol, fatty aldehyde, or fatty acid 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 and / or enzymatic conversion to convert the product to other compounds, if desired.

[0187] 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. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of fatty alcohol, fatty aldehyde, or fatty acid.

[0188] In addition to active and selective enzymes producing fatty alcohols, fatty aldehydes, or fatty acids at high yield, titer and productivity, a robust host organism that can efficiently direct carbon and reducing equivalents to fatty alcohol, fatty aldehyde and fatty acid biosynthesis can be beneficial. Host modifications described herein are particularly useful in combination with selective enzymes described herein that favor formation of the desired fatty alcohol, fatty aldehyde,, or fatty acid product. Several host modifications described herein entail introducing heterologous enzyme activities into the host organism. Other modifications involve overexpressing or elevating enzyme activity relative to wild type levels. Yet other modifications include disrupting endogenous genes or attenuating endogenous enzyme activities.

[0189] In one embodiment of the invention, the microbial organisms efficiently directs carbon and energy sources into production of acetyl-CoA, which is used as both a primer and extension unit in the MI-FAE cycle. In one embodiment of the invention, the microbial organisms efficiently directs carbon and energy sources into production of malonyl-CoA, which is used as both a primer and extension unit in the MD-FAE cycle. In unmodified microbial organism, fatty alcohol, fatty aldehyde and fatty acid production in the cytosol relies on the native cell machinery to provide the necessary precursors. Thus, high concentrations of cytosolic acetyl-CoA and / or malonyl-CoA are desirable for facilitating deployment of a cytosolic fatty alcohol, fatty aldehyde or fatty acid production pathway that originates from acetyl-CoA or malonyl-CoA. Metabolic engineering strategies for increasing cytosolic acetyl-CoA and malonyl-CoA are disclosed herein.

[0190] Since many eukaryotic organisms synthesize most of their acetyl-CoA in the mitochondria during growth on glucose, increasing the availability of acetyl-CoA in the cytosol can be obtained by introduction of a cytosolic acetyl-CoA biosynthesis pathway. Accordingly, acetyl-CoA biosynthesis pathways are described herein. In one embodiment, utilizing the pathways shown in Figure 3, acetyl-CoA can be synthesized in the cytosol from a pyruvate or threonine precursor. In other embodiment, acetyl-CoA can be synthesized in the cytosol from phosphoenolpyruvate (PEP) or pyruvate (Figure 4). In yet another embodiment acetyl-CoA can be synthesized in cellular compartments and transported to the cytosol. For example, one mechanism involves converting mitochondrial acetyl-CoA to a metabolic intermediate such as citrate or citramalate, transporting those intermediates to the cytosol, and then regenerating the acetyl-CoA (see Figures 5 and 6). Exemplary acetyl-CoA pathways and corresponding enzymes are further described in Examples V-VII.

[0191] In another embodiment, increasing cytosolic acetyl-CoA availability for fatty alcohol, fatty aldehyde, or fatty acid biosynthesis is to disrupt or attenuate competing enzymes and pathways that utilize acetyl-CoA or its precursors. Exemplary competing enzyme activities include, but are not limited to, pyruvate decarboxylase, lactate dehydrogenase, short-chain aldehyde and alcohol dehydrogenases, acetate kinase, phosphotransacetylase, glyceraldehyde-3-phosphate dehydrogenases, pyruvate oxidase and acetyl-CoA carboxylase. Exemplary acetyl-CoA consuming pathways whose disruption or attenuation can improve fatty alcohol, fatty aldehyde,, or fatty acid production include the mitochondrial TCA cycle, fatty acid biosynthesis, ethanol production and amino acid biosynthesis. These enzymes and pathways are further described herein.

[0192] Yet another strategy for increasing cytosolic acetyl-CoA production is to increase the pool of CoA available in the cytoplasm. This can be accomplished by overexpression of CoA biosynthetic enzymes in the cytosol. In particular, expression of pantothenate kinase (EC 2.7.1.33) can be used. This enzyme catalyzes the first step and rate-limiting enzyme of CoA biosynthesis. Exemplary pantothenate kinase variants resistant to feedback inhibition by CoA are well known in the art (Rock et al, J Bacterial 185: 3410-5 (2003)) and are described in the below table. Protein Accession # GI number Organism coaAAAC769521790409Escherichia coliCAB1NP_010820.3398366683Saccharomyces cerevisiaeKLLA0C00869gXP_452233.150304555Kluyveromyces lactisYALI0D25476gXP_503275.150551601Yarrowia lipolyticaANI_1_3272024XP_001400486.2317028058Aspergillus niger

[0193] Competing enzymes and pathways that divert acyl-CoA substrates from production of fatty alcohols, fatty aldehydes or fatty acids of the invention can also be attenuated or disrupted. Exemplary enzymes for attenuation include acyltransferases, carnitine shuttle enzymes and negative regulators of MI-FAE cycle, MD-FAE cycle, FAACPE cycle or termination pathway enzymes.

[0194] Disruption or attenuation of acyltransferases that transfer acyl moieties from CoA to other acceptors such as ACP, glycerol, ethanol and others, can increase the availability of acyl-CoA for fatty alcohol, fatty aldehyde or fatty acid production. For example, Acyl-CoA:ACP transacylase (EC 2.3.1.38; 2.3.1.39) enzymes such as fabH (KASIII) of E. coli transfer acyl moieties from CoA to ACP. FabH is active on acetyl-CoA and butyryl-CoA (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-deficientLactococcus 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)). 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, FEES Lett 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 herein) also catalyze this activity. Other exemplary acyltransferases include diacylglycerol acyltransferases such as LRO1 and DGA1 of S. cerevisiae and DGA1 and DGA2 of Yarrowia lipolytica, glycerolipid acyltransferase enzymes such as plsB of E. coli (GenBank: AAC77011.2, GI:87082362; Heath and Rock, J Bacteriol 180:1425-30 (1998)), sterol acyltransferases such as ARE1 and ARE2 of S. cerevisiae, ethanol acyltransferases (EEB1, EHT1), putative acyltransferases (YMR210W) and others. Protein GenBank ID GI Number Organism fabHAAC74175.11787333Escherichia colifadANP_824032.129829398Streptomyces avermitillisfabHAAC63960.13746429Plasmodium falciparumSynthetic constructACX34097.1260178848Plasmodium falciparumfabHCAL98359.1124493385Lactococcus lactisfabDAAC74176.11787334Escherichia colifabDCAB45522.15139348Brassica napsusLRO1NP_014405.16324335Saccharomyces cerevisiaeDGA1NP_014888.16324819Saccharomyces cerevisiaeDGA1CAG79269.149649549Yarrowia lipolyticaDGA2XP_504700.150554583Yarrowia lipolyticaARE1NP_009978.16319896Saccharomyces cerevisiaeARE2NP_014416.16324346Saccharomyces cerevisiaeEEB1NP_015230.16325162Saccharomyces cerevisiaeEHT1NP_009736.3398365307Saccharomyces cerevisiaeYMR210WNP_013937.16323866Saccharomyces cerevisiaeALE1NP_014818.16324749Saccharomyces cerevisiae

[0195] Increasing production of fatty alcohols, fatty aldehydes or fatty acids may necessitate disruption or attenuation of enzymes involved in the trafficking of acetyl-CoA and acyl-CoA molecules from the cytosol to other compartments of the organism such as mitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes. In these compartments, the acyl-CoA intermediate can be degraded or used as building blocks to synthesize fatty acids, cofactors and other byproducts.

[0196] Acetyl-CoA and acyl-CoA molecules localized in the cytosol can be transported into other cellular compartments with the aid of the carrier molecule carnitine via carnitine shuttles (van Roermund et al., EMBO J 14:3480-86 (1995)). Acyl-camitine shuttles between cellular compartments have been characterized in yeasts such as Candida albicans (Strijbis et al, JBiol Chem 285:2433546 (2010)). In these shuttles, the acyl moiety of acyl-CoA is reversibly transferred to carnitine by acylcamitine transferase enzymes. Acetylcamitine can then be transported across the membrane by organelle-specific acylcamitine / camitine translocase enzymes. After translocation, the acyl-CoA is regenerated by acetylcamitine transferase. Enzymes suitable for disruption or attenuation include carnitine acyltransferase enzymes, acylcamitine translocases, acylcamitine carrier proteins and enzymes involved in carnitine biosynthesis.

[0197] Carnitine acetyltransferase (CAT, EC 2.3.1.7) reversibly links acetyl units from acetyl-CoA to the carrier molecule, carnitine. Candida albicans encodes three CAT isozymes: Cat2, Yat1 and Yat2 (Strijbis et al., JBiol Chem 285:2433546 (2010)). Cat2 is expressed in both the mitochondrion and the peroxisomes, whereas Yat1 and Yat2 are cytosolic. The Cat2 transcript contains two start codons that are regulated under different carbon source conditions. The longer transcript contains a mitochondrial targeting sequence whereas the shorter transcript is targeted to peroxisomes. Cat2 of Saccharomyces cerevisiae and AcuJ of Aspergillus nidulans employ similar mechanisms of dual localization (Elgersma et al., EMBO J 14:3472-9 (1995); Hynes et al., Euk Cell 10:547-55 (2011)). The cytosolic CAT of A. nidulans is encoded by facC. Other exemplary CAT enzymes are found in Rattus norvegicus and Homo sapiens (Cordente et al., Biochem 45:613341 (2006)). Exemplary carnitine acyltransferase enzymes (EC 2.3.1.21) are the Cpt1 and Cpt2 gene products of Rattus norvegicus (de Vries et al., Biochem 36:5285-92 (1997)). Protein Accession # GI number Organism Cat2AAN31660.123394954Candida albicansYat1AAN31659.123394952Candida albicansYat2XP_711005.168490355Candida albicansCat2CAA88327.1683665Saccharomyces cerevisiaeYat1AAC09495.1456138Saccharomyces cerevisiaeYat2NP_010941.16320862Saccharomyces cerevisiaeAcuJCBF69795.1259479509Aspergillus nidulansFacCAAC82487.12511761Aspergillus nidulansCratAAH83616.153733439Rattus norvegicusCratP43155.5215274265Homo sapiensCpt1AAB48046.11850590Rattus norvegicusCpt2AAB02339.11374784Rattus norvegicus

[0198] Camitine-acylcamitine translocases can catalyze the bidirectional transport of carnitine and camitine-fatty acid complexes. The Cact gene product provides a mechanism for transporting acyl-camitine substrates across the mitochondrial membrane (Ramsay et al Biochim Biophys Acta 1546:2142 (2001)). A similar protein has been studied in humans (Sekoguchi et al., JBiol Chem 278:38796-38802 (2003)). The Saccharomyces cerevisiae mitochondrial carnitine carrier is Crc1 (van Roermund et al., supra; Palmieri et al., Biochimica et Biophys Acta 1757:1249-62 (2006)). The human carnitine translocase was able to complement a Crc1-deficient strain of S. cerevisiae (van Roermund et al., supra). Two additional carnitine translocases found in Drosophila melanogaster and Caenorhabditis elegans were also able to complement Crc1-deficient yeast (Oey et al.,Mol Genet Metab 85:121-24 (2005)). Four mitochondrial camitine / acetylcamitine carriers were identified in Trypanosoma brucei based on sequence homology to the yeast and human transporters (Colasante et al.,Mol Biochem Parasit 167:104-117 (2009)). The carnitine transporter of Candida albicans was also identified by sequence homology. An additional mitochondrial carnitine transporter is the acuH gene product of Aspergillus nidulans, which is exclusively localized to the mitochondrial membrane (Lucas et al., FEMS Microbiol Lett 201:193-8 (2006)). Protein GenBank ID GI Number Organism CactP97521.12497984Rattus norvegicusCaclNP_001034444.186198310Homo sapiensCa019.2851XP_715782.168480576Candida albicansCrc1NP_014743.16324674Saccharomyces cerevisiaeDif-1CAA88283.1829102Caenorhabditis eleganscoltCAA73099.11944534Drosophila melanogasterTh11.02.2960EAN79492.170833990Trypanosoma bruceiTb11.03.0870EAN79007.170833505Trypanosoma bruceiTb11.01.5040EAN80288.170834786Trypanosoma bruceiTb927.8.5810AAX69329.162175181Trypanosoma bruceiacuHCAB44434.15019305Aspergillus nidulans

[0199] Transport of carnitine and acylcamitine across the peroxisomal membrane has not been well-characterized. Specific peroxisomal acylcamitine carrier proteins in yeasts have not been identified to date. However, mitochonidrial carnitine translocases can also function in the peroxisomal transport of carnitine and acetylcamitine. Experimental evidence suggests that the OCTN3 protein of Mus musculus is a peroxisomal camitine / acylcamitine translocase.

[0200] Yet another possibility is that acyl-CoA or acyl-carnitine are transported across the peroxisomal or mitochondrial membranes by an acyl-CoA transporter such as the Pxa1 and Pxa2 ABC transporter of Saccharomyces cerevisiae or the ALDP ABC transporter of Homo sapiens (van Roermund et al., FASEB J 22:4201-8 (2008)). Pxa1 and Pxa2 (Pat1 and Pat2) form a heterodimeric complex in the peroxisomal membrane and catalyze the ATP-dependent transport of fatty acyl-CoA esters into the peroxisome (Verleur et al., Eur J Biochem 249: 657-61 (1997)). The mutant phenotype of a pxa1 / pxa2 deficient yeast can be rescued by heterologous expression of ALDP, which was shown to transport a range of acyl-CoA substrates (van Roermund et al., FASEB J 22:4201-8 (2008)). Deletion of the Pxa12 transport system, in tandem with deletion of the peroxisomal fatty acyl-CoA synthetase (Faa2) abolished peroxisomal beta-oxidation in S. cerevisiae. Yet another strategy for reducing transport of pathway intermediates or products into the peroxisome is to attenuate or eliminate peroxisomal function, by interfering with systems involved in peroxisomal biogenesis. An exemplary target is Pex10 of Yarrowia lipolytica and homologs. Protein Accession # GI number Organism OCTN3BAA78343.14996131Mus musculusPxa1AAC49009.1619668Saccharomyces cerevisiaePxa2AAB51597.11931633Saccharomyces cerevisiaeFaa2NP_010931.3398364331Saccharomyces cerevisiaeALDPNP_000024.27262393Homo sapiensPex10BAA99413.19049374Yarrowia lipolytica

[0201] Carnitine biosynthetic pathway enzymes are also suitable candidates for disruption or attenuation. In Candida albicans, for example, carnitine is synthesized from trimethyl-L-lysine in four enzymatic steps (Strijbis et al., FASEB J 23:2349-59 (2009)). The carnitine pathway precursor, trimethyllysine (TML), is produced during protein degradation. TML dioxygenase (CaO13.4316) hydroxylates TML to form 3-hydroxy-6-N-trimethyllysine. A pyridoxal-5'-phoshpate dependent aldolase (Ca019.6305) then cleaves HTML into 4-trimethylaminobutyraldehyde. The 4-trimethylaminobutyraldehyde is subsequently oxidized to 4-trimethylaminobutyrate by a dehydrogenase (CaO19.6306). In the final step, 4trimethylaminobutyrate is hydroxylated to form carnitine by the gene product of CaO19.7131. Flux through the carnitine biosynthesis pathway is limited by the availability of the pathway substrate and very low levels of carnitine seem to be sufficient for normal carnitine shuttle activity (Strejbis et al., IUBMB Life 62:357-62 (2010)). Protein Accession # GI number Organism CaO19.4316XP_720623.168470755Candida albicansCaO19.6305XP_711090.168490151Candida albicansCaO19.6306XP_711091.168490153Candida albicansCaO19.7131XP_715182.168481628Candida albicans

[0202] Carbon flux towards production of fatty alcohols, fatty aldehydes or fatty acids can be improved by deleting or attenuating competing pathways. Typical fermentation products of yeast include ethanol, glycerol and CO 2 . The elimination or reduction of these byproducts can be accomplished by approaches described herein. For example, carbon loss due to respiration can be reduced. Other potential byproducts include lactate, acetate, formate, fatty acids and amino acids.

[0203] The conversion of acetyl-CoA into ethanol can be detrimental to the production of fatty alcohols, fatty aldehyes, fatty acids or isopropanol because the conversion process can draw away both carbon and reducing equivalents from the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway or isopropanol pathway. Ethanol can be formed from pyruvate in two enzymatic steps catalyzed by pyruvate decarboxylase and ethanol dehydrogenase. Saccharomyces cerevisiae has three pyruvate decarboxylases (PDC1, PDC5 and PDC6). PDC1 is the major isozyme and is strongly expressed in actively fermenting cells. PDC5 also functions during glycolytic fermentation, but is expressed only in the absence of PDC1 or under thiamine limitating conditions. PDC6 functions during growth on nonfermentable carbon sources. Deleting PDC1 and PDC5 can reduce ethanol production significantly; however these deletions can lead to mutants with increased PDC6 expression. Deletion of all three eliminates ethanol formation completely but also can cause a growth defect because of inability of the cells to form sufficient acetyl-CoA for biomass formation. This, however, can be overcome by evolving cells in the presence of reducing amounts of C2 carbon source (ethanol or acetate) (van Maris et al, AFM 69:2094-9 (2003)). It has also been reported that deletion of the positive regulator PDC2 of pyruvate decarboxylases PDC1 and PDC5, reduced ethanol formation to ~10% ofthat made by wild-type (Hohmann et al, Mol Gen Genet 241:657-66 (1993)). Protein sequences and identifiers of PDC enzymes are listed in Example V.

[0204] Alternatively, alcohol dehydrogenases that convert acetaldehyde into ethanol and / or other short chain alcohol dehydrogenases can be disrupted or attenuated to provide carbon and reducing equivalents for the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway or isopropanol pathway. To date, seven alcohol dehydrogenases, ADHI-ADHVII, have been reported in S. cerevisiae (de Smidt et al, FEMS Yeast Res 8:967-78 (2008)). ADH1 (GI:1419926) is the key enzyme responsible for reducing acetaldehyde to ethanol in the cytosol under anaerobic conditions. It has been reported that a yeast strain deficient in ADH1 cannot grow anaerobically because an active respiratory chain is the only alternative path to regenerate NADH and lead to a net gain of ATP (Drewke et al, J Bacteriol 172:3909-17 (1990)). This enzyme is an ideal candidate for downregulation to limit ethanol production. ADH2 is severely repressed in the presence of glucose. In K. lactis, two NAD-dependent cytosolic alcohol dehydrogenases have been identified and characterized. These genes also show activity for other aliphatic alcohols. The genes ADH1 (GI:113358) and ADHII (GIA 1704293) are preferentially expressed in glucose-grown cells (Bozzi et al, Biochim Biophys Acta 1339:133-142 (1997)). Cytosolic alcohol dehydrogenases are encoded by ADH1 (GI:608690) in C. albicans, ADH1 (GI:3810864) in S. pombe, ADH1 (GI:5802617) in Y. lipolytica, ADH1 (GI:2114038) and ADHII (GI:2143328)in Pichia stipitis or Scheffersomyces stipitis (Passoth et al, Yeast 14:1311-23 (1998)). Candidate alcohol dehydrogenases are shown the table below. Protein GenBank ID GI number Organism SADHBAA24528.12815409Candida parapsilosisADH1NP_014555.16324486Saccharomyces cerevisiae s288cADH2NP_014032.16323961Saccharomyces cerevisiae s288cADH3NP_013800.16323729Saccharomyces cerevisiae s288cADH4NP_011258.2269970305Saccharomyces cerevisiae s288cADH5 (SFA1)NP_010113.16320033Saccharomyces cerevisiae s288cADH6NP_014051.16323980Saccharomyces cerevisiae s288cADH7NP_010030.16319949Saccharomyces cerevisiae s288cadhPCAA44614.12810Kluyveromyces lactisADH1P20369.1113358Kluyveromyces lactisADH2CAA45739.12833Kluyveromyces lactisADH3P49384.251704294Kluyveromyces lactisADH1CAA57342.1608690Candida albicansADH2CAA21988.13859714Candida albicansSADXP_712899.168486457Candida albicansADH1CAA21782.13810864Schizosaccharomyces pombeADH1AAD51737.15802617Yarrowia lipolyticaADH2AAD51738.15802619Yarrowia lipolyticaADH3AAD51739.15802621Yarrowia lipolyticaAlcBAAX53105.161696864Aspergillus nigerANI_1_282024XP_001399347.1145231748Aspergillus nigerANI_1_126164XP_001398574.2317037131Aspergillus nigerANI_1_1756104XP_001395505.2317033815Aspergillus nigerADH2CAA73827.12143328Scheffersomyces stipitis

[0205] Attenuation or disruption of one or more glycerol-3-phosphatase or glycerol-3-phosphate (G3P) dehydrogenase enzymes can eliminate or reduce the formation of glycerol, and thereby conserving carbon and reducing equivalents for production of fatty alcohols, fatty aldehydes, fatty acids or isopropnaol.

[0206] G3P phosphatase catalyzes the hydrolysis of G3P to glycerol. Enzymes with this activity include the glycerol-1-phosphatase (EC 3.1.3 .21) enzymes of Saccharomyces cerevisiae (GPP1 and GPP2), Candida albicans and Dunaleilla parva (Popp et al, Biotechnol Bioeng 100:497-505 (2008); Fan et al, FEMS Microbiol Lett 245:107-16 (2005)). The D. parva gene has not been identified to date. These and additional G3P phosphatase enzymes are shown in the table below. Protein GenBankID GI Number Organism GPP1DAA08494.1285812595Saccharomyces cerevisiaeGPP2NP_010984.16320905Saccharomyces cerevisiaeGPP1XP_717809.168476319Candida albicansKLLA0C08217gXP_452565.150305213Kluyveromyces lactisKLLA0C11143gXP_452697.150305475Kluyveromyces lactisANI_1_380074XP_001392369.1145239445Aspergillus nigerANI_1_444054XP_001390913.2317029125Aspergillus niger

[0207] S. cerevisiae has three G3P dehydrogenase enzymes encoded by GPD1 and GDP2 in the cytosol and GUT2 in the mitochondrion. GPD2 is known to encode the enzyme responsible for the majority of the glycerol formation and is responsible for maintaining the redox balance under anaerobic conditions. GPD1 is primarily responsible for adaptation of S. cerevisiae to osmotic stress (Bakker et al., FEMS Microbiol Rev 24:15-37 (2001)). Attenuation of GPD1, GPD2 and / or GUT2 will reduce glycerol formation. GPD1 and GUT2 encode G3P dehydrogenases in Yarrowia lipolytica (Beopoulos et al, AEM74:7779-89 (2008)). GPD1 and GPD2 encode for G3P dehydrogenases in S pombe. Similarly, G3P dehydrogenase is encoded by CTRG_02011 in Candida tropicalis and a gene represented by GI:20522022 in Candida albicans. Protein GenBank ID GI number Organism GPD1CAA98582.11430995Saccharomyces cerevisiaeGPD2NP_014582.16324513Saccharomyces cerevisiaeGUT2NP_012111.16322036Saccharomyces cerevisiaeGPD1CAA22119.16066826Yarrowia lipolyticaGUT2CAG83113.149646728Yarrowia lipolyticaGPD1CAA22119.13873542Schizosaccharomyces pombeGPD2CAA91239.11039342Schizosaccharomyces pombeANI_1_786014XP_001389035.2317025419Aspergillus nigerANI_1_1768134XP_001397265.1145251503Aspergillus nigerKLLA0C04004gXP_452375.150304839Kluyveromyces lactisCTRG_02011XP_002547704.1255725550Candida tropicalisGPD1XP_714362.168483412Candida albicansGPD2XP_713824.168484586Candida albicans

[0208] Enzymes that form acid byproducts such as acetate, formate and lactate can also be attenuated or disrupted. Such enzymes include acetate kinase, phosphotransacetylase and pyruvate oxidase. Disruption or attenuation of pyruvate formate lyase and formate dehydrogenase could limit formation of formate and carbon dioxide. These enzymes are described in further detail in Example V.

[0209] Alcohol dehydrogenases that convert pyruvate to lactate are also candidates for disruption or attenuation. Lactate dehydrogenase enzymes include ldhA of E. coli and ldh from Ralstonia eutropha (Steinbuchel and Schlegel, Eur. J. Biochem. 130:329-334 (1983)). Other alcohol dehydrogenases listed above may also exhibit LDH activity. Protein GenBank ID GI number Organism ldhANP_415898.116129341Escherichia coliLdhYP_725182.1113866693Ralstonia eutropha

[0210] Tuning down activity of the mitochondrial pyruvate dehydrogenase complex will limit flux into the mitochondrial TCA cycle. Under anaerobic conditions and in conditions where glucose concentrations are high in the medium, the capacity of this mitochondrial enzyme is very limited and there is no significant flux through it. However, in some embodiments, this enzyme can be disrupted or attenuated to increase fatty alcohol, fatty aldehyde or fatty acid production. Exemplary pyruvate dehydrogenase genes include PDB1, PDA1, LAT1 and LPD1. Accession numbers and homologs are listed in Example V.

[0211] Another strategy for reducing flux into the TCA cycle is to limit transport of pyruvate into the mitochondria by tuning down or deleting the mitochondrial pyruvate carrier. Transport of pyruvate into the mitochondria in S. cerevisiae is catalyzed by a heterocomplex encoded by MPC1 and MPC2 (Herzig et al, Science 337:93-6 (2012); Bricker et al, Science 337:96-100 (2012)). S. cerevisiae encodes five other putative monocarboxylate transporters (MCH1-5), several of which may be localized to the mitochondrial membrane (Makuc et al, Yeast 18:1131-43 (2001)). NDT1 is another putative pyruvate transporter, although the role of this protein is disputed in the literature (Todisco et al, JBiol Chem 20:1524-31 (2006)). Exemplary pyruvate and monocarboxylate transporters are shown in the table below: Protein GenBank ID GI number Organism MPC1NP_011435.16321358Saccharomyces cerevisiaeMPC2NP_012032.16321956Saccharomyces cerevisiaeMPC1XP_504811.150554805Yarrowia lipolyticaMPC2XP_501390.150547841Yarrowia lipolyticaMPC1XP_719951.168471816Candida albicansMPC2XP_716190.168479656Candida albicansMCH1NP_010229.16320149Saccharomyces cerevisiaeMCH2NP_012701.2330443640Saccharomyces cerevisiaeMCH3NP_014274.16324204Saccharomyces cerevisiaeMCH5NP_014951.2330443742Saccharomyces cerevisiaeNDT1NP_012260.16322185Saccharomyces cerevisiaeANI_1_1592184XP_001401484.2317038471Aspergillus nigerCaJ7_0216XP_888808.177022728Candida albicansYALI0E16478gXP_504023.150553226Yarrowia lipolyticaKLLA0D14036gXP_453688.150307419Kluyveromyces lactis

[0212] Disruption or attenuation of enzymes that synthesize malonyl-CoA and fatty acids can increase the supply of carbon available for fatty alcohol, fatty aldehyde or fatty acid biosynthesis from acetyl-CoA. Exemplary enzymes for disruption or attenuation include fatty acid synthase, acetyl-CoA carboxylase, biotin:apoenzyme ligase, acyl carrier protein, thioesterase, acyltransferases, ACP malonyltransferase, fatty acid elongase, acyl-CoA synthetase, acyl-CoA transferase and acyl-CoA hydrolase.

[0213] Another strategy to reduce fatty acid biosynthesis is expression or overexpression of regulatory proteins which repress fatty acid forming genes. Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the first step of fatty acid biosynthesis in many organisms: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This enzyme utilizes biotin as a cofactor. Exemplary ACC enzymes are encoded by accABCD of E. coli (Davis et al, JBiol Chem 275:28593-8 (2000)), ACC1 of Saccharomyces cerevisiae and homologs (Sumper et al, Methods Enzym 71:34-7 (1981)). The mitochondrial acetyl-CoA carboxylase of S. cerevisiae is encoded by HFA1. Acetyl-CoA carboxylase holoenzyme formation requires attachment of biotin by a biotin:apoprotein ligase such as BPL1 of S. cerevisiae. Protein GenBank ID GI Number Organism ACC1CAA96294.11302498Saccharomyces cerevisiaeKLLA0F06072gXP_455355.150310667Kluyveromyces lactisACC1XP_718624.168474502Candida albicansYALI0C11407pXP_501721.150548503Yarrowia lipolyticaANI_1_1724104XP_001395476.1145246454Aspergillus nigeraccAAAC73296.11786382Escherichia coliaccBAAC76287.11789653Escherichia coliaccCAAC76288.11789654Escherichia coliaccDAAC75376.11788655Escherichia coliHFA1NP_013934.16323863Saccharomyces cerevisiaeBPL1NP_010140.16320060Saccharomyces cerevisiae

[0214] Proteins participating in the synthesis of fatty acids are shown below. The fatty acid synthase enzyme complex of yeast is composed of two multifunctional subunits, FAS1 and FAS2, which together catalyze the net conversion of acetyl-CoA and malonyl-CoA to fatty acids (Lomakin et al, Cell 129: 319-32 (2007)). Additional proteins associated with mitochondrial fatty acid synthesis include OAR1, Mct1, ETR1, ACP1 and PPT2. ACP1 is the mitochondrial acyl carrier protein and PPT2 encodes a phosphopantetheine transferase, which pantetheinylates mitochondrial ACP and is required for fatty acid biosynthesis in the mitochondria (Stuible et al, J Biol Chem: 273: 22334-9 (1998)). A non-genetic strategy for reducing activity of fatty acid synthases is to add an inhibitor such as cerulenin. Global regulators of lipid biosynthesis can also be altered to tune down endogenous fatty acid biosynthesis pathways during production of long chain alcohols or related products. An exemplary global regulator is SNF1 of Yarrowia lipolytica and Saccharomyces cerevisiae. Protein GenBank ID GI Number Organism FAS1NP_012739.16322666Saccharomyces cerevisiaeFAS2NP_015093.16325025Saccharomyces cerevisiaeFAS1XP_451653.150303423Kluyveromyces lactisFAS2XP_452914.150305907Kluyveromyces lactisFAS1XP_716817.168478392Candida albicansFAS2XP_723014.168465892Candida albicansFAS1XP_500912.150546885Yarrowia lipolyticaFAS2XP_501096.150547253Yarrowia lipolyticaFAS1XP_001393490.2317031809Aspergillus nigerFAS2XP_001388458.1145228299Aspergillus nigerOAR1NP_012868.16322795Saccharomyces cerevisiaeMCT1NP_014864.4398365823Saccharomyces cerevisiaeETR1NP_009582.16319500Saccharomyces cerevisiaeACP1NP_012729.16322656Saccharomyces cerevisiaePPT2NP_015177.237362701Saccharomyces cerevisiaeSNF1CAG80498.149648180Yarrowia lipolyticaSNF1P06782.1134588Saccharomyces cerevisiae

[0215] Disruption or attenuation of elongase enzymes which convert acyl-CoA substrates to longer-chain length fatty acid derivatives longer than the product of interest can also be used to increase fatty alcohol, fatty aldehyde or fatty acid production. Elongase enzymes are found in compartments such as the mitochondria, endoplasmic reticulum, proteoliposomes and peroxisomes. For example, some yeast such as S. cerevisiae are able to synthesize long-chain fatty acids of chain length C16 and higher via a mitochondrial elongase which accepts exogenous or endogenous acyl-CoA substrates (Bessoule et al, FEBS Lett 214: 158-162 (1987)). This system requires ATP for activity. The endoplasmic reticulum also has an elongase system for synthesizing very long chain fatty acids (C18+) from acyl-CoA substrates of varying lengths (Kohlwein et al, Mol Cell Biol 21:109-25 (2001)). Genes involved in this system include TSC13, ELO2 and ELO3. ELO1 catalyzes the elongation of C12 acyl-CoAs to C16-C18 fatty acids. Protein Accession # GI number Organism ELO2NP_009963.16319882Saccharomyces cerevisiaeELO3NP_013476.3398366027Saccharomyces cerevisiaeTSC13NP_010269.16320189Saccharomyces cerevisiaeELO1NP_012339.16322265Saccharomyces cerevisiae

[0216] Native enzymes converting acyl-CoA pathway intermediates to acid byproducts can also reduce fatty alcohol, fatty aldehyde or fatty acid yield. For example, CoA hydrolases, transferases and synthetases can act on acyl-CoA intermediates to form short-, medium- or long chain acids. Disruption or attenuation of endogenous CoA hydrolases, CoA transerases and / or reversible CoA synthetases can be used to increase fatty alcohol, fatty aldehyde or fatty acid yield. Exempalry enzymes are shown in the table below. Protein GenBank ID GI number Organism Tes1NP_012553.16322480Saccharomyces cerevisiae s288cACH1NP_009538.16319456Saccharomyces cerevisiae s288cEHD3NP_010321.16320241Saccharomyces cerevisiae s288cYALI0F14729pXP_505426.150556036Yarrowia lipolyticaYALI0E30965pXP_504613.150554409Yarrowia lipolyticaKLLA0E16523gXP_454694.150309373Kluyveromyces lactisKLLA0E10561gXP_454427.150308845Kluyveromyces lactisACH1P83773.2229462795Candida albicansCaO19.10681XP_714720.168482646Candida albicansANI_1_318184XP_001401512.1145256774Aspergillus nigerANI_1_1594124XP_001401252.2317035188Aspergillus nigertesBNP_414986.116128437Escherichia colitesBNP_355686.2159185364Agrobacterium tumefaciensatoA2492994P76459.1Escherichia coliatoD2492990P76458.1Escherichia coli

[0217] Enzymes that favor the degradation of products, MI-FAE cycle intermediates, MD-FAE cycle intermediates, FAACPE cycle intermediates, termination pathway intermediates, or isopropanol pathway intermediates can also be disrupted or attenuated. Examples include aldehyde dehydrogenases, aldehyde decarbonylases, oxidative alcohol dehydrogenases, and irreversible fatty acyl-CoA degrading enzymes.

[0218] For production of fatty alcohols, fatty aldehydes, or fatty acids of the invention, deletion or attenuation of non-specific aldehyde dehydrogenases can improve yield. For production of fatty acids, expression of such an enzyme may improve product formation. Such enzymes can, for example, convert acetyl-CoA into acetaldehyde, fatty aldehydes to fatty acids, or fatty alcohols to fatty acids. Acylating aldehyde dehydrogenase enzymes are described in Example IV. Acid-forming aldehyde dehydrogenase are described in Examples VI and XII.

[0219] The pathway enzymes that favor the reverse direction can also be disrupted or attenuated, if they are detrimental to fatty alcohol, fatty aldehyde, or fatty acid production. An example is long chain alcohol dehydrogenases (EC 1.1.1.192) that favor the oxidative direction. Exemplary long chain alcohol dehydrogenases are ADH1 and ADH2 of Geobacillus thermodenitrificans, which oxidize alcohols up to a chain length of C30 (Liu et al, Physiol Biochem 155:2078-85 (2009)). These and other exemplary fatty alcohol dehydrogenase enzymes are listed in Examples IV and V. If an alcohol-forming acyl-CoA reductase is utilized for fatty alcohol, fatty aldehyde or fatty acid biosynthesis, deletion of endogenous fatty alcohol dehydrogenases will substantially reduce backflux.

[0220] Beta-oxidation enzymes may be reversible and operate in the direction of acyl-CoA synthesis. However, if they are irreversible or strongly favored in the degradation direction they are candidates for disruption or attenuation. An enzyme that fall into this category includes FOX2 of S. cerevisiae, a multifunctional enzyme with 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activity (Hiltunen et al, JBiol Chem 267: 6646-6653 (1992)). Additional genes include degradative thiolases such as POT1 and acyl-CoA dehydrogenases that utilize cofactors other than NAD(P)H (EG. EC 1.3.8.-) such as fadE of E. coli. Protein GenBank ID GI Number Organism POT1NP_012106.16322031Saccharomyces cerevisiaeFOX2NP_012934.16322861Saccharomyces cerevisiaefadEAAC73325.287081702Escherichia coli

[0221] Fatty acyl-CoA oxidase enzymes such as POX1 of S. cerevisiae catalyze the oxygen-dependent oxidation of fatty acyl-CoA substrates. Enzymes with this activity can be disrupted or attenuated, if they are expressed under fatty alcohol, fatty aldehyde or fatty acid producing conditions. POX1 (EC 1.3.3.6) genes and homologs are shown in the table below. POX1 is subject to regulation by OAF1, which also activates genes involved in peroxisomal beta-oxidation, organization and biogenesis (Luo et al, JBiol Chem 271:12068-75 (1996)). Regulators with functions similar to OAF1, and peroxisomal fatty acid transporters PXA1 and PXA2 are also candidates for deletion. Protein GenBank ID GI Number Organism POX1NP_011310.16321233Saccharomyces cerevisiaeOAF1NP_009349.3330443370Saccharomyces cerevisiaePXA1NP_015178.16325110Saccharomyces cerevisiaePXA2NP_012733.16322660Saccharomyces cerevisiaeYALI0F10857gXP_505264.150555712Yarrowia lipolyticaYALI0D24750pXP_503244.150551539Yarrowia lipolyticaYALI0E32835pXP_504703.150554589Yarrowia lipolyticaYALI0E06567pXP_503632.150552444Yarrowia lipolyticaYALI0E27654pXP_504475.150554133Yarrowia lipolyticaYALI0C23859pXP_502199.150549457Yarrowia lipolyticaPOXXP_455532.150311017Kluyveromyces lactisPOX104XP_721610.168468582Candida albicansPOX105XP_717995.168475844Candida albicansPOX102XP_721613.168468588Candida albicans

[0222] Another candidate for disruption or attenuation is an acyl-CoA binding protein. The acyl binding protein ACB1 of S. cerevisiae, for example, binds acyl-CoA esters and shuttles them to acyl-CoA utilizing processes (Schjerling et al, J Biol Chem 271: 22514-21 (1996)). Deletion of this protein did not impact growth rate and lead to increased accumulation of longer-chain acyl-CoA molecules. Acyl-CoA esters are involved in diverse cellular processes including lipid biosynthesis and homeostatis, signal transduction, growth regulation and cell differentiation (Rose et al, PNAS USA 89: 11287-11291 (1992)). Protein GenBank ID GI Number Organism ACB1P31787.3398991Saccharomyces cerevisiaeKLLA0B05643gXP_451787.2302309983Kluyveromyces lactisYALI0E23185gXP_002143080.1210076210Yarrowia lipolyticaANI_1_1084034XP_001390082.1145234867Aspergillus niger

[0223] To achieve high yields of fatty alcohols, fatty aldehydes fatty acids or isopropanol, it is desirable that the host organism can supply the cofactors required by the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, the termination pathway and / or isopropanol pathway in sufficient quantities. In several organisms, in particular eukaryotic organisms, such as several Saccharomyces, Kluyveromyces, Candida, Aspergillus, and Yarrowia species, NADH is more abundant than NADPH in the cytosol as it is produced in large quantities by glycolysis. NADH can be made even more abundant by converting pyruvate to acetyl-CoA by means of heterologous or native NAD-dependant enzymes such as NAD-dependant pyruvate dehydrogenase, NAD-dependant formate dehydrogenase, NADH:ferredoxin oxidoreductase, or NAD-dependant acylating acetylaldehyde dehydrogenase in the cytosol. Given the abundance of NADH in the cytosol of most organisms, it can be beneficial for all reduction steps of the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, terminatio pathway and / or isopropanol pathway to accept NADH as the reducing agent preferentially over other reducing agents such as NADPH. High yields of fatty alcohols, fatty aldehydes or fatty acids can thus be accomplished by, for example: 1) identifying and implementing endogenous or exogenous MI-FAE cycle, MD-FAE cycle and / or termination pathway enzymes with a stronger preference for NADH than other reducing equivalents such as NADPH; 2) attenuating one or more endogenous MI-FAE cycle, MD-FAE cycle or termination pathway enzymes that contribute NADPH-dependant reduction activity; 3) altering the cofactor specificity of endogenous or exogenous MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway or isopropanol pathway enzymes so that they have a stronger preference for NADH than their natural versions; or 4) altering the cofactor specificity of endogenous or exogenous MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway or isopropanol pathway enzymes so that they have a weaker preference for NADPH than their natural versions.

[0224] Strategies for engineering NADH-favoring MI-FAE cycle, MD-FAE cycle, FAACPE cycle termination pathways and / or isopropanol pathways are described in further detail in Example VIII. Methods for changing the cofactor specificity of an enzyme are well known in the art, and an example is described in Example IX.

[0225] If one or more of the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway and / or isopropanol pathway enzymes utilizes NADPH as the cofactor, it can be beneficial to increase the production of NADPH in the host organism. In particular, if the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway and / or isopropanol pathway is present in the cytosol of the host organism, methods for increasing NADPH production in the cytosol can be beneficial. Several approaches for increasing cytosolic production of NADPH can be implemented including channeling an increased amount of flux through the oxidative branch of the pentose phosphate pathway relative to wild-type, channeling an increased amount of flux through the Entner Doudoroff pathway relative to wild-type, introducing a soluble or membrane-bound transhydrogenase to convert NADH to NADPH, or employing NADP-dependant versions of the following enzymes: phosphorylating or non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase. These activities can be augmented by disrupting or attenuating native NAD-dependant enzymes including glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, or acylating acetylaldehyde dehydrogenase. Strategies for engineering increased NADPH availability are described in Example X.

[0226] Synthesis of fatty alcohols, fatty aldehyes, or fatty acids in the cytosol can be dependent upon the availability of sufficient carbon and reducing equivalents. Therefore, without being bound to any particular theory of operation, increasing the redox ratio of NAD(P)H to NAD(P) can help drive the MI-FAE cycle, MD-FAE cycle, FAACPE cycle, termination pathway and / or isopropanol pathway in the forward direction. Methods for increasing the redox ratio of NAD(P)H to NAD(P) include limiting respiration, attenuating or disrupting competing pathways that produce reduced byproducts such as ethanol and glycerol, attenuating or eliminating the use of NADH by NADH dehydrogenases, and attenuating or eliminating redox shuttles between compartments.

[0227] One exemplary method to provide an increased number of reducing equivalents, such as NAD(P)H, for enabling the formation of fatty alcohols, fatty aldehydes, fatty acids or isopropanol is to constrain the use of such reducing equivalents during respiration. Respiration can be limited by: reducing the availability of oxygen, attenuating NADH dehydrogenases and / or cytochrome oxidase activity, attenuating G3P dehydrogenase, and / or providing excess glucose to Crabtree positive organisms.

[0228] Restricting oxygen availability by culturing the non-naturally occurring eukaryotic organisms in a fermenter is one exmaple for limiting respiration and thereby increasing the ratio of NAD(P)H to NAD(P). The ratio of NAD(P)H / NAD(P) increases as culture conditions become more anaerobic, with completely anaerobic conditions providing the highest ratios of the reduced cofactors to the oxidized ones. For example, it has been reported that the ratio of NADH / NAD = 0.02 in aerobic conditions and 0.75 in anaerobic conditions in E. coli (de Graes et al, J Bacteriol 181:2351-57 (1999)).

[0229] Respiration can also be limited by reducing expression or activity ofNADH dehydrogenases and / or cytochrome oxidases in the cell under aerobic conditions. In this case, respiration can be limited by the capacity of the electron transport chain. Such an approach has been used to enable anaerobic metabolism of E. coli under completely aerobic conditions (Portnoy et al, AEM 74:7561-9 (2008)). S. cerevisiae can oxidize cytosolic NADH directly using external NADH dehydrogenases, encoded by NDE1 and NDE2. One such NADH dehydrogenase in Yarrowia lipolytica is encoded by NDH2 (Kerscher et al, J Cell Sci 112:2347-54 (1999)). These and other NADH dehydrogenase enzymes are listed in the table below. Protein GenBank ID GI number Organism NDE1NP_013865.16323794Saccharomyces cerevisiae s288cNDE2NP_010198.16320118Saccharomyces cerevisiae s288cNDH2AJ006852.13718004Yarrowia lipolyticaANI_1_610074XP_001392541.2317030427Aspergillus nigerANI_1_2462094XP_001394893.2317033119Aspergillus nigerKLLA0E21891gXP_454942.150309857Kluyveromyces lactisKLLA0C06336gXP_452480.150305045Kluyveromyces lactisNDE1XP_720034.168471982Candida albicansNDE2XP_717986.168475826Candida albicans

[0230] Cytochrome oxidases of Saccharomyces cerevisiae include the COX gene products. COX 1-3 are the three core subunits encoded by the mitochondrial genome, whereas COX4-13 are encoded by nuclear genes. Attenuation or disruption of any of the cytochrome genes results in a decrease or block in respiratory growth (Hermann and Funes, Gene 354:43-52 (2005)). Cytochrome oxidase genes in other organisms can be inferred by sequence homology. Protein GenBank ID GI number Organism COX1CAA09824.14160366Saccharomyces cerevisiae s288cCOX2CAA09845.14160387Saccharomyces cerevisiae s288cCOX3CAA09846.14160389Saccharomyces cerevisiae s288cCOX4NP_011328.16321251Saccharomyces cerevisiae s288cCOX5ANP_014346.16324276Saccharomyces cerevisiae s288cCOX5BNP_012155.16322080Saccharomyces cerevisiae s288cCOX6NP_011918.16321842Saccharomyces cerevisiae s288cCOX7NP_013983.16323912Saccharomyces cerevisiae s288cCOX8NP_013499.16323427Saccharomyces cerevisiae s288cCOX9NP_010216.16320136Saccharomyces cerevisiae s288cCOX12NP_013139.16323067Saccharomyces cerevisiae s288cCOX13NP_011324.16321247Saccharomyces cerevisiae s288c

[0231] Cytosolic NADH can also be oxidized by the respiratory chain via the G3P dehydrogenase shuttle, consisting of cytosolic NADH-linked G3P dehydrogenase and a membrane-bound G3P:ubiquinone oxidoreductase. The deletion or attenuation of G3P dehydrogenase enzymes will also prevent the oxidation of NADH for respiration. Enzyme candidates encoding these enzymes are described herein.

[0232] Additionally, in Crabtree positive organisms, fermentative metabolism can be achieved in the presence of excess of glucose. For example, S cerevisiae makes ethanol even under aerobic conditions. The formation of ethanol and glycerol can be reduced / eliminated and replaced by the production of fatty alcohol, fatty aldehyde, or fatty acid in a Crabtree positive organism by feeding excess glucose to the Crabtree positive organism. In another aspect, provided herein is a method for producing fatty alcohols, fatty aldehydes, fatty acids, isopropanol comprising culturing a non-naturally occurring eukaryotic organism under conditions and for a sufficient period of time to produce fatty alcohol, fatty aldehyde, or fatty acid, wherein the eukaryotic organism is a Crabtree positive organism, and wherein the eukaryotic organism is in a culture medium comprising excess glucose.

[0233] Preventing formation of reduced fermentation byproducts will increase the availability of both carbon and reducing equivalents for fatty alcohol, fatty aldehyde, or fatty acid production. The two key reduced byproducts under anaerobic and microaerobic conditions are ethanol and glycerol. Ethanol is typically formed from pyruvate in two enzymatic steps catalyzed by pyruvate decarboxylase and ethanol dehydrogenase. Glycerol is formed from the glycolytic intermediate dihydroxyacetone phosphate by the enzymes glycerol-3-phsophate dehydrogenase and glycerol-3-phosphate phosphatase. Attenuation of one or more of these enzyme activities will increase the yield of fatty alcohols, fatty aldehydes, fatty acids or isopropanol. Strain engineering strategies for reducing or eliminating ethanol and glycerol formation are described herein.

[0234] Yeast such as S. cerevisiae can produce glycerol to allow for regeneration of NAD(P) under anaerobic conditions. Another way to reduce or eliminate glycerol production is by oxygen-limited cultivation (Bakker et al, supra). Glycerol formation only sets in when the specific oxygen uptake rates of the cells decrease below the rate that is required to reoxidize the NADH formed in biosynthesis.

[0235] In addition to the redox sinks listed above, malate dehydrogenase can potentially draw away reducing equivalents when it functions in the reductive direction. Several redox shuttles believed to be functional in S. cerevisiae utilize this enzyme to transfer reducing equivalents between the cytosol and the mitochondria. This transfer of redox can be prevented by attenuating malate dehydrogenase and / or malic enzyme activity. The redox shuttles that can be blocked by the attenuation of mdh include (i) malate-asparate shuttle, (ii) malate-oxaloacetate shuttle, and (iii) malate-pyruvate shuttle. Genes encoding malate dehydrogenase and malic enzymes are listed in the table below. Protein GenBank ID GI Number Organism MDH1NP_012838.16322765Saccharomyces cerevisiaeMDH2NP_014515.2116006499Saccharomyces cerevisiaeMDH3NP_010205.16320125Saccharomyces cerevisiaeMAE1NP_012896.16322823Saccharomyces cerevisiaeMDH1XP_722674.168466384Candida albicansMDH2XP_718638.168474530Candida albicansMAE1XP_716669.168478574Candida albicansKLLA0F25960gXP_456236.150312405Kluyveromyces lactisKLLA0E18635gXP_454793.150309563Kluyveromyces lactisKLLA0E07525gXP_454288.150308571Kluyveromyces lactisYALI0D16753pXP_502909.150550873Yarrowia lipolyticaYALI0E18634pXP_504112.150553402Yarrowia lipolyticaANI_1_268064XP_0013913...

Claims

1. A non-naturally occurring microbial organism producing a compound of Formula (I): wherein R1 is C1-24 linear alkyl; R2 is CH2OH, or CHO; R3 is H, OH, or oxo (=O); and ------ represents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, the organism having: (i) a formaldehyde fixation pathway; (ii) a formate assimilation pathway; and (iii) a methanol metabolic pathway, wherein said formaldehyde fixation pathway comprises: (1) a) a 3-hexulose-6-phosphate synthase and b) a 6-phospho-3-hexuloisomerase; wherein said formate assimilation pathway comprises: (2) a) a formyltetrahydrofolate synthetase, b) a methenyltetrahydrofolate cyclohydrolase, c) a methylenetetrahydrofolate dehydrogenase, d) a glycine cleavage system, e) a serine hydroxymethyltransferase, and f) a serine deaminase; wherein said methanol metabolic pathway comprises: (3) a methanol dehydrogenase; the microbial organism further comprising: A) a malonyl-CoA independent fatty acyl-CoA elongation (MI-FAE) cycle and / or a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway, wherein said MI-FAE cycle comprises one or more thiolase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said MD-FAE cycle comprises one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase, wherein said termination pathway is selected from: (4A) an acyl-CoA reductase (aldehyde forming); (5A) a) an acyl-CoA reductase (aldehyde forming) and b) an alcohol dehydrogenase; or (6A) an acyl-CoA reductase (alcohol forming), wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the MI-FAE cycle, the MD-FAE cycle or the termination pathway is encoded by at least one exogenous nucleic acid; or B) a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway, wherein said FAACPE cycle comprises one or more β-ketoacyl-ACP synthase, one or more β-ketoacyl-ACP reductase, one or more β-hydroxyacyl-ACP reductase, and one or more enoyl ACP-reductase, wherein said termination pathway is selected from: (4B) a fatty acyl-ACP reductase; (5B) a) a thioesterase and b) a carboxylic acid reductase (CAR); or (6B) a) a thioesterase, b) a CAR, and c) a fatty aldehyde reductase, wherein an enzyme of the formaldehyde fixation pathway, the formate assimilation pathway, the methanol metabolic pathway, the FAACPE cycle or the termination pathway is encoded by at least one exogenous nucleic acid, wherein the organism is a species of bacteria, wherein the species of bacteria is E. coli.

2. A method for producing a compound of Formula (I): wherein R1 is C1-24 linear alkyl; R2 is CH2OH, or CHO; R3 is H, OH, or oxo (=O); andrepresents a single or double bond with the proviso that the valency of the carbon atom to which R3 is attached is four, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions for a sufficient period of time to produce said compound of Formula (I).

3. The non-naturally occurring microbial organism of claim 1, wherein each of said formaldehyde fixation pathway, said formate assimilation pathway, and said methanol metabolic pathway comprises at least one exogenous nucleic acid encoding at least one metabolic pathway enzyme.

4. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MI-FAE cycle and an acyl-CoA reductase (aldehyde forming).

5. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MI-FAE cycle, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase.

6. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MI-FAE cycle and an acyl-CoA reductase (alcohol forming).

7. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MD-FAE cycle and an acyl-CoA reductase (aldehyde forming).

8. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MD-FAE cycle, an acyl-CoA reductase (aldehyde forming) and an alcohol dehydrogenase.

9. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an MD-FAE cycle and an acyl-CoA reductase (alcohol forming).

10. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism comprises an FAACPE cycle and a fatty acyl-ACP reductase.

11. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an FAACPE cycle, a thioesterase, and a CAR.

12. The non-naturally occurring microbial organism of claim 1, wherein the microbial organism has an FAACPE cycle, a thioesterase, a CAR, and a fatty aldehyde reductase.

13. The method of claim 2, wherein R3 is H.