AU2013202923A1 - Microorganism for producing primary alcohols and related compounds and methods related thereto - Google Patents

Abstract

The invention provides a non-naturally occurring microbial organism having a microbial organism having at least one exogenous gene insertion and/or one or more gene disruptions that confer production of primary alcohols and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of the primary alcohol by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2. A method for producing long chain alcohols includes culturing these non-naturally occurring microbial organisms.

Description

MICROORGANISM FOR PRODUCING PRIMARY ALCOHOLS AND RELATED COMPOUNDS AND METHODS RELATED THERETO The entire disclosure in the complete specification of our Australian Patent Application No. 2012273005 is by this cross-reference incorporated into the present specification. 5 The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created June 18, 2012, is named 871943-228150_PCTSequenceListing.txt and is 77,825 bytes in size 10 BACKGROUND OF THE INVENTION This invention relates generally to biosynthetic processes and, more specifically to organisms having primary alcohol biosynthetic capability. 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 15 used to make a large number of additional industrial products including polymers and surfactants. For example, higher primary alcohols (C 4

-C

20 ) 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 20 lubricating oil additives. Long-chain primary alcohols, such as octanol and hexanol, have useful organoleptic properties and have long been employed as fragrance and flavor materials. Smaller chain (C4-C8) higher primary alcohols (e.g., butanol) are used as chemical intermediates for production of derivatives such as acrylates used in paints, coatings, and adhesives applications. 25 Primary 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 primary alcohols directly from the oxidation of petroleum-based linear hydrocarbons (n-paraffins). This impracticality is because the oxidation of n-paraffins produces primarily secondary 30 alcohols, tertiary alcohols or ketones, or a mixture of these compounds, but does not produce high yields of primary alcohols. Additionally, currently known methods for producing primary 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. 5 LCA 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 LCAs and other fatty acid derivatives. There is currently a great deal of improvement that can be achieved to provide more efficient biosynthesis pathways for LCA production with significantly higher theoretical product and energy yields. 10 Thus, there exists a need for alternative means for effectively producing commercial quantities of primary alcohols. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION In some aspects, embodiments disclosed herein relate to a non-naturally occurring microbial 15 organism having a microbial organism having a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway having at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of the primary alcohol by (i) enhancing carbon 20 fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2, the malonyl-CoA-independent FAS pathway having ketoacyl-CoA acyltransferase or ketoacyl CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase, the acyl-reduction pathway having an acyl-CoA reductase and an alcohol 25 dehydrogenase. In other aspects, embodiments disclosed herein relate to a method for producing a primary alcohol. The method includes culturing a non-naturally occurring microbial organism have having a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway having at least one exogenous nucleic acid encoding a malonyl-CoA-independent 30 FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol and at least one exogenous nucleic acid that encodes an enzyme that increases the yields of the 2 primary alcohol by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C02, and/or H2, under substantially anaerobic conditions for a sufficient period of time to produce the primary alcohol, the malonyl-CoA-independent FAS 5 pathway having ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase, the acyl-reduction pathway having an acyl-CoA reductase and an alcohol dehydrogenase. In some aspects, embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes one or more gene disruptions occurring in genes encoding enzymes 10 that couple long-chain alcohol (LCA) production to growth of the non-naturally occurring microbial organism. In other embodiments, LCA production can be accomplished during non-growth phases using the same disruption strategies. The one or more gene disruptions reduce the activity of the enzyme, whereby the gene disruptions confer production of LCA onto the non-naturally occurring microbial organism. 15 In other aspects, embodiments disclosed herein relate to a method for producing LCA that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions. The one or more gene disruptions occur in genes encoding an enzyme that confers LCA production in the organism. In some aspects, embodiments disclosed herein relate to a non-naturally occurring eukaryotic 20 organism, that includes one or more gene disruptions. The one or more gene disruptions occur in genes that encode enzymes such as a cytosolic pyruvate decarboxylase, a mitochondrial pyruvate dehydrogenase, a cytosolic ethanol-specific alcohol dehydrogenase and a mitochondrial ethanol-specific alcohol dehydrogenase. These disruptions confer production of long chain alcohols in the cytosol of the organism. 25 In some aspects, embodiments disclosed herein relate to a non-naturally occurring eukaryotic organism that includes one or more gene disruptions. The one or more gene disruptions occur in genes encoding enzymes such as a cytosolic pyruvate decarboxylase, a cytosolic ethanol-specific alcohol dehydrogenase, and a mitochondrial ethanol-specific alcohol dehydrogenase. These disruptions confer production of long chain alcohols in the 30 mitochondrion of said organism. 3 In other aspects, embodiments disclosed herein relate to a method for producing long chain alcohols, including culturing these non-naturally occurring eukaryotic organisms. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the malonyl-CoA-independent fatty acid synthesis and reduction (MI-LCA) 5 pathway to produce LCAs. Figure 2 shows the contrasted hypothetical production envelopes of an OptKnock-designed strain against a typical non-growth-coupled production strain. The potential evolutionary trajectories of the OptKnock strain lead to a high producing phenotype. Figure 3 shows the growth-coupled LCA production characteristics of strain design I 10 (alternating dotted and dashed) compared with those of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. Figure 4 shows the growth-coupled LCA production characteristics of strain designs II (alternating dotted and dashed), III-V (dashed), and VI-XI (dotted) compared with those of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. 15 Figure 5 shows the growth-coupled LCA production characteristics of strain designs XII (alternating dotted and dashed) and XIII-XV (dashed) compared with those of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. Figure 6 shows the growth-coupled LCA production characteristics of strain designs XVI XVIII (alternating dotted and dashed) and XIX-XXI (dashed) compared with those of wild 20 type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. Figure 7 shows the growth-coupled LCA production characteristics of Designs I (alternating dotted and dashed), V (dashed), and VA (dotted) compared with those of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is assumed. Point A refers to the dodecanol production rate at maximum growth of a strain engineered according to design 25 VA and point B refers to the minimal dodecanol production rate required for growth. Figure 8 shows the growth-coupled LCA production characteristics of Designs I (alternating dotted and dashed, XII (long dashed), XIIA (short dashed), and XIIB (dotted) compared with those of wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/hr is 4 assumed. Point A refers to the dodecanol production rate at maximum growth of a strain engineered according to design XIIB and point B refers to the minimal dodecanol production rate required for growth. Figure 9a shows the formation of dodecanol in the cytosol by relying on the AMP-forming 5 acetyl CoA synthetase for the formation of acetyl CoA for dodecanol production. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 9b shows the growth-coupled production envelopes for the production of dodecanol in S. cerevisiae in the scenario where acetyl CoA synthetase is used for acetyl CoA production in the cytosol. The black curve shows the production envelope for the wild-type network 10 under aerobic conditions, and the dark gray curve shows the growth-coupled production characteristics for the mutant network. A glucose uptake rate of 10 mmol/gDCW.hr is assumed. Figure 1Oa shows the formation of dodecanol in the cytosol by relying on the ADP-forming acetate CoA ligase for the formation of acetyl CoA for dodecanol production. The gray 15 arrow represents the addition of a heterologous enzyme. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure lOb shows the growth-coupled production envelopes for the production of dodecanol in S. cerevisiae in the scenario where acetate CoA ligase is employed for acetyl-CoA production in the cytosol. The black curve shows the production envelope for the wild-type 20 network under aerobic conditions. The light gray curve shows the increase in feasible space after acetate CoA ligase is added to the network and the dark gray curve shows the growth coupled production characteristics for the mutant network in the presence of oxygen. A glucose uptake rate of 10 mmol/gDCW.hr is assumed. Figure 1 la shows the formation of dodecanol in the cytosol by relying on the acylating 25 acetaldehyde dehydrogenase for the formation of acetyl CoA for dodecanol production. The gray arrow shows a heterologous enzyme. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 1 lb shows the growth-coupled production envelopes for the anaerobic production of dodecanol in S. cerevisiae. The black curve shows the production capabilities for the wild 30 type network, the light gray dotted curve shows the production characteristics when acylating 5 acetaldehyde dehydrogenase is added to the network and the dark gray curve shows the growth-coupling when alcohol dehydrogenase is deleted from the augmented network. Note the increase in the theoretical maximum when acylating acetaldehyde dehydrogenase is functional. A glucose uptake rate of 10 mmol/gDCW.hr is assumed. 5 Figure 12 shows the formation of dodecanol in the cytosol by relying on a cytosolic pyruvate dehydrogenase for acetyl CoA and NADH production. This can be accomplished by introducing a heterologous cytosolic enzyme (shown in gray) or by retargeting the native mitochondrial enzyme to the cytosol. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. 10 Figure 13 shows the formation of dodecanol in the cytosol by relying on a cytosolic pyruvate:NADP oxidoreductase for acetyl CoA and NADH production. This can be accomplished by introducing a heterologous enzyme in the cytosol (shown in gray). The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 14 shows the formation of dodecanol in the cytosol by the introduction of a 15 heterologous pyruvate formate lyase (shown in gray) in the cytosol. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 15a shows the formation of dodecanol in the mitochondrion by using the pyruvate dehydrogenase for the formation of acetyl-CoA. The dotted arrows depict the flow of the majority of the carbon flux in this production scenario. 20 Figure 15b shows the growth-coupled production envelopes for the production of dodecanol in S. cerevisiae mitochondrion. The black curve shows the production capabilities for the wild-type network under anaerobic conditions and the dark gray curve shows the production characteristics in the absence of oxygen when pyruvate decarboxylase is deleted from the network. A glucose uptake rate of 10 mmol/gDCW.hr is assumed. 25 Figure 16 shows the formation of dodecanol in the mitochondrion by using the pyruvate:NADP oxidoreductase for formation of acetyl CoA. The gray arrow shows the heterologous enzyme and the dotted arrows depict the flow of the majority of the carbon flux in this production scenario. 6 Figure 17 shows the formation of dodecanol in the mitochondrion by using the pyruvate formate lyase for formation of acetyl CoA. The gray arrow shows the heterologous enzyme and the dotted arrows depict the flow of the majority of the carbon flux in this production scenario. 5 Figure 18 shows the formation of dodecanol in the mitochondrion by adding the mitochondrial acylating acetaldehyde dehydrogenase. The gray arrow shows the heterologous enzyme(s) and the dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 19a shows the formation of dodecanol in the mitochondrion by using the acetyl CoA 10 synthetase for formation of acetyl CoA. The gray arrow shows the heterologous enzyme(s) and the dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 19b shows the growth-coupled production envelopes for the production of dodecanol in S. cerevisiae mitochondrion when acetyl-CoA is formed through the mitochondrial acetyl 15 CoA synthetase. The black curve shows the production envelope for the wild-type network under aerobic conditions, the light dark gray curve shows the production characteristics when the deletions have been imposed upon the network. The growth coupling can be improved further (dark gray curve) when flux through the oxidative part of the pentose phosphate pathway is decreased. A glucose uptake rate of 10 mmol/gDCW.hr is assumed. 20 Figure 20 shows the formation of dodecanol in the mitochondrion by using the acetate CoA ligase for formation of acetyl CoA. The gray arrows show the heterologous enzyme(s) and the dotted arrows depict the flow of the majority of the carbon flux in this production scenario. Figure 21 shows the reverse TCA cycle for fixation of CO 2 on carbohydrates as substrates. 25 The enzymatic transformations are carried out by the enzymes as shown. Figure 22 shows the pathway for the reverse TCA cycle coupled with carbon monoxide dehydrogenase and hydrogenase for the conversion of syngas to acetyl-CoA. Figure 23 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. thermoacetica 7 CODH (Moth_1202/1203) or Mtr (Moth 1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng). Figure 24 shows CO oxidation assay results. Cells (M. thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts 5 prepared. Assays were performed at 55'C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. Figure 25, panels A and B, show exemplary pathways for fixation of CO 2 to acetyl-CoA using the reductive TCA cycle and exemplary pathways for the biosynthesis of long chain acyl-CoA's, acids, aldehydes, and alcohols from acetyl-CoA. 10 Figure 26, panels A and B, show exemplary pathways for fixation of CO 2 to acetyl-CoA using the reductive TCA cycle and exemplary pathways for the biosynthesis of long chain acyl-ACP's, acyl-CoA's, acids, aldehydes, and alcohols from acetyl-CoA. Figure 27A shows the nucleotide sequence (SEQ ID NO: 1) of carboxylic acid reductase from Nocardia iowensis (GNM__720), and Figure 27B shows the encoded amino acid 15 sequence (SEQ ID NO: 2). Figure 28A shows the nucleotide sequence (SEQ ID NO: 3) of phosphpantetheine transferase, which was codon optimized, and Figure 28B shows the encoded amino acid sequence (SEQ ID NO: 4). Figure 29A shows the nucleotide sequence (SEQ ID NO: 5) of carboxylic acid reductase 20 from Mycobacterium smegmatis mc(2)155 (designated 890), and Figure 29B shows the encoded amino acid sequence (SEQ ID NO: 6). Figure 30A shows the nucleotide sequence (SEQ ID NO: 7) of carboxylic acid reductase from Mycobacterium a vium subspecies paratuberculosis K- 10 (designated 891), and Figure 30B shows the encoded amino acid sequence (SEQ ID NO: 8). 25 Figure 3 1A shows the nucleotide sequence (SEQ ID NO: 9) of carboxylic acid reductase from Mycobacterium marinum M (designated 892), and Figure 3 1B shows the encoded amino acid sequence (SEQ ID NO: 10). 8 Figure 32A shows the nucleotide sequence (SEQ ID NO: 11) of carboxylic acid reductase designated 891GA, and Figurc 32B shows the encoded amino acid sequence (SEQ ID NO: 12). DETAILED DESCRIPTION OF THE INVENTION 5 The invention is directed, in part, to recombinant microorganisms capable of synthesizing the primary alcohols using a malonyl-CoA-independent fatty acid synthesis and reduction pathway. The modified microorganisms of the invention also are capable of secreting the resultant primary alcohol into the culture media or fermentation broth for further manipulation or isolation. Recombinant microorganisms of the invention can be engineered 10 to produce commercial quantities of a variety of different primary alcohols having different chain lengths between 4 (C4) and 24 (C24) or more carbon atoms. Production of primary alcohols through the modified pathways of the invention is particularly useful because it results in higher product and ATP yields than through naturally occurring biosynthetic pathways such as the well-documented malonyl-CoA dependent fatty acid synthesis pathway. 15 Using acetyl-CoA as a C2 extension unit instead of malonyl-acyl carrier protein (malonyl ACP) saves one ATP molecule per unit flux of acetyl-CoA entering the elongation cycle. The elongation cycle results in acyl-CoA instead of acyl-ACP, and precludes the need of the ATP-consuming acyl-CoA synthase reactions for the production of octanol and other primary alcohols. The primary alcohol producing organisms of the invention can additionally allow 20 the use of biosynthetic processes to convert low cost renewable feedstock for the manufacture of chemical products. In one specific embodiment, the invention utilizes a heterologous malonyl-CoA-independent fatty acid synthesis pathway coupled with an acyl-CoA reduction pathway to form primary alcohol species. The coupling of these two pathways will convert a carbon or energy source 25 into acetyl-CoA, which is used as both primer and extension unit in biosynthetic elongation cycle. The elongation cycle includes ketoacyl-CoA thiolase (or ketoacyl-CoA acyltransferase), 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. Each cycle results in the formation of an acyl-CoA extended by one C2 unit compared to the acyl-CoA substrate entering the elongation cycle. Carbon chain-length of 30 the primary alcohols can be controlled by chain-length specific enoyl-CoA reductase, ketoacyl-CoA thiolase and/or acyl-CoA reductase. Acyl-CoA products with desired chain lengths are funneled into a reduction pathway and reduced through the combination of acyl 9 CoA reductase and alcohol dehydrogenase or the fatty alcohol forming acyl-CoA reductase to form desired primary alcohol. These reduction steps serve as another mechanism for control of chain length, for example, through the use of chain-length specific acyl-CoA reductases. As used herein, the term "non-naturally occurring" when used in reference to a microbial 5 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 10 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 15 porteins within a malonyl-CoA-independent fatty acid biosynthetic pathway and enzymes within an acyl-reduction pathway. A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments 20 thereof. Exemplary metabolic modifications are disclosed herein. As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The 25 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 30 microbes and microbes cultured in a medium that is non-naturally occurring. 10 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, 5 archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. As used herein, the term "primary alcohol" is intended to mean an alcohol which has the hydroxyl radical connected to a primary carbon. The term includes an alcohol that possesses 10 the group -CH 2 OH which can be oxidized so as to form a corresponding aldehyde and acid having the same number of carbon atoms. Alcohols include any of a series of hydroxyl compounds, the simplest of which are derived from saturated hydrocarbons, have the general formula C.H 2 n+1OH, and include ethanol and methanol. Exemplary primary alcohols include butanol, hexanol, heptanol, octanol, nananol, decanol, dodecanol, tetradecanol, and 15 hexadecanol. 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, for example, in certain condensing enzymes, acts in acetyl or other acyl group 20 transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation. 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% 25 oxygen. "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic 30 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 11 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 5 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 10 host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed 15 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. 20 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 25 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 30 nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism. 12 As used herein, the term "growth-coupled" when used in reference to the production of a biochemical is intended to mean that the biosynthesis of the referenced biochemical is a product produced during the growth phase of a microorganism. "Non-growth-coupled" when used in reference to the production of a biochemical is intended to mean that the biosynthesis 5 of the referenced biochemical is a product produced during a non-growth phase of a microorganism. Production of a biochemical product can be optionally obligatory to the growth of the organism. As used herein, the term "metabolic modification" is intended to refer to a biochemical reaction that is altered from its naturally occurring state. Metabolic modifications can 10 include, for example, elimination of a biochemical reaction activity by functional disruptions of one or more genes encoding an enzyme participating in the reaction. Sets of exemplary metabolic modifications are illustrated in Table 1. Individual reactions specified by such metabolic modifications and their corresponding gene complements are exemplified in Table 2 for Escherichia coli. Reactants and products utilized in these reactions are exemplified in 15 Table 3. 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. 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 20 truncated gene product or by any of various mutation strategies that inactivate the encoded gene product. 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. The term "gene disruption" is also intended to mean a genetic alteration that lowers the activity of a given gene product relative to its 25 activity in a wild-type organism. This attenuation of activity can be due to, for example, a deletion in a portion of the gene which results in a truncated gene product or any of various mutation strategies that render the encoded gene product less active than its natural form, replacement or mutation of the promoter sequence leading to lower or less efficient expression of the gene, culturing the organism under a condition where the gene is less highly 30 expressed than under normal culture conditions, or introducing antisense RNA molecules that interact with complementary mRNA molecules of the gene and alter its expression. 13 As used herein, the term "stable" when used in reference to growth-coupled production of a biochemical product is intended to refer to microorganism that can be cultured for greater than five generations without loss of the coupling between growth and biochemical synthesis. Generally, stable growth-coupled biochemical production will be greater than 10 generations, 5 particularly stable growth-coupled biochemical production will be greater than about 25 generations, and more particularly, stable growth-coupted biochemical production will be greater than 50 generations, including indefinitely. Stable growth-coupled production of a biochemical can be achieved, for example, by disruption of a gene encoding an enzyme catalyzing each reaction within a set of metabolic modifications. The stability of growth 10 coupled production of a biochemical can be enhanced through multiple disruptions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism 15 such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli 20 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. 25 As used herein, the term "confers production" refers not only to organisms that lack operational metabolic pathways for the production of LCAs, but also to organisms that may have some level of production of LCAs. Thus, an organism that already generates LCAs can benefit from improved production conferred onto the organism by the disruption of one or more genes. 30 As used herein, the term "eukaryotic organism" refers to any organism having a cell type having specialized organelles in the cytoplasm and a membrane-bound nucleus enclosing genetic material organized into chromosomes. The term is intended to encompass all 14 eukaryotic organisms including eukaryotic microbial organisms such as yeast and fungi. The term also includes cell cultures of any eukaryotic species that can be cultured for the production of a biochemical where the eukaryotic species need not be a microbial organism. A "eukaryotic microbial organism," "microbial organism" or "microorganism" is intended to 5 mean any eukaryotic organism that exists as a microscopic cell that is included within the domain of eukarya. 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 10 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 15 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 20 activator and elastase, are considered to have arisen by vertical descent from a common ancestor. Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into 25 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 30 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 seine protease activity, into distinct molecules as 15 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. 5 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 10 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. 15 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 20 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 25 includes, for example, a paralog or an unrelated gene. Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species 30 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 16 a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes to ensure that any functional redundancy in enzymatic activities do not short circuit the designed metabolic modifications. Orthologs, paralogs and nonorthologous gene displacements can be determined by methods 5 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, 10 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 15 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 20 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. 25 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 30 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 17 increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences. The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refer to microorganisms that can be cultured for greater than five 5 generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. Those skilled in the art will understand that the genetic alterations, including metabolic 10 modifications exemplified herein are described with reference to Euglena gracilis, E. coli and S. cerevisiae genes and their corresponding metabolic reactions. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli 15 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. 20 In some embodiments, the invention provides a non-naturally occurring microbial organism having a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway having at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol, said malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or 25 ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl CoA reductase, said acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase. Malonyl-CoA-independent fatty acid synthesis is a metabolic process used by photosynthetic flagellate such as Euglena gracilis (Inui et al., Euro. J. Biochem. 96:931-34 (1984). These 30 single cell organisms exhibit both algae and protozoan characteristics and, depending on conditions, can utilize either light energy (photosynthesis) or chemical energy (eating) for 18 biochemical processes. Under anaerobic conditions, E. gracilis converts paramylon, the reserve beta-1,2-glucan polysaccharide, into wax ester with concomitant generation of ATP, a phenomenon named wax ester fermentation (Inui et al., supra, 1982; Inui et al., Agricultural and Biological Chemistry 47:2669-2671 (1983)). Fatty acid synthesis through the malonyl 5 CoA-independent pathway results in a net gain of ATP, whereas other fatty acid synthesis systems can not support the net gain of ATP. ATP also can be produced under aerobic conditions (Inui et al., Archives Biochemistry and Biophysics 237:423-29 (1985)). In the absence of oxygen, acetyl-CoA is generated from pyruvate via an oxygen-sensitive pyruvate:NADP+ oxidoreductase (Inui et al., supra, 1984; Inui et al., supra, 1985; Inui et al., 10 Archives of Biochemistry and Biophysics 280:292-98 (1990); Inui et al., Journal of Biological Chemistry 262:9130-35 (1987)), and serves as the terminal electron acceptor of glucose oxidation via the malonyl-CoA-independent fatty acid synthesis to form wax ester (Inui et al., supra, (1985)). E. gracilis contains five different systems of fatty acid synthesis, including four fatty acid synthesis systems located in different compartments, and the mitochondrial 15 malonyl-CoA-independent FAS system involved in anaerobic wax ester fermentation (Hoffmeister et al., J. ofBiological Chemistry 280:4329-38 (2005)). The malonyl-CoA independent FAS system has been shown to produce C8-C18 fatty acids. A fatty acid is reduced to alcohol, esterified with another fatty acid, and deposited in the cytosol as waxes (Inui et al., Febs Letters 150:89-93 (1982); Inui et al., European Journal ofBiochemistry 20 142:121-126 (1984)). The wax can constitute approximately 50% of the total lipid in dark grown cells (Rosenberg, A., Biochemistry 2:1148 (1963)). A particularly useful embodiment of the invention harness the malonyl-CoA-independent fatty acid synthesis (FAS) system under anaerobic conditions to produce large quantities of alcohols using the modified biosynthetic pathways described herein. 25 The malonyl-CoA-independent fatty acid synthesis pathway is similar to the reversal of fatty acid oxidation and is referred as the fatty acid synthesis in mitochondria or acyl-carrier protein (ACP)-independent fatty acid synthesis as it is known in the art. Compared to the malonyl-CoA-dependent fatty acid synthesis (a.k.a. ACP dependent fatty acid synthesis; Smith et al., Progress in Lipid Research 42:289-317 (2003); White et al., Annual Review of 30 Biochemistry 74:791-831 (2005)), there are several differences. First, acetyl-CoA is used as the extension unit instead of malonyl-ACP. Utilization of acetyl-CoA as elongation substrate in the malonyl-CoA-independent pathway eliminates the need for acetyl-CoA carboxylase 19 complex (ACC), which converts acetyl-CoA to malonyl-CoA, and thus conserves one ATP molecule per unit flux of acetyl-CoA entering the elongation cycle. Second, all of the intermediates in the elongation cycle are attached to CoA instead of ACP. The elongation cycle can include (i) ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase, EC 2.3.1.16), 5 (ii) 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 and 1.1.1.211), (iii) enoyl-CoA hydratase (EC 4.2.1.17 and 4.2.1.74), and (iv) enoyl-CoA reductase (EC 1.3.1.44 and 1.3.1.38). Third, the product from the elongation cycle is acyl-CoA, which can be utilized directly by acyl-CoA reductase, followed by a dehydrogenase for conversion to alcohol, or by fatty acid forming acyl-CoA reductase (FAR), which converts acyl-CoA directly to alcohol. 10 Therefore, thioesterase and acyl-CoA synthase are not required for the production of primary alcohols, as is the case with the malonyl-CoA-dependent pathways. For example, the microorganisms of the invention utilize the malonyl-CoA-independent fatty acid synthesis pathway coupled with the reduction of the fatty acid to form primary alcohol as illustrated in Figure 1. The microorganism can additionally be modified to convert, for 15 example, renewable feedstock to acetyl-CoA. In the bioengineered pathways of the invention, acetyl-CoA can be used as both a primer and an extension unit in the elongation cycle described above. At the end of each elongation cycle, an acyl-CoA is formed that is one C2 unit longer than the acyl-CoA entering the elongation cycle. Coupling the above synthesis pathway to a reduction pathway yields the primary alcohol products of the 20 invention. Particularly useful is the coupling of acyl-CoA having a desired chain-length to a reduction pathway that uses the combination of chain-length specific acyl-CoA reductase (EC 1.2.1.50) and alcohol dehydrogenase (1.1.1.1) or the fatty alcohol forming acyl-CoA reductase (FAR, EC 1.1.1) to form desired primary alcohol. Carbon chain-length of the primary alcohols can be controlled by chain-length specific enoyl-CoA reductase, ketoacyl 25 CoA thiolase and/or acyl-CoA reductase. The microorganisms of the invention having the coupled biosynthetic pathways described above can produce primary alcohols at very high levels. For example, the maximum theoretical yield for octanol using the malonyl-CoA-independent fatty acid biosynthetic pathway and the associated energetics were calculated by adding the malonyl-CoA 30 independent fatty acid synthesis, acyl-CoA reductase and alcohol dehydrogenase reactions to a predictive E. coli metabolic stoichiometric network using the in silico metabolic modeling system known in the art as SimPhenyTM (see, for example, U.S. Patent Application Serial No. 20 10/173,547, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003). The model assumes that the secretion of octanol does not require energy. Table 4 shows the maximum theoretical yield for octanol under both aerobic and anaerobic conditions. The malonyl-CoA-independent fatty acid biosynthetic 5 pathway is much more energy-efficient than the malonyl-CoA-dependent fatty acid synthesis pathways, and allows for a maximum theoretical yield of 0.5 mole octanol/mole of glucose and maximum ATP yield of 2.125 mole/mole of glucose under both aerobic and anaerobic conditions. Table 4: Comparison of the maximum theoretical yield of octanol using (1) the malonyl 10 CoA-independent fatty acid synthesis and acyl-reduction pathway and (2) the ACP-dependent fatty acid synthesis and pathway. Malonyl-CoA-independent Typical fatty acid fatty acid biosynthetic and biosynthetic and reduction pathway reduction pathway Anaerobic Aerobic Anaerobic Aerobic Octanol Yield (mole/mole glucose) 0.5 0.5 0.375 0.48 Max ATP Yield @ max octanol 2.125 2.125 0 0 yield (mole/mole glucose) I A non-naturally occurring microbial organism of the invention employs combinations of metabolic reactions for biosynthetically producing a target primary alcohol or a target 15 mixture of primary alcohols of the invention. The combination of metabolic reactions can be engineered in a variety of different alternatives to achieve exogenous expression of a malonyl-CoA-independent FAS pathway in sufficient amounts to produce a primary alcohol. The non-naturally occurring microbial organisms will express at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme. In certain embodiments, 20 the non-naturally occurring microbial organisms of the invention will be engineered to exogenously express more than one, including all, nucleic acids encoding some or all of the enzymes for the complete pathway of malonyl-CoA independent FAS pathway enzymes. Some or all of the enzymes for acyl-reduction also can be exogenously expressed. Exogenous expression should be at levels sufficient to produce metabolically utilizable gene 25 product and result in the production of a target primary alcohol or set of alcohols. 21 The biochemical reactions for formation of primary alcohols from a carbon or other energy source through a malonyl-CoA independent FAS pathway is shown in Figure 1. The malonyl-CoA independent FAS pathway produces acyl-CoA. Concomitant utilization of this intermediate product to produce target primary alcohols by an acyl-reduction pathway also is 5 shown in Figure 1. This invention is also directed, in part to engineered biosynthetic pathways to improve carbon flux through a central metabolism intermediate en route to a primary alcohol, a fatty acyl CoA, a fatty ester, or a wax. The present invention provides non-naturally occurring microbial organisms having one or more exogenous genes encoding enzymes that can 10 catalyze various enzymatic transformations en route to a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax. In some embodiments, these enzymatic transformations are part of the reductive tricarboxylic acid (RTCA) cycle and are used to improve product yields, including but not limited to, from carbohydrate-based carbon feedstock. In numerous engineered pathways, realization of maximum product yields based on 15 carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents and/or carbon to byproducts. In accordance with some embodiments, the present invention increases the yields of a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such 20 as CO, C0 2 , and/or H 2 . In addition to syngas, other sources of such gases include, but are not listed to, the atmosphere, either as found in nature or generated. The C0 2 -fixing reductive tricarboxylic acid (RTCA) cycle is an endergenic anabolic pathway of CO 2 assimilation which uses reducing equivalents and ATP (Figure 21). One turn of the RTCA cycle assimilates two moles of CO 2 into one mole of acetyl-CoA, or four moles of 25 CO 2 into one mole of oxaloacetate. This additional availability of acetyl-CoA improves the maximum theoretical yield of product molecules derived from carbohydrate-based carbon feedstock. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose and glycerol. In some embodiments, the reductive TCA cycle, coupled with carbon monoxide 30 dehydrogenase and/or hydrogenase enzymes, can be employed to allow syngas, C0 2 , CO, H 2 , and/or other gaseous carbon source utilization by microorganisms. Synthesis gas (syngas), in 22 particular is a mixture of primarily H 2 and CO, sometimes including some amounts of C0 2 , that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter. Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids 5 full combustion, of organic materials at high temperatures (500-1500*C) to provide syngas as a 0.5:1-3:1 H2/CO mixture. In addition to coal, biomass of many types has been used for syngas production and represents an inexpensive and flexible feedstock for the biological production of renewable chemicals and fuels. Carbon dioxide can be provided from the atmosphere or in condensed from, for example, from a tank cylinder, or via sublimation of 10 solid CO 2 . Similarly, CO and hydrogen gas can be provided in reagent form and/or mixed in any desired ratio. Other gaseous carbon forms can include, for example, methanol or similar volatile organic solvents. The components of synthesis gas and/or other carbon sources can provide sufficient C0 2 , reducing equivalents, and ATP for the reductive TCA cycle to operate. One turn of the 15 RTCA cycle assimilates two moles of CO 2 into one mole of acetyl-CoA and requires 2 ATP and 4 reducing equivalents. CO and/or H 2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively. Reducing equivalents can come in the form of NADH, NADPH, FADH, reduced quinones, reduced ferredoxins, thioredoxins, and reduced flavodoxins. The reducing equivalents, particularly 20 NADH, NADPH, and reduced ferredoxin, can serve as cofactors for the RTCA cycle enzymes, for example, malate dehydrogenase, fumarate reductase, alpha ketoglutarate:ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase), pyruvate:ferredoxin oxidoreductase and isocitrate dehydrogenase. The electrons from these 25 reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions, protons, or an electrode. The ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme. The reductive TCA cycle was first reported in the green sulfur photosynthetic bacterium 30 Chlorobium limicola (Evans et al., Proc. Nati. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways have been characterized in some prokaryotes (proteobacteria, green sulfur bacteria and thermophillic Knallgas bacteria) and sulfur-dependent archaea (Hugler et al., J. 23 Bacterial. 187:3020-3027 (2005; Hugler et al., Environ. Microbiol. 9:81-92 (2007). In some cases, reductive and oxidative (Krebs) TCA cycles are present in the same organism (Hugler et al., supra (2007); Siebers et al., J. Bacterial. 186:2179-2194 (2004)). Some methanogens and obligate anaerobes possess incomplete oxidative or reductive TCA cycles that may 5 function to synthesize biosynthetic intermediates (Ekiel et al., J. Bacterial. 162:905-908 (1985); Wood et al., FEMS Microbiol. Rev. 28:335-352 (2004)). The key carbon-fixing enzymes of the reductive TCA cycle are alpha ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase and isocitrate dehydrogenase. Additional carbon may be fixed during the conversion of 10 phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase or by conversion of pyruvate to malate by malic enzyme.. Many of the enzymes in the TCA cycle are reversible and can catalyze reactions in the reductive and oxidative directions. However, some TCA cycle reactions are irreversible in 15 vivo and thus different enzymes are used to catalyze these reactions in the directions required for the reverse TCA cycle. These reactions are: (1) conversion of citrate to oxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate, and (3) conversion of succinyl-CoA to alpha-ketoglutarate. In the TCA cycle, citrate is formed from the condensation of oxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrate to oxaloacetate and 20 acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, or citryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyase can be coupled to acetyl-CoA synthetase, an acetyl-CoA transferase, or phosphotransacetylase and acetate kinase to form acetyl-CoA and oxaloacetate from citrate. The conversion of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse reaction is catalyzed by fumarate reductase. In the 25 TCA cycle succinyl-CoA is formed from the NAD(P)* dependent decarboxylation of alpha ketoglutarate by the alpha-ketoglutarate dehydrogenase complex. The reverse reaction is catalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase. An organism capable of utilizing the reverse tricarboxylic acid cycle to enable production of acetyl-CoA-derived products on 1) CO, 2) CO 2 and H 2 , 3) CO and C0 2 , 4) synthesis gas 30 comprising CO and H 2 , and 5) synthesis gas or other gaseous carbon sources comprising CO, C0 2 , and H 2 can include any of the following enzyme activities: ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, aconitase, isocitrate dehydrogenase, alpha 24 ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoA synthetase, acetyl-CoA transferase, pyruvate:ferredoxin oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, hydrogenase, and 5 ferredoxin (see Figure 23). Enzymes and the corresponding genes required for these activities are described herein above. Carbon from syngas or other gaseous carbon sources can be fixed via the reverse TCA cycle and components thereof. Specifically, the combination of certain carbon gas-utilization pathway components with the pathways for formation of a primary alcohol, a fatty acyl-CoA, 10 a fatty ester, or a wax from acetyl-CoA results in high yields of these products by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously or produced endogenously from CO, into acetyl-CoA. In some embodiments, a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway in a non-naturally occurring microbial organism of the invention can utilize any combination of 15 (1) CO, (2) C0 2 , (3) H 2 , or mixtures thereof to enhance the yields of biosynthetic steps involving reduction, including addition to driving the reductive TCA cycle. In some embodiments a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic 20 acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha ketoglutarate:ferredoxin oxidoreductase; and at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C0 2 , (3) 25 H2, (4) CO 2 and H 2 , (5) CO and C0 2 , (6) CO and H 2 , or (7) CO, C0 2 , and H2. In some embodiments a method includes culturing a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway also comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, 30 a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. Additionally, such an 25 organism can also include at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C0 2 , (3) H 2 , (4) CO 2 and H 2 , (5) CO and C0 2 , (6) CO and H 2 , or (7) CO, C0 2 , and H 2 to produce a product. 5 In some embodiments a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA 10 lyase, a fumarate reductase, a pyruvate:ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability 15 of reducing equivalents in the presence of carbon monoxide and/or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. The at least one exogenous nucleic acid is selected from a carbon monoxide dehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin. In some embodiments, the present invention provides a method for enhancing the availability of 20 reducing equivalents in the presence of carbon monoxide or hydrogen thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock, such as sugars or gaseous carbon sources, the method includes culturing this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax. 25 In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway includes two exogenous nucleic acids, each encoding a reductive TCA pathway enzyme. In some embodiments, the non naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway includes three exogenous nucleic acids each encoding a reductive 30 TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding an ATP-citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the 26 non-naturally occurring microbial organism includes three exogenous nucleic acids encoding a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non-naturally occurring microbial organism includes four exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase; a 5 phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase, a CO dehydrogenase; and an H 2 hydrogenase. In some embodiments, the non-naturally occurring microbial organism includes two exogenous nucleic acids encoding a CO dehydrogenase and an 12 hydrogenase. In some embodiments, the non-naturally occurring microbial organisms having a primary 10 alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further include an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, and combinations 15 thereof. In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further includes an exogenous nucleic acid encoding an enzyme selected from carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin, NAD(P)H:ferredoxin oxidoreductase and combinations thereof. 20 In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway utilizes a carbon feedstock selected from (1) CO, (2) CO 2 , (3) CO 2 and H2, (4) CO and H 2 , or (5) CO, CO 2 , and 112. In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway utilizes hydrogen for reducing equivalents. In 25 some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway utilizes CO for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway utilizes combinations of CO and hydrogen for reducing equivalents. 30 In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further includes one or more 27 nucleic acids encoding an enzyme selected from a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a pyruvate carboxylase, and a malic enzyme. In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further includes one or more 5 nucleic acids encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA synthetase, and a succinyl-CoA transferase. In some embodiments, the non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax pathway further includes at least one exogenous nucleic acid encoding a citrate lyase, an ATP-citrate lyase, a citryl-CoA 10 synthetase, a citryl-CoA lyase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, and a ferredoxin. It is understood by those skilled in the art that the above-described pathways for increasing product yield can be combined with any of the pathways disclosed herein, including those 15 pathways depicted in the figures. One skilled in the art will understand that, depending on the pathway to a desired product and the precursors and intermediates of that pathway, a particular pathway for improving product yield, as discussed herein above and in the examples, or combination of such pathways, can be used in combination with a pathway to a desired product to increase the yield of that product or a pathway intermediate. 20 In one embodiment, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a primary alcohol pathway comprising at least one exogenous nucleic acid encoding a primary alcohol pathway enzyme expressed in a sufficient amount to produce a primary alcohol; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic 25 acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a 30 pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at 28 least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an

H

2 hydrogenase, and combinations thereof; and wherein said primary alcohol pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA 5 hydratase and enoyl-CoA reductase; and an acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase. In a specific embodiment, said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, 10 an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate 15 dehydrogenase, and combinations thereof. In another specific embodiment, said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme. In another specific embodiment, said microbial organism comprises four exogenous nucleic acids encoding malonyl-CoA-independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase 20 or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and two exogenous nucleic acids encoding acyl-reduction pathway enzymes comprising an acyl-CoA reductase and an alcohol dehydrogenase. In another specific embodiment, said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another 25 specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. In 30 another specific embodiment, said exogenous nucleic acid encoding an acyl-reduction pathway enzyme in said non-naturally occurring microbial organism comprises an enzyme having acyl-CoA reductase and alcohol dehydrogenase activity, e.g., fatty alcohol forming acyl-CoA reductase (FAR). In another embodiment, said non-naturally occurring microbial organism further comprises an acyl-reduction pathway comprising an acyl-CoA hydrolase, an 29 acyl-CoA transferase or an acyl-CoA ligase; a carboxylic acid reductase and an alcohol dehydrogenase. In another specific embodiment, said primary alcohol is produced in amounts at least 10% greater levels compared to a microbial organism lacking said exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme. In 5 another specific embodiment, said primary alcohol comprises an alcohol having between 4 24 carbon atoms, e.g., said primary alcohol is selected from butanol, hexanol, heptanol, octanol, nonanol, decanol, dodecanol, tetradecanol or hexadecanol. In another embodiment, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a fatty acyl-CoA pathway comprising at least one 10 exogenous nucleic acid encoding a fatty acyl-CoA pathway enzyme expressed in a sufficient amount to produce a fatty acyl-CoA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA 15 lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at 20 least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an

H

2 hydrogenase, and combinations thereof; and wherein said fatty acyl-CoA pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoAreductase. In a specific embodiment, said microbial organism 25 comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another 30 specific embodiment, said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, said microbial 30 organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme. In another specific embodiment, said microbial organism comprises four exogenous nucleic acids encoding malonyl-CoA-independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase 5 or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoAreductase. In another specific embodiment, said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA 10 pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, the invention provides a non-naturally occurring microbial organism, 15 comprising a microbial organism having a fatty ester pathway comprising at least one exogenous nucleic acid encoding a fatty ester pathway enzyme expressed in a sufficient amount to produce a fatty ester; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid 20 is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a 25 phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an

H

2 hydrogenase, and combinations thereof; and wherein said fatty ester pathway comprises a pathway selected from: (a) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises 30 ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an alcohol acetyltransferase, wherein said malonyl-CoA independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl- CoA 31 thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. In a specific embodiment, said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a 5 succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a 10 succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme. In another specific embodiment, said microbial organism comprises (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis 15 (FAS) pathway enzymes and one exogenous nucleic acid encoding a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl CoA hydratase and an enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one exogenous 20 nucleic acid encoding an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl- CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase. In another specific embodiment, said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another 25 specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. 30 In another embodiment, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a wax pathway comprising at least one exogenous nucleic acid encoding a wax pathway enzyme expressed in a sufficient amount to produce a wax; said non-naturally occurring microbial organism further comprising: (i) a reductive 32 TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway 5 comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and 10 combinations thereof; and wherein said wax pathway comprises a pathway selected from: (a) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis 15 (FAS) pathway and an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl- CoA thiolase, 3 hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. In a specific embodiment, said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an 20 aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof In another specific embodiment, said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an 25 aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme. In another specific embodiment, said microbial organism comprises 30 (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one nucleic acid encoding a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and and enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA 33 independent fatty acid synthesis (FAS) pathway enzymes and one nucleic acid encoding an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase. In another specific 5 embodiment, said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring 10 microbial organism is a heterologous nucleic acid. In another specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having an acyl-ACP pathway comprising at least one exogenous nucleic acid encoding an acyl-ACP pathway enzyme expressed in a sufficient 15 amount to produce acyl-ACP; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a 20 reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an 25 H 2 hydrogenase, and combinations thereof; and wherein said acyl-ACP pathway comprises a pathway selected from: (A) acetyl-CoA carboxylase and a fatty acid synthase; and (B) a fatty acid synthase. In a specific embodiment, said non-naturally occurring microbial organism further comprises a fatty acid pathway comprising an exogenous nucleic acid encoding a thioesterase expressed in a sufficient amount to produce fatty acid. In another 30 specific embodiment, said non-naturally occurring microbial organism further comprises an acyl-CoA pathway comprising an exogenous nucleic acid encoding an acyl-CoA synthetase or an acyl-CoA ligase expressed in a sufficient amount to produce acyl-CoA. In another specific embodiment, said non-naturally occurring microbial organism further comprises a 34 fatty aldehyde pathway comprising an exogenous nucleic acid enconding an acyl-CoA reductase (aldehyde forming) or carboxylic acid reductase expressed in a sufficient amount to produce a fatty aldehyde and/or further comprising a fatty alcohol pathway comprising an exogenous nucleic acid encoding an acyl-CoA reductase (alcohol forming) expressed in a 5 sufficient amount to produce a fatty alcohol. In another specific embodiment, said non naturally occurring microbial organism further comprises a fatty alcohol pathway comprising an exogenous nucleic acid encoding an aldehyde reductase expressed in a sufficient amount to produce a fatty alcohol. In another specific embodiment, said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected 10 from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, said microbial organism comprising (ii) further comprises an 15 exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a acyl-ACP, fatty acid, acyl-CoA, fatty adehyde or fatty alcohol pathway enzyme. In another 20 specific embodiment, said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said said at least one exogenous nucleic acid is a heterologous nucleic 25 acid. In another specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, 30 an acyl-ACP, a fatty acid, acyl-CoA, a fatty aldehyde or a fatty alcohol or any primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake 35 sources can provide isotopic enrichment for any atom present in the product primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate, or for side products generated in 5 reactions diverging away from a primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. In some embodiments, the uptake sources can be selected to alter the carbon- 12, carbon-13, 10 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, 15 and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios. In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural 20 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 25 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 30 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 36 atmospheric carbon source, such as C0 2 , which can possess a larger amount of carbon-14 than its petroleum-derived counterpart. The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 1012 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon 5 is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ("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". 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 10 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. 15 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 20 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. The biobased content of a compound is estimated by the ratio of carbon-14 ("C) to carbon- 12 (1 2 C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S B)/(M-B), where B, S and M represent the "C/1 2 C ratios of the blank, the sample and the 25 modern reference, respectively. Fraction Modern is a measurement of the deviation of the 4

C/

12 C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to 6'CVPDB=- 19 per mil (Olsson, The use of Oxalic acid as a Standard. in, Radiocarbon Variations and Absolute Chronology, 30 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 37 definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to S 1 CvpDB=-1 9 per mil This is equivalent to an absolute (AD 1950) 14

C/

12 C ratio of 1,176 0.0 10 x 1012 (Karlen et al., Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one istope with respect to another, for example, 5 the preferential uptake in biological systems of C 12 over C 13 over C' 4 , and these corrections are reflected as a Fm corrected for 6s. 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 10 from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modem standard (see discussion of original vs. currently available oxalic acid standards in 15 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. 20 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 D6866-1 1. 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 25 environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old. ASTM D6866 quantifies the biobased content relative to the material's total organic content 30 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) 38 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 5 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. 10 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 15 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). 20 Accordingly, in some embodiments, the present invention provides primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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. 25 For example, in some aspects the primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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 last 60%, at 30 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 primary alcohol, fatty acyl-CoA, fatty 39 ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate that has a carbon- 12, carbon- 13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the primary alcohol, fatty acyl-CoA, 5 fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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 10 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 primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that is 15 obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon 12, carbon- 13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources. Further, the present invention relates to the biologically produced primary alcohol, fatty acyl 20 CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate as disclosed herein, and to the products derived therefrom, wherein the primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a primary alcohol, fatty acyl-CoA, fatty 25 ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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 primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a bioderived primary alcohol, fatty acyl-CoA, fatty ester, wax, 30 acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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 40 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 primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a bioderived primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty 5 acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described 10 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 15 materials or acrylates are generated directly from or in combination with bioderived primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol or a bioderived primary alcohol, fatty acyl-CoA, fatty ester, wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate as disclosed herein. Primary alcohols, fatty esters, waxs, fatty acids, fatty aldehydes and fatty alcohols are 20 chemicals 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, 25 flavor materials or acrylates comprising one or more bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. As used herein, the term "bioderived" means derived from or synthesized by a biological 30 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 41 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 5 product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock. In some embodiments, the invention provides a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate comprising bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or 10 bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate, wherein the bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate includes all or part of the primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or primary alcohol, fatty ester, wax, fatty acid, 15 fatty aldehyde or fatty alcohol intermediate used in the production of 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, fragrance, flavor material or acrylate comprising at least 2%, at least 3%, at least 5%, at least 10%, at 20 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/o, at least 95%, at least 98% or 100% bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate as disclosed herein. Additionally, in some aspects, the invention provides a 25 biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate wherein the primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate used in its production is a combination of bioderived and petroleum derived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty 30 alcohol or primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate. For example, a biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate can be produced using 50% bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or 42 fatty alcohol and 50% petroleum derived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10//90% of bioderived/petroleum derived precursors, so long as at least a portion of the product 5 comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate using the bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol or bioderived primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol 10 intermediate of the invention are well known in the art. In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or 15 protein that converts a substrate to a product selected from the group consisting of p ketoacyl-CoA to p-hydroxyacyl-CoA, p-hydroxyacyl-CoA to trans-2-enoyl-CoA, trans-2 enoyl-CoA to acyl-CoA, acyl-CoA to p-ketoacyl-CoA, acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, acyl-CoA to a fatty acid, a fatty acid to a fatty aldehyde, oxaloacetate to malate, malate to fumarate, fumarate to succinate, succinate to succinyl-CoA, 20 succinyl-CoA to a-ketoclutarate, a-ketoclutarate to D-isocitrate, D-isocitrate to citrate, citrate to acetate, citrate to oxaloacetate, acetate to acetyl-CoA, citrate to acetyl-CoA, acetyl-CoA to malonly-CoA, acetyl-CoA and malonyl-CoA to acyl-ACP, acyl-ACP to a fatty acid, a fatty acid to acyl-CoA, acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, a fatty acid to a fatty aldehyde and an acyl-CoA to a fatty alcohol. One skilled in the art will 25 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 30 enzyme or protein, where the enzyme or protein converts the substrates and products of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway, such as that shown in Figure 1, 9-22, 25 and 26. 43 While generally described herein as a microbial organism that contains a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway, it is understood that the invention additionally provides a non naturally occurring microbial organism comprising at least one exogenous nucleic acid 5 encoding a a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway enzyme expressed in a sufficient amount to produce an intermediate of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway. For example, as disclosed herein, a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty 10 acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway is exemplified in Figures 1, 9-22, 25 and 26. Therefore, in addition to a microbial organism containing a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway that produces a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty-alcohol, the invention additionally 15 provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway enzyme, where the microbial organism produces a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate. 20 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, 9-22, 25 and 26, 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 25 expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate can be utilized to produce the intermediate as a desired product. 30 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 44 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 hereint, reference to a reactant or product also references the reaction, and reference to any of these metabolic 5 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 10 products of the reaction. As disclosed herein, the product fatty acid, as well as other intermediates, are carboxylic acids, which can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix "-ate," or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated 15 form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that carboxylate products or intermediates includes ester forms of carboxylate products or pathway intermediates, such as O-carboxylate and S carboxylate esters. 0- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates. Some such 0- or S-carboxylates include, without limitation, 20 methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl 0- or S carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl 0- or S-carboxylates. O-carboxylates can be the product of a biosynthetic pathway. Exemplary O-carboxylates accessed via biosynthetic pathways can include, without limitation, methyl fatty acid, ethyl fatty acid, and n-propyl 25 fatty acid. Other biosynthetically accessible O-carboxylates can include medium to long chain groups, that is C7-C22, 0-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stcaryl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations. O-carboxylate esters can also be 30 accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an 0- or S-carboxylate. S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters. 45 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthetic pathways. 5 Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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) 10 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthesis. Thus, a non-naturally occurring 15 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl 20 CoA, fatty aldehyde or fatty alcohol. Depending on the a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol 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 a primary alcohol, a fatty acyl 25 CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more [a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthetic pathways. For example, a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or 30 fatty alcohol 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway, exogenous 46 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 a primary alcohol, a fatty acyl-CoA, a fatty ester, a 5 wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol can be included, such as a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl CoA, fatty aldehyde or fatty alcohol . Generally, a host microbial organism is selected such that it produces the precursor of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, 10 fatty aldehyde or fatty alcohol pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol is produced naturally in a host organism such as E. 15 coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway. 20 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 primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthetic pathway onto the microbial organism. 25 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 primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl CoA, fatty aldehyde or fatty alcohol biosynthetic capability. For example, a non-naturally occurring microbial organism having a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, 30 an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of fatty acid synthase and a thioesterase, or alternatively a aconitase and a 47 aldehyde reductase, or alternatively an alpha-detoglutarate ferredoxin oxidoreductase and a fatty alcohol forming acyl-CoA 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 5 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 CO dehydrogenase, a fumarase and a enoyl-CoA reductase, or alternatively an acyl-CoA reductase, and alcohol dehydrogenase and a hydrogenase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in 10 production of the corresponding desired product. Similarly, any combination of four, a CO dehydrogenase, a ATP citrate lyase, a carboxyl acid reductase and a alcohol dehydrogenase, or alternatively an acetyl-CoA carboxylase, an fatty acid synthease, a thioesterase and a isocitratedehydrogenase, 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 15 desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. In addition to the biosynthesis of primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol as described herein, the non naturally occurring microbial organisms and methods of the invention also can be utilized in 20 various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol other than use of the primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or 25 fatty alcohol producers is through addition of another microbial organism capable of converting a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate to primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehydc or fatty alcohol. One such procedure includes, for example, the fermentation of a microbial organism 30 that produces a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate. The primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate can then be used as a substrate for a second microbial 48 organism that converts the primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl ACP, fatty acid, acyl-CoA, fatty aldchyde or fatty alcohol pathway intermediate to primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol. The primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an 5 acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate can be added directly to another culture of the second organism or the original culture of the primary alcohol, a fatty acyl-CoA, a fatty ester, a, wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second 10 organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, 15 acyl-CoA, fatty aldehyde or fatty alcohol. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 20 primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol also can be 25 biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate and the second microbial organism converts the intermediate to primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty 30 acid, acyl-CoA, fatty aldehyde or fatty alcohol. Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring 49 microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, 5 acyl-CoA, fatty aldehyde or fatty alcohol. Microbial organisms other than Euglena gracilis generally lack the capacity to synthesize acyl-CoA through a malonyl-CoA independent FAS pathway. Moreover, organisms having all of the requisite metabolic enzymatic capabilities are not known to produce acyl-CoA from the enzymes described and biochemical pathways exemplified herein. Rather, 10 microorganisms having the enzymatic constituents of malonyl-CoA independent FAS pathway operate to degrade short, medium, and long chain fatty-acyl-CoA compounds to acetyl-CoA. E. gracilis, having a malonyl-CoA independent FAS pathway, utilizes this pathway to produce acylglycerols, trihydric sugar alcohols, phospholipids, wax esters and/or fatty acids. In contrast, the non-naturally occurring microbial organisms of the invention 15 generate acyl-CoA as a product of the malonyl-CoA independent FAS pathway and funnel this product into an acyl-reduction pathway via favorable thermodynamic characteristics. Product biosynthesis of using the non-naturally occurring organisms of the invention is not only particularly useful for the production of primary alcohols, it also allows for the further biosynthesis of compounds using acyl-CoA and/or primary alcohols as an intermediate 20 reactant. The non-naturally occurring primary alcohol-producing microbial organisms of the invention are generated by ensuring that a host microbial organism includes functional capabilities for the complete biochemical synthesis of a malonyl-CoA independent fatty acid biosynthetic pathway and for an acyl-reduction pathway of the invention. Ensuring complete functional 25 capabilities for both pathways will confer primary alcohol biosynthesis capability onto the host microbial organism. The enzymes participating in a malonyl-CoA independent FAS pathway include ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. The enzymes participating in an acyl-reduction pathway include an acyl-CoA reductase and an alcohol dehydrogenase or 30 an enzyme having dual reductase and dehydrogenase activity. The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes participating in 50 the malonyl-CoA independent FAS and/or acyl-reduction pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of these biosynthetic pathways can be expressed. For example, if a chosen host is deficient in all of the enzymes in the malonyl-CoA independent FAS pathway, then expressible nucleic acids 5 for each of the four enzymes ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3 hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase are introduced into the host for subsequent exogenous expression. Alternatively, for example, if the chosen host is deficient less than all four of the above enzymes, then all that is needed is to express nucleic acids encoding the deficient enzymes. For example, if a host is deficient 10 in 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase a functionally complete malonyl-CoA independent FAS pathway can be engineererd by introduction of nucleic acids encoding these two enzymes. In like fashion, where endogenous host biosynthetic machinery is complete for an acyl reduction pathway, then genetic modification is unnecessary. However, if host capabilities 15 are deficient in either or both of the acyl-CoA reductase and/or alcohol dehydrogenase activities, then introduction of the deficient activity by expression of an exogenous encoding nucleic acid is needed. Accordingly, depending on the malonyl-CoA independent FAS and acyl-reduction pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously 20 expressed malonyl-CoA independent FAS pathway-encoding nucleic acid and up to all six malonyl-CoA independent FAS and acyl-reduction pathway encoding nucleic acids. 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 parallel the malonyl-CoA independent FAS and acyl-reduction pathway deficiencies of the selected host 25 microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five or six encoding nucleic acids encoding the above enzymes constituting the malonyl-CoA independent FAS pathway, an acyl-reduction pathway or both the malonyl-CoA independent FAS and acyl-reduction biosynthetic pathways. In some embodiments, the non-naturally occurring microbial organisms also can 30 include other genetic modifications that facilitate or optimize acyl-CoA and/or primary alcohol 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 51 or more of the malonyl-CoA independent FAS pathway precursors such as acetyl-CoA, p ketoacyl-CoA, p-hydroxyacyl-CoA, trans-2-enoyl-CoA and/or fatty aldehyde. In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize acyl-CoA through 5 a malonyl-CoA independent FAS pathway, or having the capability to catalyze one or more of the enzymatic steps within the malonyl-CoA independent FAS and/or acyl-reduction pathways. In these specific embodiments it can be useful to increase the synthesis or accumulation of a malonyl-CoA independent FAS pathway product or an acyl-reduction pathway product to, for example, efficiently drive malonyl-CoA independent FAS and/or 10 acyl-reduction pathway reactions toward primary alcohol production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described malonyl-CoA independent FAS and/or acyl-reduction pathway enzymes. Overexpression of the desired pathway enzyme or enzymes can occur, for example, through exogenous expression of the endogenous gene or genes, or through 15 exogenous expression of a heterologous gene or genes. Therefore, naturally occurring organisms can readily be generated to be non-naturally primary alcohol producing microbial organisms of the invention through overexpression of one, two, three, four, five or all six nucleic acids encoding a malonyl-CoA independent FAS and/or a acyl-reduction pathway enzymes. In addition, a non-naturally occurring organism can be generated by mutagenesis 20 of an endogenous gene that results in an increase in activity of an enzyme in the malonyl CoA independent FAS and/or acyl-reduction biosynthetic pathways. 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 25 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. For example, activation offadB, an E. coli gene having malonyl-CoA independent FAS activity corresponding to 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities can 30 be accomplished by genetically knocking out a negative regulator,fadR, and co-expressing a heterologous ketothiolase (phaA from Ralstonia eutropha; Sato et al., Journal ofBioscience and Bioengineering 103:38-44 (2007)). Thus, an endogenous gene having a naturally 52 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 5 regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism. Additionally, for example, if an endogenous enzyme or enzymes operate in a reverse direction to the desired malonyl-CoA independent FAS pathway, genetic modifications can be made to attenuate or eliminate such activities. For example, within the malonyl-CoA 10 independent FAS pathway, the ketothiolase, dehydrogenase, and enoyl-CoA hydratase steps are reversible whereas the enoyl-CoA reductase step is primarily oxidative under physiological conditions (Hoffneister et al., Journal ofBiological Chemistry 280:4329-4338 (2005); Campbell, J. W. and J. E. Cronan, Jr., JBacteriol. 184:3759-3764 (2002)). To accomplish reduction of a 2-enoyl-CoA intermediate a genetic modification can be 15 introduced to attenuate or eliminate the reverse oxidative reaction. Sources of encoding nucleic acids for a malonyl-CoA independent FAS and/or acyl-reduction pathway enzyme 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, 20 and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Acetobacter aceti, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp., Acinetobacter sp. Strain M-1, Actinobacillus succinogenes, Aedes aegypti, Aeropyrum pernix ,Alcanivorax borkumensis SK2 ,Alkaliphilus metalliredigens QYMF, Alkaliphilus oremlandii OhILAs, 25 Allochromatium vinosum DSM 180, Anaerobiospirillum succiniciproducens, Anopheles gambiae str. PEST, Apis melhfera, Aquifex aeolicus, Arabidopsis thaliana, Archaeoglobus fulgidus, Archaeoglobusfulgidus DSM 4304, Aramatoleum aromaticum EbNJ, Aspergillus nidulans, Aspergillus niger, Azoarcus sp. T, Azotobacter vinelandii DJ, Bacillus megaterium, Bacillus sp. SG-1, Bacillus sphaericus, Bacillus subtilis, Bacillus weihenstephanensis 30 KBAB4, Balnearium lithotrophicum, Bifidobacterium bifidum, Bifidobacterium longum NCC2705, Brassica juncea, Brevibacterium sp., Burkholderia multivorans A TCC 17616, butyrate-producing bacterium L2-50, Caenorhabditis briggsae AFJ6, Caenorhabditis 53 elegans, Campylobacter curvus 525.92, Campylobacterjejuni, Candida albicans, Candida Antarctica, Canis lupus.familiaris (dog), Carboxydothermus hydrogenoformans, Chlorobium phaeobacteroides DSM 266, Chlorobium limicola, Citrobacter youngae A TCC 29220, Clostridium acetobutylicum, Clostridium carboxidivorans P7, Clostridium cellulolyticum 5 Hi0, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium pasteurianum, Clostridium saccharoperbutylacetonicum, Clostridium tetani E88, Colwellia psychrerythraea 34H, Corynebacterium glutamicum, Corynebacterium u/cerans, Cryptococcus neoformans var, Cyanobium PCC7001, Danio rerio, Desulfatibacillum alkenivorans AK-01, Desu/fococcus oleovorans Hxd3, Desulfovibrio africanus, Desufo Vibrio desu/furicans G20, 10 Desulfovibrio desulfuricans subsp. desulfuricans str. A TCC 27774, Desu/fovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Dictyostelium discoideum, Dictyostelium discoideum AX4, Escherichia coli K12, Escherichia coli K12 MGJ 655, Escherichia coli str. K-12 substr. MG1655, Euglena gracilis, Flavobacteria bacterium BAL38, Fragaria x ananassa, Fusarium oxysporum, Fusarium proliferatum, Geobacter 15 metallireducens GS-15, Geobacter su/furreducens, Haemophilus influenza, Haloarcula marismortui, Helianthus annuus, Helicobacter pylori, Helicobacter pylori 26695, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumonia, Lactococcus lactis, Leuconostoc mesenteroides, Macaca mulatta, Magnetospirillum magneticum AMB-1, Mannheimia succiniciproducens, Marinobacter aquaeolei VT8, Marinobacter 20 hydrocarbonoclasticus, Methanosarcina thermophila, Methanothermobacter thermautotrophicus, Methanothermobacter thermautotrophicus, Methylobacterium extorquens, Monosiga brevicollis MXJ, Moorella thermoacetica, Mus musculus, Mycobacterium avium subsp. paratuberculosis K-1 0, Mycobacterium bovis BCG, Mycobacterium kansasii ATCC 12478, Myvcobacterium marinum M, Mycobacterium 25 marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacterium sp. MCS, Mycobacterium tuberculosis H3 7Rv, Myxococcus xanthus DK 1622, Nematostella vectensis, Nocardiafarcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120, Opitutaceae bacterium TA V2, Pelobacter carbinolicus DSM 2380, Pelotomaculum thermopropionicum, Penicillium chrysogenum, Photobacterium sp. 30 SKA34, Picea sitchensis, Pseudomonas aeruginosa PA01, Pseudomonasfluorescens, Pseudomonas mendocina, Pseudomonas putida, Pseudomonas putida GB-1, Pseudomonas sp, Pseudomonas sp. CF600, Pseudomonas stutzeri A1501, Pyrobaculum aerophilum str. IM2, Ralstonia eutropha, Ralstonia eutropha H16, Rattus norvegicus, Reinekea sp. MED297, Rhizobium etli CFN 42, Rhizobium leguminosarum, Rhodobacter capsulatus, Rhodococcus 54 erythropolis SK121, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA 009, Rhadospirillum rubrum, Rosa hybrid cultivar, Roseovarius sp. HTCC2601, Saccharomyces cerevisiae, Salmonella enteric, Salmonella enterica subsp. arizonae serva, Salmonella typhimurium, Schizosaccharomycespombe, Simmondsia chinensis, Simmandsia chinensis, 5 Sordaria macrospora, Stenotrophomonas maltophilia, Streptomyces griseus subsp. griseus NBRC 13350, Strongylocentratus purpuratus, Sulfolobus acidocalarius, Sulfolobus sp. strain 7, Sulfurihydrogenibium subterraneum, Sulfurimonas denitrificans, Synechocystis str. PCC 6803, Synthetic variant, Tetraodon nigroviridis, Thauera aromatic, Thermotoga maritime, Thermus thermophilus, Thiobacillus denitrificans, Thiocapsa roseopersicina, Treponema 10 denticola, Tribolium castaneum, Trichomonas vaginalis G3, Trypanosoma brucei, Trypanosoma brucei, Trypanosoma cruzi strain CL Brener, Tsukamurella paurometabola DSM 20162, Vibrio cholerae V51, Xanthomonas campestris, Xenopus tropicalis, Yarrowia lipolytica, Yersinia intermedia A TCC 29909, and Zea mays, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, 15 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 malonyl-CoA independent FAS and/or acyl-reduction biosynthetic activity for one or more genes in related or distant species, including for 20 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 enabling biosynthesis of primary alcohols of the invention described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic 25 organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms. In some instances, such as when an alternative malonyl-CoA independent FAS constituent enzyme or pathway exists in an unrelated species, primary alcohol biosynthesis can be 30 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 55 actual genes 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 5 species of interest that will synthesize the primary alcohol products of the invention. Encoding nucleic acids and species that can be used as sources for conferring malonyl-CoA independent FAS and/or acyl-reduction pathway capability onto a host microbial organism are exemplified further below. In one exemplary embodiment, the genesfadA andfadB encode a multienzyme complex that exhibits three constituent activities of the malonyl-CoA 10 independent FAS pathway, namely, ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activities (Nakahigashi, K. and H. Inokuchi, Nucleic Acids Research 18:4937 (1990); Yang et al, Journal of Bacteriology 173:7405-7406 (1991); Yang et al, Journal ofBiological Chemistry 265:10424-10429 (1990); Yang et al., Biochemistry 30:6788-6795 (1990)). Thefad! andfadJ genes encode similar activities which 15 can substitute for the above malonyl-CoA independent FAS conferring genesfadA andfadB. These genes are naturally expressed under anaerobic conditions (Campbell and Cronan, supra, (2002)). The nucleic acid sequences for each of the abovefad genes are well known in the art and can be accessed in the public databases such as Genbank using the following accession numbers. 20 fadA YP_026272.1 Escherichia coli fadB NP_418288.1 Escherichia coli fadI NP_416844.1 Escherichia coli fadJ NP_416843.1 Escherichia coli fadR NP_415705.1 Escherichia coli 25 Other exemplary genes for the ketothiolase step include atoB which can catalyze the reversible condensation of 2 acetyl-CoA molecules (Sato et al., supra, 2007), and its homolog yqeF. Non-E. coli genes that can be used include phaA from R. eutropha (Jenkins, L. S. and W. D. Nunn. Journal ofBacteriology 169:42-52 (1987)), and the two ketothiolases, thiA and thiB, from Clostridium acetobutylicum (Winzer et al., Journal of Molecular Microbiology 30 and Biotechnology 2:531-541 (2000)). The sequences for these genes can be found at the following Genbank accession numbers: atoB NP_416728.1 Escherichia coli yqeF NP_417321.2 Escherichia coli 56 phaA Y P_725941 Ralstonia eutropha thiA N P_349476.1 Clostridium acetobutylicum thiB NP_149242.1 Clostridium acetobutylicum 5 An exemplary gene from E. coli which can be used for conferring 3-hydroxyacyl-CoA dehydrogenase transformation activity is paaH (Ismail et al., European Journal of Biochemistry 270:3047-3054 (2003)). Non-E. coli genes applicable for conferring this activity include AA072312.1 from E. gracilis (Winkler et al., Plant Physiology 131:753-762 (2003)), paaC from Pseudomonas putida (Olivera et al., PNAS USA 95:6419-6424 (1998)), 10 paaC from Pseudomonasfluorescens (Di Gennaro et al., Archives ofMicrobiology 188:117 125 (2007)), and hbd from C. acetobutylicum (Atsumi et al., Metabolic Engineering (2007) and Boynton et al., Journal ofBacteriology 178:3015-3024 (1996)). The sequences for each of these exemplary genes can be found at the following Genbank accession numbers: paaH NP_415913.1 Escherichia coli 15 AA072312.1 Euglena gracilis paaC NP_745425.1 Pseudomonas putida paaC ABF82235.1 Pseudomonasfluorescens hbd NP_349314.1 Clostridium acetobutylicum 20 Exemplary genes encoding the enoyl-CoA hydratase step include, for example, maoC (Park and Lee, Journal Bacteriology 185:5391-5397 (2003)), paaF (Ismail et al., European Journal of Biochemistry 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346 (2004) and Park and Yup, Biotechnol. Bioeng. 86:681-686 (2004)), and paaG (Ismail et al., European Journal ofBiochemistry 270:3047-3054 (2003); Park and Lee, 25 Appl. Biochem. Biotechnol. 113-116:335-346 (2004) and Park and Yup, Biotechnol. Bioeng. 86:681-686 (2004)). Other genes which can be used to produce the gene product catalyzing this step , for example, paaA, paaB, and paaN from P. putida (Olivera et al., PNAS USA 95:6419-6424 (1998)) and P. fluorescens (Di Gennaro et al., Archives ofMicrobiology 188:117-125 (2007)). The gene product of crt from C. acetobutylicum also can be used 30 (Atsumi et al., Metabolic Engineering (2007) and Boynton et al., Journal ofBacteriology 178: 3015-3024 (1996. The sequences for each of these exemplary genes can be found at the following Genbank accession numbers: maoC NP_415905.1 Escherichia coli paaF NP_415911.1 Escherichia coli 35 paaG NP_415912.1 Escherichia coli paaA NP_745427.1 Pseudomonas putida paaA ABF82233.1 Pseudomonasfluorescens 57 paaB NP_745426.1 Pseudomonas putida paaB ABF82234.1 Pseudomonasfluorescens paaN NP_745413.1 Pseudomonasputida paaN ABF82246. 1 Pseudomonasfluorescens 5 crt NP_349318.1 Clostridium acetobutylicum An exemplary gene which can be introduced into a non-naturally occurring microbial organism of the invention to confer enoyl-CoA reductase activity is the mitochondrial enoyl CoA reductase from E. gracilis Hoffineister et al., supra (2005)). A construct derived from 10 this sequence following the removal of its mitochondrial targeting leader sequence has been cloned and expressed in E. coli. This approach for heterologous expression of membrane targeted polypeptides in a soluble form is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this 15 gene, TDE0597, from the prokaryote Treponema denticola represents also can be employed to confer enoyl-CoA reductase activity (Tucci and Martin, FEBSLetters 581:1561-1566 (2007)). Butyryl-CoA dehydrogenase, encoded by bed from C. acetobutylicum, is a further exemplary enzyme that can be used to confer enoyl-CoA reductase activity onto a host microbial organism of the invention (Atsumi et al., Metabolic Engineering (2007) and 20 Boynton et al., Journal ofBacteriology 178: 3015-3024 (1996)). Alternatively, E. coli genes exhibiting this activity can be obtained using methods well known in the art (see, for example, Mizugaki et al., Chemical & Pharmaceutical Bulletin 30:206-213 (1982) and Nishimaki et al., Journal ofBiochemistry 95:1315-1321 (1984)). The sequences for each of the above exemplary genes can be found at the following Genbank accession numbers: 25 TER Q5EU90.1 Euglena gracilis TDE0597 NP_971211.1 Treponema denticola bed NP_349317.1 Clostridium acetobutylicum At least three mitochondrial enoyl-CoA reductase enzymes exist in E. gracilis that similarly are applicable for use in the invention. Each enoyl-CoA reductase enzyme exhibits a unique 30 chain length preference (Inui et al., European Journal ofBiochemistry 142:121-126 (1984)), which is particularly useful for dictating the chain length of the desired primary alcohol products of the invention. EST's ELL00002199, ELL00002335, and ELL00002648, which are all annotated as mitochondrial trans-2-enoyl-CoA reductases, can be used to isolate these additional enoyl-CoA reductase genes as described further below. 58 Those skilled in the art also can obtain nucleic acids encoding any or all of the malonyl-CoA independent FAS pathway or acyl-reduction pathway enzymes by cloning using known sequences from available sources. For example, any or all of the encoding nucleic acids for the malonyl-CoA independent FAS pathway can be readily obtained using methods well 5 known in the art from E. gracilis as this pathway has been well characterized in this organism. E. gracilis encoding nucleic acids can be isolated from, for example, an E. gracilis cDNA library using probes of known sequence. The probes can be designed with whole or partial DNA sequences from the following EST sequences from the publically available sequence database TBestDB (http://tbestdb.bcm.umontreal.ca). The nucleic acids generated 10 from this process can be inserted into an appropriate expression vector and transformed into E. coli or other microorganisms to generate primary alcohol production organisms of the invention. ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase) ELL00002550 15 ELL00002493 ELL00000789 3-hydroxyacyl-CoA dehydrogenase ELL00000206 ELL00002419 20 ELL00006286 ELL00006656 enoyl-CoA hydratase ELL00005926 ELL0000 1952 25 ELL00002235 ELL00006206 enoyl-CoA reductase ELL00002199 ELL00002335 30 ELL00002648 Alternatively, the above EST sequences can be used to identify homologue polypeptides in GenBank through BLAST search. The resulting homologue polypeptides and their corresponding gene sequences provide additional encoding nucleic acids for transformation into E. coli or other microorganisms to generate the primary alcohol producing organisms of 35 the invention. Listed below are exemplary homologue polypeptide and their gene accession numbers in GenBank which are applicable for use in the non-naturally occurring organisms of the invention. 59 ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase) YP_001530041 Desulfococcus oleovorans Hxd3 ZP_02133627 Desulfatibacillum alkenivorans AK-01 ZP_01860900 Bacillus sp. SG-1 5 YP_001511817 A lkaliphilus oremlandii OhILAs NP_781017 Clostridium tetani E88 Y P_001646648 Bacillus weihenstephanensis KBAB4 Y P_001322360 Alkaliphilus metalliredigens QYMF Y P_001397054 Clostridium kluyveri DSM 555 10 NP_070026 Archaeoglobusfulgidus DSM 4304 YP_001585327 Burkholderia multivorans ATCC 17616 3-hydroxyacyl-CoA dehydrogenase AA072312 Euglena gracilis XP_001655993 Aedes aegypti 15 NP 001011073 Xenopus tropicalis NP_001003515 Danio rerio XP_973042 Tribolium castaneum XP_001638329 Nematostella vectensis CAG 11476 Tetraodon nigroviridis 20 X P_787188 Strongylocentrotus purpuratus XP_001749481 Monosiga brevicollis MXI NP_509584 Caenorhabditis elegans XP_572875 Cryptococcus neoformans var enoyl-CoA hydratase 25 XP_844077 Trypanosoma brucei XP_802711 Trypanosoma cruzi strain CL Brener XP_806421 Trypanosoma cruzi strain CL Brener. YP_001669856 Pseudomonas putida GB-I YP_641317 Mycobacterium sp. MCS 30 YP_959434 Marinobacter aquaeolei VT8 ABK24445 Picea sitchensis XP_640315 Dictyostelium discoideum YP_633978 Myxococcus xanthus DK 1622 YP_467905 Rhizobium etli CFN 42 35 YP_419997 Magnetospirillum magneticum AMB-1 YP_001172441 Pseudomonas stutzeri A1501 enoyl-CoA reductase. XP_642118 Dictyostelium discoideum AX4 XP_001639469 Nematostella vectensis 40 XP_001648220 Aedes aegypti XP_974428 Tribolium castaneum XP 535334 Canis lupusfamiliaris (dog) NP_001016371 Xenopus tropicalis XP_320682 Anopheles gambiae str. PEST 45 ZP_01645699 Stenotrophomonas maltophilia XP_001679449 Caenorhabdiis briggsae AF16 ZP_01443601 Roseovarius sp. HTCC2601 XP_395130 Apis mellifera XP_001113746 Macaca mulatta 50 ZP_01485509 Vibrio cholerae V51 60 ZP_02012479 Opitutaceae bacterium TAV2 ZP_01163033 Photobacterium sp. SKA34 Y P_267463 Colwellia psychrerythraea 34H ZP_01114282 Reinekea sp. MED297 5 ZP_01732824 Flavobacteria bacterium BAL38 As described previously, after the malonyl-CoA independent elongation cycle, the resulting acyl-CoA can be reduced to produce a primary alcohol by either a single enzyme or pair of 10 enzymes that exhibit acyl-CoA reductase and alcohol dehydrogenase activities. Exemplary genes that encode enzymes for catalyzing the reduction of an acyl-CoA to its corresponding aldehyde include the Acinetobacter calcoaceticus acri encoding a fatty acyl-CoA reductase (Reiser and Somerville, Journal ofBacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M- 1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 15 (2002)), and the sucD gene from Clostridium kluyveri (Sohling and Gottschalk, Journal Bacteriology 178:871-880 (1996)). acri YP_047869.1 Acinetobacter calcoaceticus AAC45217 Acinetobacter baylyi BAB85476.1 Acinetobacter sp. Strain M-1 20 sucD P38947.1 Clostridium kluyveri Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol. 25 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., Nature 451:86 89 (2008)), and yqhD from E. coli which has preference for molecules longer than C3 (Suizenbacher et al., Journal ofMolecular Biology 342:489-502 (2004)). aIrA BAB12273.1 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 Saccharymyces cerevisiae 30 yqhD NP_417484.1 Escherichiacoli Alternatively, the fatty acyl-CoA can be reduced in one step by a fatty alcohol forming acyl CoA reductase or any other enzyme with dual acyl-CoA reductase and alcohol dehydrogenase activity. For example, the jojoba (Simmondsia chinensis) FAR encodes an 35 alcohol-forming fatty acyl-CoA reductase and its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al., Plant Physiology 122:635-644 (2000)). The reductase with narrow substrate chain-length specificities will also function as additional control for product chain-length. Additional gene candidates include the E. coli 61 adhE (Kessler et al., FEBS Letters 281:59-63 (2000)) and C. acetobutylicum bdh I and bdh II (Walter et al., Journal ofBacteriology 174:7149-7158 (1992)) which can reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. FAR AAD38039.1 Simmondsia chinensis 5 adhE NP 415757.1 Escherichia coli bdh I NP_349892.1 Clostridium acetobutylicum bdh II NP_349891.1 Clostridium acetobutylicum In addition, the E. gracilis nucleic acid sequences encoding enzymes for the reduction step can be obtained and transformed into a host as described previously for the malonyl-CoA 10 independent FAS pathway encoding nucleic acids. Isolated from an E. gracilis cDNA library using probes, designed with whole or partial DNA sequences from the following EST sequences from TBestDB (http://tbestdb.bcm.umontreal.ca) can be performed as described previously. aldehyde dehydrogenase 15 ELL00002572 ELL00002581 ELLOOOO0 108 In addition to the above exemplary encoding nucleic acids, nucleic acids other than those within the malonyl-CoA independent FAS and/or acyl-reduction pathways of the invention 20 also can be introduced into a host organism for further production of primary alcohols. For example, the Ralstonia eutropha BktB and PhbB genes catalyze the condensation of butyryl CoA and acetyl-CoA to form B-keto-hexanoyl-CoA and the reduction of p-keto-hexanoyl CoA to 3-hydroxy-hexanoyl-CoA (Fukui et al., Biomacromolecules 3:618-624 (2002)). To improve the production of primary alcohols, exogenous DNA sequences encoding for these 25 specific enzymes can be expressed in the production host of interest. Furthermore, the above described enzymes can be subjected to directed evolution to generate improved versions of these enzymes with high activity and high substrate specificity. A similar approach also can be utilized with any or all other enzymatic steps in the primary alcohol producing pathways of the invention to, for example, improve enzymatic activity and/or specificity and/or to 30 generate long chain alcohols of a predetermined chain length or lengths. In addition, fatty acyl-CoA and fatty alcohols generated as described above can be applied to produce esters of various lengths. These esters can be formed between: 1) fatty acyl-CoA and short-chain alcohols such as methanol, ethanol, propanol, etc.; 2) fatty alcohols and 62 short-chain acyl-CoA such as formyl-CoA, acetyl-CoA, and propionyl-CoA, etc.; 3) fatty acyl-CoA and fatty alcohols as shown in the following equations. fatty acyl-CoA + short-chain alcohols 4 fatty esters + CoA fatty alcohols + short-chain acyl-CoA 4 fatty esters + CoA 5 fatty acyl-CoA + fatty alcohols 4 wax + CoA The fatty (or long-chain) alcohols can be synthesized intracellularly by the pathways described herein or can be added to the medium and taken up by the engineered microbe. Similarly, short-chain alcohols can be added to the medium or produced endogenously. 10 Ethanol is an exemplary short chain alcohol that is naturally produced by many microorganisms including Escherichia coli and Saccahyromyces cerevisiae. Exemplary fatty esters include, but not limited to, fatty acid methyl esters (FAMEs), fatty acid ethyl esters (FAEEs), acetyl esters, and wax. Such molecules have broad applications including in food, personal care, coatings, surfactants, and biodiesel (Gerhard Knothe, Energy & Fuels 2008, 15 22, 1358-1364). Fatty esters, in this context, are differentiated from wax by the size of the hydrocarbon chain on each side of the ester bond. Waxes have long chain hydrocarbons on each side of the ester bond, whereas fatty esters have one short chain and one long chain hydrocarbon on each side of the ester bond, respectively. Esters can be produced chemically, for example, by heating the acid in the presence of an 20 alcohol or multiple alcohols in the presence of a dehydrating agent such as an acid catalyst. Enzymes with ester-forming activity can also be applied to form esters directly from the acids and alcohols. The ester-forming enzymes can be targeted to the cytosol or another cellular compartment to enable intracellular conversion of alcohols and acids to esters. Alternatively, the ester-forming enzymes can be secreted into the fermentation medium to enable 25 extracellular conversion of alcohols and acids to esters. Another option is to add ester forming enzymes to a mixture containing acids and alcohols such as a fermentation broth.Several enzymes with ester forming activity are described below. The amidase from Brevibacterium sp. R312 (EC 3.5.1.4) is a likely enzyme with ester forming activity. This enzyme was shown to hydrolyze ethylacrylate (Thiery et al., J. Gen. 30 Microbiol., 132:2205-8, 1986; Soubrier et al., Gene, 116:99-104,1992). The microsomal epoxide hydrolase from Rattus norvegicus (EC 3.3.2.9) has activity on hydrolyzing glycidyl methacrylate and is another suitable enzyme (Guengerich et al., Rev. Biochem. Toxicol. 4:5 30, 1982). The protein sequences of these genes are provided below. 63 Gene GenBank ID GI Number Organism amiE JC1174 98711 Brevibacterium sp. Eph-1 P07687.1 123928 Rattus norvegicus The reactions to produce these esters can be catalyzed by enzymes with acyl-CoA:alcohol transacylase activities. Exemplary enzymes for catalyzing the formation of fatty esters include the acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS, EC 2.3.1.75) and 5 acetyl-CoA:alcohol 0-acetyltransferase (EC 2.3.1.84). Exemplary genes coding for these enzymes include the Acinetobacter sp. ADPI atfA encoding a bifunctional enzyme with both wax ester synthase (WS) and acyl-CoA: diacylglycerol acyltransferase (DGAT) activities (Kalscheuer et al. AJ Biol Chem 2003, 278: 8075-8082.); the Simmondsia chinensis gene AAD38041 encoding a enzyme required for the accumulation of waxes in jojoba seeds 10 (Lardizabal et al. Plant Physiology 2000, 122: 645-655.); the Alcanivorax borkumensis atfAl and atfA2 encoding bifunctional WS/DGAT enzymes (Kalscheuer et &\. J Bacteriol 2007, 189: 918-928.); ths Fragaria x ananassa AA T encoding an alcohol acetyltransferasae (Noichinda et al. FoodSci Technol Res 1999, 5: 239-242.); the Rosa hybrid cultivar AA Tl encoding an alcohol acetyltransferase (Guterman et al. Plant Mol Biol 2006, 60: 555-563.); 15 and the Saccharomyces cerevisiae A TFl and A TF2 encoding alcohol acetyltransferases (Mason et al. Yeast 2000, 16: 1287-1298.); and Wsl and Ws2 from Marinobacter hydrocarbonoclasticus (Holtzapple,E. and Schmidt-Dannert,C., J. Bacteriol. 189 (10), 3804 3812, 2007). The protein sequences of the enzymes encoded by these genes are provided below. Gene GenBank ID GI Number Organism atfA Q8GGG1 81478805 Acinetobacter sp. ADPl A.AD38041 5020219 Simmondsia chinensis atfAl YP694462 110835603 Alcanivorax borkumensis SK2 atfA2 YP693524 110834665 Alcanivorax borkumensis SK2 AAT AAG13130 110834665 Fragaria x ananassa AA Tl Q516B5 75105208 Rosa hybrid cultivar ATFI P40353 2506980 Saccharomyces cerevisiae A TF2 P53296 1723729 Saccharomyces cerevisiae Ws2 ABO21021.1 126567232 Marinobacter hydrocarbonoclasticus 64 WsI ABO21020.1 126567230 Marinobacter hydrocarbonoclasticus The Homo sapiens paraoxonase enzymes PONI, PON] (G3C9), and PON3 (EC 3.1.8.1) possess both arylesterase and organophosphatase activities and also may possess ester forming activity. PONI has a common polymorphic site at residue 192, glutamine (R) or 5 arginine (Q), that results in qualitative differences. For example, the R isozyme has a higher esterase activity on GBL than the S isozyme (Billecke et al,, Drug Metab Dispos. 28:1335 1342 (2000)). In H. sapiens cells, PON 1 resides on high-density lipoprotein (HDL) particles, and its activity and stability require this environment. Wild type and recombinant PONI enzymes have been functionally expressed in other organisms (Rochu et al., 10 Biochem,Soc. Trans. 35:1616-1620 (2007); Martin et al., Appl.Environ.Microbiol. (2009)). A directed evolution study of PONI yielded several mutant enzymes with improved solubility and catalytic properties in E. coli (nucleotide accession numbers AY499188-AY499199) (Aharoni et al., Proc.Nat.Acad.Sci. U.S.A 101:482-487 (2004)). One recombinant variant from this study, G3C9 (Aharoni et al., Proc.Natl.Acad.Sci. U.S.A 101:482-487 (2004)), was 15 recently used in an integrated bioprocess for the pH-dependent production of 4-valerolactone from levulinate (Martin et al., Appl.Environ.Microbiol. (2009)). Human PON3 is yet another suitable enzyme that may possess ester-forming activity (Draganov et al., JLipid Res. 46:1239-1247 (2005)). Gene Accession No. GI No. Organism PON1 NP_000437.3 19923106 Homo sapiens PON] (G3C9) AAR95986.1 40850544 Synthetic variant PON3 NP_000931.1 29788996 Homo sapiens 20 Additionally, the Candida antarctica lipase B is another suitable candidate enzyme with ester-forming activity (Efe et al., Biotechnol.Bioeng, 99:1392-1406 (2008)). The esterase from Pseudomonasfluorescens, encoded by EstF1, is yet another suitable enzyme (Khalameyzer et al., Appl.Environ.Microbiol. 65:477-482 (1999)). Other lipase enzymes from organisms such as Pseudomonasfluorescens and Bacillus subtilis may also catalyze this 25 transformation. The B. subtilis and P.fluorescens genes encode triacylglycerol lipase enzymes which have been cloned and characterized in E. coli (Dartois et al., 65 Biochim.Biophys.Acta 1131:253-260 (1992); Tan et al., Appl.Environ.Microbiol. 58:1402 1407 (1992)). Gene Accession No. GI No. Organism caIB P41365.1 1170790 Candida Antarctica EstFl AAC36352.1 3641341 Pseudomonasfluorescens lipB P41773.1 1170792 Pseudomonasfluorescens estA P37957.1 7676155 Bacillus subtilis Formation of esters may also be catalyzed by enzymes in the 3.1.1 family that act on 5 carboxylic ester bonds molecules for the interconversion between cyclic lactones and the open chain hydroxycarboxylic acids. The L-lactonase from Fusarium proliferatum ECU2002 exhibits lactonase and esterase activities on a variety of lactone substrates (Zhang et al., Appl.Microbiol.Biotechnol. 75:1087-1094 (2007)). The 1,4-lactone hydroxyacylhydrolase (EC 3.1.1.25), also known as 1,4-lactonase or gamma-lactonase, is specific for 1,4-lactones 10 with 4-8 carbon atoms. The gamma lactonase in human blood and rat liver microsomes was purified (Fishbein et al., JBiol Chem 241:4835-4841 (1966)) and the lactonase activity was activated and stabilized by calcium ions (Fishbein et al., JBiol Chem 241:4842-4847 (1966)). The optimal lactonase activities were observed at pH 6.0, whereas high pH resulted in hydrolytic activities (Fishbein and Bessman, JBiol Chem 241:4842-4847 (1966)). Genes 15 from Xanthomonas campestris, Aspergillus niger and Fusarium oxysporum have been annotated as 1,4-lactonase and can be utilized to catalyze the transformation of 4 hydroxybutyrate to GBL (Zhang et al., Appl Microbiol Biotechnol 75:1087-1094 (2007)). Gene Accession No. GI No. Organism EU596535.1:1..1206 ACC61057.1 183238971 Fusarium proliferatum xccb100_2516 YP_001903921.1 188991911 Xanthomonas campestris An16g06620 CAK46996.1 134083519 Aspergillus niger BAA34062 BAA34062.1 3810873 Fusarium oxysporum Host microbial organisms can be selected from, and the non-naturally occurring microbial 20 organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species 66 selected from E. coli, Rhodococcus opacus, Ralstonia eutropha, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, 5 Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonasfluorescens, Pseudomonas putida and E. gracilis. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. Methods for constructing and testing the expression levels of a non-naturally occurring 10 primary alcohol-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); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). For example, nucleic acids encoding enzymes in the 15 malonyl-CoA independent FAS and/or acyl-reduction pathway 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, for example, mitochondrial genes will encode an N-terminal targeting signals, which can be 20 removed before transformation into host cells. For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of the targeting sequence, or alternatively, can be targeted to mitochondrion with the addition of mitochondrial targeting signal functional in the host organism. Furthermore, genes can be subjected for codon optimization with techniques well known in the art, to achieve optimal 25 expression of the one or more malonyl-CoA independent FAS and/or acyl-reduction pathway gene products. An expression vector or vectors can be constructed to include one or more malonyl-CoA independent FAS and/or acyl-reduction pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. 30 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 67 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 5 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 nucleic acids encoding 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 10 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) 15 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 20 using methods well known in the art and as disclosed herein. Primary alcohol production can be detected and/or monitored using methods well known to those skilled in the art. For example, final product of primary alcohol and/or intermediates such as acyl-CoA and organic acids can be analyzed by HPLC, GC-MS and LC-MS. For example, primary alcohols can be separated by HPLC using a Spherisorb 5 ODSI column 25 and a mobile phase of 70% 10 mM phosphate buffer (pH=7) and 30% methanol, and detected using a UV detector at 215 nm (Hennessy et al. 2004, J. Forensic Sci. 46(6):1-9). The release or secretion of primary alcohol into the culture medium or fermentation broth also can be detected using these procedures. Activities of one or more enzymes in the malonyl-CoA independent FAS and/or acyl-reduction pathway also can be measured using methods well 30 known in the art. The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified above to exogenously express at least one 68 nucleic acid encoding a malonyl-CoA independent FAS pathway enzyme in sufficient amounts to produce primary alcohol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of greater than that which can be synthesized in naturally occurring organisms. Generally, the 5 intracellular concentration of, for example, octanol is about 54pg/L and decanol is about 148 pg/L. As described further below, one exemplary growth condition for achieving biosynthesis of primary alcohols includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be 10 sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions 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 15 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. The invention further provides a method for the production of primary alcohols. The method 20 includes culturing a non-naturally occurring microbial organism have having a malonyl-CoA independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway comprising at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol under substantially anaerobic conditions for a sufficient period of time to produce said primary alcohol, said 25 malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl CoA reductase, said acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase. Any of the non-naturally occurring microbial organisms described previously can be cultured 30 to produce the biosynthetic products of the invention. For example, the primary alcohol producers can be cultured for the biosynthetic production of its engineered target primary 69 alcohol. The primary alcohol can be isolated or isolated and further utilized in a wide variety of products and procedures. In one embodiment, provided herein is a method for producing a primary alcohol, comprising culturing a non-naturally occurring microbial organism described herein as capable of 5 producing a primary alcohol under conditions and for a sufficient period of time to produce a primary alcohol. For example, in one embodiment, the invention provides a method for producing a primary alcohol using a non-naturally occurring microbial organism, comprising a microbial organism having a primary alcohol pathway comprising at least one exogenous nucleic acid encoding a primary alcohol pathway enzyme expressed in a sufficient amount to 10 produce a primary alcohol; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a 15 reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, 20 an H2 hydrogenase, and combinations thereof; and wherein said primary alcohol pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and an acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase. In a specific embodiment, the invention provides a 25 method for producing a primary alcohol using said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin 30 oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, the invention provides a method for producing a primary alcohol using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA 70 transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol pathway enzyme. In another specific embodiment, the invention provides a method for producing a primary alcohol using 5 said microbial organism comprises four exogenous nucleic acids encoding malonyl-CoA independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase or ketoacyl CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and two exogenous nucleic acids encoding acyl-reduction pathway enzymes comprising an acyl-CoA reductase and an alcohol dehydrogenase. In another specific 10 embodiment, the invention provides a method for producing a primary alcohol using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, the invention provides a method for producing a primary alcohol using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA 15 pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another specific embodiment, said exogenous nucleic acid encoding an acyl-reduction pathway enzyme in said non-naturally occurring microbial 20 organism comprises an enzyme having acyl-CoA reductase and alcohol dehydrogenase activity, e.g., fatty alcohol forming acyl-CoA reductase (FAR). In another embodiment, said non-naturally occurring microbial organism further comprises an acyl-reduction pathway comprising an acyl-CoA hydrolase, an acyl-CoA transferase or an acyl-CoA ligase; a carboxylic acid reductase and an alcohol dehydrogenase. In another specific embodiment, 25 said primary alcohol is produced in amounts at least 10% greater levels compared to a microbial organism lacking said exogenous nucleic acid encoding a malonyl-CoA independent FAS pathway enzyme. In another specific embodiment, said primary alcohol comprises an alcohol having between 4-24 carbon atoms, e.g., said primary alcohol is selected from butanol, hexanol, heptanol, octanol, nonanol, decanol, dodecanol, tetradecanol 30 or hexadecanol. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, provided herein is a method for producing a fatty acyl-CoA, comprising culturing a non-naturally occurring microbial organism described herein as 71 capable or proaucing a ratty acyi-uoin unaer conaions ano ior a suicien penoa or time to produce a fatty acyl-CoA. For example, in one embodiment, the invention provides a method for producing a fatty acyl-CoA using a non-naturally occurring microbial organism, comprising a microbial organism having a fatty acyl-CoA pathway comprising at least one 5 exogenous nucleic acid encoding a fatty acyl-CoA pathway enzyme expressed in a sufficient amount to produce a fatty acyl-CoA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA 10 lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) 15 at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; and wherein said fatty acyl-CoA pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoAreductase. In a specific embodiment, the invention provides a 20 method for producing a fatty acyl-CoA using said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin 25 oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, the invention provides a method for producing a fatty acyl-CoA using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthctase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another 30 specific embodiment, said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a fatty acyl-CoA pathway enzyme. In another specific embodiment, the invention provides a method for producing a fatty acyl-CoA using said microbial organism comprises four exogenous nucleic acids encoding malonyl-CoA independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase or ketoacyl 72 CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl CoAreductase. In another specific embodiment, the invention provides a method for producing a fatty acyl-CoA using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In 5 another specific embodiment, said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. In a 10 specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, provided herein is a method for producing a fatty ester, comprising culturing a non-naturally occurring microbial organism described herein as capable of producing a fatty ester under conditions and for a sufficient period of time to produce a fatty 15 ester. For example in one embodiment, the invention provides a method for producing a fatty ester using a non-naturally occurring microbial organism, comprising a microbial organism having a fatty ester pathway comprising at least one exogenous nucleic acid encoding a fatty ester pathway enzyme expressed in a sufficient amount to produce a fatty ester; said non naturally occurring microbial organism further comprising: (i) a reductive TCA pathway 20 comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least 25 one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; and wherein said fatty ester pathway comprises a pathway selected from: (a) a 30 malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis 73 (FAS) pathway and an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl- CoA thiolase, 3 hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. In a specific embodiment, the invention provides a method for producing a fatty ester using said 5 microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof In another 10 specific embodiment, the invention provides a method for producing a fatty ester using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method for producing a fatty ester 15 using said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a fatty ester enzyme. In another specific embodiment, the invention provides a method for producing a fatty ester using said microbial organism comprises (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one exogenous nucleic acid encoding a wax ester synthase, wherein 20 said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl CoA hydratase and an enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one exogenous nucleic acid encoding an alcohol acetyltransferase, wherein said malonyl-CoA-independent 25 FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl- CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase. In another specific embodiment, the invention provides a method for producing a fatty ester using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, the 30 invention provides a method for producing a fatty ester using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific embodiment, said non-naturally occurring microbial 74 organism is in a substantially anaerobic culture medium. In a specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, provided herein is a method for producing a wax, comprising culturing a non-naturally occurring microbial organism described herein as capable of 5 producing a wax under conditions and for a sufficient period of time to produce a wax. For example, In one embodiment, the invention provides a method for producing a wax using a non-naturally occurring microbial organism, comprising a microbial organism having a wax pathway comprising at least one exogenous nucleic acid encoding a wax pathway enzyme expressed in a sufficient amount to produce a wax; said non-naturally occurring microbial 10 organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic 15 acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof, and wherein said wax 20 pathway comprises a pathway selected from: (a) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3 hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an alcohol 25 acetyltransferase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl CoA acyltransferase or ketoacyl- CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl CoA hydratase and enoyl-CoA reductase. In a specific embodiment, the invention provides a method for producing a wax using said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin 30 oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, the invention 75 provides a method for producing a wax using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the 5 invention provides a method for producing a wax using said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme. In another specific embodiment, the invention provides a method for producing a wax using said microbial organism comprises (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis 10 (FAS) pathway enzymes and one nucleic acid encoding a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and and enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA independent fatty acid synthesis (FAS) pathway enzymes and one nucleic acid encoding an 15 alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase. In another specific embodiment, the invention provides a method for producing a wax using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding 20 a reductive TCA pathway enzyme. In another specific embodiment, the invention provides a method for producing a wax using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid. In another specific 25 embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, provided herein is a method for producing acyl-ACP, comprising culturing a non-naturally occurring microbial organism described herein as capable of 30 producing acyl-ACP under conditions and for a sufficient period of time to produce acyl ACP. For example, in one embodiment, the invention provides a method for producing acyl ACP using a non-naturally occurring microbial organism, comprising a microbial organism having an acyl-ACP pathway comprising at least one exogenous nucleic acid encoding a 76 acyl-ACP pathway enzyme expressed in a sufficient amount to produce acyl-ACP; said non naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a 5 citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO 10 dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; and wherein said acyl-ACP pathway comprises a pathway selected from: (A) acetyl CoA carboxylase and a fatty acid synthase; and (B) a fatty acid synthase. In another specific embodiment, the invention provides a method for producing acyl-ACP using said microbial 15 organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof In another 20 specific embodiment, the invention provides a method for producing acyl-ACP usingsaid microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method for producing acyl-ACP 25 using said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a acyl-ACP pathway enzyme. In another specific embodiment, the invention provides a method for producing acyl-ACP using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, the invention provides a method for 30 producing acyl-ACP using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said said at least one exogenous nucleic acid is a heterologous nucleic acid. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 77 In another embodiment, provided herein is a method for producing a fatty acid, comprising culturing a non-naturally occurring microbial organism described herein as capable of producing a fatty acid under conditions and for a sufficient period of time to produce a fatty acid. For example, in one embodiment, the invention provides a method for producing a 5 fatty acid using a non-naturally occurring microbial organism having an acyl-ACP pathway as disclosed herein further comprises a fatty acid pathway comprising an exogenous nucleic acid encoding a thioesterase expressed in a sufficient amount to produce fatty acid. In another specific embodiment, the invention provides a method for producing a fatty acid using said microbial organism comprising (i) further comprises an exogenous nucleic acid 10 encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, the invention provides a method for producing a fatty acid 15 using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method for producing a fatty acid using said microbial organism comprises two, three, four, five, or six 20 exogenous nucleic acids each encoding a acyl-ACP, fatty acid, acyl-CoA, fatty adehyde or fatty alcohol pathway enzyme. In another specific embodiment, the invention provides a method for producing a fatty acid using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, the invention provides a method for producing a fatty acid 25 using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said said at least one exogenous nucleic acid is a heterologous nucleic acid. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 30 In another embodiment, provided herein is a method for producing acyl-CoA, comprising culturing a non-naturally occurring microbial organism described herein as capable of producing acyl-CoA under conditions and for a sufficient period of time to produce acyl CoA. For example, in one embodiment, the invention provides a method for producing a 78 fatty acid using said non-naturally occurring microbial organism having a fatty acid pathway as disclosed herein further comprises an acyl-CoA pathway comprising an exogenous nucleic acid encoding an acyl-CoA synthetase or an acyl-CoA ligase expressed in a sufficient amount to produce acyl-CoA. In another specific embodiment, said microbial organism comprising 5 (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another specific embodiment, the 10 invention provides a method for producing acyl-CoA using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method for producing acyl-CoA using said 15 microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding acyl-CoA pathway enzyme. In another specific embodiment, the invention provides a method for producing acyl-CoA using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said microbial organism comprising (ii) 20 comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said said at least one exogenous nucleic acid is a heterologous nucleic acid. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In another embodiment, provided herein is a method for producing a fatty aldehyde, 25 comprising culturing a non-naturally occurring microbial organism described herein as capable of producing fatty aldehyde under conditions and for a sufficient period of time to produce fatty aldehyde. For example, in one embodiment, the invention provides a method for producing a fatty aldehyde using said non-naturally occurring microbial organism having an acyl-CoA pathway as disclosed herein further comprises a fatty aldehyde pathway 30 comprising an exogenous nucleic acid enconding an acyl-CoA reductase (aldehyde forming) or carboxylic acid reductase expressed in a sufficient amount to produce a fatty aldehyde. In another specific embodiment, the invention provides a method for producing a fatty aldehyde using said microbial organism comprising (i) further comprises an exogenous nucleic acid 79 encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. 5 In another specific embodiment, the invention provides a method for producing a fatty aldehyde using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method 10 for producing a fatty aldehyde using said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a fatty adehyde pathway enzyme. In another specific embodiment, the invention provides a method for producing a fatty aldehyde using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, the 15 invention provides a method for producing a fatty aldehyde using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said said at least one exogenous nucleic acid is a heterologous nucleic acid. In a specific embodiment, said non naturally occurring microbial organism is in a substantially anaerobic culture medium. 20 In another embodiment, provided herein is a method for producing a fatty alcohol, comprising culturing a non-naturally occurring microbial organism described herein as capable of producing fatty alcohol under conditions and for a sufficient period of time to produce fatty alcohol. For example, in one embodiment, the invention provides a method for producing a fatty alcohol using said non-naturally occurring microbial organism having a 25 fatty aldehyde pathway as disclosed herein further comprises a fatty alcohol pathway comprising an exogenous nucleic acid encoding an aldehyde reductase expressed in a sufficient amount to produce a fatty alcohol. In another embodiment, the invention provide a method for producing a fatty alcohol using said non-naturally occurring microbial organism an acyl-CoA pathway as disclosed herein further comprising a fatty alcohol pathway 30 comprising an exogenous nucleic acid encoding an acyl-CoA reductase (alcohol forming) expressed in a sufficient amount to produce a fatty alcohol. In another specific embodiment, the invention provides a method for producing a fatty alcohol using said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected 80 from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In another 5 specific embodiment, the invention provides a method for producing a fatty alcohol using said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In another specific embodiment, the invention provides a method for 10 producing a fatty alcohol using said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a fatty alcohol pathway enzyme. In another specific embodiment, the invention provides a method for producing a fatty alcohol using said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said 15 microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In another specific embodiment, said at least one exogenous nucleic acid is a heterologous nucleic acid. In a specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 20 The primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl CoA, fatty aldehyde or fatty alcoholcan 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, 25 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. In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously 30 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. Exemplary anaerobic conditions for fermentation processes are described 81 below and are well known in the art. 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. In addition to the culturing and fermentation conditions disclosed herein, growth condition 5 for achieving biosynthesis of primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcoholcan 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 10 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. 15 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 20 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. The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described further below in the 25 Examples, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions. 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. 30 Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of primary alcohols. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of primary alcohols will 82 include culturing a non-naturally occurring primary alcohol producing organism of the invention in sufficient neutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can be include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include 1 week, 2, 5 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. Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic 10 production of primary alcohol products of the invention 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 well known in the art are exemplified further below in the Examples. In a further embodiment, the primary alcohol producing microbial organisms of the invention 15 utilize renewable feedstocks and carbon-containing gas as carbon sources for growth. Employing these alternative materials as a feedstock is particularly useful because they are beneficial from an environmental standpoint and lower production costs of bioprocess derived products such as the primary alcohols of the invention. Renewable feedstocks useful for growth of the primary alcohol producing organisms of the 20 invention, including fermentation processes with the modified organisms of the invention, can include any regenerative raw material which can be used by the cell as a supply a carbon or other energy source. In general, renewable feedstock are derived from living organisms or their metabolic byproducts including material derived from biomass, often consisting of underutilized components like chaff. Agricultural products specifically grown for use as 25 renewable feedstocks and useful in the methods of the invention include, for example, corn, soybeans and cotton; flaxseed and rapeseed; sugar cane and palm oil. Renewable feedstocks that can be used therefore include an array of carbohydrates, fats and proteins derived from agricultural and/or animal matter which can be harnessed by the primary alcohol producing organisms of the invention as a source for carbon. 30 Plant-derived biomass which is available as an energy source on a sustainable basis includes, for example, herbaceous and woody energy crops, agricultural food and feed crops, 83 agricultural crop wastes and residues, wood wastes and residues, aquatic plants, and other waste materials including some municipal wastes (see, for example, the URL 1.eere.energy.gov/biomass/information resources.html, which includes a database describing more than 150 exemplary kinds of biomass sources). Exemplary types of biomasses that can 5 be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks 10 and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of a wide variety of primary alcohols. In addition to renewable feedstocks such as those exemplified above, the primary alcohol 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 15 expressed in the primary alcohol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source. Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H 2 and CO and can be obtained from the 20 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, C0 2 . 25 The Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO 2 and CO 2

/H

2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H 2 -dependent conversion of CO 2 to acetate by microorganisms was recognized long before it was revealed 30 that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO 2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents 84 (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation: 2 CO 2 + 4 H 2 + n ADP + n Pi -+. CH 3 COOH + 2 H 2 0 + n ATP Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway 5 can utilize CO 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products. The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl 10 branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following 15 enzymes: cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase. Following the teachings and guidance provided above for introducing a sufficient number of encoding nucleic acids to complete the either or both the malonyl-CoA independent FAS and/or the acyl-reduction pathway, those skilled in the art will understand 20 that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability. 25 The invention is also directed, in part, to the design and creation of cells and organisms having growth-coupled production of LCA. In one embodiment, the invention utilizes optimization-based approaches based on in silico stoichiometric model of Escherichia coli metabolism that identify metabolic designs for optimal production of LCA. A bilevel programming framework, OptKnock, is applied within an iterative algorithm to predict 30 multiple sets of gene disruptions, that collectively result in the growth-coupled production of LCA. The results described herein indicate that combinations of strategically placed gene deletions or functional disruptions of genes significantly improve the LCA production 85 capabilities of Escherichia coli and other cells or organisms. The strain design strategies are equally applicable if an organism other than E. coli is chosen as the production host, even if the organism naturally lacks the activity or exhibits low activity of a subset of the gene products marked for disruption. In those cases, disruptions must only be introduced to 5 eliminate or lessen the enzymatic activities of the gene products that are naturally present in the chosen production host. Growth-coupled production of LCA for the in silico designs are confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment growth-coupled product production. 10 The invention is also directed, in part, to the design and creation of cells and organisms that produce long chain alcohols, LCAs based on in silico stoichiometric model of Saccharomyces cerevisiae metabolism. One skilled in the art will recognize the ability to also produce LCAs by non-growth-coupled production by providing a non-producing growth phase, followed by a non-growth production phase, for example. The results described herein 15 indicate that combinations of gene deletions or functional disruptions of genes significantly improve the LCA production capabilities of Saccharomyces cerevisaie and other cells of eukaryotic organisms and eukaryotic microbial organisms. The strain design pathways are equally applicable if a eukaryotic microbial organism other than S. cerevisiae is chosen as the production host, even if the organism naturally lacks the activity or exhibits low activity of a 20 subset of the gene products marked for disruption. In the latter case, disruptions can be introduced to eliminate or lessen the enzymatic activities of the gene products that are naturally present in the chosen production host. In some embodiments, growth-coupled production of LCA for the in silico determined metabolic pathways is confirmed by construction of strains having the designed metabolic genotype. These metabolically 25 engineered cells or organisms can also be subjected to adaptive evolution to further augment growth-coupled product production. In some embodiments, the engineered cells or organisms can also incorporate additional copies of beneficial genes to increase flux through a particular metabolic pathway. Alternatively, exogenous gene insertions from another organism can be used to install functionality that is not present in the host organism. 30 In some embodiments, the designed LCA production pathway utilizes a malonyl-CoA independent fatty acid synthesis pathway coupled with reduction of the fatty acid to form primary alcohol as shown in Figure 1. The malonyl-CoA independent LCA production 86 pathway (MI-LCA pathway) comprises the malonyl-CoA-independent fatty acid synthesis steps and the acyl-CoA reduction steps. An engineered microorganism possessing the MI LCA pathway will convert low cost renewable feedstocks, such as glucose and sucrose, to acetyl-CoA through glycolysis. Acetyl-CoA then is used as both primer and extension units 5 in an elongation cycle that involves the ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase. At the end of each elongation cycle, an acyl-CoA is formed that is one C 2 unit longer than the acyl-CoA entering the elongation cycle. The acyl-CoA with a desired chain-length is then reduced through the combination of acyl-CoA reductase and alcohol dehydrogenase or the fatty alcohol forming 10 acyl-CoA reductase to form the desired primary alcohol. The carbon chain-length of the LCA can be controlled by chain-length specific enoyl-CoA reductase, ketoacyl-CoA thiolase, and/or acyl-CoA reductase. The MI-LCA pathway has the advantage of better product and ATP yields than that through the typical energy-intensive fatty acid synthesis pathways for LCA production. For example, 15 the maximum theoretical yield for dodecanol (C 12 ) using the MI-LCA pathway is 0.333 mol per mol of glucose consumed under both aerobic and anaerobic conditions: 3C 6

H

12 0 6

C

12

H

26 0 + 6CO 2 + 5H 2 0 Additionally, the energy and redox characteristics of the MI-LCA pathway make it suited for 20 the creation of strains that couple LCA production to growth using OptKnock algorithms (Burgard, A.P., P. Pharkya, and C.D. Maranas, Optknock: a bilevelprogrammingframework for identifying gene knockout strategies for microbial strain optimization. Biotechnol Bioeng, 2003. 84(6): p. 647-57; Pharkya, P., A.P. Burgard, and C.D. Maranas, Exploring the overproduction of amino acids using the bilevel optimization framework OptKnock. 25 Biotechnol Bioeng, 2003. 84(7): p. 887-99; Pharkya, P., A.P. Burgard, and C.D. Maranas, OptStrain: a computationalframework for redesign of microbial production systems. Genome Res, 2004. 14(11): p. 2367-76.). The resulting growth-coupled production strains will be inherently stable, self-optimizing, and suited for batch, fed-batch, and continuous process designs. 30 In some embodiments, the invention is directed to an integrated computational and engineering platform for developing metabolically altered microorganism strains having enhanced LCA producing characteristics. Strains identified via the computational component 87 of the platform are put into actual production by genetically engineering the predicted metabolic alterations which lead to the enhanced production of LCA. Production of the desired product is coupled to optimal growth of the microorganism to optimize yields of this product during fermentation. In yet another embodiment, strains exhibiting growth-coupled 5 production of LCA are further subjected to adaptive evolution to further augment product biosynthesis. The levels of growth-coupled product production following adaptive evolution also can be predicted by the computational component of the system where, in this specific embodiment, the elevated product levels are realized only following evolution. In some embodiments, the invention provides a non-naturally occurring microbial organism, 10 that includes one or more gene disruptions. The disruptions occur in genes encoding an enzyme that couples LCA production to growth of the organism when the gene disruption reduces the activity of the enzyme, such that the gene disruptions confer stable growth coupled production of LCA onto the non-naturally occurring organism. In particular embodiments, the invention provides a non-naturally occurring eukaryotic 15 organism, that includes one or more gene disruptions. The one or more gene disruptions occur in genes that encode enzymes that include, for example a cytosolic pyruvate decarboxylase, a mitochondrial pyruvate dehydrogenase, a cytosolic ethanol-specific alcohol dehydrogenase or a mitochondrial ethanol-specific alcohol dehydrogenase. These gene disruptions confer production of long chain alcohols in the cytosol or mitochondrion (vide 20 infra)of the organism. Further, the present invention provides methods of producing such non-naturally microbial organisms having stable growth-coupled production of LCA. For LCA production, for example, the method includes: (a) identifying in silico a set of metabolic modifications requiring LCA production during cell growth, and (b) genetically modifying a microorganism 25 to contain the set of metabolic modifications requiring LCA production. One consideration for bioprocessing is whether to use a batch or continuous fermentation scheme. One difference between the two schemes that will influence the amount of product produced is the presence of a preparation, lag, and stationary phase for the batch scheme in addition to the exponential growth phase. In contrast, continuous processes are kept in a state 30 of constant exponential growth and, if properly operated, can run for many months at a time. For growth-associated and mixed-growth-associated product formation, continuous processes 88 provide much higher productivities (i.e., dilution rate times cell mass) due to the elimination of the preparation, lag, and stationary phases. For example, given the following reasonable assumptions: Monod kinetics (i.e., p = pmS/(Ks+S)) 5 p,=1.0 hr' final cell concentration/initial cell concentration = 20 tprp + tig + tstat = 5 hr feed concentration of limiting nutrient >> Ks increased productivity from a continuous process has been estimated at 8-fold, Shuler et al, 10 Prentice Hall, Inc.: Upper Saddle River, NJ., 245-247. Despite advantages in productivity, many more batch processes are in operation than continuous processes for a number of reasons. First, for non-growth associated product formation (e.g., penicillin), the productivity of a batch system may significantly exceed that of a continuous process because the latter would have to operate at very low dilution rates. 15 Next, production strains generally have undergone modifications to their genetic material to improve their biochemical or protein production capabilities. These specialized strains are likely to grow less rapidly than their parental complements whereas continuous processes such as those employing chemostats (fermenters operated in continuous mode) impose large selection pressures for the fastest growing cells. Cells containing recombinant DNA or 20 carrying point mutations leading to the desired overproduction phenotype are susceptible to back-mutation into the original less productive parental strain. It also is possible for strains having single gene deletions to develop compensatory mutations that will tend to restore the wild-type growth phenotype. The faster growing cells usually out-compete their more productive counterparts for limiting nutrients, drastically reducing productivity. Batch 25 processes, on the other hand, limit the number of generations available by not reusing cells at the end of each cycle, thus decreasing the probability of the production strain reverting back to its wild-type phenotype. Finally, continuous processes are more difficult to operate long term due to potential engineering obstacles such as equipment failure and foreign organism contamination. The consequences of such failures also are much more considerable for a 30 continuous process than with a batch culture. For small-volume production of specialty chemicals and/or proteins, the productivity increases of continuous processes rarely outweigh the risks associated with strain stability and reliability. However, for the production of large-volume, growth-associated products 89 such as LCA, the increases in productivity for a continuous process can result in significant economic gains when compared to a batch process. Although the engineering obstacles associated with continuous bioprocess operation would always be present, the strain stability concerns can be overcome through metabolic engineering strategies that reroute metabolic 5 pathways to reduce or avoid negative selective pressures and favor production of the target product during the exponential growth phase. One computational method for identifying and designing metabolic alterations favoring growth-coupled production of a product is the OptKnock computational framework, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003). OptKnock is a metabolic modeling and 10 simulation program that suggests gene disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become a byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed 15 gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. The concept of growth-coupled biochemical production can be visualized in the context of the biochemical production envelopes of a typical metabolic network calculated using an in 20 silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating the maximum and minimum rates of biochemical production at each attainable level of growth. Although exceptions exist, typically the production of a desired biochemical is in direct competition with biomass formation for intracellular resources. Thus, enhanced rates of biochemical 25 production will necessarily result in sub-maximal growth rates. The disruptions suggested by OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain as depicted in Figure 2. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point should lie within its calculated solution 30 boundary. Plots such as these enable one to visualize how close strains are to their performance limits or, in other words, how much room is available for improvement. The OptKnock framework has already been able to identify promising gene disruption strategies 90 for biochemical overproduction, (Burgard, A.P., P. Pharkya, and C.D. Maranas, Biotechnol Bioeng, 84(6):647-657 (2003); Pharkya, P., A.P. Burgard, and C.D. Maranas, Biotechnol Bioeng, 84(7):887-899 (2003)) and establishes a systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks. 5 Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and 10 methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic 15 networks in the presence of gene additions or disruptions. OptKnock computational framework allows the construction of model formulations that enable an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. Patent 20 Application Serial No. 10/043,440, filed January 10, 2002, and in International Patent No. PCT/USO2/00660, filed January 10, 2002. Another computational method for identifying and designing metabolic alterations favoring growth-coupled production of a product is metabolic modeling and simulation system termed SimPheny*. This computational method and system is described in, for example, U.S. Patent 25 Application Serial No. 10/173,547, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny* is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities 30 of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the 91 solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or 5 of its biochemical components. Analysis methods such as convex analysis, linear programming and the calculation of extreme pathways as described, for example, in Schilling et al., J. Theor. Biol. 203:229-248 (2000); Schilling et al., Biotech. Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Prog. 15:288-295 (1999), can be used to determine such phenotypic capabilities. 10 As described above, one constraints-based method used in the computational programs applicable to the invention is flux balance analysis. Flux balance analysis is based on flux balancing in a steady state condition and can be performed as described in, for example, Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approaches have been applied to reaction networks to simulate or predict systemic properties of, for example, 15 adipocyte metabolism as described in Fell and Small, J. Biochem. 138:781-786 (1986), acetate secretion from E. coli under ATP maximization conditions as described in Majewski and Domach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeast as described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996). Additionally, this approach can be used to predict or simulate the growth of S. cerevisiae on a variety of single-carbon sources 20 as well as the metabolism of H. influenzae as described in Edwards and Palsson, Proc. Nati. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio. Chem. 274:17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130 (2001). Once the solution space has been defined, it can be analyzed to determine possible solutions under various conditions. This computational approach is consistent with biological realities 25 because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a 30 reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. 92 Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement growth-coupled production of a biochemical product. Such metabolic modeling and simulation methods include, for example, the computational systems 5 exemplified above as SimPheny* and OptKnock. For simplicity in illustrating the invention, the methods and strains will be described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods 10 well known in the art. The ability of a cell or organism to couple growth to the production of a biochemical product can be illustrated in the context of the biochemical production limits of a typical metabolic network calculated using an in silico model. These limits are obtained by fixing the uptake rate(s) of the limiting substrate(s) to their experimentally measured value(s) and calculating 15 the maximum and minimum rates of biochemical production at each attainable level of growth. As shown in Figure 2, the production of a desired biochemical generally is in direct competition with biomass formation for intracellular resources. Under these circumstances, enhanced rates of biochemical production will necessarily result in sub-maximal growth rates. The disruptions suggested by the above metabolic modeling and simulation programs 20 such as OptKnock are designed to restrict the allowable solution boundaries forcing a change in metabolic behavior from the wild-type strain as depicted in Figure 2. Although the actual solution boundaries for a given strain will expand or contract as the substrate uptake rate(s) increase or decrease, each experimental point will lie within its calculated solution boundary. Plots such as these enable accurate predictions of how close the designed strains are to their 25 performance limits which also indicates how much room is available for improvement. The OptKnock mathematical framework is exemplified herein for pinpointing gene disruptions leading to growth-coupled biochemical production as illustrated in Figure 2. The procedure builds upon constraint-based metabolic modeling which narrows the range of possible phenotypes that a cellular system can display through the successive imposition of 30 governing physico-chemical constraints, Price et al., Nat Rev Microbiol, 2: 886-97 (2004). As described above, constraint-based models and simulations are well known in the art and 93 generally invoke the optimization of a particular cellular objective, subject to network stoichiometry, to suggest a likely flux distribution. Briefly, the maximization of a cellular objective quantified as an aggregate reaction flux for a steady state metabolic network comprising a set N = {1,..., N} of metabolites and a set M = 5 {1,..., M}of metabolic reactions is expressed mathematically as follows: maximize Vce1Wa,.objective M subject to SVj = 0, VieN j=1 Vs.bsarate V,.bs,.,, plake mmo/gD W-hr V i e {limiting substrate(s)} 10 v 0 ,> v , main mmol/gDW-hr v> 0, V j e (irrev. reactions) where S,; is the stoichiometric coefficient of metabolite i in reaction, vj is the flux of reaction j, vsubrat,,_ptake represents the assumed or measured uptake rate(s) of the limiting substrate(s), 15 and vap_main is the non-growth associated ATP maintenance requirement. The vector v includes both internal and external fluxes. In this study, the cellular objective is often assumed to be a drain of biosynthetic precursors in the ratios required for biomass formation, Neidhardt, F.C. et al., 2nd ed. 1996, Washington, D.C.: ASM Press. 2 v. (xx, 2822, lxxvi ). The fluxes are generally reported per 1 gD W-hr (gram of dry weight times hour) such that 20 biomass formation is expressed as g biomass produced/gD W-hr or 1/hr. The modeling of gene deletions, and thus reaction elimination, first employs the incorporation of binary variables into the constraint-based approach framework, Burgard et al., Biotechnol Bioeng, 74: 364-375 (2001), Burgard et al., Biotechnoi Prog, 17: 791-797 (2001). These binary variables, 2 1, if reaction flux vj is active 25 yj = 0, if reaction flux v, is not active ,VjEM assume a value of 1 if reaction j is active and a value of 0 if it is inactive. The following constraint, 94 vja - , j v, Vi v"" - V ,Vj ensures that reaction flux v; is set to zero only if variable y is equal to zero. Alternatively, when; is equal to one, vj is free to assume any value between a lower vj'in and an upper v;" bound. Here, vj"' and vj"" are identified by minimizing and maximizing, respectively, every 5 reaction flux subject to the network constraints described above, Mahadevan et al., Metab Eng, 5: 264-76 (2003), Optimal gene/reaction disruptions are identified by solving a bilevel optimization problem that chooses the set of active reactions (yj = 1) such that an optimal growth solution for the resulting network overproduces the chemical of interest. Schematically, this bilevel 10 optimization problem is illustrated in Figure 2. Mathematically, this bilevel optimization problem is expressed as the following bilevel mixed-integer optimization problem: maximize vchemical (OptKnock) yj subject to maximize vbi,,s vi 15 subject to s 0, V i E N Vsubstrate v br uptake V i E (limiting substrate(s)) Vatp vatp_main Vbb ,! - bioma.~ 20 ~ mi !"" -y v, v"- V j E= M 20 ViVji ,Mj jeM *" y; E (O,1), VjEM where venemic, is the production of the desired target product, for example LCA or other biochemical product, and K is the number of allowable knockouts. Note that setting K equal 25 to zero returns the maximum biomass solution of the complete network, while setting K equal to one identifies the single gene/reaction knockout (y; = 0) such that the resulting network involves the maximum overproduction given its maximum biomass yield. The final 95 constraint ensures that the resulting network meets a minimum biomass yield. Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), provide a more detailed description of the model formulation and solution procedure. Problems containing hundreds of binary variables can be solved in the order of minutes to hours using CPLEX 8.0, GAMS: The Solver Manuals. 2003: 5 GAMS Development Corporation, accessed via the GAMS, Brooke et al., GAMS Development Corporation (1998), modeling environment on an IBM RS6000-270 workstation. The OptKnock framework has already been able to identify promising gene disruption strategies for biochemical overproduction, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), Pharkya et al., Biotechnol Bioeng, 84: 887-899 (2003), and establishes a 10 systematic framework that will naturally encompass future improvements in metabolic and regulatory modeling frameworks. Any solution of the above described bilevel OptKnock problem will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in LCA as a product during the growth phase of the organism. 15 Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction 20 database having a relationship between enzymes and encoding genes. Once identified, the set of reactions that are to be disrupted in order to achieve growth coupled LCA production are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. As described previously, one particularly useful means to achieve functional disruption of the reaction set 25 is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid 30 assessments of the product coupling are desired or when genetic reversion is less likely to occur. 96 To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the growth-coupled production of LCA, or other biochemical products, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively 5 solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for 10 disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions: y, + y2 + y3 1. The integer cut method is well known in the art and can be found described in, for example, reference, Burgard et al., Biotechnol Prog, 17: 791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic 15 modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny. Constraints of the above form preclude identification of larger reaction sets that include previously identified sets. For example, employing the integer cut optimization method 20 above in a further iteration would preclude identifying a quadruple reaction set that specified reactions 1, 2, and 3 for disruption since these reactions had been previously identified. To ensure identification of all possible reaction sets leading to growth-coupled production of a product, a modification of the integer cut method was employed. Briefly, the modified integer cut procedure begins with iteration 'zero' which calculates the 25 maximum production of the desired biochemical at optimal growth for a wild-type network. This calculation corresponds to an OptKnock solution with K equaling 0. Next, single disruptions are considered and the two parameter sets, objstoreer and ystoreuerj, are introduced to store the objective function (veseical) and reaction on-off information (yj), respectively, at each iteration, iter. The following constraints are then successively added to 30 the OptKnock formulation at each iteration. chemical objstoreiter + 6 - M yyorej 97 In the above equation, E and Mare a small and a large numbers, respectively. In general, C can be set at about 0.01 and M can be set at about 1000. However, numbers smaller and/or larger then these numbers also can be used. M ensures that the constraint can be binding only for previously identified disruption strategies, while c ensures that adding disruptions to a 5 previously identified strategy must lead to an increase of at least s in biochemical production at optimal growth. The approach moves onto double disruptions whenever a single disruption strategy fails to improve upon the wild-type strain. Triple disruptions are then considered when no double disruption strategy improves upon the wild-type strain, and so on. The end result is a ranked list, represented as desired biochemical production at optimal 10 growth, of distinct disruption strategies that differ from each other by at least one disruption. This optimization procedure as well as the identification of a wide variety of reaction sets that, when disrupted, lead to the growth-coupled production of a biochemical product are exemplified in detail further below. Given the teachings and guidance provided herein, those skilled in the art will understand that the methods and metabolic engineering designs 15 exemplified herein are applicable to the coupling of cell or microorganism growth to any biochemical product. Employing the methods exemplified above, the methods of the invention enable the construction of cells and organisms that couple the production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic 20 alterations. In this regard, metabolic alterations have been identified that obligatorily couple the production of LCA to organism growth. Microbial organism strains constructed with the identified metabolic alterations produce elevated levels of LCA during the exponential growth phase. These strains can be beneficially used for the commercial production of LCA in continuous fermentation process without being subjected to the negative selective 25 pressures described previously. Therefore, the methods of the invention provide a set of metabolic modifications that are identified by an in silico method selected from OptKnock. The set of metabolic modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. For LCA production metabolic modifications can 30 be selected from the set of metabolic modifications listed in Table 1. Also provided is a method of producing a non-naturally occurring microbial organism having stable growth-coupled production of LCA. The method includes: (a) identifying in silico a 98 set of metabolic modifications requiring LCA production during exponential growth; (b) genetically modifying an organism to contain the set of metabolic modifications requiring product production, and culturing the genetically modified organism. Culturing can include adaptively evolving the genetically modified organism under conditions requiring product 5 production. The methods of the invention are applicable to bacterium, yeast and fungus as well as a variety of other cells and microorganism. Exemplary bacteria include species selected from E. coli, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus 10 plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonasfluorescens, and Pseudomonasputida. Exemplary eukaryotic organisms include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Rhizopus arrhizus, Rhizopus oryzae, and Pichia pastoris. 15 A microbial organism produced by the methods of the invention is further provided. Additionally, the invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions encoding an enzyme associated with growth coupled production of LCA and exhibiting stable growth-coupled production of these products. The non-naturally occurring microbial organism of the invention includes one or 20 more gene disruptions occurring in genes encoding an enzyme obligatorily coupling LCA production to growth of the microbial organism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers stable growth-coupled production of LCA onto the non-naturally occurring microbial organism. The non-naturally occurring microbial organism can have one or more gene disruptions 25 included in a metabolic modification listed in Table 1. The one or more gene disruptions can be a deletion. The non-naturally occurring microbial organism of the invention can be selected from a group of microbial organism having a metabolic modification listed in Tables 1. Non-naturally occurring microbial organisms of the invention include bacteria, yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes. 30 Exemplary bacteria include species selected from E. coli, A. succiniciproducens, A. succinogenes, M. succiniciproducens, R. etli, Bacillus subtilis, C. glutamicum, G. oxydans, Z. mobilis, L. lactis, L. plantarum, S. coelicolor, C. acetobutylicum, P. fluorescens, and P. 99 putida. Exemplary eukaryotic organisms include species selected from S. cerevisiae, S. pombe, K. lactis, K. marxianus, A. terreus, A. niger, R. arrhizus, R. oryzae, and P. pastoris. The microbial organisms having growth-coupled LCA production are exemplified herein with reference to an Escherichia coli genetic background. However, with the complete 5 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 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 10 organisms is routine and well known in the art. Accordingly, the metabolic alterations enabling growth-coupled production of LCA described herein with reference to a particular organism such as Escherichia coli can be readily applied to other microorganisms. 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. 15 As described previously, homologues can include othologs and/or nonorthologous gene displacements. In some instances, such as when a substitute metabolic pathway exists in the species of interest, functional disruption can be accomplished by, for example, deletion of a paralog that catalyzes a similar, yet non-identical metabolic reaction which replaces the referenced reaction. Because certain differences among metabolic networks between 20 different organisms, those skilled in the art will understand that the actual genes disrupted between different organisms may differ. However, the given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to all microorganisms to identify the cognate metabolic alterations between organisms and to construct an organism in a species of interest that will enhance the coupling 25 of LCA biosynthesis to growth. The invention will be described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more genes 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 30 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 that reference to any of these metabolic constitutes also references the gene or genes encoding the enzymes 100 that catalyze the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes as well as the reactants and products of the reaction. As described previously and further below, 5 exemplary reactions, reaction nomenclature, reactants, products, cofactors and genes encoding enzymes catalyzing a reaction involved in the growth-coupled production of LCA are set forth in Tables 2 and 3. The invention provides non naturally occurring microbial organisms having growth-coupled production of LCA. Product production is obligatorily linked to the exponential growth 10 phase of the microorganism by genetically altering the metabolic pathways of the cell. The genetic alterations make the desired product a product during the growth phase. Sets of metabolic alterations or transformations that result in elevated levels of LCA biosynthesis are exemplified in Table 1, respectively. Each alteration within a set corresponds to the requisite metabolic reaction that should be functionally disrupted. Functional disruption of all 15 reactions within each set results in the production of LCA by the engineered strain during the growth phase. The corresponding reactions to the referenced alterations and the gene or genes that potentially encode them in Escherichia coli, are set forth in Table 2. The various metabolites, their abbreviations and location are set forth in Table 3. For example, for each strain exemplified in Table 1, the metabolic alterations that can be 20 generated for growth coupled LCA production are shown in each row. These alterations include the functional disruption of from one to six or more reactions. In particular, 995 strains are exemplified in Table 1 that have non-naturally occurring metabolic genotypes. Each of these non-naturally occurring alterations result in an enhanced level of LCA production during the exponential growth phase of the microbial organism compared to a 25 wild-type strain, under appropriate culture conditions. Appropriate conditions include, for example, those exemplified further below in the Example I such as particular carbon sources or reactant availabilities and/or adaptive evolution. Given the teachings and guidance provided herein, those skilled in the art will understand that to disrupt an enzymatic reaction it is necessary to disrupt the catalytic activity of the one or 30 more enzymes involved in the reaction. Disruption can occur by a variety of means including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption 101 can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity disruption can occur by a genetic alteration that reduces or destroys the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, 5 disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products. Destruction of activity can be accomplished by loss of the binding activity of one or more subunits in order to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic 10 reaction of the invention. Such other functions are well known to those skilled in the art. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the 15 invention can be disrupted so long as the targeted reaction is reduced or destroyed. Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can 20 lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will 25 understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the growth coupled product production. Herein below are described the designs identified for increasing LCA production in 30 Escherichia coli. The OptKnock algorithm identified designs based on a stoichiometric model of Escherichia coli metabolism. Assumptions include (i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic or microaerobic conditions; and (iii) a minimum non-growth 102 associated maintenance requirement of 3 mmol/gdw/hr. Dodecanol, a C 12 molecule, was chosen as an exemplary long chain alcohol whose production can be coupled to growth following the teachings of this invention. Although glucose was assumed to be the growth substrate, it is understood that the strategies are applicable to any substrate including glucose, 5 sucrose, xylose, arabinose, or glycerol. The complete set of growth-coupled LCA productions designs are listed in Table 1. The enzyme names, their abbreviations, and the corresponding reaction stoichiometries are listed in Table 2. Finally, metabolites names corresponding to the abbreviations in the reaction equations are listed in Table 3. Although the designs were identified using a metabolic model of E. coli metabolism, and the gene 10 names listed in Table 2 are specific to E. coli, the method of choosing the metabolic engineering strategies and also the-designs themselves are applicable to any LCA-producing organism. Thus the designs are essentially lists of enzymatic transformations whose activity must be either eliminated, attenuated, or initially absent from a microorganism to enable the growth coupled production of long chain alcohols. 15 One criterion for prioritizing the final selection of designs was the growth-coupled yield of dodecanol. To examine this, production cones were constructed for each strategy by first maximizing and, subsequently minimizing the dodecanol yields at different rates of biomass formation (as described in the previous section). If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum 20 yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the dodecanol in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs were given a lower priority. A short list of the highest priority OptKnock 25 designs is provided here in Table I which represents a subset of the designs of Table 1. 103 TABLE I Design Enzyne activity Abbreviation Other notes Design Enzyme activity Abbreviation Other notes I Acetaldehyde-CoA dehydrogenase ADHEr XI Acetaldehyde-CoA dehydrogenase ADHEr Design V + THD2 D-lactate dehydroeenase LDH D D-lactate debydrogenase LDH_D II Acetaldehyde-CoA dehydrogenase ADHEr Design I + PFL Pyruvate fomate lyase PFLi D-lactate dehydrogenase LDH_D Malate dehydrogenase MDH Pymuvate format lyase PFLi NAD(P) transhydroeenase THD2 III Acetaldehyde-CoA dehydrogenase ADHEr Design II + FRD2 XII Acetaldehyde-CoA dehydrogenase ADHEr Design I + PTAr and/or ACKr D-lactate dehydrogenase LDH D D-lactate dehydrogenase LDH_D Pyruvate formate lyase PFLI Phosphotrasacetylasc sd/or Acetate kinase PTAx and/or ACKr Fumarate reductase FRD2 XIII Acetaldehyde-CoA dehydrogenase ADHEr Design XII + FRD2 IV Acetaldebyde-CoA debydrogenase ADHEr Design 11 + FLM D-lactate dehydrogenase LDH_D D-lactate dehydrogenase LDH_D Phosphotransacetylase and/or Acetate kinase PTAr and/or ACKr Pyruvate format lyase PFLi Fumarate reductase FRD2 Fumarase FUM XIV Acetaldehyde-CoA dehydrogenase ADHEr Design XII + FUM V Acetaldehyde-CoA dehydrogenase ADHEr Design II + MDH D-lactate dehydrogenase LDHD D-lactate dehydrogenase LDH_D Phosphotransacetylase and/or Acetate kiase PTAr and/or ACKr Pymuvate formate lyase PFLi Fumarase FUM Malate dehydrogenase MDH XV Acetaldehyde-CoA dehydrogenase ADHEr Design XII + MDH VI Acetaldehyde-CoA dehydrogenase ADHEr Design III + GLUDy D-lactate dehydrogenase LDH_D D-lactate dehydrogenase LDH_D Phosphotransacetyse and/or Acetate kinase PTAr and/or ACKr Pyruvate formate lyase PFLi Malate dehydmgenase MDH Fumarate reductase FRD2 XVI Acetaldehyde-CoA debydrogenase ADHEr Design I + FRD Glutamate dehydrogzenase GLUDy D-lactate dehydrogenase LDHD VII Acetaldehyde-CoA dehydrogenase ADHEr Design IV + GLUDy Fumarate reductase FRD2 D-lactate dehydrogenase LDHD XVII Acetaldehyde-CoA dehydrogenase ADHEr Design I + FUM Pymvate formate lyase PFLi D-lactate dehydrogenase LDH D Fumarase FUM Fumarase FUM Glutamate dehydroienase GLUDY XVIII Acetaldehyde-CoA dehydrogenase ADHEr Design I + MDH VIII Acetaldehyde-CoA dehydrogenase ADHEr Design V + GLUTDy D-lactate dehydrogenase LDH_D D-lactate dehydrogenase LDHD Malate dehydrogenase MDH Pyruvate formate Iyase PFLi XIX Acetaldehyde-CoA dehydrogenase ADHEr Design XVI + ATPS4r Malate dehydrogenase MDH D-lactate dehydrogenase LDH_D Glutamate dehvdroeenase GLUDy Fumarate reductase FRD2 IX Acetaldehyde-CoA dehydrogenase ADHEr Design III + THD2 ATP synthase ATPS4r D-lactate dehydrogenase LDH D XX Acetaldehyde-CoA dehydrogenase ADHEr Design XVII + ATPS4r Pymsvate formate lyase PFLi D-lactate dehydrogenase LDH_D Fumarate reductase FRD2 Fumarase FUM NAD(P) transhydrogenase THD2 ATP synthase ATPS4r X Acetaldehyde-CoA dehydrogenase ADHEr Design IV + THD2 XXI Acetaldehyde-CoA dehydrogenase ADHEr Design XVIII + ATPS4r D-lactate dehydrogenase LDHD D-Iactate dehydrogenase LDH_D Pyivate formate lyase PFLi Fumarate reductase MDH Fumarase FUM Al? synthase ATPS4r NAD(P) transhydrogenase THD2 104 All growth coupled designs in this document build upon Design I which calls for the joint disruption of acetylaldehyde-CoA dehydrogenase (ADHEr) and lactate dehydrogenase (LDHD) activities to reduce the formation of ethanol and lactate, respectively. A dodecanol yield of 0.14 mol/mol glucose is predicted to be attained upon achieving a maximum growth rate of 0.20 1/hr (Design I, Figure 3). Design II specifies the removal, attenuation, or absence of ADHEr, LDH_D, and pyruvate formate lyase (PFLi) and is predicted to result in a dodecanol yield of 0.28 mol/mol glucose at maximum growth as shown in Figure 4. A tighter coupling of LCA production to growth is attained by the further disruption of fumarate reductase (FRD2), fumarase (FUM), or malate dehydrogenase (MDH) activity as indicated by the solution boundary of Designs III-V in Figure 4. An even tighter coupling of production to growth is attained by the further disruption of glutamate dehydrogenase (GLUDy) or NADP transhydrogenase (THD2) activity as shown in solution boundary of Designs VI - XI in Figure 4. Designs VI - XI actually require a non-insignificant yield of LCA, specifically, 0.05 mol dodecanol/mol glucose, to enable a minimal amount of cell growth. Design XII calls for the disruption of phosphotransacetylase (PTAr) and/or acetate kinase (ACKr) activity in addition to ADHEr and LDH_D to prevent or lessen the production of acetate, ethanol, and lactate, respectively. A dodecanol yield of 0.28 mol/mol is required to attain a maximum growth rate of 0.16 1/hr assuming a glucose uptake rate of 10 mmol/gDW/hr as shown in Figure 5. A tighter coupling of LCA production to growth is attained by the further disruption of FRD2, FUM, or MDH as indicated by the solution boundary of Designs XIII - XV. Designs XVI - XVIII specify that the disruption of FRD2, FUM, or MDH activity in addition to ADHEr and LDHD results in a tighter coupling of dodecanol production to cell growth as compared to Design I as shown in Figure 6. Further disrupting ATP synthase activity in designs XIX - XXI is predicted to result in a dodecanol yield of 0.30 mol/mol at a maximum growth rate of 0.13 1/hr as shown in Figure 6. The disruption of this activity forces the organism to rely on the MI-LCA pathway for energy generation. Accordingly, a minimum dodecanol yield of 0.05 mol/mol is required for any growth to be attained assuming that the organism lacks the activities listed in Designs XIX - XXI. It is understood that the disruption of certain activities in addition to those listed by Designs I XXI can lead to even higher production yields as illustrated in the following examples. Design 105 V_A involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDHD), malate dehydrogenase (MDH), pyruvate formate lyase (PFLi), L-aspartase (ASPT), pyruvate kinase (PYK), glucose 6-phosphate dehydrogenase (G6PDHy), and dihydroxyacetone phosphotransferase (DHAPT). Upon addition of the MI-LCA pathway, an engineered strain containing disruptions in these activities is predicted to have a growth-coupled dodecanol yield of 0.327 mol/mol glucose at the maximum growth rate of 0.02 1/hr (Figure 7, point A). This corresponds to 98% of the maximum theoretical yield of 0.333 mol dodecanol/mol glucose. The maximum growth rate of such a strain is predicted to be approximately 10% of the wide type strain while a minimum dodecanol yield of 0.09 mol/mol is required for growth (Figure 7, point B). A recombinant strain containing reduced activity ofthese functionalities can be constructed in a single step or in subsequent steps by, for example, disrupting 2-3 activities each step. For example, one can engineer E. coli for growth coupled LCA production by first removing genes encoding ADHEr and LDHD activities resulting in Design I. Design V is then constructed by further deleting genes responsible for MDH and PFLi activities. Design VA is then constructed by deleting genes encoding ASPT, PYK, G6PDHy, and DHAPT activities. Finally, note that several activities (i.e., 6-phosphogluconolactonase (PGL), phosphogluconate dehydratase (PGDHY), or 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA)) can replace G6PDHy for disruption and yield the same characteristics as Design V_A. Design XIIA involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDHD), acetate kinase (ACKr) and/or phosphotransacetylase (PTAr), glutamate dehydrogenase (NADP) (GLUDy), phosphogluconate dehydrogenase (PGDH), and glucose-6-phosphate isomerase (PGI). Design XIIB involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDHD), acetate kinase (ACKr) and/or phosphotransacetylase (PTAr), glutamate dehydrogenase (NADP) (GLUDy), phosphogluconate dehydrogenase (PGDH), glucose-6-phosphate isomerase (PGI), and D-glucose transport via PEP:Pyr PTS (GLCpts). Upon addition of the MI-LCA pathway, an engineered strain lacking the activities specified by Design XIIB is predicted to have a growth-coupled dodecanol yield of 0.322 mol/mol glucose at the maximum growth rate of 0.04 1/hr (Figure 8, point A). This corresponds to 97% of the maximum theoretical yield of 0.333 mol dodecanol/mol glucose. The maximum growth rate of such a strain is predicted to be approximately 20% of the wild type strain while a minimum dodecanol yield of 0.05 mol/mol is required for growth (Figure 8, point 106 B). A recombinant strain containing reduced activity of these functionalities can be constructed in a single step or in subsequent steps by, for example, removing additional activities each step. For example, one can engineer E. coli for growth coupled LCA production by first removing genes encoding ADHEr and LDH_D activities resulting in Design I. Design XII is then constructed by further deleting genes encoding PTAr and/or ACKr activities. Design XIIA is then constructed by deleting the genes responsible for GLUDy, PGDH, and PGI activities. Finally, Design XIIB is constructed by further deleting a gene essential for GLCpts activity. Accordingly, the invention also provides a non-naturally occurring microbial organism having a set of metabolic modifications coupling LCA production to growth of the organism, the set of metabolic modifications includes disruption of one or more genes selected from the set of genes encoding proteins that include an acetylaldehyde-CoA dehydrogenase and a lactate dehydrogenase. The present invention also provides a strain lacking the activities listed for Design I above that further lack at least one of the following activities: pyruvate formate lyase (PFLi), phosphotransacetylase (PTAr), acetate kinase (ACKr), fumarate reductase (FRD2), fumarase (FUM), or malate dehydrogenase (MDH) as exemplified by Designs II, XII, XVI, XVII, and XVIII. In further embodiments, the invention provides a strain lacking the activities listed for Design II above and further lacks at least one of the following activities: fumarate reductase (FRD2), fumarase (FUM), or malate dehydrogenase (MDH) as exemplified by Designs III, IV, and V. In still further embodiments, the invention provides strains lacking the activities listed for Designs III, IV, or V, above and further lack glutamate dehydrogenase (GLUDy) activity as exemplified by Designs VI, VII, and VIII. The invention also provides strains lacking the activities listed for designs III, IV, or V, above and further lack NAD(P) transhydrogenase (THD2) activity as exemplified by Designs IX, X, and XI. In yet further embodiments, the invention provides a strain lacking the activities listed for Design XII above and further lack at least one of the following activities: fumarate reductase (FRD2), 107 fumarase (FUM), or malate dehydrogenase (MDH) as exemplified by Designs XIII, XIV, and XV. Finally, the invention provides strains lacking the activities listed for designs XVI, XVII, and XVIII, above and further lack ATP synthase (ATPS4r) activity as exemplified by Designs XIX, XX, and XXI. Herein below are described the pathways identified for increasing LCA production in S. cerevisiae. The OptKnock algorithm, described herein further below, identified designs based on a stoichiometric model of Saccharomyces cerevisaiemetabolism. Assumptions include (i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic or microaerobic conditions; and (iii) a minimum non-growth associated maintenance requirement of 3 mmol/gdw/hr. Dodecanol, a C 12 molecule, was chosen as an exemplary long chain alcohol whose production can be coupled to growth following the teachings of this invention. Although glucose was assumed to be the growth substrate, it is understood that the methods are applicable to any substrate including glucose, sucrose, xylose, arabinose, or glycerol. Although the designs were identified using a metabolic model of S. cerevisiae metabolism the method of choosing the metabolic engineering pathways and also the designs themselves are applicable to any LCA-producing eukaryotic organism. Thus, the designs are essentially lists of enzymatic transformations whose activity must be either eliminated, attenuated, or initially absent from a microorganism to enable the production of long chain alcohols. One criterion for prioritizing the final selection of pathways was the yield of dodecanol. To examine this, production cones were constructed for each set of pathways by first maximizing and, subsequently minimizing the dodecanol yields at different rates of biomass formation. If the rightmost boundary of all possible phenotypes of the mutant network is a single point, it implies that there is a unique optimum yield of the product at the maximum biomass formation rate possible in the network. In other cases, the rightmost boundary of the feasible phenotypes is a vertical line, indicating that at the point of maximum biomass the network can make any amount of the dodecanol in the calculated range, including the lowest amount at the bottommost point of the vertical line. Such designs were given a lower priority. 108 The organisms of the present invention can be cultured in a substantially anaerobic culture medium or a microaerobic culture medium as detailed herein below further. Such organisms have one or more gene disruptions which may include complete deletion in some embodiments, or disruption by removal or changes in functional portions encoded by fragments of the entire gene. In some embodiments, the present invention provides non-naturally occurring eukaryotic microbial organisms that produce LCAs in the cytosol. Note that cytosol herein refers to any compartment outside the mitochondrion. In such embodiments, one or more gene disruptions in the eukaryotic organism encoding an enzyme include, for example, a cytosolic pyruvate decarboxylase, a mitochondrial pyruvate dehydrogenase, a cytosolic ethanol-specific alcohol dehydrogenase and a mitochondrial ethanol-specific alcohol dehydrogenase. Exemplary genes endocing these enzymes include, for example, YLR044C, YLR134W, YGRO87C, PDC3, YNL07IW, YER178W, YBR221C, YGR193C, YFLO18C, YBR145W, YGL256W, YOLO86C, YMR303, YMR083W, YPLO88W, YAL061W, YMR318C, YCR105W, and YDL168W. Other gene disruptions encoding an enzyme include, for example, a cytosolic malate dehydrogenase, a glycerol-3-phospate dehydrogenase shuttle, an external NADH dehydrogenase, and an internal mitochondrial NADH dehydrogenase can also be effected. Exemplary genes of the later include, for example, YOL126C, YDL022W, YOL059W, YIL155C, YMR145C, YDLO85W, and YML120C. These organisms can also include an exogenous nucleic acid encoding an enzyme in the cytosol including, for example, an acetyl-CoA synthetase (AMP-forming), an ADP-dependent acetate CoA ligase, an acylating acetaldehyde dehydrogenase, a pyruvate dehydrogenase, a pyruvate:NADP oxidoreductase, and a pyruvate formate lyase, or their corresponding gene regulatory regions. An exogenous nucleic acid encoding a cytosolic transhydrogenase or its gene regulatory region can also be incorporated. In some embodiments these gene products may be natively expressed in the cytosol, while in other embodiments, they may be overexpressed by, for example, adding copies of the gene from the same source or from other organisms, or by introducing or changing gene regulatory regions. Such gene regulatory regions include, for example, alternate promoters, inducible promoters, variant promoters or enhancers to enhance 109 gene expression. Functional disruption of negative regulatory elements such as repressors and/or silencers also can be employed to enhance gene expression. Similar modifications can be made to translation regulatory regions to enhance polypeptide synthesis and include, for example, substitution of a ribosome binding site with an optimal or consensus sequence and/or removing secondary structures. These organisms maximize the availability of acetyl CoA, ATP and reducing equivalents (NADH) for dodecanol production. Acetyl CoA is the primary carbon precursor for the production of LCA via the proposed MI-LCA route. All the reactions enabling the formation of dodecanol via the malonyl-CoA independent pathway are operational in the cytosol. Specifically, ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase function in the appropriate direction to form acyl CoA which is then reduced to fatty aldehyde and dodecanol via acyl CoA reductase and alcohol dehydrogenase. Introduction of the MiI-LCA pathway in the cytosol prevented any flux through the native pyruvate dehydrogenase in silico. 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 deleted or attenuated to increase LCA production. In one embodiment, LCA production in the cytosol uses the AMP-forming acetyl-CoA synthetase. Dodecanol production in the cytosol relies on the native cell machinery to provide the precursors needed in LCA production. A majority of the pyruvate flux generated by glycolysis is channeled into the formation of acetyl CoA via the pyruvate dehydrogenase bypass comprised of the pyruvate decarboxylase, the acetaldehyde dehydrogenase and the AMP forming acetyl-CoA synthetase (Figure 9a). This bypass is reported to have significant flux through it even under aerobic conditions at high concentrations of glucose (Pronk et al., Yeast 12:1607-1633 (1996)). The last step of the bypass that converts acetate into acetyl-CoA is catalyzed by acetyl-CoA synthetase, encoded by the ACS] and ACS2 genes. Since ACS2 is constitutively expressed on glucose and is present in cytosol among other compartments, in some embodiments the non naturally occurring eukarotyic organism is engineered to overexpress ACS2. In other 110 embodiments the A CS2 gene is replaced with a mutant A CS from Salmonellas enterica (Genbank id NP_807785.1) that is not subject to post-translational modification and has higher activity in S. cerevisiae as compared to A CS1 or A CS2 (Shiba et al., Metab Eng. 9:160-168 (2007)). The AMP-generating acetyl CoA synthetase uses two ATP equivalents for the conversion of each molecule of acetate into acetyl CoA (CoA+ acetate + ATP 4 acetyl-CoA + PPi + AMP). Under anaerobic conditions, when energy is available only through substrate-level phosphorylation, the production of dodecanol via the AMP-forming acetyl CoA synthetase is not energetically favorable. Therefore, a small amount of oxygen is made available to the cell to fulfill its energetic requirements, simultaneously increasing the conversion of acetate into acetyl CoA. The production of dodecanol can be improved by disruption of ethanol-specific alcohol dehydrogenases to prevent acetyl-CoA and NADH from being used for ethanol production. Additionally, the production of LCA benefits from preventing NADH from being used in the respiratory electron-transport chain. Thus, disruptions in the internal mitochondrial NADH dehydrogenase, the glycerol-3-phosphate dehydrogenase shuttle (consisting of cytosolic NADH linked glycerol-3-phosphate dehydrogenase and a membrane-bound glycerol-3 phosphate:ubiquinone oxidoreductase) (Bakker et al., FEMS Microbiol. Rev. 25:15-37 (2001)) and the external NADH dehydrogenase are introduced in some embodiments. Further, cytosolic malate dehydrogenase that can potentially draw NADH away from dodecanol production is also disrupted. A growth-coupled production envelope after imposing these disruptions is shown in dark gray in Figure 9b and compared with the dodecanol production characteristics under aerobic conditions. In some embodiments, the non-naturally occurring eukaryotic organism incorporates an exogenous gene encoding an ADP-fonning acetate CoA ligase. In this embodiment, the AMP forming acetyl CoA synthetase in the cytosol is replaced by the ADP-forming acetate CoA ligase (CoA+ acetate + ATP + acetyl-CoA + Pi + ADP) (Figure 10a). Exogenous genes to introduce acetate CoA ligase include, for example, acdA and acdB from Pyrococcusfuriosus (Glasemacher et al., Eur. J. Biochem. 244:561-567 (1997)) (Mai and Adams, J. Bacteriol. 178: 5897-5903 (1996)). The introduction of this enzyme that uses one equivalent of ATP for formation of each 111 molecule of acetyl CoA (as opposed to 2 ATP equivalents) allows the production of dodecanol to be energetically neutral. In this embodiment, a small amount of oxygen or other electron acceptor respiration is used to generate energy to support growth. Such small amounts of oxygen are referred to as microaerobic conditions, as described further below. In some embodiments, the ethanol-specific alcohol dehydrogenases is disrupted to prevent ethanol formation. In embodiments incorporating CoA ligase, one or more of the following knockouts can be introduced for LCA production: cytosolic malate dehydrogenase, glycerol-3-phospate dehydrogenase shuttle, the external NADH dehydrogenase, and the internal mitochondrial NADH dehydrogenase. The growth-coupled production after imposition of these disruptions is shown in Figure l0b in dark gray. The black curve shows the production envelope for the wild type strain under aerobic conditions and the light gray curve shows the envelope when the network is augmented with acetate-CoA ligase. Note the increase in the maximum theoretical yield of dodecanol after introduction of this enzyme. In some embodiments, the non-naturally occurring eukaryotic organism incorporates an exogenous gene encoding an acylating acetaldehyde dehydrogenase. Improvement in the energetics of the dodecanol process can be accomplished by using the acylating acetaldehyde dehydrogenase (acetaldehyde + CoA + NAD -- > acetyl-CoA + NADH) for the conversion of acetaldehyde into acetyl CoA (Figure l la). The benefits of using this enzyme are that (i) no energy is expended for production of acetyl CoA, and (ii) one molecule of NADH is formed for every molecule of acetyl CoA formed. Thus, the reducing equivalents needed for the production of acetyl CoA can also be generated. The introduction of this enzyme allows production of LCA under anaerobic conditions. Acylating acetaldehyde dehydrogenase has been reported in several bacteria, including Acetobacterium woodii (Mai and Adams, J. Bacteriol. 178:5897-5903 (1996)), Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. U. S. A 105:2128-2133 (2008); Smith and Kaplan, Arch. Biochem. Biophys. 203:663-675(1980)), Clostridium beijerinckii (Yan et al., Appl. Environ. Microbiol 56:2591-2599 (1990)), and in species of Pseudomonas such as strain CF600 (Lei et al., Biochemistry 47:6870-6882 (2008); Manjasetty et al., Acta Crystallogr. D. Biol. Crystallogr. 57:582-585 (2001)). The Genbank ids of genes are shown in Table 5 below. 112 Table 5 Aid YP 001394464.1 Clostridium kluyveri dmpF CAA43226.1 Pseudomonas sp. CF600 | bphG BAA03892.1 Pseudomonas sp NP 4148 Escherichia coli K12 hpD 84.1 MG1655 In some embodiments each of the strains above can be supplemented with additional disruptions. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis and can also be disrupted. The anaerobic growth-coupled production of dodecanol (or any LCA) can be accomplished by disrupting ethanol-specific alcohol dehydrogenase activity. The introduction of an acylating acetaldehyde dehydrogenase, with its favorable energetics, prevents or reduces carbon flux through the native acetaldehyde dehydrogenase and the acetyl-CoA synthetase. The production envelope is shown in Figure 1lb. The wild-type S. cerevisiae (black) network can form only small amounts of dodecanol as an byproduct of growth under anaerobic conditions. When the network is augmented with acylating dehydrogenase, there is an increase in the theoretical maximum yield in the network, but no growth-coupling is observed (dotted light gray curve). However, disruption of ethanol-specific alcohol dehydrogenase from the augmented network shows that dodecanol production is coupled to growth at the maximum feasible biomass in the network (dark gray curve). In some embodiments, the non-naturally occurring eukaryotic organism uses a cytosolic pyruvate dehydrogenase for dodecanol production. Cytosolic pyruvate dehydrogenase for generating the precursors for the MI-LCA pathway are shown in Figure 12. In such embodiments, (i) pyruvate is directly converted into acetyl CoA in the cytosol without the expenditure of energy, and (ii) more reducing equivalents are available to the cell. In some embodiments, the non-naturally occurring eukaryotic organism is engineered to retarget the native mitochondrial pyruvate dehydrogenase to the cytosol. In other embodiments, a heterologous cytosolic enzyme is introduced into the organism. The retargeting of an enzyme to 113 a different compartment can be accomplished by changing the targeting sequence of the protein (van Loon and Young, EMBO J. 5:161-165 (1986)). Disruption of the native pyruvate decarboxylase enables a majority of the carbon flux to be introduced into the cytosol for processing by cytosolic pyruvate dehydrogenase. This also allows the production of dodecanol under anaerobic conditions. The growth-coupled production envelope is similar to that depicted in Figure 1 lb. Note that pyruvate decarboxylase is disrupted instead of alcohol dehydrogenase to achieve growth-coupling in the network. In some embodiments, the non-naturally occurring eukaryotic organism uses a cytosolic pyruvate:NADP oxidoreductase. Pyruvate: NADP oxidoreductase allows for the production of acetyl CoA and reducing equivalents in the cytosol as shown in Figure 13. The addition of this enzyme allows for the production of acetyl CoA without expending energy that would otherwise have been required by acetyl CoA synthetase. The enzyme has been purified from the mitochondrion of Euglena gracilis and is oxygen-sensitive (Inui ct al., Journal ofBiochemistry 96:931-934 (1984); Inui et al., Archives ofBiochemistry and Biophysics 237:423-429 (1985); Inui et al., Archives of Biochemistry and Biophysics 274:434-442 (1989); Inui et al., Archives of Biochemistry and Biophysics 280:292-298 (1990)). It is used for generating acetyl CoA from pyruvate, simultaneously producing NADPH. The corresponding gene is pno and its Genbank id is: CAC37628.1. It can be targeted to the cytosol by removing the mitochondrial targeting sequence. In some embodiments, a transhydrogenase is also added. This enzyme can be introduced as an exogenous gene from an organism such as E coli to convert the generated NADPH into NADH (Nissen et al., Yeast 18:19-32 (2001)). With its low ATP requirements, the pathway is energetically favorable even under anaerobic conditions. To prevent or reduce the utilization of NADH and pyruvate for ethanol production, pyruvate decarboxylase activity can be disrupted. This leads to a growth-coupled production of dodecanol similar to that shown in Figure 11 b. In some embodiments, a non-naturally occurring eukaryotic organism uses a pyruvate formate lyase. In such embodiments, a heterologous cytosolic pyruvate fornate lyase (pfl) is used to generate both acetyl CoA and NADH as shown in Figure 14. This enzyme is active typically under anaerobic conditions in organisms such as E. coli. The lack of energy requirement for 114 conversion of pyruvate into acetyl CoA makes the production of dodecanol feasible under anaerobic conditions. The conversion of pyruvate into acetyl CoA is accompanied by the production of formate. This is metabolized by the native formate dehydrogenase, leading to additional generation of reducing equivalents in stoichiometric quantities. In some embodiments that use this strain design, one or more of the three pyruvate decarboxylases, PDC1, PDC5 and PDC6, can be disrupted. The Genbank ids of exemplary genes encoding pyruvate formate lyase are shown in Table 6 below. Table 6 pflB NP 415423.1 Escherichia coli pfl YP 001588758. Lactococcus lactis pfl |YP 001394497.1 Clostridium kluyveri The disruption of pyruvate decarboxylase along with the introduction of a heterologous pyruvate formate lyase in the network leads to a growth-coupled production of dodecanol. The production curve is similar to what is shown in Figure 1 lb. While the non-naturally occurring eukaryotic organisms described above produce LCAs in the cytosol, it is also possible to produce LCAs in the mitochondrion. Exemplary designs for the distribution of the carbon flux towards dodecanol production are detailed herein below. Organisms that produce LCAs in the mitochondrion include one or more disruptions in genes that encode enzymes such as a cytosolic pyruvate decarboxylase, a cytosolic ethanol-specific alcohol dehydrogenase, and amitochondrial ethanol-specific alcohol dehydrogenase. Exemplary genese encoding these enzymes include, for example, YLR044C, YLR1 34W, YGR087C, PDC3, YBR145W, YGL256W, YOL086C, YMR303, YMR083W, YPLO88W, YAL061W, YMR318C, YCR1 05W, and YDL 168W. Other genes disruptions include those encoding an enzyme suchas a cytosolic malate dehydrogenase, glycerol-3-phospate dehydrogenase shuttle, catalyzed by, the external NADH dehydrogenase, and internal NADH dehydrogenase. Exemplary genes of the latter include, for example, YOL126C, YDL022W, YOLO59W, YIL155C, YMR145C, YDL085W, and YML120C. 115 Organisms that produce LCAs in the mitochondrion can also include an exogenous nucleic acid encoding an enzyme such as a pyruvate dehydrogenase, a pyruvate: NADP oxidoreductase, a pyruvate formate lyase, an acylating acetaldehyde dehydrogenase, an acetate CoA ligase, and an AMP-forming acetyl CoA synthetase or their corresponding gene regulatory regions as described above. Additionally, such organisms benefit from enhanced NADH transporting shuttle systems for transport of NADH from the cytosol into the mitochondrion. Other exogenous nucleic acids encoding an enzyme that can be inserted in such organisms include a transhydrogenase, formate dehydrogenase, a pyruvate decarboxylase, and a pyruvate oxidase, all in the mitochondrion, or their corresponding gene regulatory regions. In one embodiment a mitochondrial pyruvate dehydrogenase is used in the non-naturally occurring eukaryotic organism. This can be the native pyruvate dehydrogenase which produces both acetyl CoA and NADH as shown in Figure 15a. Since, there is no energy requirement for the conversion of pyruvate to acetyl CoA via this route; the production of dodecanol, for example, is energetically favorable even under anaerobic conditions. The mitochondrial pyruvate dehydrogenase is known to be active in both aerobic and anaerobic conditions in S. cerevisiae (Pronk et al., Yeast 12:1607-1633 (1996)). In some embodiments the enzyme is overexpressed in its native or a heterologous form. The native enzyme can be overexpressed by using a stronger promoter. Additionally, mutations can be introduced aimed at increasing its activity under anaerobic conditions (Kim et al., J Bacteriol. 190:3851-3858 (2008)). Reducing equivalents generated in the cytosol are made available in the mitochondrion for dodecanol production by using the redox shuttles present in S. cerevisiae. Note that these shuttles transport NADH into the mitochondrion for energy generation under respiratory conditions (Overkamp et al., J. Bacteriol. 182:2823-2830 (2000)). For growth-coupled production, pyruvate decarboxylase activity is disrupted to allow for pyruvate flux to be directed towards pyruvate dehydrogenase and to inhibit ethanol formation. The production curve for the mutant network is shown in Figure 15b. In some embodiments, a non-naturally occurring eukaryotic organism uses a heterologous pyruvate:NADP-oxidoreductase. The production of dodecanol in the mitochondrion can be achieved by introduction of the pyruvate:NADP oxidoreductase in the mitochondrion as shown 116 in Figure 16. This enzyme is purified from E. gracilis. Since the enzyme is naturally present in mitochondrion and is active under anaerobic conditions, it is possible to get high activity of the enzyme under anaerobic conditions. The introduction of this enzyme provides the precursor acetyl CoA for dodecanol production and also reducing equivalents. The NADPH generated by the enzyme is converted into NADH by a transhydrogenase, which can be introduced into the mitochondrion. For additional reducing equivalents, the redox shuttles need to transport NADH from the cytosol to the mitochondrion. The growth-coupled production of LCA using this enzyme can be obtained by disruption of pyruvate decarboxylase. The production curve of the mutant strain is very similar to the one shown in Figure 15b. In some embodiments, a non-naturally occurring eukaryotic organism uses a heterologous pyruvate formate lyase. The production of dodecanol using a pyruvate formate lyase in mitochondrion is shown in Figure 17. These genes have been outlined herein above. In such embodiments, the native formate dehydrogenase is retargeted to the mitochondrion to allow for further metabolizing formate and generating more reducing equivalents. This strain can be adopted to carry sufficient flux to sustain high yield and productivity of LCA production in the mitochondrion in the absence of oxygen. Anaerobic growth conditions are feasible for the production of dodecanol using this strain design. Redox shuttles can be overexpressed to transport NADH generated in the cytosol to the mitochondrion. Production in this scenario is possible by disrupting the cytosolic pyruvate decarboxylase activity. The production characteristics of the mutant strain are similar to that shown in Figure 15b. In some embodiments, a non-naturally occurring eukaryotic organism uses a heterologous acetaldehyde dehydrogenase (acylating). In such embodiments, an acylating acetaldehyde dehydrogenase is introduced into the mitochondrion to provide both acetyl-CoA and NADH for LCA production as shown in Figure 18. A pyruvate decarboxylase isozyme is retargeted to the mitochondrion to convert pyruvate into acetaldehyde in some embodiments. The expression of these two activities in the mitochondrion is equivalent to the activity of pyruvate dehydrogenase. The growth-coupled production curve is the same as that shown in Figure 15b. The growth coupled production strain has the native mitochondrial acetaldehyde dehydrogenase (Pronk et al., 117 Yeast 12:1607-1633 (1996)) and the cytosolic pyruvate decarboxylase disrupted in some embodiments. In other embodiments, the mitochondrial ethanol-specific alcohol dehydrogenase is also disrupted to prevent the conversion of acetaldehyde into ethanol. In some embodiments, a non-naturally occurring eukaryotic organism uses a mitochondrial acetyl CoA synthetase (AMP-forming). As discussed above, the expression of this enzyme requires oxygen for favorable energetics. ACS 1, an isozyme of acetyl CoA synthetase is expressed in S. cerevisiae in the mitochondrion under aerobic conditions but is repressed by glucose. This enzyme can be mutated to eliminate the repression or a heterologous enzyme that is expressed under the conditions of interest can be introduced. Additionally, pyruvate decarboxylase also can be expressed in the mitochondrion to form acetate. S. cerevisiae, for example, already possesses a mitochondrial acetaldehyde dehydrogenase (Pronk et al., Yeast 12:1607-1633 (1996)). Alternatively, enzymes such as pyruvate oxidase can be heterologously expressed to convert pyruvate into acetate. One such enzyme candidate is pyruvate oxidase from E. coli (Genbank id: NP_451392.1). This enzyme is naturally expressed in the presence of oxygen. The production of LCA using this strain design benefits from one or more of the following disrupted enzymes: cytosolic malate dehydrogenase, the glycerol-3-phospate dehydrogenase shuttle, the external NADH dehydrogenase, and the internal mitochondrial NADH dehydrogenase. The glycerol-3 -phosphate shuttle is comprised of the cytosolic glycerol-3 phosphate dehydrogenase and the membrane-bound glycerol-3-phosphate:ubiquionone oxidoreductase, with the latter also functioning as the mitochondrial glycerol-3-phosphate dehydrogenase. In some embodiments, the mitochondrial ethanol-specific alcohol dehydrogenase is also disrupted to prevent or reduce the conversion of acetaldehyde into ethanol. The production curve for the wild type strain with a mitochondrial pyruvate decarboxylase added to the network is shown in black in Figure 19b. This curve is shown for aerobic conditions. The production characteristics when the aforementioned disruptions are imposed on the network are shown in light gray. The downregulation of the oxidative part of the pentose phosphate pathway, especially the committing step, glucose-6-phosphate dehydrogenase, further improves the LCA production characteristics of the network. 118 In some embodiments, a non-naturally occurring eukaryotic organism uses a mitochondrial acetate CoA ligase (ADP-forming). Mitochondrial LCA production can also be accomplished using an acetate-CoA ligase to convert acetate into acetyl-CoA as shown in Figure 20. As described above, the use of this enzyme is energetically favorable and LCA production is energetically neutral unless oxygen is supplied. The mitochondrial expression of pyruvate decarboxylase is used in such embodiments. LCA production is obtained by imposing disruptions in cytosolic malate dehydrogenase, the glycerol-3-phospate dehydrogenase shuttle, the external NADH dehydrogenase, and the internal NADH dehydrogenase. The down regulation of the oxidative part of the pentose phosphate pathway further improves the growth coupled production characteristics to yield a production curve similar to the one shown in Figure 19b. In some embodiments, the mitochondrial ethanol-specific alcohol dehydrogenase is also disrupted to prevent or reduce the conversion of acetaldehyde into ethanol. The design strategies described herein are useful not only for enhancing growth-coupled production, but they are also well-suited for enhancing non-growth coupled production because they link the production of long chain alcohols to energy generation and/or redox balance. Exemplary non-growth coupled production methods include implementing an aerobic growth phase followed by an anaerobic production phase. For example, Vemuri et al. J. Ind. Microbiol. Biotechnol. (6):325-332, (2002) describe a dual-phase process for the production of succinate in E. Coli. Okino et al. Apple. Microbiol. Biotechnol. Sep 6. (2008) [Currently available in online edition]. describe a similar non-growth couple production process in a strain of Corynebacterium glutamicum strain. Another such method involves withholding an essential nutrient from a propogated cell culture, thereby limiting growth, but not precluding production as described in Durner et al. Apple. Environ. Microbiol. (8):3408-3414( 2000). Yet another strategy aimed at decoupling growth from production involves replacing the growth substrate with another compound that is more slowly metabolizable as described in Altamirano et al. Biotechnol. Bioeng. 76:351-360 (2001). Growth decoupled-product formation can also be brought about by specific genetic modifications as described in Blombach et al. Apple. Microbiol. Biotechnol. 79:471-9 (2008). 119 Some microbial organisms capable of LCA production are exemplified herein with reference to an Saccharomyces cerevisaie genetic background. However, with the complete genome sequence available now for 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 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 LCA described herein with reference to a particular organism such as Saccharomyces cerevisaie can be readily applied to other microorganisms. 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. The methods of the invention are applicable to various eukarotic organisms such as yeast and fungus. The yeast can include S. cerevisiae and Rhizopus arrhizus, for example. Exemplary eukaryotic species include those selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergilus terreus, Aspergillus niger, Rhizopus arrhizus, Rhizopus oryzae, Candida albicans, Candida boidinii and Pichia pastoris. Additionally, select cells from larger eukaryotic organisms are also applicable to methods of the present invention. Genes can be inserted into S. cerevisiae, using several methods; some of these are plasmid-based whereas others allow for the incorporation of the gene in the chromosome. The latter approach employs an integrative promoter based expression vector, for example, the pGAPZ or the pGAPZa vector based on the GAP promoter. The expression vector constitutes the GAP promoter, the HIS4 wild-type allele for integration and the 3' AOX transcription termination region of P. pastoris in addition to a KanMX cassette, flanked by loxP sites enabling removal and recycling of the resistance marker. The vectors are commercially available from Invitrogen. The details of which are elaborated in the Example below. The engineered strains are characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for 120 a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose, alcohols, and other organic acid byproducts in the culture supernatant will be determined by analytical methods including HPLC using an HPX-87H column (BioRad), or GC-MS, and used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures. The invention also provides a method for producing long chain alcohols by culturing the non naturally occurring eukaryotic organism described herein above. The one or more gene disruptions occur in genes encoding an enzyme to coupling long chain alcohol production to growth of the organism when the gene disruption reduces an activity of the enzyme. The one or more gene disruptions confers stable growth-coupled production of long chain alcohols onto the organism. In alternate embodiments the gene disruptions can enhance LCA production in a non growth dependent manner. Each of the strains presented herein may be supplemented with additional disruptions if it is determined that the predicted strain designs do not sufficiently couple the formation of LCAs with biomass formation. However, the list of gene disruption sets provided here serves as an excellent starting point for the construction of high-yielding growth-coupled LCA production strains. Each of the proposed strains can be supplemented with additional disruptions if it is determined that the predicted strain designs do not sufficiently couple the formation of the product with biomass formation. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis and can also be disrupted. However, the list of gene disruption sets provided here serves as a starting point for construction of high-yielding growth-coupled LCA production strains. The non-naturally occurring microbial organisms of the invention can be employed in the growth-coupled production of LCA. Essentially any quantity, including commercial quantities, can be synthesized using the growth-coupled LCA producers of the invention. Because the organisms of the invention obligatorily couple LCA to continuous growth or near-continuous growth processes are particularly useful for biosynthetic production of LCA. Such continuous 121 and/or near continuous growth processes are described above and exemplified below in the Example I. Continuous and/or near-continuous microorganism growth processes also are well known in the art. Briefly, continuous and/or near-continuous growth processes involve maintaining the microorganism in an exponential growth or logarithmic phase. Procedures include using apparatuses such as the Evolugator" evolution machine (Evolugate LLC, Gainesville, FL), fermentors and the like. Additionally, shake flask fermentation and grown under microaerobic conditions also can be employed. Given the teachings and guidance provided herein those skilled in the art will understand that the growth-coupled LCA producing microorganisms can be employed in a variety of different settings under a variety of different conditions using a variety of different processes and/or apparatuses well known in the art. Generally, the continuous and/or near-continuous production of LCA will include culturing a non-naturally occurring growth-coupled LCA 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 be grown, for example, for a day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous cultures can include time durations of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. 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. In particular embodiments, culturing is conducted in a substantially anaerobic culture medium. LCA can be harvested or isolated at any time point during the continuous and/or near-continuous culture period exemplified above. As exemplified below, the longer the microorganisms are maintained in a continuous and/or near-continuous growth phase, the proportionally greater amount of LCA can be produced. Therefore, the invention provides a method for producing LCA that includes culturing a non naturally occurring microbial organism that includes one or more gene disruptions. The disruptions can occur in genes encoding an enzyme to coupling LCA production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, such that the disruptions confer stable growth-coupled production of LCA onto the non-naturally microbial organism. 122 In some embodiments, the gene disruption can include a complete gene deletion. In some embodiments other means to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it may confer to the non-naturally occurring organism from reverting to its wild-type. In particular, the gene disruptions are selected from the gene set that includes genes detailed herein above. The metabolic engineering strategies listed in this disclosure assume that the organism can produce long chain alcohols via the malonyl-CoA independent pathway. The construction of a recombinant host organism capable of producing long chain alcohols via the malonyl-CoA independent pathway involves engineering a non-naturally occurring microbial organism having a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an acyl-reduction pathway having at least one exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme expressed in sufficient amounts to produce a primary alcohol. Such a malonyl-CoA independent FAS pathway includes a ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3 hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase. The acyl reduction pathway includes an acyl-CoA reductase and an alcohol dehydrogenase. In order to validate the computational predictions presented herein, the strains must be constructed, evolved, and tested. Escherichia coli K-12 MG1655 housing the MI-LCA pathway will serve as the strain into which the disruptions will be introduced. The disruptions will be constructed by incorporating in-frame deletions using homologous recombination via the X Red recombinase system of Datsenko and Wanner (Datsenko, K.A. and B.L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A, 2000. 97(12): p. 6640-5.). The approach involves replacing a chromosomal sequence (i.e., the gene targeted for removal) with a selectable antibiotic resistance gene, which itself is later removed. Knockouts are integrated one by one into the recipient strain. No antibiotic resistance markers remain after each deletion allowing accumulation of multiple mutations in each target strain. The deletion technology completely removes the gene targeted for removal so as to substantially reduce the possibility of the constructed mutants reverting back to the wild-type. 123 As intermediate strains are being constructed, strain performance will be quantified by performing shake flask fermentations. Anaerobic conditions will be obtained by sealing the flasks with a rubber septum and then sparging the medium with nitrogen. For strains where growth is not observed under strict anaerobic conditions, microaerobic conditions can be applied by covering the flask with foil and poking a small hole for limited aeration. All experiments are performed using M9 minimal medium supplemented with glucose unless otherwise stated. Pre cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. LCAs, ethanol, and organic acids are analyzed by GC-MS or HPLC using routine procedures. Triplicate cultures are grown for each strain. The performance of select strains is tested in anaerobic, pH-controlled batch fermentations. This enables reliable quantification of the growth, glucose uptake, and formation rates of all products, as well as ensuring that the accumulation of acidic fermentation products will not limit cell growth. In addition, it allows accurate determination of LCA volumetric productivity and yield, two important parameters in benchmarking strain performance. Fermentations are carried out in 1-L bioreactors with 600 mL working volume, equipped with temperature and pH control. The reactor is continuously sparged with N 2 at approximately 0.5 L/min to ensure that DO levels remain below detection levels. The culture medium is the same as described above, except that the glucose concentration is increased in accordance with the higher cell density achievable in a fermentation vessel. Chemostat experiments will be conducted to obtain a direct measure of how the switch in fermentation mode from batch to continuous affects LCA yield and volumetric productivity. The bioreactors described above using batch mode are operated in chemostat mode through continuous supply of medium and removal of spent culture. The inlet flow rate is set to maintain a constant dilution rate of 80% of the maximum growth rate observed for each strain in batch, and the outlet flow is controlled to maintain level. Glucose is the limiting nutrient in the medium, and set to achieve the desired optical density in the vessel. 124 The recombinant strains are initially expected to exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To enable this adjustment, the strains are adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong, S.S. and B.O. Palsson, Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes. Nat Genet, 2004. 36(10): p. 1056-8.). The OptKnock-generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli (Fong, S.S. and B.O. Palsson, Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes. Nat Genet, 2004. 36(10): p. 1056-8; Fong, S.S., J.Y. Marciniak, and B.O. Palsson, Description and interpretation of adaptive evolution of Escherichia coli K-12 MGJ655 by using a genome scale in silico metabolic model. J Bacteriol, 2003. 185(21): p. 6400-8; Ibarra, R.U., J.S. Edwards, and B.O. Palsson, Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth. Nature, 2002. 420(6912): p. 186-189.) that could potentially result in one strain having superior production qualities over the others. Evolutions are run for a period of 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained. The growth-coupled biochemical production concept behind the OptKnock approach results in the generation of genetically stable overproducers. The engineered strains can be characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate can be determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose and other organic acid byproducts in the culture supernatant are determined by HPLC using an HPX-87H column (BioRad), and used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures. 125 Following the adaptive evolution process, the new strains are characterized again by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results will be compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes in the above figures. The most successful OptKnock design/evolution combinations are chosen to pursue further, and are characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers. Thus, the cultures are maintained in continuous mode for one month to evaluate long-term stability. Periodic samples are taken to ensure that yield and productivity are maintained throughout the experiment. As previously mentioned, one computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). The framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become a byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product. Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux 126 balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or disruptions. OptKnock computational framework allows the construction of model formulations that enable an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. patent application serial No. 11/891,602, filed August 10, 2007. Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny@. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny@ is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components. These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. 127 Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny@ and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art. The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as a product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes. Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur. To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the 128 biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny@. The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny@. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene disruption combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction disruptions requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the 129 resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). An in silico stoichiometric model of Escherichia coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene disruptions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of disruptions. To enumerate all meaningful solutions, that is, all sets of disruptions leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above. Adaptive evolution is a powerful experimental technique that can be used to increase growth rates of mutant or engineered microbial strains, or of wild-type strains growing under unnatural environmental conditions. It is especially useful for strains designed via the OptKnock formalism, which results in growth-coupled product formation. Therefore, evolution toward optimal growing strains will indirectly optimize production as well. Unique strains of E. coli K 12 MG1655 were created through gene knockouts and adaptive evolution. (Fong, S. S. and B. 0. Palsson, Nat.Genet. 36:1056-1058 (2004).) In this work, all adaptive evolutionary cultures were maintained in prolonged exponential growth by serial passage of batch cultures into fresh medium before the stationary phase was reached, thus rendering growth rate as the primary selection pressure. The genes that were selected for this knockout study were ackA, frdA, pckA, ppc, tpiA, and zwf Knockout strains were constructed and evolved on minimal medium supplemented with different carbon substrates (four for each knockout strain). Evolution cultures were carried out in duplicate or triplicate, giving a total of 50 evolution knockout strains. The evolution cultures were maintained in exponential growth until a stable growth rate was reached. The computational predictions were accurate (ie., within 10%) at predicting the post evolution growth rate of the knockout strains in 38 out of the 50 cases examined. Furthermore, a combination of OptKnock design with adaptive evolution has led to improved lactic acid 130 production strains. (Fong, S. S., A. P. Burgard, C. D. Herring, E. M. Knight, F. R. Blattner, C. D. Maranas, and B. 0. Palsson, Biotechnol Bioeng 91:643-648 (2005).) The guidance of these teachings relevant to E. coli can be applied to other organisms. There are a number of developed technologies for carrying out adaptive evolution. Exemplary methods are provided herein below. In some embodiments, optimization of a non-naturally occurring organism of the present invention includes subject the use of any of the these adaptive evolution techniques. Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured organisms have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature, (Lenski, R. E. and M. Travisano, Proc Natl Acad Sci US.A. 91:6808-6814 (1994).) in experiments which clearly demonstrated consistent improvement in reproductive rate over period of years. In the experiments performed in the Palsson lab described above, transfer is usually performed during exponential phase, so each day the transfer volume is precisely calculated to maintain exponential growth through the next 24 hour period. This process is usually done manually, with considerable labor investment, and is subject to contamination through exposure to the outside environment. Furthermore, since such small volumes are transferred each time, the evolution is inefficient and many beneficial mutations are lost. On the positive side, serial dilution is inexpensive and easy to parallelize. In continuous culture the growth of cells in a chemostat represents an extreme case of dilution in which a very high fraction of the cell population remains. As a culture grows and becomes saturated, a small proportion of the grown culture is replaced with fresh media, allowing the culture to continually grow at close to its maximum population size. Chemostats have been used to demonstrate short periods of rapid improvement in reproductive rate. (Dykhuizen, D. E., Methods Enzymol 613-631 (1993).) The potential power of these devices was recognized, but traditional chemostats were unable to sustain long periods of selection for increased reproduction rate, due to the unintended selection of dilution-resistant (static) variants. These variants are able to resist dilution by adhering to the surface of the chemostat, and by doing so, outcompete less sticky individuals including those that have higher reproductive rates, thus obviating the intended 131 purpose of the device. (Chao, L. and G. Ramsdell J.Gen.Microbiol 20:132-138 (1985).) One possible way to overcome this drawback is the implementation of a device with two growth chambers, which periodically undergo transient phases of sterilization, as described in the patent by the Pasteur Institute (Marliere and Mutzel, US Patent 6,686,194, filed 1999). Evolugator" is a continuous culture device developed by Evolugate, LLC (Gainesville, FL) exhibits significant time and effort savings over traditional evolution techniques. (de Crecy, E., Metzgar, D., Allen, C., Penicaud, M., Lyons, B., Hansen, C.J., de Crecy-Lagard, V. AppL. Microbiol. Biotechnol. 77:489-496 (2007).) The cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat in evolution of cell fitness. For example, a mutant of Acinetobacter sp ADP1 deficient in a component of the translation apparatus, and having severely hampered growth, was evolved in 200 generations to 80% of the wild-type growth rate. However, in contrast to the chemostat which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. The transfer volume is adjustable, and normally set to about 50%. As disclosed herein, a nucleic acid encoding a desired activity of a primary alcohol, a fatty acyl CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl CoA, fatty aldehyde or fatty alcohol pathway enzyme or protein to increase production of primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator. One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or 132 alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >104). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol.Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol.Eng 22:1-9 (2005).; and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (Km), including broadening substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen. A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a primary alcohol, a fatty acyl-CoA, a fatty ester, a wax, an acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Theor.Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 133 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)). Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al,, Nucleic Acids Res 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which 134 mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et aL, Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem, 341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode "all genetic diversity in targets" and allows a very high diversity for the shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:el 17 (2005)). Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation MutagenesisTM (GSSMTM), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al., 135 Apple. Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)). Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassemblyrM (TGRTm) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. A cad. Sci. USA 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLE I Primary Alcohol Biosynthesis This Example describes the generation of a microbial organism capable of producing primary alcohols using a malonyl-CoA independent FAS metabolic pathway and acyl-reduction metabolic pathways. 136 Escherichia coli is used as a target organism to engineer a malonyl-CoA-independent FAS and acyl-reduction pathway as shown in Figure 1. E. coli provides a good host for generating a non naturally occurring microorganism capable of producing primary alcohol, such as octanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, effectively under anaerobic conditions. To generate an E. coli strain engineered to produce primary alcohol, nucleic acids encoding the enzymes utilized in the malonyl-CoA-independent FAS and acyl-reduction pathway as described previously, are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra, 1989). In particular, thefadI/fadJgenes (NP_416844.1 and NP_416843.1), encoding the multienzyme complex with ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activities under anaerobic conditions, and the TDE0597 (NP_97121 1.1), encoding enoyl-CoA reductase, are cloned into the pZE 13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The acrlgene (YP_047869.1), encoding acyl-CoA reductase, and the alrA gene (BAB12273.1), encoding alcohol dehydrogenase, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of plasmids are transformed into E, coli strain MG 1655 to express the proteins and enzymes required for the malonyl-CoA-independent FAS and acyl-reduction pathway. The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of malonyl-CoA-independent FAS and acyl-reduction pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities (see, for example, Tucci, supra, 2007; Hoffmeister et al., 2005; Inui et al., supra, 1984; Winkler, 2003; Tani, 2000; Reiser, 1997; Ishige, 2000). The ability of the engineered E. coli strain to produce primary alcohol, such as octanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS). 137 Microbial strains engineered to have a functional malonyl-CoA-independent FAS and acyl reduction pathway is further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers. To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also 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 in 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 primary alcohols. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of primary alcohols. Adaptive evolution also can be used to generate better producers of, for example, the acetyl-CoA intermediate or the primary alcohol product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the primary alcohol producer to further increase their production. For large-scale production of primary alcohols, the above malonyl-CoA independent FAS pathway-containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be 138 quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng., 775-779 (2005). Isolation of the product primary alcohol is performed based their insolubility in water. In particular, a two-phase fermentation process is used for separation of these product alcohols where they can either form a separate phase or be readily extracted in an organic phase from the fermentation broth. Residual cells and any other insoluble impurities are removed by filtration, allowing a continuous or semi-continuous fermentation process. EXAMPLE 11 Microorganisms Having Growth-couipled Production of LCA This Example describes the construction in silico designed strains for the growth-coupled production of LCA. E. coli K-12 MG1655 serves as the wild-type strain into which the disruptions are introduced. The disruptions are constructed by incorporating in-frame deletions using homologous recombination via the X Red recombinase system of Datsenko and Wanner. (Datsenko, K.A. and B.L. Wanner, Proc NatlAcadSci USA., 97(12):6640-5 (2000).) The approach involves replacing a chromosomal sequence (i.e., the gene targeted for removal) with a selectable antibiotic resistance gene, which itself is later removed. Knockouts are integrated one by one into the recipient strain. No antibiotic resistance markers will remain after each deletion allowing accumulation of multiple mutations in each target strain. The deletion technology completely removes the gene targeted for removal so as to substantially reduce the possibility of the constructed mutants reverting back to the wild-type. As described further below, one exemplary growth condition for achieving biosynthesis of LCA includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organism of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions 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 139 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 /C0 2 mixture or other suitable non-oxygen gas or gases. The engineered strains are characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose, LCA, and other organic acid byproducts in the culture supernatant are determined by HPLC using an HPX-87H column (BioRad), and are used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures. The recombinant strains can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To enable this adjustment, the strains are adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model. (Fong, S.S. and B.O. Palsson, Nat Genet, 36(10):1056-8 (2004).) These teachings can be applied to Escherichia coli. Should the OptKnock predictions prove successful; the growth improvements brought about by adaptive evolution will be accompanied by enhanced rates of LCA production. The OptKnock generated strains are adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli (Fong, S.S. and B.O. Palsson, Nat Genet, 36(10):1056-8 (2004); Fong, S.S., J.Y. Marciniak, and B.O. Palsson, JBacteriol, 185(21):6400-8 (2003); Ibarra, R.U., J.S. Edwards, and B.O. Palsson, Nature, 420(6912):186-189 (2002)) that could potentially result in one strain having superior production qualities over the others. Evolutions are run for a period of 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained. 140 The adaptive evolution procedure involves maintaining the cells in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. Briefly, one procedure allows cells to reach mid-exponential growth (A 6 oo=0.5) before being diluted and passed to fresh medium (i.e., M9 minimal media with 2 g/L carbon source). This process is repeated, allowing for about 500 generations for each culture. Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded for each day throughout the course of the evolutions. The evolutions are performed in triplicate due to differences in the evolutionary patterns witnessed previously Donnelly et al., Appl Biochem Biotechnol 70-72: 187-98 (1998); Vemuri et al., Appl Environ Microbiol 68:1715-27 (2002), that could potentially result in one strain having superior production qualities over the others. The adaptive evolution step can take up to about two months or more. The adaptive evolution step also can be less than two months depending on the strain design, for example. Another process can evolve cells using automation technology and is commercially available by Evolugate, LLC (Gainesville, FL) under a service contract. The procedure employs the Evolugatorm evolution machine which results in significant time and effort savings over non automated evolution techniques. Cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat for evolution of cell fitness. For example, a mutant of Acinetobacter sp ADP1 deficient in a component of the translation apparatus, and having severely hampered growth, was evolved in 200 generations to 80% of the wild-type growth rate. However, in contrast to the chemostat which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall growth. The transfer volume is adjustable, and normally set to about 50%. In contrast to a chemostat, which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded each day throughout the course of the evolutions. The Evolugator is used for each strain until a stable growth rate is achieved. Growth 141 rate improvements of nearly 50% have been observed in two weeks using this device. The above-described strains are adaptively evolved in triplicate (running in parallel). At ten day intervals, culture samples are taken from the Evolugator, purified on agar plates, and cultured in triplicate as discussed above to assess strain physiology. EvolugatorTm is a continuous culture device that exhibits significant time and effort savings over traditional evolution techniques. (de Crecy et al., Appi. Microbiol. Biotechnol. 77:489-496 (2007)). Following the adaptive evolution process, the new strains are again characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes. The most successful OptKnock design/evolution combinations are chosen to pursue further, and is characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers. Thus, the cultures can be maintained in continuous mode for one month to evaluate long-term stability. Periodic samples will be taken to ensure that yield and productivity are maintained throughout the experiment. EXAMPLE III Microorganisms Havin2 Growth-coupled Production of LCA This Example describes the construction in silico designed strains for the growth-coupled production of LCA. Gene deletions are introduced into S. cerevisiae by homologous recombination of the gene interrupted by the KanMX cassette, flanked by loxP sites enabling removal and recycling of the resistance marker (e.g. URA3) (Wach, A., et al., PCR-based gene targeting in Saccharomyces cerevisiae, in Yeast Gene Analysis, M.F. Tuite, Editor. 1998, Academic Press: San Diego). Starting with a loxP-kanMX-loxP sequence on a plasmid, an artificial construct with this sequence flanked by fragments of the gene of interest are created by PCR using primers containing both 45-50 bp target sequence followed by a region homologous to the above cassette. This linear DNA is transformed into wild-type S, cerevisiae, and recombinants are selected by geneticin resistance (Wach, A., et al. supra]. Colonies are purified and tested for 142 correct double crossover by PCR. To remove the KanMX marker, a plasmid containing the Cre recombinase and bleomycin resistance are introduced, promoting recombination between the loxP sites (Gueldener, U., et al., Nucleic Acids Res. e23 (2002))]. Finally, the resulting strain is cured of the Cre plasmid by successive culturing on media without any antibiotic present. The final strain will have a markerless gene deletion, and thus the same method can be used to introduce multiple deletions in the same strain. The strains are constructed, evolved, and tested by methods disclosed herein. Genes can be inserted into S. cerevisiae, for example, using several methods. These methods can be plasmid based whereas others allow for the incorporation of the gene in the chromosome. The latter approach employs an integrative promoter based expression vector, for example, the pGAPZ or the pGAPZa vector based on the GAP promoter. The expression vector constitutes the GAP promoter, the HIS4 wild-type allele for integration and the 3' AOX transcription termination region of P. pastoris in addition to a KanMX cassette, flanked by loxP sites enabling removal and recycling of the resistance marker. Both of these vectors are commercially available from Invitrogen (Carlsbad, CA). The method entails the synthesis and amplification of the gene of interest with suitable primers, followed by the digestion of the gene at a unique restriction site, such as that created by the EcoRlIXhoI enzymes (Vellanki et al., Biotechnol. Lett. 29:313-318 (2007)). The gene is inserted at the EcoRI and XhoI sites in the expression vector, downstream of the GAP promoter. The gene insertion is verified by PCR and/or DNA sequence analysis. The recombinant plasmid is then linearized with NarI for histidine integration, purified and integrated into the chromosomal DNA of S. cerevisiae using an appropriate transformation method. The cells are plated on the YPD medium with the appropriate selection marker (e.g., kanamycin) and incubated for 2-3 days. The transformants will then be analyzed for the requisite gene insert by colony PCR. To remove the antibiotic marker, a plasmid containing the Cre recombinase is introduced, promoting recombination between the loxP sites (Gueldener et al., supra). Finally, the resulting strain is cured of the Cre plasmid by successive culturing on media without any antibiotic present. The final strain will have a markerless gene deletion, and thus the same method can be used to introduce multiple insertions in the same strain. 143 The engineered strains are characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. Cultures are grown overnight and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate is determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose, alcohols, and other organic acid byproducts in the culture supernatant are determined by analytical methods including HPLC using an HPX-87H column (BioRad), or GC-MS, and used to calculate uptake and secretion rates. All experiments are performed with triplicate cultures. The knockout strains are initially anticipated to exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To enable this adjustment, the strains will be adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells will be compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model. The growth improvements brought about by adaptive evolution can be accompanied by enhanced rates of LCA production. The OptKnock-generated strains can be adaptively evolved in triplicate (running in parallel) due to differences in the evolutionary patterns witnessed previously in E. coli that could potentially result in one strain having superior production qualities over the others. Evolutions can be run for a period of 2-6 weeks, or longer depending upon the rate of growth improvement attained. In general, evolutions can be stopped once a stable phenotype is obtained. The adaptive evolution procedure involves maintaining the cells in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. Briefly, one procedure allows cells to reach mid-exponential growth (A 6 ooO=0.5) before being diluted and passed to fresh medium (i.e., M9 minimal media with 2 g/L carbon source). This process is repeated, allowing for about 500 generations for each culture. Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded for each day throughout the course of the evolutions. The evolutions are performed in triplicate due to differences in the evolutionary patterns witnessed previously Donnelly et al., Appl Biochem 144 Biotechnol. 70-72: 187-98 (1998); Vemuri et al., Appl Environ Microbiol. 68:1715-27 (2002), that could potentially result in one strain having superior production qualities over the others. The adaptive evolution step can take up to about two months or more. The adaptive evolution step also can be less than two months depending on the strain design, for example. Another process can evolve cells using automation technology and is commercially available by Evolugate, LLC (Gainesville, FL) under a service contract. The procedure employs the Evolugatorm evolution machine which results in significant time and effort savings over non automated evolution techniques. Cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat for evolution of cell fitness. In contrast to a chemostat, which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. Culture samples are taken, frozen with liquid nitrogen, and the optical culture density recorded each day throughout the course of the evolutions. The Evolugator is used. for each strain until a stable growth rate is achieved. Growth rate improvements of nearly 50% have been observed in two weeks using this device. The above-described strains are adaptively evolved in triplicate (running in parallel). At ten day intervals, culture samples are taken from the Evolugator, purified on agar plates, and cultured in triplicate as discussed above to assess strain physiology. Following the adaptive evolution process, the new strains are again characterized by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the OptKnock predictions by plotting actual growth and production yields along side the production envelopes. The most successful OptKnock design/evolution combinations are chosen to pursue further, and is characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproducers. Thus, the cultures can be maintained in continuous mode for one month to evaluate long-term stability. Periodic samples will be taken to ensure that yield and productivity are maintained throughout the experiment. 145 Described herein above, is the application of the OptKnock methodology for generating useful gene disruption targets. Multiple disruption strategies were enumerated for establishing the coupling between LCA production and Escherichia coli growth. This methodology is applicable to a wide variety of cells and microorganisms other than Escherichia coli and also can utilize metabolic modeling and simulation systems other than OptKnock. The combined computational and engineering platform described herein is generally applicable to any stoichiometric model organism and the teachings and guidance provided herein will allow those skilled in the art to design and implement sets of genetic manipulations for metabolic alterations that lead to the growth-coupled production of any biochemical product. The present disclosure provides gene disruption strategies for growth-coupled production of LCA in Escherichia coli under anaerobic conditions. The suggested strategies can increase product yields significantly over the reported yields for each of these products. A comprehensive list of the strategies is listed in Table 1 for LCA production. The associated genes and stoichiometries for each of the reaction disruption are catalogued in Table 2. Table 3 lists metabolite abbreviations and their corresponding names along with their location. Table 1: The list of all disruption strategies identified by OptKnock that are most likely to provide growth-coupled LCA produciton. 1 ADHEr LDHD ASPT MDH PFLi PGDHY PYK DHAPT 2 ADHEr LDHD ASPT MDH PFLi PGL PYK DHAPT 3 ADHEr LDH_D ASPT G6PDHy MDH PFLi PYK DHAPT 4 ADHEr LDHD ASPT EDA MDH PFLi PYK DHAPT 5 ADHEr LDHD GLCpts GLUDy PGDH PGI PTAr 6 ADHEr LDHD ACKr GLCpts GLUDy PGDH PGI 7 ADHEr LDHD GLCpts GLUDy PGI PTAr TAL 8 ADHEr LDHD GLCpts GLUDy PGI PTAr TKT1 9 ADHEr LDH_D ACKr GLCpts GLUDy PGI TKT1 10 ADHEr LDHD ACKr GLCpts GLUDy PGI TAL 11 ADHEr LDH_D FBA GLCpts GLUDy PTAr 12 ADHEr LDH_D GLCpts GLUDy PTAr TPI 13 ADHEr LDHD ACKr GLCpts GLUDy TPI 14 ADHEr LDH_D GLCpts GLUDy PFK PTAr 15 ADHEr LDH_D ACKr FBA GLCpts GLUDy 16 ADHEr LDHD ACKr GLCpts GLUDy PFK 146 17 ADHEr LDHD ACKr GLCpts GLUDy PGI RPE 18 ADHEr LDHD GLCpts GLLTDy PGI PTAr RPE 19 ADHEr LDHD GLCpts GLUDy PGI PTAr TKT2 20 ADHEr LDHD ACKr GLCpts GLUDy PGI TKT2 21 ADHEr LDHD ACKr GLCpts PGDH PGI 22 ADHEr LDHD GLCpts PGDH PGI PTAr 23 ADHEr LDHD ACKr GLCpts PGI TKT1 24 ADHEr LDHD ACKr GLCpts PGI TAL 25 ADHEr LDHD GLCpts PGI PTAr TAL 26 ADHEr LDHD GLCpts PGI PTAr TKT1 27 ADHEr LDHD ACKr GLCpts PFK 28 ADHEr LDHD ACKr GLCpts TPI 29 ADHEr LDHD ACKr FBA GLCpts 30 ADHEr LDHD FBA GLCpts PTAr 31 ADHEr LDHD GLCpts PFK PTAr 32 ADHEr LDHD GLCpts PTAr TPI 33 ADHEr LDHD ACKr GLCpts PGI RPE 34 ADHEr LDHD GLCpts PGI PTAr RPE 35 ADHEr LDHD FRD2 GLCpts GLUDy PFLi PGI 36 ADHEr LDHD ACKr GLCpts PGI TKT2 37 ADHEr LDHD GLCpts PGI PTAr TKT2 38 ADHEr LDHD FRD2 GLCpts GLUDy PFLi TPI 39 ADHEr LDHD FBA FRD2 GLCpts GLUDy PFLi 40 ADHEr LDHD FRD2 GLCpts GLUDy PFK PFLi 41 ADHEr LDHD ASPT ATPS4r FUM NADH6 PGI 42 ADHEr LDHD ASPT ATPS4r MDH NADH6 PGI 43 ADHEr LDHD ASPT ATPS4r MDH NADH6 PFK 44 ADHEr LDHD ASPT ATPS4r FBA FUM NADH6 45 ADHEr LDHD ASPT ATPS4r FUM NADH6 TPI 46 ADHEr LDHD ASPT ATPS4r MDH NADH6 TPI 47 ADHEr LDHD ASPT ATPS4r FBA MDH NADH6 48 ADHEr LDHD ASPT ATPS4r FUM NADH6 PFK 49 ADHEr LDHD FUM GLCpts GLUDy PFLi PGI 50 ADHEr LDHD GLCpts GLUDy MDH PFLi PGI 51 ADHEr LDHD GLCpts P.FLi PGI SUCD4 52 ADHEr LDHD GLCpts NADH6 PFLi PGI 53 ADHEr LDHD FRD2 GLCpts PFLi PGI 54 ADHEr LDHD ACKr GLUDy HEX1 PGDH PGI 55 ADHEr LDHD GLUDy HEX1 PGDH PGI PTAr 56 ADHEr LDHD FUM GLCpts GLUDy PFK PFLi 57 ADHEr LDHD FBA FUM GLCpts GLUDy PFLi 58 ADHEr LDHD GLCpts GLUDy MDH PFK PFLi 59 ADHEr LDHD FUM GLCpts GLUDy PFLi TPI 60 ADHEr LDHD FBA GLCpts GLUDy MDH PFLi 61 ADHEr LDHD GLCpts GLUDy MDH PFLi TPI 62 ADHEr LDHD GLCpts NADH6 PFLi TPI 147 63 ADHEr LDHD FRD2 GLCpts PFLi TPI 64 ADHEr LDHD FBA FRD2 GLCpts PFLi 65 ADHEr LDHD FBA GLCpts NADH6 PFLi 66 ADHEr LDHD FBA GLCpts PFLi SUCD4 67 ADHEr LDHD FRD2 GLCpts PFK PFLi 68 ADHEr LDHD GLCpts PFLi SUCD4 TPI 69 ADHEr LDHD GLCpts PFK PFLi SUCD4 70 ADHEr LDHD GLCpts NADH6 PFK PFLi 71 ADHEr LDHD ASPT GLCpts MDH PFLi PGI 72 ADHEr LDHD ASPT FUM GLCpts PFLi PGI 73 ADHEr LDHD ASPT ATPS4r MDH PGI PPS 74 ADHEr LDHD ASPT ATPS4r FUM PGI PPS 75 ADHEr LDHD GLUDy HEX1 PGI PTAr TAL 76 ADHEr LDHD ACKr GLUDy HEXI PGI TAL 77 ADHEr LDHD ACKr GLUDy HEX1 PGI TKT1 78 ADHEr LDHD GLUDy HEX1 PGI PTAr TKT1 79 ADHEr LDHD ACKr GLUDy HEX1 TPI 80 ADHEr LDHD ACKr GLUDy HEX1 PFK 81 ADHEr LDHD GLUDy HEXI PTAr TPI 82 ADHEr LDHD GLUDy HEX1 PFK PTAr 83 ADHEr LDHD ACKr FBA GLUDy HEX1 84 ADHEr LDHD FBA GLUJDy HEX1 PTAr 85 ADHEr LDHD ASPT GLCpts MDH PFLi TPI 86 ADHEr LDHD ASPT FBA GLCpts MDH PFLi 87 ADHEr LDHD ASPT GLCpts MDH PFK PFLi 88 ADHEr LDHD ASPT FUM GLCpts PFK PFLi 89 ADHEr LDHD ASPT FUM GLCpts PFLi TPI 90 ADHEr LDHD ASPT FBA FUM GLCpts PFLi 91 ADHEr LDHD GLUDy HEX PGI PTAr RPE 92 ADHEr LDHD ACKr GLUDy HEX1 PGI RPE 93 ADHEr LDHD ASPT ATPS4r FUM GLUDy PGI 94 ADHEr LDHD ASPT ATPS4r GLJDy MDH PGI 95 ADHEr LDHD ASPT ATPS4r FBA MDH PPS 96 ADHEr LDHD ASPT ATPS4r FUM PFK PPS 97 ADHEr LDHD ASPT ATPS4r MDH PFK PPS 98 ADHEr LDHD ASPT ATPS4r MDH PPS TPI 99 ADHEr LDHD ASPT ATPS4r FUM PPS TPI 100 ADHEr LDHD ASPT ATPS4r FBA FUM PPS 101 ADHEr LDHD ACKr GLUDy HEX] PGI TKT2 102 ADHEr LDHD GLUDy HEX1 PGI PTAr TKT2 103 ADHEr LDHD ASPT ATPS4r FBA FUM GLUDy 104 ADHEr LDHD ASPT ATPS4r GLUDy MDH PFK 105 ADHEr LDHD ASPT ATPS4r FBA GLUDy MDH 106 ADHEr LDHD ASPT ATPS4r FUM GLLTDy TPI 107 ADHEr LDHD ASPT ATPS4r FUM GLUDy PFK 108 ADHEr LDHD ASPT ATPS4r GLUDy MDH TPI 148 109 ADHEr LDHD ACKr GLLDy PGDH PGI 110 ADHEr LDHD GLUDy PGDH PGI PTAr 111 ADHEr LDHD ACKr GLUDy PGI TAL 112 ADHEr LDHD GLUDy PGI PTAr TKT1 113 ADHEr LDHD ACKr GLUDy PGI TKT1 114 ADHEr LDHD GLUDy PGI PTAr TAL 115 ADHEr LDHD ACKr GLUDy TPI 116 ADHEr LDHD GLUDy PFK PTAr 117 ADHEr LDHD FBA GLUDy PTAr 118 ADHEr LDHD ACKr FBA GLUDy 119 ADHEr LDHD ACKr GLUDy PFK 120 ADHEr LDHD GLUDy PTAr TPI 121 ADHEr LDHD ACKr GLUDy PGI RPE 122 ADHEr LDHD GLUDy PGI PTAr RPE 123 ADHEr LDHD GLUDy PGI PTAr TKT2 124 ADHEr LDHD ACKr GLUDy PGI TKT2 125 ADHEr LDHD HEX PGDH PGI PTAr 126 ADHEr LDHD ACKr HEX PGDH PGI 127 ADHEr LDHD ASPT ATPS4r CBMK2 FUM PGI 128 ADHEr LDHD ASPT ATPS4r CBMK2 MDH PGI 129 ADHEr LDHD HEX PGI PTAr TAL 130 ADHEr LDHD HEXi PGI PTAr TKT1 131 ADHEr LDHD ACKr HEXI PGI TKT1 132 ADHEr LDHD ACKr HEX PGI TAL 133 ADHEr LDHD GLUDy HEX PFLi PGI SUCD4 134 ADHEr LDHD FRD2 GLLTDy HEX1 PFLi PGI 135 ADHEr LDHD GLUDy HEX1 NADH6 PFLi PGI 136 ADHEr LDHD ACKr FBA HEX 137 ADHEr LDHD FBA HEX PTAr 138 ADHEr LDHD HEX] PFK PTAr 139 ADHEr LDH D ACKr HEX PFK 140 ADHEr LDHD ACKr HEX1 TPI 141 ADHEr LDHD HEX1 PTAr TPI 142 ADHEr LDHD HEX] PGI PTAr RPE 143 ADHEr LDHD ACKr HEX1 PGI RPE 144 ADHEr LDHD ASPT ATPS4r CBMK2 FBA FUM 145 ADHEr LDHD ASPT ATPS4r CBMK2 FBA MDH 146 ADHEr LDHD ASPT ATPS4r CBMK2 MDH TPI 147 ADHEr LDHD ASPT ATPS4r CBMK2 FUM PFK 148 ADHEr LDHD ASPT ATPS4r CBMK2 FUM TPI 149 ADHEr LDHD ASPT ATPS4r CBMK2 MDH PFK 150 ADHEr LDHD FBA GLUDy HEX1 NADH6 PFLi 151 ADHEr LDHD GLUDy HEX1 NADH6 PFK PFLi 152 ADHEr LDHD FBA GLUDy HEX PFLi SUCD4 153 ADHEr LDHD FRD2 GLUDy HEX PFK PFLi 154 ADHEr LDHD GLUDy HEX PFK PFLi SUCD4 149 155 ADHEr LDHD GLUDy HEX1 NADH6 PFLi TPI 156 ADHEr LDHD FBA FRD2 GLUDy HEXI PFLi 157 ADHEr LDHD GLUDy HEXi PFLi SUCD4 TPI 158 ADHEr LDHD FRD2 GLUDy HEXI PFLi TPI 159 ADHEr LDH_D GLUDy HEX1 MDH PFLi PGI 160 ADHEr LDHD FUM GLUDy HEX1 PFLi PGI 161 ADHEr LDHD HEXI PGI PTAr TKT2 162 ADHEr LDHD ACKr HEX PGI TKT2 163 ADHEr LDHD ATPS4r GLUDy HEXI MDH PFK 164 ADHET LDHD ATPS4r FBA GLUDy HEX1 MDH 165 ADHEr LDHD ATPS4r GLUDy HEX1 MDH TPI 166 ADHEr LDHD ATPS4r FBA FUM GLUDy HEXI 167 ADHEr LDHD ATPS4r FUM GLUDy HEX1 PFK 168 ADHEr LDHD ATPS4r FUM GLUDy HEX1 TPI 169 ADHEr LDHD FBA FUM GLUDy HEX1 PFLi 170 ADHEr LDHD FUM GLUDy HEX1 PFLi TPI 171 ADHEr LDHD GLUDy HEX1 MDH PFLi TPI 172 ADHEr LDHD GLUDy HEX1 MDH PFK PFLi 173 ADHEr LDHD FBA GLUDy HEXI MDH PFLi 174 ADHEr LDHD FUM GLUDy HEX1 PFK PFLi 175 ADHEr LDHD ATPS4r FUM GLUDy HEX1 PGI 176 ADHEr LDHD ATPS4r GLUDy HEX1 MDH PGI 177 ADHEr LDHD ASPT ATPS4r MDH PGI 178 ADHEr LDHD ASPT ATPS4r FUM PGI 179 ADHEr LDHD ATPS4r GLUDy MDH NADH6 PGI 180 ADHEr LDHD ATPS4r FUM GLUDy NADH6 PGI 181 ADHEr LDHD ATPS4r GLUDy HEX1 PGDH PGI 182 ADHEr LDHD PGDH PGI PTAr 183 ADHEr LDHD ACKr PGDH PGI 184 ADHEr LDHD ATPS4r GLUDy HEX1 PFLi PGI 185 ADHEr LDHD ASPT ATPS4r MDH TPI 186 ADHEr LDHD ASPT ATPS4r FUM TPI 187 ADHEr LDHD ASPT ATPS4r MDH PFK 188 ADHEr LDHD ASPT ATPS4r FBA FUM 189 ADHEr LDHD ASPT ATPS4r FBA MDH 190 ADHEr LDHD ASPT ATPS4r FUM PFK 191 ADHEr LDHD ACKr PGI TKT1 192 ADHEr LDHD PGI PTAr TAL 193 ADHEr LDHD PGI PTAr TKT1 194 ADHEr LDHD ACKr PGI TAL 195 ADHEr LDHD ATPS4r FBA GLUDy MDH NADH6 196 ADHEr LDHD ATPS4r GLUDy MDH NADH6 PFK 197 ADHEr LDHD ATPS4r GLUDy MDH NADH6 TPI 198 ADHEr LDHD ATPS4r FUM GLUDy NADH6 TPI 199 ADHEr LDHD ATPS4r FUM GLUDy NADH6 PFK 200 ADHEr LDHD ATPS4r FBA FUM GLUDy NADH6 150 201 ADHEr LDHD ATPS4r GLUDy HEXi PGI TAL 202 ADHEr LDHD ATPS4r GLUDy HEX1 PGI TKT1 203 ADHEr LDHD ATPS4r GLUDy HEXi PFK 204 ADHEr LDHD ATPS4r GLUDy HEX TPI 205 ADHEr LDHD ATPS4r FBA GLUDy HEXi 206 ADHEr LDHD GLUDy PTAr PYK SUCD4 207 ADHEr LDHD ACKr GLUDy PYK SUCD4 208 ADHEr LDHD FRD2 GLUDy PTAr PYK 209 ADHEr LDHD ACKr FRD2 GLUDy PYK 210 ADHEr LDHD FDH2 GLUDy NADH6 PTAr PYK 211 ADHEr LDHD ACKr FDH2 GLUDy NADH6 PYK 212 ADHEr LDHD PFK PTAr 213 ADHEr LDHD ACKr TPI 214 ADHEr LDHD ACKr FBA 215 ADHEr LDHD PTAr TPI 216 ADHEr LDHD FBA PTAr 217 ADHEr LDH D ACKr PFK 218 ADHEr LDHD FRD2 GLUDy PFLi PGI 219 ADHEr LDHD GLUDy PFLi PGI PROlz SUCD4 220 ADHEr LDHD ACKr PGI RPE .221 ADHEr LDHD PGI PTAr RPE 222 ADHEr LDHD ACKr PGI TKT2 223 ADHEr LDHD PGI PTAr TKT2 224 ADHEr LDHD ATPS4r GLUDy HEXi PGI RPE 225 ADHEr LDHD FRD2 GLUDy PFLi TPI 226 ADHEr LDHD FRD2 GLUDy PFK PFLi 227 ADHEr LDHD FBA FRD2 GLUDy PFLI 228 ADHEr LDHD GLUDy PFK PFLi PROlz SUCD4 229 ADHEr LDHD GLUDy PFLi PROlz SUCD4 TPI 230 ADHEr LDHD FBA GLUDy PFLi PROlz SUCD4 231 ADHEr LDHD GLUDy MDH PFLi PGI SUCD4 232 ADHEr LDHD FUM GLUDy NADH6 PFLi PGI 233 ADHEr LDHD GLUDy MDH NADH6 PFLi PGI 234 ADHEr LDHD FUM GLUDy PFLi PGI SUCD4 235 ADHEr LDHD ASPT GLUDy MDH PFLi PGI 236 ADHEr LDHD ASPT FUM GLUDy PFLi PGI 237 ADHEr LDHD ATPS4r GLUDy HEXi PGI TKT2 238 ADHEr LDHD FUM GLUDy PFK PFLi SUCD4 239 ADHEr LDHD GLUDy MDH NADH6 PFK PFLi 240 ADHEr LDHD FUM GLUDy PFLi SUCD4 TPI 241 ADHEr LDH D FUM GLUDy NADH6 PFK PFLi 242 ADHEr LDHD FBA FUM GLUDy PFLi SUCD4 243 ADHEr LDHD GLUDy MDH PFLi SUCD4 TPI 244 ADHEr LDHD GLUDy MDH PFK PFLi SUCD4 245 ADHEr LDHD FBA FUM GLUDy NADH6 PFLi 246 ADHEr LDHD FBA GLUDy MDH PFLi SUCD4 151 247 ADHEr LDHD FBA GLUDy MDH NADH6 PFLi 248 ADHEr LDHD GLUDy MDH NADH6 PFLi TPI 249 ADHEr LDHD FUM GLUDy NADH6 PFLi TPI 250 ADHEr LDHD ASPT ATPS4r FUM NADH6 PYK 251 ADHEr LDHD ASPT ATPS4r MDH NADH6 PYK 252 ADHEr LDHD GLCpts GLUDy PFLi PGI PTAr 253 ADHEr LDHD ACKr GLCpts GLUDy PFLi PGI 254 ADHEr LDHD ASPT FBA GLUDy MDH PFLi 255 ADHEr LDHD ASPT GLUDy MDH PFK PFLi 256 ADHEr LDHD ASPT FBA FUM GLUDy PFLi 257 ADHEr LDHD ASPT GLUDy MDH PFLi TPI 258 ADHEr LDHD ASPT FUM GLUDy PFLi TPI 259 ADHEr LDHD ASPT FUM GLUDy PFK PFLi 260 ADHEr LDHD ME2 PGL PTAr PYK SUCD4 261 ADHEr LDHD FRD2 G6PDHy ME2 PTAr PYK 262 ADHEr LDHD ACKr ME2 PGL PYK SUCD4 263 ADHEr LDHD ACKr FRD2 ME2 PGL PYK 264 ADHEr LDHD FRD2 ME2 PGL PTAr PYK 265 ADHEr LDHD G6PDHy ME2 PTAr PYK SUCD4 266 ADHEr LDHD ACKr FRD2 G6PDHy ME2 PYK 267 ADHEr LDH-D ACKr G6PDHy ME2 PYK SUCD4 268 ADHEr LDHD G6PDHy MDH PTAr PYK SUCD4 269 ADHEr LDHD ACKr G6PDHy MDH NADH6 PYK 270 ADHEr LDHD FRD2 G6PDHy MDH PTAr PYK 271 ADHEr LDHD FRD2 MDH PGL PTAr PYK 272 ADHEr LDHD ACKr G6PDHy MDH PYK SUCD4 273 ADHEr LDHD ACKr MDH PGL PYK SUCD4 274 ADHEr LDHD MDH NADH6 PGL PTAr PYK 275 ADHEr LDHD ACKr MDH NADH6 PGL PYK 276 ADHEr LDHD ACKr FRD2 G6PDHy MDH PYK 277 ADHEr LDHD MDH PGL PTAr PYK SUCD4 278 ADHEr LDHD ACKr FRD2 MDH PGL PYK 279 ADHEr LDHD G6PDHy MDH NADH6 PTAr PYK 280 ADHEr LDHD ATPS4r GLLTDy NADH6 PGI 281 ADHEr LDHD FUM GLUDy PTAr PYK 282 ADHEr LDHD ACKr GLUDy MDH PYK 283 ADHEr LDHD ACKr FUM GLUDy PYK 284 ADHEr LDHD GLUTDy MDH PTAr PYK 285 ADHEr LDHD ATPS4r HEXI PGDH PGI 286 ADHEr LDHD ATPS4r GLUDy NADH6 TPI 287 ADHEr LDHD ATPS4r GLUDy NADH6 PFK 288 ADHEr LDHD ATPS4r FBA GLUDy NADH6 289 ADHEr LDHD HEXI PFLi PGI 290 ADHEr LDHD ASPT ATPS4r GLUDy MDH PYK 291 ADHEr LDHD ASPT ATPS4r FUM GLUDy PYK 292 ADHEr LDHD ATPS4r HEXI PGI TKT1 152 293 ADHEr LDHD ATPS4r HEXI PGI TAL 294 ADHEr LDHD ATPS4r HEX1 PFK 295 ADHEr LDHD ATPS4r FBA HEX1 296 ADHEr LDHD ATPS4r HEX1 TPI 297 ADHEr LDHD HEXI PFLi TPI 298 ADHEr LDHD HEXI PFK PFLi 299 ADHEr LDHD FBA HEXI PFLi 300 ADHEr LDHD ATPS4r HEX1 PGI RPE 301 ADHEr LDHD ACKr GLUDy NADH6 PGI PYK 302 ADHEr LDHD GLUDy NADH6 PGI PTAr PYK 303 ADHEr LDHD ATPS4r HEXI PGI TKT2 304 ADHEr LDHD ACKr FRD2 PYK 305 ADHEr LDHD ACKr PYK SUCD4 306 ADHEr LDHD FRD2 PTAr PYK 307 ADHEr LDHD PTAr PYK SUCD4 308 ADHEr LDHD ACKr FDH2 NADH6 PYK 309 ADHEr LDHD FDH2 NADH6 PTAr PYK 310 ADHEr LDHD ATPS4r NADH6 PGI 311 ADHEr LDHD ACKr GLCpts PFLi PGI 312 ADHEr LDHD GLCpts PFLi PGI PTAr 313 ADHEr LDHD FRD2 GLUDy PFLi PYK 314 ADHEr LDHD ATPS4r FUM GLUDy PGDH PGI 315 ADHEr LDHD ATPS4r GLUDy MDH PGDH PGI 316 ADHEr LDHD FUM GLUDy PFLi PGI 317 ADHEr LDHD GLUDy MDH PFLi PGI 318 ADHEr LDHD ATPS4r FBA NADH6 319 ADHEr LDHD ATPS4r NADH6 PFK 320 ADHEr LDHD ATPS4r NADH6 TPI 321 ADHEr LDHD ATPS4r FBA FUM GLUDy 322 ADHEr LDHD ATPS4r FUM GLUDy PFK 323 ADHEr LDHD ATPS4r FBA GLUDy MDH 324 ADHEr LDHD ATPS4r GLUDy MDH TPI 325 ADHEr LDHD ATPS4r FUM GLUDy TPI 326 ADHEr LDHD ATPS4r GLUDy MDH PFK 327 ADHEr LDHD FRD2 G6PDHy ME2 PFLi PYK 328 ADHEr LDHD FRD2 ME2 PFLi PGL PYK 329 ADHEr LDHD EDA FRD2 ME2 PFLi PYK 330 ADHEr LDHD FRD2 ME2 PFLi PGDHY PYK 331 ADHEr LDHD GLUDy MDH PFK PFLi 332 ADHEr LDHD FBA GLUDy MDH PFLi 333 ADHEr LDHD GLUDy MDH PFLi TPI 334 ADHEr LDHD FBA FUM GLUDy PFLi 335 ADHEr LDHD FUM GLUDy PFLi TPI 336 ADHEr LDHD FUM GLUDy PFK PFLi 337 ADHEr LDHD PFLi PGI SUCD4 338 ADHEr LDHD FRD2 PFLi PGI 153 339 ADHEr LDHD NADH6 PFLi PGI 340 ADHEr LDHD FRD2 MDH PFLi PGL PYK 341 ADHEr LDHD FRD2 G6PDHy MDH PFLi PYK 342 ADHEr LDHD FRD2 MDH PFLi PGDHY PYK 343 ADHEr LDHD EDA FRD2 MDH PFLi PYK 344 ADHEr LDH_D ACKr ASPT MDH PYK 345 ADHEr LDHD ASPT MDH PTAr PYK 346 ADHEr LDHD ACKr ASPT FUM PYK 347 ADHEr LDHD ASPT FUM PTAr PYK 348 ADHEr LDHD ATPS4r GLUDy MDH PGI 349 ADHEr LDHD ATPS4r FUM GLLDy PGI 350 ADHEr LDHD FBA PFLi SUCD4 351 ADHEr LDHD FRD2 PFK PFLi 352 ADHEr LDHD PFLi SUCD4 TPI 353 ADHEr LDHD FBA FRD2 PFLi 354 ADHEr LDHD PFK PFLi SUCD4 355 ADHEr LDHD FRD2 PFLi TPI 356 ADHEr LDHD NADH6 PFLi TPI 357 ADHEr LDHD FBA NADH6 PFLi 358 ADHEr LDHD NADH6 PFK PFLi 359 ADHEr LDHD ASPT MDH PFLi PGI 360 ADHEr LDHD ASPT FUM PFLi PGI 361 ADHEr LDHD ASPT GLUDy MDH PFLi PYK 362 ADHEr LDHD ASPT FUM GLUDy PFLi PYK 363 ADHEr LDHD ASPT ATPS4r CBMK2 FUM PYK 364 ADHEr LDHD ASPT MDH PFLi TPI 365 ADHEr LDHD ASPT FUM PFLi TPI 366 ADHEr LDHD ASPT FBA MDH PFLi 367 ADHEr LDHD ASPT FBA FUM PFLi 368 ADHEr LDHD ASPT MDH PFK PFLi 369 ADHEr LDHD ASPT FUM PFK PFLi 370 ADHEr LDHD ACKr NADH6 PGI PYK 371 ADHEr LDHD NADH6 PGI PTAr PYK 372 ADHEr LDHD ASPT ATPS4r FUM PYK 373 ADHEr LDHD ASPT ATPS4r MALS MDH PYK 374 ADHEr LDHD ASPT ATPS4r ICL MDH PYK 375 ADHEr LDHD GLUDy PFLi PGDH PGI 376 ADHEr LDHD ATPS4r GLUDy PFLi PGI 377 ADHEr LDHD FBA GLUDy PFLi 378 ADHEr LDHD GLUDy PFLi TPI 379 ADHEr LDHD GLUDy PFK PFLi 380 ADHEr LDHD GLUDy PFLi PGI TAL 381 ADHEr LDHD GLUDy PFLi PGI TKT1 382 ADHEr LDHD GLUDy PFLi PROlz PYK SUCD4 383 ADHEr LDHD GLUDy MDH NADH6 PFLi PYK 384 ADHEr LDHD GLUDy MDH PFLi PYK SUCD4 154 385 ADHEr LDHD FUM GLUDy PFLi PYK SUCD4 386 ADHEr LDHD FLM GLUDy NADH6 PFLi PYK 387 ADHEr LDHD GLUDy PFLi PGI 388 ADHEr LDHD EDA MDH PFLi PYK SUCD4 389 ADHEr LDHD MDH PFLi PGDHY PYK SUCD4 390 ADHEr LDHD MDH PFLi PGL PYK SUCD4 391 ADHEr LDHD G6PDHy MDH PFLi PYK SUCD4 392 ADHEr LDHD ATPS4r GLUDy MDH NADH6 PYK 393 ADHEr LDHD ATPS4r FUM GLUDy NADH6 PYK 394 ADHEr LDHD ACKr AKGD ATPS4r GLUDy PYK 395 ADHEr LDHD AKGD ATPS4r GLUDy PTAr PYK 396 ADHEr LDHD FRD2 PFLi PYK 397 ADHEr LDHD ALAR PFLi PROlz PYK SUCD4 398 ADHEr LDHD DAAD PFLi PROlz PYK SUCD4 399 ADHEr LDHD PFLi PGDH PGI 400 ADHEr LDHD ATPS4r PFLi PGI 401 ADHEr LDH_D ATPS4r FUM GLUDy PFLi PYK 402 ADHEr LDHD ATPS4r GLUDy MDH PFLi PYK 403 ADHEr LDHD PFLi TPI 404 ADHEr LDHD FBA PFLi 405 ADHEr LDHD PFK PFLi 406 ADHEr LDHD ASPT FUM PFLi PYK 407 ADHEr LDHD ASPT MDH PFLi PYK 408 ADHEr LDHD PFLi PGI TKT1 409 ADHEr LDHD PFLi PGI TAL 410 ADHEr LDHD ASPT ATPS4r FUM GLUDy NADH6 411 ADHEr LDHD ASPT ATPS4r GLUDy MDH NADH6 412 ADHEr LDHD G6PDHy ME2 PFLi PYK SUCD4 413 ADHEr LDHD EDA ME2 PFLi PYK SUCD4 414 ADHEr LDHD ME2 PFLi PGDHY PYK SUCD4 415 ADHEr LDHD ME2 PFLi PGL PYK SUCD4 416 ADHEr LDHD MDH NADH6 PFLi PGDHY PYK 417 ADHEr LDHD G6PDHy MDH NADH6 PFLi PYK 418 ADHEr LDHD EDA MDH NADH6 PFLi PYK 419 ADHEr LDHD MDH NADH6 PFLi PGL PYK 420 ADHEr LDHD ASPT ATPS4r CBMK2 MDH NADH6 421 ADHEr LDHD ASPT ATPS4r CBMK2 FUM NADH6 422 ADHEr LDH D CBMK2 PFLi PGI RPE 423 ADHEr LDHD ASNS2 GLU5K PFLi PGI RPE 424 ADHEr LDHD ASNS2 G5SD PFLi PGI RPE 425 ADHEr LDHD ASPT ATPS4r GLUDy MDH PTAr 426 ADHEr LDHD ASPT ATPS4r FUM GLUDy PTAr 427 ADHEr LDHD PFLi PGI 428 ADHEr LDHD ASPT ATPS4r FUM GLUDy 429 ADHEr LDHD ASPT ATPS4r GLUDy MDH 430 ADHEr LDHD ACKr AKGD ATPS4r PYK 155 431 ADHEr LDHD AKGD ATPS4r PTAr PYK 432 ADHEr LDH D ASPT ATPS4r MDH NADH6 433 ADHEr LDHD ASPT ATPS4r FUM NADH6 434 ADHEr LDHD G6PDHy GLCpts GLUDy PTAr 435 ADHEr LDHD ACKr GLCpts GLUDy PGL 436 ADHEr LDHD GLCpts GLUDy PGDH PTAr 437 ADHEr LDHD GLCpts GLUDy PGL PTAr 438 ADHEr LDHD ACKr G6PDHy GLCpts GLUDy 439 ADHEr LDHD ACKr GLCpts GLUDy PGDH 440 ADHEr LDHD GLCpts GLUDy PTAr TKT1 441 ADHEr LDHD GLCpts GLUDy PTAr TAL 442 ADHEr LDHD ACKr GLCpts GLUDy TKT1 443 ADHEr LDHD ACKr GLCpts GLUDy TAL 444 ADHEr LDHD ACKr GLCpts GLUDy RPE 445 ADHEr LDHD GLCpts GLUDy PTAr RPE 446 ADHEr LDHD ACKr GLCpts GLUDy TKT2 447 ADHEr LDHD GLCpts GLUDy PTAr TKT2 448 ADHEr LDHD GLCpts PGDH PTAr THD2 449 ADHEr LDHD G6PDHy GLCpts PTAr THD2 450 ADHEr LDHD ACKr G6PDHy GLCpts THD2 451 ADHEr LDHD ACKr GLCpts PGL THD2 452 ADHEr LDHD ACKr GLCpts PGDH THD2 453 ADHEr LDHD GLCpts PGL PTAr THD2 454 ADHEr LDHD ACKr GLCpts THD2 TKT1 455 ADHEr LDHD ACKr GLCpts TAL THD2 456 ADHEr LDHD GLCpts PTAr TAL THD2 457 ADHEr LDHD GLCpts PTAr THD2 TKT1 458 ADHEr LDHD ASPT ATPS4r MDH 459 ADHEr LDHD ASPT ATPS4r FUM 460 ADHEr LDHD GLCpts PTAr RPE THD2 461 ADHEr LDHD ACKr GLCpts RPE THD2 462 ADHEr LDHD ACKr ATPS4r PYK SUCOAS 463 ADHEr LDHD ATPS4r PTAr PYK SUCOAS 464 ADHEr LDHD FRD2 GLCpts GLUDy PFLi 465 ADHEr LDHD GLCpts PTAr THD2 TKT2 466 ADHEr LDHD ACKr GLCpts THD2 TKT2 467 ADHEr LDHD FRD2 GLCpts PFLi THD2 468 ADHEr LDHD ACKr GLUDy PGDH THD2 469 ADHEr LDHD GLUDy PGL PTAr THD2 470 ADHEr LDHD G6PDHy GLUDy PTAr THD2 471 ADHEr LDHD GLUDy PGDH PTAr THD2 472 ADHEr LDHD ACKr GLUDy PGL THD2 473 ADHEr LDHD ACKr G6PDHy GLUDy THD2 474 ADHEr LDHD FRD2 GLUDy PFLi THD2 475 ADHEr LDHD GLUDy PTAr THD2 TKT1 476 ADHEr LDHD GLUDy PTAr TAL THD2 156 477 ADHEr LDHD ACKr GLUDy TAL THD2 478 ADHEr LDHD ACKr GLLTDy THD2 TKT1 479 ADHEr LDHD ACKr GLCpts PGDH 480 ADHEr LDHD ACKr GLCpts PGL 481 ADHEr LDHD GLCpts PGDH PTAr 482 ADHEr LDHD GLCpts PGL PTAr 483 ADHEr LDHD ACKr G6PDHy GLCpts 484 ADHEr LDHD G6PDHy GLCpts PTAr 485 ADHEr LDHD GLUDy PTAr RPE THD2 486 ADHEr LDHD ACKr GLUDy RPE THD2 487 ADHEr LDHD GLCpts GLUDy PTAr 488 ADHEr LDHD ACKr GLCpts GLLDy 489 ADHEr LDHD GLCpts PTAr TKT1 490 ADHEr LDHD GLCpts PTAr TAL 491 ADHEr LDHD ACKr GLCpts TAL 492 ADHEr LDHD ACKr GLCpts TKT1 493 ADHEr LDHD NADH6 PFLi PTAr PYK 494 ADHEr LDHD ACKr NADH6 PFLi PYK 495 ADHEr LDHD ACKr GLUDy THD2 TKT2 496 ADHEr LDHD GLUDy PTAr THD2 TKT2 497 ADHEr LDHD ACKr GLCpts RPE 498 ADHEr LDHD GLCpts PTAr RPE 499 ADHEr LDHD ACKr GLCpts TKT2 500 ADHEr LDHD GLCpts PTAr TKT2 501 ADHEr LDHD ACKr GLUDy PGDH 502 ADHEr LDH_D GLUDy PGL PTAr 503 ADHEr LDHD ACKr GLUDy PGL 504 ADHEr LDHD ACKr G6PDHy GLUDy 505 ADHEr LDH_D GLUDy PGDH PTAr 506 ADHEr LDHD G6PDHy GLUDy PTAr 507 ADHEr LDHD GLUDy PTAr TKT1 508 ADHEr LDHD ACKr GLUDy TKT1 509 ADHEr LDHD ACKr GLUDy TAL 510 ADHEr LDHD GLUDy PTAr TAL 511 ADHEr LDHD GLUDy PTAr RPE 512 ADHEr LDHD ACKr GLUJDy RPE 513 ADHEr LDHD GLUDy PTAr TKT2 514 ADHEr LDHD ACKr GLUDy TKT2 515 ADHEr LDHD PGDH PTAr THD2 516 ADHEr LDHD ACKr PGDH THD2 517 ADHEr LDHD G6PDHy PTAr THD2 518 ADHEr LDHD PGL PTAr THD2 519 ADHEr LDHD ACKr PGL THD2 520 ADHEr LDHD ACKr G6PDHy THD2 521 ADHEr LDHD PTAr TAL THD2 522 ADHEr LDHD ACKr THD2 TKT1 157 523 ADHEr LDHD ACKr TAL THD2 524 ADHEr LDHD PTAr THD2 TKT1 525 ADHEr LDHD PTAr RPE THD2 526 ADHEr LDHD ACKr RPE THD2 527 ADHEr LDH~D FRD2 GLUDy PFLi 528 ADHEr LDHD GLUDy PFLi PROlz SUCD4 529 ADHEr LDHD FRD2 GLCpts PFLi 530 ADHEr LDHD PTAr THD2 TKT2 531 ADHEr LDHD ACKr THD2 TKT2 532 ADHEr LDH D ACKr GLCpts 533 ADHEr LDHD GLCpts PTAr 534 ADHEr LDHD FRD2 PFLi THD2 535 ADHEr LDHD ATPS4r FUM GLUDy 536 ADHEr LDHD ATPS4r GLUDy MDH 537 ADHEr LDHD FUM GLCpts PFLi SUCD4 538 ADHEr LDHD GLCpts MDH PFLi SUCD4 539 ADHEr LDHD FUM GLUDy PFLi SUCD4 540 ADHEr LDHD GLUDy MDH PFLi SUCD4 541 ADHEr LDHD GLUDy MDH NADH6 PFLi 542 ADHEr LDHD FUM GLUDy NADH6 PFLi 543 ADHEr LDHD MDH PFLi SUCD4 THD2 544 ADHEr LDHD FUM PFLi SUCD4 THD2 545 ADHEr LDHD ASPT FUM GLCpts PFLi 546 ADHEr LDHD ASPT GLCpts MDH PFLi 547 ADHEr LDHD ASPT FLTM GLUDy PFLi 548 ADHEr LDHD ASPT GLLTDy MDH PFLi 549 ADHEr LDHD GLCpts PFLi SUCD4 THD2 550 ADHEr LDHD PGDH PTAr 551 ADHEr LDHD PGL PTAr 552 ADHEr LDHD ACKr PGL 553 ADHEr LDHD G6PDHy PTAr 554 ADHEr LDHD ACKr G6PDHy 555 ADHEr LDHD ACKr PGDH 556 ADHEr LDHD ASPT FUM PFLi THD2 557 ADHEr LDHD ASPT MDH PFLi THD2 558 ADHEr LDHD ACKr GLUDy 559 ADHEr LDHD GLUDy PTAr 560 ADHEr LDHD PTAr TAL 561 ADHEr LDHD ACKr TAL 562 ADHEr LDHD ACKr TKT1 563 ADHEr LDHD PTAr TKT1 564 ADHEr LDHD ACKr RPE 565 ADHEr LDHD PTAr RPE 566 ADHEr LDHD GLCpts GLUDy PFLi SUCD4 567 ADHEr LDHD FUM GLCpts GLUDy PFLi 568 ADHEr LDHD GLCpts GLUDy MDH PFLi 158 569 ADHEr LDHD ACKr TKT2 570 ADHEr LDHD PTAr TKT2 571 ADHEr LDHD GLUDy PFLi SUCD4 THD2 572 ADHEr LDHD FUM GLUDy PFLi THD2 573 ADHEr LDHD GLUDy MDH PFLi THD2 574 ADHEr LDHD GLCpts GLUDy NADH6 PFLi 575 ADHEr LDHD ATPS4r GLUDy NADH6 PFLi 576 ADHEr LDHD GLCpts MDH PFLi THD2 577 ADHEr LDHD FUM GLCpts PFLi THD2 578 ADHEr LDHD ACKr CBMK2 FRD2 PFLi 579 ADHEr LDHD CBMK2 FRD2 PFLi PTAr 580 ADHEr LDHD MDH PTAr SUCD4 581 ADHEr LDHD FRD2 MDH PTAr 582 ADHEr LDHD ACKr MDH SUCD4 583 ADHEr LDHD ACKr FRD2 MDH 584 ADHEr LDHD FDH2 MDH NADH6 PTAr 585 ADHEr LDHD ACKr FDH2 MDH NADH6 586 ADHEr LDHD GLCpts NADH6 PFLi THD2 587 ADHEr LDHD GLCpts PFLi SUCD4 588 ADHEr LDHD GLCpts NADH12 NADH6 PFLi 589 ADHEr LDHD ATPS4r FUM PGL 590 ADHEr LDHD ATPS4r MDH PGDH 591 ADHEr LDHD ATPS4r FUM PGDH 592 ADHEr LDHD ATPS4r FUM G6PDHy 593 ADHEr LDHD GLCpts MDH NADH6 PFLi 594 ADHEr LDHD FUM GLCpts NADH6 PFLi 595 ADHEr LDHD FRD2 PFLi 596 ADHEr LDHD ALAR PFLi PROlz SUCD4 597 ADHEr LDHD DAAD PFLi PRO~z SUCD4 598 ADHEr LDHD ACKr 599 ADHEr LDHD PTAr 600 ADHEr LDHD FUM PFLi SUCD4 601 ADHEr LDHD MDH PFLi SUCD4 602 ADHEr LDHD FUM NADH12 NADH6 PFLi 603 ADHEr LDHD MDH NADH12 NADH6 PFLi 604 ADHEr LDHD ATPS4r MDH TKT1 605 ADHEr LDHD ATPS4r FUM TKT1 606 ADHEr LDHD ATPS4r MDH TAL 607 ADHEr LDHD ATPS4r FUM TAL 608 ADHEr LDHD ATPS4r NADH6 PFLi PYK 609 ADHEr LDHD ASPT FUM PFLi 610 ADHEr LDHD ASPT MDH PFLi 611 ADHEr LDHD ATPS4r MDH RPE 612 ADHEr LDHD ATPS4r FUM RPE 613 ADHEr LDHD PFLi SUCD4 THD2 614 ADHEr LDHD NADH12 NADH6 PFLi THD2 159 615 ADHEr LDHD FUM NADH6 PFLi THD2 616 ADHEr LDHD MDH NADH6 PFLi THD2 617 ADHEr LDHD ATPS4r MDH TKT2 618 ADHEr LDHD ATPS4r FUM TKT2 619 ADHEr LDHD GLCpts NADH6 PFLi 620 ADHEr LDHD GLUDy NADH6 PFLi THD2 621 ADHEr LDHD GLUDy PFLi SUCD4 622 ADHEr LDHD GLJDy NADH12 NADH6 PFLi 623 ADHEr LDHD FUM GLUDy PFLi 624 ADHEr LDHD GLLTDy MDH PFLi 625 ADHEr LDHD ATPS4r FUM NADH6 626 ADHEr LDHD ATPS4r MDH NADH6 627 ADHEr LDHD ATPS4r G6PDHy GLUDy NADH6 628 ADHEr LDHD ATPS4r GLUDy NADH6 PGDH 629 ADHEr LDHD ATPS4r GLUDy NADH6 PGL 630 ADHEr LDHD ATPS4r MDH PFLi THD2 631 ADHEr LDHD ATPS4r FUM PFLi THD2 632 ADHEr LDHD ATPS4r GLUDy NADH6 TKT1 633 ADHEr LDHD ATPS4r GLUDy NADH6 TAL 634 ADHEr LDHD ATPS4r GLUDy PFLi THD2 635 ADHEr LDHD GLCpts MDH PFLi 636 ADHEr LDHD FUM GLCpts PFLi 637 ADHEr LDHD GLUDy NADH6 PFLi 638 ADHEr LDHD ATPS4r GLUDy NADH6 RPE 639 ADHEr LDHD ATPS4r GLUDy NADH6 TKT2 640 ADHEr LDHD FUM PFLi THD2 641 ADHEr LDHD MDH PFLi THD2 642 ADHEr LDHD NADH6 PFLi THD2 643 ADHEr LDHD PFLi SUCD4 644 ADHEr LDHD NADH12 NADH6 PFLi 645 ADHEr LDHD ATPS4r NADH6 PFLi 646 ADHEr LDHD FUM NADH6 PFLi 647 ADHEr LDHD MDH NADH6 PFLi 648 ADHEr LDHD ATPS4r NADH6 PGL 649 ADHEr LDHD ATPS4r NADH6 PGDH 650 ADHEr LDHD ATPS4r G6PDHy NADH6 651 ADHEr LDHD ATPS4r NADH6 TAL 652 ADHEr LDHD ATPS4r NADH6 TKT1 653 ADHEr LDHD CBMK2 GLU5K NADH6 PFLi 654 ADHEr LDHD CBMK2 G5SD NADH6 PFLi 655 ADHEr LDHD ASNS2 CBMK2 NADH6 PFLi 656 ADHEr LDHD ATPS4r PFLi THD2 657 ADHEr LDHD NADH6 PFLi 658 ADHEr LDHD ATPS4r NADH6 RPE 659 ADHEr LDHD ATPS4r NADH6 TKT2 660 ADHEr LDHD CBMK2 FUM G5SD PFLi 160 661 ADHEr LDHD CBMK2 GLU5K MDH PFLi 662 ADHEr LDHD CBMK2 FUM GLU5K PFLi 663 ADHEr LDHD CBMK2 G5SD MDH PFLi 664 ADHEr LDHD ASNS2 CBMK2 FUM PFLi 665 ADHEr LDHD ASNS2 CBMK2 MDH PFLi 666 ADHEr LDHD MDH PFLi 667 ADHEr LDHD FUM PFLi 668 ADHEr LDHD ATPS4r GLUDy PFLi RPE 669 ADHEr LDHD ATPS4r GLUDy PFLi TAL 670 ADHEr LDHD ATPS4r GLUDy PFLi TKT1 671 ADHEr LDHD ATPS4r GLUDy PFLi TKT2 672 ADHEr LDHD ATPS4r GLUDy PFLi 673 ADHEr LDHD ATPS4r GLUDy NADH6 674 ADHEr LDHD ATPS4r PFLi RPE 675 ADHEr LDHD ATPS4r PFLi TAL 676 ADHEr LDHD ATPS4r PFLi TKT1 677 ADHEr LDHD ATPS4r PFLi TKT2 678 ADHEr LDHD ATPS4r CBMK2 PFLi 679 ADHEr LDHD ATPS4r PFLi 680 ADHEr LDHD ASPT MDH PGDHY PYK 681 ADHEr LDHD ASPT EDA MDH PYK 682 ADHEr LDHD ATPS4r CBMK2 NADH6 683 ADHEr LDHD ATPS4r NADH6 684 ADHEr LDHD ATPS4r HEXI PGI PPS 685 ADHEr LDHD G6PDHy ME2 THD2 686 ADHEr LDHD ME2 PGL THD2 687 ADHEr LDHD ME2 PGDH PGDHY THD2 688 ADHEr LDHD EDA ME2 PGDH THD2 689 ADHEr LDHD EDA ME2 TAL THD2 690 ADHEr LDHD ME2 PGDHY TAL THD2 691 ADHEr LDHD ME2 PGDHY THD2 TKT1 692 ADHEr LDHD EDA ME2 THD2 TKT1 693 ADHEr LDHD ME2 PGDHY RPE THD2 694 ADHEr LDHD EDA ME2 RPE THD2 695 ADHEr LDHD MDH PGL THD2 696 ADHEr LDHD G6PDHy MDH THD2 697 ADHEr LDHD EDA MDH PGDH THD2 698 ADHEr LDHD MDH PGDH PGDHY THD2 699 ADHEr LDHD ME2 PGDHY THD2 TKT2 700 ADHEr LDHD EDA ME2 THD2 TKT2 701 ADHEr LDHD MDH PGDHY THD2 TKT1 702 ADHEr LDHD EDA MDH THD2 TKT1 703 ADHEr LDHD MDH PGDHY TAL THD2 704 ADHEr LDHD EDA MDH TAL THD2 705 ADHEr LDHD ATPS4r GLUDy HEXI PGI 706 ADHEr LDHD MDH PGDHY RPE THD2 161 707 ADHEr LDHD EDA MDH RPE THD2 708 ADHEr LDHD MDH PGDHY THD2 TKT2 709 ADHEr LDH D EDA MDH THD2 TKT2 710 ADHEr LDHD ATPS4r HEXI PGI 711 ADHEr LDHD FRD2 HEXI MDH PGI 712 ADHEr LDHD HEXI MDH PGI SUCD4 713 ADHEr LDHD HEXI PGI SUCOAS 714 ADHEr LDH D HEXI MDH NADH6 PGI 715 ADHEr LDHD FUM HEXI NADH6 PGI 716 ADHET LDHD FRD2 FUM HEXI PGI 717 ADHEr LDHD HEXI PGI 718 ADHEr LDHD SUCOASTHD2 719 ADHEr LDHD THD2 720 ADHEr LDHD GLCpts SUCOAS TKT2 TPI 721 ADHEr LDHD GLCpts PFK SUCOAS TKT2 722 ADHEr LDHD FBA GLCpts SUCOAS TKT2 723 ADHEr LDHD GLCpts GLUDy TKT2 TPI 724 ADHEr LDHD FBA GLCpts GLUDy TKT2 725 ADHEr LDHD GLCpts GLUDy PFK TKT2 726 ADHEr LDHD GLCpts PGI SUCOAS 727 ADHEr LDHD GLCpts GLUDy PGI 728 ADHEr LDHD GLCpts PFK RPE SUCOAS 729 ADHEr LDHD GLCpts RPE SUCOAS TPI 730 ADHEr LDHD FBA GLCpts RPE SUCOAS 731 ADHEr LDHD GLCpts GLUDy RPE TPI 732 ADHEr LDHD FBA GLCpts GLUDy RPE 733 ADHEr LDHD GLCpts GLUDy PFK RPE 734 ADHEr LDHD FBA GLUDy SUCOAS TKT2 735 ADHEr LDHD GLUDy PFK SUCOAS TKT2 736 ADHEr LDHD GLUDy SUCOAS TKT2 TPI 737 ADHEr LDHD GLCpts GLUDy PFK SUCOAS 738 ADHEr LDHD GLCpts GLUDy SUCOAS TPI 739 ADHEr LDHD FBA GLCpts GLUDy SUCOAS 740 ADHEr LDHD GLCpts PFK TKT2 741 ADHEr LDHD FBA GLCpts TKT2 742 ADHEr LDHD GLCpts TKT2 TPI 743 ADHEr LDHD GLUDy PGI SUCOAS 744 ADHEr LDHD PGDHY PGI 745 ADHEr LDHD EDA PGI 746 ADHEr LDHD GLCpts PGI 747 ADHEr LDHD GLUDy PFK RPE SUCOAS 748 ADHEr LDHD GLUDy RPE SUCOAS TPI 749 ADHEr LDHD FBA GLUDy RPE SUCOAS 750 ADHEr LDHD GLCpts RPE TPI 751 ADHEr LDHD GLCpts PFK RPE 752 ADHEr LDH_D FBA GLCpts RPE 162 753 ADHEr LDHD PFK SUCOAS TKT2 754 ADHEr LDHD FBA SUCOAS TKT2 755 ADHEr LDHD SUCOAS TKT2 TPI 756 ADHEr LDHD GLCpts SUCOAS TPI 757 ADHEr LDHD GLCpts PFK SUCOAS 758 ADHEr LDHD FBA GLCpts SUCOAS 759 ADHEr LDHD FBA GLCpts GLUDy 760 ADHEr LDHD GLCpts GLUDy TPI 761 ADHEr LDHD GLCpts GLUDy PFK 762 ADHEr LDHD GLUDy PFK TKT2 763 ADHEr LDHD FBA GLUDy TKT2 764 ADHEr LDHD GLUDy TKT2 TPI 765 ADHEr LDHD PGI SUCOAS 766 ADHEr LDHD GLUDy PGI 767 ADHEr LDHD ASPT G6PDHy MDH PYK 768 ADHEr LDHD ASPT MDH PGL PYK 769 ADHEr LDH D FBA RPE SUCOAS 770 ADHEr LDH D PFK RPE SUCOAS 771 ADHEr LDH D RPE SUCOAS TPI 772 ADHEr LDHD HEXI PFK SUCOAS TKT1 773 ADHEr LDHD FBA HEXI SUCOAS TAL 774 ADHEr LDHD HEXI PFK SUCOAS TAL 775 ADHEr LDHD HEXI SUCOAS TKT1 TPI 776 ADHEr LDHD FBA HEXI SUCOAS TKT1 777 ADHEr LDHD HEXI SUCOAS TAL TPI 778 ADHEr LDHD GLUDy RPE TPI 779 ADHEr LDHD FBA GLUDy RPE 780 ADHEr LDHD GLUDy PFK RPE 781 ADHEr LDHD GLUDy HEX TKT1 TPI 782 ADHEr LDHD GLUDy HEXI PFK TKT1 783 ADHEr LDHD FBA GLUDy HEX1 TKT1 784 ADHEr LDHD GLUDy HEXI TAL TPI 785 ADHEr LDHD FBA GLUDy HEXI TAL 786 ADHEr LDHD GLUDy HEXI PFK TAL 787 ADHEr LDHD GLUDy SUCOAS TPI 788 ADHEr LDHD GLUDy PFK SUCOAS 789 ADHEr LDHD FBA GLUDy SUCOAS 790 ADHEr LDHD FRD2 PYK SUCOAS TKT2 791 ADHEr LDHD PYK SUCD4 SUCOAS TKT2 792 ADHEr LDHD GLCpts TPI 793 ADHEr LDHD GLCpts PFK 794 ADHEr LDHD FBA GLCpts 795 ADHEr LDHD FRD2 GLUDy PYK TKT2 796 ADHEr LDHD GLUDy PYK SUCD4 TKT2 797 ADHEr LDHD PFK TKT2 798 ADHEr LDHD FBA TKT2 163 799 ADHEr LDHD TKT2 TPI 800 ADHEr LDHD CBMK2 SUCOAS TAL TPI 801 ADHEr LDHD CBMK2 FBA SUCOAS TAL 802 ADHEr LDHD CBMK2 FBA SUCOAS TKT1 803 ADHEr LDHD CBMK2 PFK SUCOAS TAL 804 ADHEr LDHD CBMK2 PFK SUCOAS TKT1 805 ADHEr LDHD CBMK2 SUCOAS TKT1 TPI 806 ADHEr LDHD CBMK2 FBA HEXI SUCOAS 807 ADHEr LDHD CBMK2 HEX SUCOAS TPI 808 ADHEr LDHD CBMK2 HEX1 PFK SUCOAS 809 ADHEr LDHD PGI 810 ADHEr LDHD HEXI PFK TAL 811 ADHEr LDHD HEX TAL TPI 812 ADHEr LDHD FBA HEXI TAL 813 ADHEr LDHD HEXI PFK TKT1 814 ADHEr LDHD HEXI TKT1 TPI 815 ADHEr LDHD FBA HEXI TKT1 816 ADHEr LDHD PYK RPE SUCD4 SUCOAS 817 ADHEr LDHD FRD2 PYK RPE SUCOAS 818 ADHEr LDHD FRD2 GLUDy PYK RPE 819 ADHEr LDHD GLUDy PYK RPE SUCD4 820 ADHEr LDH_D RPE TPI 821 ADHEr LDHD PFK RPE 822 ADHEr LDHD FBA RPE 823 ADHEr LDHD SUCOAS TPI 824 ADHEr LDHD PFK SUCOAS 825 ADHEr LDHD FBA SUCOAS 826 ADHEr LDIHD GLUDy TPI 827 ADHEr LDHD FBA GLUDy 828 ADHEr LDHD GLUDy PFK 829 ADHEr LDHD FRD2 GLUDy PYK SUCOAS 830 ADHEr LDHD GLUDy PYK SUCD4 SUCOAS 831 ADHEr LDHD HEXI MDH PFK SUCD4 832 ADHEr LDHD HEX1 MDH SUCD4 TPI 833 ADHEr LDHD FBA HEXI MDH SUCD4 834 ADHEr LDHD FRD2 HEXI MDH TPI 835 ADHEr LDHD FBA FRD2 HEX MDH 836 ADHEr LDHD FRD2 HEXI MDH PFK 837 ADHEr LDHD FRD2 MDH TKT1 TPI 838 ADHEr LDHD FRD2 MDH TAL TPI 839 ADHEr LDHD MDH PFK SUCD4 TKT1 840 ADHEr LDHD MDH PFK SUCD4 TAL 841 ADHEr LDHD FBA MDH SUCD4 TKT1 842 ADHEr LDHD FBA MDH SUCD4 TAL 843 ADHEr LDHD MDH SUCD4 TAL TPI 844 ADHEr LDHD FRD2 MDH PFK TKT1 164 845 ADHEr LDHD FRD2 MDH PFK TAL 846 ADHEr LDHD FBA FRD2 MDH TAL 847 ADHEr LDHD MDH SUCD4 TKTI TPI 848 ADHEr LDHD FBA FRD2 MDH TKTI 849 ADHEr LDHD PYK SUCD4 TKT2 850 ADHEr LDHD FRD2 PYK TKT2 851 ADHEr LDHD FDH2 NADH6 PYK TKT2 852 ADHEr LDHD CBMK2 PFK TAL 853 ADHEr LDHD CBMK2 TAL TPI 854 ADHEr LDHD CBMK2 FBA TKT1 855 ADHEr LDHD CBMK2 TKT1 TPI 856 ADHEr LDHD CBMK2 FBA TAL 857 ADHEr LDHD CBMK2 PFK TKT1 858 ADHEr LDHD CBMK2 HEXI PFK 859 ADHEr LDHD CBMK2 HEX1 TPI 860 ADHEr LDHD CBMK2 FBA HEXI 861 ADHEr LDHD GLU5K TAL TPI 862 ADHEr LDHD G5SD TAL TPI 863 ADHEr LDH D FBA GLU5K TKT1 864 ADHEr LDHD G5SD TKT1 TPI 865 ADHEr LDHD G5SD PFK TKT1 866 ADHEr LDHD GLU5K PFK TAL 867 ADHEr LDHD FBA G5SD TAL 868 ADHEr LDHD FBA G5SD TKT1 869 ADHEr LDHD G5SD PFK TAL 870 ADHEr LDHD GLU5K TKT1 TPI 871 ADHEr LDHD GLU5K PFK TKT1 872 ADHEr LDHD FBA GLU5K TAL 873 ADHEr LDHD GLU5K HEXI TPI 874 ADHEr LDHD GLU5K HEXI PFK 875 ADHEr LDHD G5SD HEXi PFK 876 ADHEr LDHD FBA G5SD HEX1 877 ADHEr LDHD FBA GLU5K HEXI 878 ADHEr LDHD G5SD HEXI TPI 879 ADHEr LDHD ASNS2 PFK TKT1 880 ADHEr LDHD ASNS2 TKT1 TPI 881 ADHEr LDHD ASNS2 PFK TAL 882 ADHEr LDHD ASNS2 FBA TKT1 883 ADHEr LDHD ASNS2 FBA TAL 884 ADHEr LDHD ASNS2 TAL TPI 885 ADHEr LDHD ASNS2 HEXI PFK 886 ADHEr LDHD ASNS2 FBA HEXI 887 ADHEr LDHD ASNS2 HEX1 TPI 888 ADHEr LDHD PYK SUCD4 SUCOAS TKTI 889 ADHEr LDHD FRD2 PYK SUCOAS TAL 890 ADHEr LDHD PYK SUCD4 SUCOAS TAL 165 891 ADHEr LDHD FRD2 PYK SUCOAS TKT1 892 ADHEr LDHD PYK RPE SUCD4 893 ADHEr LDHD FRD2 PYK RPE 894 ADHEr LDHD FDH2 NADH6 PYK RPE 895 ADHEr LDHD GLUDy MDH PYK TKT2 896 ADHEr LDHD FUM GLUDy PYK TKT2 897 ADHEr LDH_D GLCpts GLUDy SUCOAS TKT2 898 ADHEr LDHD GLUDy PYK SUCD4 899 ADHEr LDHD FRD2 GLUDy PYK 900 ADHEr LDHD FDH2 GLUDy NADH6 PYK 901 ADHEr LDHD FBA 902 ADHEr LDHD TPI 903 ADHEr LDHD PFK 904 ADHEr LDHD PYK SUCD4 SUCOAS 905 ADHEr LDH D FRD2 PYK SUCOAS 906 ADHEr LDHD FDH2 NADH6 PYK SUCOAS 907 ADHEr LDH_D FRD2 ME2 PGDHY PYK 908 ADHEr LDHD EDA FRD2 ME2 PYK 909 ADHEr LDHD FRD2 ME2 PGL PYK 910 ADHEr LDHD EDA ME2 PYK SUCD4 911 ADHEr LDHD ME2 PGDHY PYK SUCD4 912 ADHEr LDHD ME2 PGL PYK SUCD4 913 ADHEr LDHD FRD2 G6PDHy ME2 PYK 914 ADHEr LDHD G6PDHy ME2 PYK SUCD4 915 ADHEr LDHD MDH NADH6 PGDHY PYK 916 ADHEr LDHD MDH PGL PYK SUCD4 917 ADHEr LDHD FRD2 MDH PGL PYK 918 ADHEr LDHD FRD2 MDH PGDHY PYK 919 ADHEr LDHD G6PDHy MDH PYK SUCD4 920 ADHEr LDHD MDH NADH6 PGL PYK 921 ADHEr LDHD EDA FRD2 MDH PYK 922 ADHEr LDHD EDA MDH PYK SUCD4 923 ADHEr LDHD MDH PGDHY PYK SUCD4 924 ADHEr LDHD EDA MDH NADH6 PYK 925 ADHEr LDHD FRD2 G6PDHy MDH PYK 926 ADHEr LDHD G6PDHy MDH NADH6 PYK 927 ADHEr LDHD GLUDy MDH PYK RPE 928 ADHEr LDHD FUM GLUDy PYK RPE 929 ADHEr LDHD FRD2 PYK TAL 930 ADHEr LDHD PYK SUCD4 TKT1 931 ADHEr LDHD PYK SUCD4 TAL 932 ADHEr LDHD FRD2 PYK TKT1 933 ADHEr LDHD FDH2 NADH6 PYK TAL 934 ADHEr LDHD FDH2 NADH6 PYK TKT1 935 ADHEr LDHD GLCpts GLUDy RPE SUCOAS 936 ADHEr LDHD GLUDy MDH PYK SUCOAS 166 937 ADHEr LDHD FUM GLUDy PYK SUCOAS 938 ADHEr LDHD FUM GLUDy NADH6 PYK 939 ADHEr LDHD GLUDy MDH NADH6 PYK 940 ADHEr LDHD GLCpts SUCOAS TKT2 941 ADHEr LDHD GLUDy SUCOAS TKT2 942 ADHEr LDH_D ASPT MDH PYK TKT2 943 ADHEr LDHD ASPT FUM PYK TKT2 944 ADHEr LDHD FRD2 PYK 945 ADHEr LDHD PYK SUCD4 946 ADHEr LDHD FDH2 NADH6 PYK 947 ADHEr LDHD GLCpts GLUDy TKT2 948 ADHEr LDHD GLCpts GLUDy SUCOAS TAL 949 ADHEr LDHD GLCpts GLUDy SUCOAS TKT1 950 ADHEr LDHD FUM GLUDy PYK 951 ADHEr LDHD GLUDy MDH PYK 952 ADHEr LDHD GLCpts RPE SUCOAS 953 ADHEr LDHD ASPT FUM PYK RPE 954 ADHEr LDHD ASPT MDH PYK RPE 955 ADHEr LDHD GLUDy RPE SUCOAS 956 ADHEr LDHD GLCpts GLUDy RPE 957 ADHEr LDHD ASPT FUM PYK SUCOAS 958 ADHEr LDHD ASPT MDH PYK SUCOAS 959 ADHEr LDHD GLCpts GLUDy SUCOAS 960 ADHEr LDHD ASPT FUM NADH6 PYK 961 ADHEr LDHD ASPT MDH NADH6 PYK 962 ADHEr LDHD SUCOAS TKT2 963 ADHEr LDHD GLCpts TKT2 964 ADHEr LDHD ASPT MDH PYK TKT1 965 ADHEr LDHD ASPT FUM PYK TAL 966 ADHEr LDHD ASPT MDH PYK TAL 967 ADHEr LDHD ASPT FUM PYK TKT1 968 ADHEr LDHD GLCpts SUCOAS TAL 969 ADHEr LDHD GLCpts SUCOAS TKT1 970 ADHEr LDHD GLUDy TKT2 971 ADHEr LDHD GLCpts GLUDy TKT1 972 ADHEr LDHD GLCpts GLUDy TAL 973 ADHEr LDHD GLUDy SUCOAS TKT1 974 ADHEr LDHD GLUDy SUCOAS TAL 975 ADHEr LDHD ASPT MDH PYK 976 ADHEr LDHD ASPT FUM PYK 977 ADHEr LDHD RPE SUCOAS 978 ADHEr LDHD GLCpts RPE 979 ADHEr LDHD GLCpts SUCOAS 980 ADHEr LDHD GLUDy RPE 981 ADHEr LDHD GLCpts GLUDy 982 ADHEr LDHD GLUDy SUCOAS 167 983 ADHEr LDHD TKT2 984 ADHEr LDHD GLCpts TAL 985 ADHEr LDHD GLCpts TKT1 986 ADHEr LDHD SUCOAS TAL 987 ADHEr LDHD SUCOAS TKT1 988 ADHEr LDHD GLUDy TKT1 989 ADHEr LDHD GLUDy TAL 990 ADHEr LDHD RPE 991 ADHEr LDHD GLCpts 992 ADHEr LDHD SUCOAS 993 ADHEr LDHD GLUDy 994 ADHEr LDHD TAL 995 ADHEr LDHD TKT1 Table 2: A list of all the reaction stoichiometries and the associated genes known to be associated with the reactions identified for disruption in the strategies listed in Tables 1. Reaction Abbreviatio n Reaction Name Reaction Stoichiometry Assigned Genes ACKr acetate kinase 1c: ac + atp ==> actp + adp b2296,_b3115 acetaldehyde-CoA [c] accoa + (2) h + (2) nadh ADHEr dehydrogenase <==> coa + etoh +2)nad b1241 2-oxoglutarate [c]: akg + coa + nad --> co2 + AKGD dehydrogenase nadh + succoa -b0727, b0726, b0116 ALAR alanine racemase Jc: ala-L <==> ala-D b4053 [c] : asp-L + atp + nh4 -- > amp ASNS2 .... aspar ie ta + asn-L + h + ppi b3744 ASPT L-aspartase ic: asp-L --> fum + nh4 b4139 Ab3738+b3736+b3737, b3739, ATP synthase (four protons adp[c] + (4) h[e] + pi[c] <> b3734+b3732+b3735+b3733+ A rPS4r for one ATP) atpjc]+(3)hc]+h2oc] b37_31 [c] : atp + co2 + nh4 --> adp + CBMK2 Carbamate kinase cbp. . +(2) h i b0323, b0521, b2874 D-Amino acid [c] : ala-D + fad + h2o -- > fadh2: DAAD dehydrogenase + nh4-+ pyr b1189 2-dehydro-3-deoxy EDA phosphogluconate aldolase Ic: 2ddg6p-->g3p+pyr b1850 fructose-bisphosphate FBA aldolase -- - Ic: fdp <==> dhap_+ g3p b1773, b2097, b2925 formate dehydrogenase for[c] + (3) h[c] + ubq8[c] -> b3893+b3894+b3892, FDH2 (quinone-8: 2 protons) co2[c] + (2) h[e] + ubq8h2[c] jb1476+b1475+b1474, b4079 168 [c] : fum + mql8 -> mqn8 + FRD2 fumarate reductase succ b4153+b4152+b4151+b4154 FUM fumarase :iJ: fum + h2o <==> mal-L b1612,1b4122 b1611 glutamate-5-semialdehyde [c] : glu5p + h + nadph -- > G5SD dehydrogenase -glu5sa + nadp+pi -------- b0243 glucose 6-phosphate [c] : g6p + nadp <==> 6pgl + h G6PDHy dehydrogenase + nadph b1852 -- -- --- --- --- --- --- --- --- -- D-glucose transport via gic-D[e] + pep[c] -- > g6p[c] + b1817, b1818, b2417, b1621, GLCpts PEP:Pyr PTS pyrIc] -b2416,b1819,b1101,b2415 GLU5K glutamate5-kinase Jc: atp + glu-L -> adp + glu5p b0242 glutamate dehydrogenase [c] glu-L + h2o + nadp <==> GLUDy .NADPakg+h+nadph+ nh4 b171--------------- [c] : atp + gic-D ->adp + g6p + HEM hexokinase (D-glucose ATP h _b2388 ICL Isocitrate lyase JcJ; icit -> glx + succ b4015 [c]: lac-D + nad <==> h + nadh: LDH D D-lactate dehydrogenase +_pyr b2133, b1380 [c] : accoa + glx + h2o --> coa MALS malate synthase + h + mal-L b4014, b2976 [c]: mal-L + nad <==> h + MDH malate dehydrogenase nadh + oaa _ b3236 [c] : mal-L + nadp -> co2 + ME2 malic enzyme (NADP) nadph +pyr- ........... -b2463 NADH dehydrogenase [c] : h + nadh + ubq8 --> nad + NADH12 (ubiquinone-8 )---------ubq8h2 b1109 b2288+b2277+b2285+b2278+ (4.5) h[c] + nadh[c] + ubq8[c] - b2276+b2286+b2287+b2279+ NADH dehydrogenase > (3.5) h[e] + nad[c] + : b2280+b2284+b2283+b2282+ NADH6 (ubiquinone-8 & 3.5_protons ubg8h2c] :b2281 PFK phosphofructokinase Ic ; atp + f6p -- > adp + fdp + h b3916,b1723 b3114, b3951+b3952, PFLi pyruvate formate lyase .. J coa + pyr -- > accoa + for b0902+b2579+b0903 phosphogluconate [c] : 6pgc + nadp -- > co2 + PGDH dehydrogenase nadph + ru5p-D b2029 phosphogluconate PGDHY dehydratase c6pgc -> 2ddg6p + h2o b1851 glucose-6-phosphate PGI isomerase Jc::g6p<-->f6p b4025 PGL 6-phosphogluconolactonase [c] : 6pgl + h2o -- > 6pgc + h b0767 169 phosphoenolpyruvate [c] : atp + h2o + pyr --> amp + PPS synthase 2)hpp + pi b1702 [c] : fad + pro-L --> 1pyr5c + PRO1z proline oxidase fadh2 + h b1014 [c] : accoa + pi <==> actp + PTAr phosphotransacetylase coa b2297 PYK pyruvate kinase Jc :adp+h+ pep -- > atp +pyr b1854, b1676 ribulose 5-phosphate 3 RPE epimerase cli*ru5p-D <==> xu5p-D b4301, b3386 [c] : fadh2 + ubq8 <==> fad + SUCD4 succinate dehyrdogenase ubg8h2 b0723+b0721+b0724+b0722 succinyl-CoA synthetase [c] : atp + coa + succ <==> adp SUCOAS (ADP-forming) +-pi + succoa b0729+b0728 TAL transaldolase JcJ;_gp +s7p <=-> e4p + f6p b2464, b0008 (2) h[e] + nadh[c] + nadp[c] THD2 NAD(P)transhydrogenase .(2) h[cl + nad[cl+ nadph[ql b1602+bl603 [c] : r5p + xu5p-D <==> g3p + TKT1 transketolase s7p b2935, b2465 [c] e4p + xu5p-D <==> f6p + TKT2 _ transketolase 3p b2935, b2465 TPI triose-phosphate isomerase [c] : dhap <==> g3p b3919 170 Table 3: List of the metabolite abbreviations, the corresponding names and locations of all the metabolites that participate in the reactions listed in Table 2. Metabolite Abbreviation Compartment Metabolite Name -1p-- - Cytoso------- 1-Pyrroline-5-carboxylate ddgp Cosl 2-Dehydro-3-deoxy-D-gluconate 6-phosphate 6pgc LCytosol ------ 6-Phospho-D-gluconate 6 p9 yto-------------- C l--------phospho-D-glucono-1 ,5-lactone c ytAcetate accoa Cytosol ActyI-CoA pgtp Cyto Acetyl phspha adp Cytos C ADP ak -- ------- Cytoso - 2-Oxoglutarate ala-D jCytosol D-Alanine ala-L -ytosol L-Alanine amp -to------------- AMP asn-L Cytosol L-Asparagine as-L Cytoso - L-Aspartate Ct- ytosol ATP cbp --------------- Cytos Carbamoyl phosphate cit Cytosol Citrate co2 Cytoso C02 ------------------ Sqto Coe------nI----zymActp Cytosol CTP dha Qytosol Dihydroxyacetone dhap Cytoso Dihydroxyacetone phosphate e p .Cytoso . D-Erythrose 4-phosphate etoh CytosoE thanol [p Cytosol D-Fructose 6-phosphate fad Cytosol FAD fadh2 Cytosol _ FADH2 fdp Cosol D-Fructose I,6-bisphosphate for Cytoso Formate fumrn Cytosl Fumarate -- p Cytoso - Glyceraldehyde -phosphate 9Op Cyo---gso-------------- solsphat gic-D --- ytoso . D-Glucose 9c- -- e----t--g-nis D-ucose 9 -5p Cytoso - L-Glutamate 5-phosphate glu5sa ------------- ytosol L-Glutamate 5-semialdehyde glu-L Cytosl L-Glutamate Cx 9ytoso . Glyoxylate h Cytosol_ i_ h[eExtra-organism H+ h2 Cytosol H2 h2o --------------- ytosol H20 icit Cytosol Isocitrate 171 k_ gytosol K+ Iac-D -ytosol D-Lactate mal-L Cytosol L-Malate mgyo-------------- ytoso----- Menaquinol 8 mqn --------------- So1 Menaquinone 8 nad Cytosol Nicotinamide adenine dnucleotide nadh - Cytosol Nicotinamide adenine dinucleotide - reduced P Ct--------------o --- Nicotinamide adenine dinucleotide phosphate nadph .. ytosol Nicotinarmide adenine dinucleotidephosphate - reduced nh4 -ytoso Ammonium o2 Cytosol 02 oaa Cytosol Oxaloacetate Rep----------- C---ytosol Phosphoenolpyruvate pi Cyos---------------------- ol Phosphate ppi--------Q-------ytosol Diphosphate pro----------------- ytosol L-Praline pyr --------------- ytosol Pyruvate r5p ytslapaDRbs--------------- 9------e phosphate p5p-D Cytoso! D.. ibulse 5-phosphate s 7 p Cytosol Sedoheptulose 7-phosphate __ succytosol Succinate suco Cytosol Succinyl-QoA Cb8ytosol Ubigtuinone-8 ubg8h2 ----------- ytosol Ubiguinol-8 xu5p-D Cytosol D-Xylulose 5-phosphate Example IV Exemplary H-ydrogenase and CO Dehydrogenase Enzymes for Extracting Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle Enzymes Enzymes of the reductive TCA cycle useful in the non-naturally occurring microbial organisms of the present invention include one or more of ATP-citrate lyase and three C0rfixing enzymes: isocitrate dehydrogenase, alpha-ketoglutarate: ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase. The presence of ATP-citrate lyase or citrate lyase and alpha ketoglutarate:ferredoxin oxidoreductase indicates the presence of an active reductive TCA cycle in an organism. Enzymes for each step of the reductive TCA cycle are shown below. ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase, catalyzes the ATP dependent cleavage of citrate to oxaloacetate and acetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied in green sulfur bacteria Chlorobium limicola and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was cloned and 172 characterized in E. coli (Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, is irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP. A recombinant ACL from Chlorobium tepidum was also expressed in E. coli and the holoenzyme was reconstituted in vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism (Kim and Tabita, J. Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in Balnearium lithotrophicum, Sulfurikydrogenibium subterraneum and other members of the bacterial phylum Aquificae (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungi as well. Exemplary organisms include Sordaria macrospora (Nowrousian et al., Curr. Genet. 3 7:189-93 (2000), Aspergillus nidulans , Yarrowia lipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039 1048, (2010) and Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Other candidates can be found based on sequence homology. Information related to these enzymes is tabulated below: Protein GenBank ID GI Number Organism acLA BAB21376.1 12407237 Chlorobium limicola aclB BAB21375.1 12407235 Chlorobium limicola acIA AAM72321.1 21647054 Chlorobium tepidum aclB AAM72322.1 21647055 Chlorobiumtepidum ac/A AB50076.1 114054981 Balnearium lithotrophicum aciB ABI50075.1 114054980 Balnearium lithotrophicum ac/A ABI50085.1 114055040 Sulfurihydrogenibium subterraneum aclB AB150084.1 114055039 Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504 Sulfurimonas denitrificans aclB AAX76835.1 62199506 Sulfurimonas denitrificans acli XP_504787.1 50554757 Yarrowia lipolytica acl2 XP_503231.1 50551515 Yarrowia lipolytica SPBC1 703.07 NP_596202.1 19112994 Schizosaccharomycespombe SPAC22A12.16 NP_593246.1 19114158 Schizosaccharomyces pombe acl] CAB76165.1 7160185 Sordaria macrospora acl2 CAB76164.1 7160184 Sordaria macrospora 173 Protein GenBank ID GI Number Organism aclA CBF86850.1 259487849 Aspergillus nidulans aciB CBF86848 259487848 Aspergillus nidulans In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl-CoA synthetase (EC 6,2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M,, Appl. Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by ccsA and csB, respectively (Aoshima et al., MoL. Micrbiol. 52:751-761 (2004)). The citryl-CoA synthetase of Aquifex aeolicus is composed of alpha and beta subunits encoded by sucC1 and sucD1 (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme is a homotrimer encoded by ccl in Hydrogenobacter thermophilus (Aoshima et al., MoL. Microbiol. 52:763-770 (2004)) and aq_150 in Aqufex aeolicus (Hugler et al., supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA have also been reported recently in Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002). Protein GenBank ID GI Number Organism ccsA BAD17844.1 46849514 Hydrogenobacter thermophilus ccsB BAD17846.1 46849517 Hydrogenobacter thermophilus sucCi AAC07285 2983723 Aquifex aeolicus sucD1 AAC07686 2984152 Aquifex aeolicus Ccl BAD17841.1 46849510 Hydrogenobacter thermophilus aq_150 AAC06486 2982866 Aquifex aeolicus CT0380 NP_661284 21673219 Chlorobium tepidum CT0269 NP_661173.1 21673108 Chlorobium tepidum CT1834 AAM73055.1 21647851 Chlorobium tepidum Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the forward and reverse direction. S. cerevisiae possesses three copies of malate 174 dehydrogenase, MDR] (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11:370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem, 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. E. coli is known to have an active malate dehydrogenase encoded by mdh. Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae Mdh NP_417703.1 16131126 Escherichia coli Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to malate. The three fumarases of E. coli, encoded byfumA,fumB andfumC, are regulated under different conditions of oxygen availability. FumB is oxygen sensitive and is active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is active under aerobic growth conditions (Tseng et al., J. Bacteriol. 183:461-467 (2001);Woods et al., Biochim. Biophys. Acta 954:14-26 (1988); Guest et al., J. Gen. Microbiol. 131:2971-2984 (1985)). S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al., J. Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes are found in Campylobacterjejuni (Smith et al., Int. J. Biochem. Cell. Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology includefuml from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)). Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570 Escherichia coli 175 Protein GenBank ID GI Number Organism fumB NP_418546.1 16131948 Escherichia coli fumC NP_416128.1 16129569 Escherichia coli FUMI NP_015061 6324993 Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacterium glutamicum fumC 069294.1 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicus MmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum thermopropionicum Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. The electron donor for this reaction is menaquinone and the two protons produced in this reaction do not contribute to the proton gradient (Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto et al., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch. Biochem, Biophys. 352:175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used during anaerobic growth on glucose (Arikawa et al., FEMSMicrobiol. Lett. 165:111-116 (1998)). Protein GenBank ID GI Number Organism FRDSJ P32614 418423 Saccharomyces cerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli frdB NP 418577.1 16131978 Escheichia coli frdC NP_418576.1 16131977 Escherichia coli frdD NP_418475.1 16131877 Escherichia coli The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2 genes of S. cerevisiae and the sucC 176 and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below: Protein GenBank ID GI Number Orgunism LSCJ NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also known as 2-oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase (OFOR), forms alpha-ketoglutarate from C02 and succinyl-CoA with concurrent consumption of two reduced ferredoxin equivalents. OFOR and pyruvate:ferredoxin oxidoreductase (PFOR) are members of a diverse family of 2 oxoacid:ferredoxin (flavodoxin) oxidoreductases which utilize thiamine pyrophosphate, CoA and iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in this class are reversible and function in the carboxylation direction in organisms that fix carbon by the RTCA cycle such as Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus and Chlorobium species (Shiba et al. 1985; Evans et al., Proc. Natl. Acad. ScL. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilus, encoded by karAB, has been cloned and expressed in E. coli (Yun et al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFOR from the same organism with strict substrate specificity for succinyl-CoA, encoded byforDABGE, was recently identified and expressed in E. coli (Yun et al. 2002). The kinetics of C02 fixation of both H. thermophilus OFOR enzymes have been characterized (Yamamoto et al., Extremophiles 14:79-85 (2010)). A C02-fixing OFOR from Chlorobium thiosulfatophilum has been purified and characterized but the genes encoding this enzyme have not been identified to date. Enzyme candidates in Chlorobium species can be inferred by sequence similarity to the H. thermophilus genes. For example, the Chlorobium limicola genome encodes two similar proteins. Acetogenic bacteria such as Moorella thermoacetica are predicted 177 to encode two OFOR enzymes. The enzyme encoded by Moth 0034 is predicted to function in the C02-assimilating direction. The genes associated with this enzyme, Moth_0034 have not been experimentally validated to date but can be inferred by sequence similarity to known OFOR enzymes. OFOR enzymes that function in the decarboxylation direction under physiological conditions can also catalyze fhe reverse reaction. The OFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7, encoded by ST2300, has been extensively studied (Zhang et al. 1996. A plasmid based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). The OFOR encoded by Ape1472/Ape1473 from Aeropyrum pernix str. Ki was recently cloned into E. coli, characterized, and found to react with 2-oxoglutarate and a broad range of 2 oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded by oorDABC in Helicobacterpylori (Hughes et al. 1998). An enzyme specific to alpha ketoglutarate has been reported in Thauera aromatica (Dorner and Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similar enzyme can be found in Rhodospirillum rubrum by sequence homology. A two subunit enzyme has also been identified in Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002)). Protein GenBank ID GI Number Organism korA BAB21494 12583691 Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacter thermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forA BAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.1 14970996 Hydrogenobacter therm ophilus forG BAB62135.1 14970997 Hydrogenobacter therm ophilus forE BAB62136.1 14970998 Hydrogenobacter thermophilus Clim0204 ACD89303.1 189339900 Chlorobium limicola Clim_0205 ACD89302.1 189339899 Chlorobium limicola Clim_1123 ACD90192.1 189340789 Chlorobium limicola Clim_1124 ACD90193.1 189340790 Chlorobium limicola 178 Moth_1984 YP_430825.1 83590816 Moorella thermoacetica Moth_1985 YP_430826.1 83590817 Moorella thermoacetica Moth_0034 YP_428917.1 83588908 Moorella thermoacetica ST2300 NP_378302.1 15922633 Sulfolobus sp. strain 7 Ape1472 BAA80470.1 5105156 Aeropyrumpernix Ape1473 BAA80471.2 116062794 Aeropyrumpernix oorD NP_207383.1 15645213 Helicobacterpylori oorA NP_207384.1 15645214 Helicobacter pylori oorB NP_207385.1 15645215 Helicobacterpylori oorC NP_207386.1 15645216 Helicobacterpylori CT0163 NP_661069.1 21673004 Chlorobium tepidum CT0162 NP_661068.1 21673003 Chlorobium tepidum korA CAA12243.2 19571179 Thauera aromatica korB CAD27440.1 19571178 Thauera aromatica Rru_A 2721 YP_427805.1 83594053 Rhodospirillum rubrum RruA 2722 YP_427806.1 83594054 Rhodospirillum rubrum Isocitrate dehydrogenase catalyzes the reversible decarboxylation of isocitrate to 2-oxoglutarate coupled to the reduction of NAD(P)+. IDH enzymes in Saccharomyces cerevisiae and Escherichia coli are encoded by IDP1 and icd, respectively (Haselbeck and McAlister-Henn, J. Bio. Chem. 266:2339-2345 (1991); Nimmo, H.G., Biochem. J. 234:317-2332 (1986)). The reverse reaction in the reductive TCA cycle, the reductive carboxylation of 2-oxoglutarate to isocitrate, is favored by the NADPH-dependent C0 2 -fixing IDH from Chlorobium limicola and was functionally expressed in E. coli (Kanao et al., Eur. J. Biochem. 269:1926-1931 (2002)). A similar enzyme with 95% sequence identity is found in the C. tepidum genome in addititon to some other candidates listed below. Protein GenBank ID GI Number Organism Icd AC184720.1 209772816 Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces cerevisiae Idh BAC00856.1 21396513 Chlorobium limicola 179 Protein GenBank ID GI Number Organism Icd AAM71597.1 21646271 Chlorobium tepidum icd NP_952516.1 39996565 Geobacter sulfurreducens icd YP_393560. 78777245 Sulfurimonas denitrificans In H. thermophilus the reductive carboxylation of 2-oxoglutarate to isocitrate is catalyzed by two enzymes: 2-oxoglutarate carboxylase and oxalosuccinate reductase. 2-Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent carboxylation of alpha-ketoglutarate to oxalosuccinate (Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a large complex composed of two subunits. Biotinylation of the large (A) subunit is required for enzyme function (Aoshima et al., Mol. Microbiol. 51:791-798 (2004)). Oxalosuccinate reductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate to D-threo-isocitrate. The enzyme is a homodimer encoded by icd in H. thermophilus. The kinetic parameters of this enzyme indicate that the enzyme only operates in the reductive carboxylation direction in vivo, in contrast to isocitrate dehydrogenase enzymes in other organisms (Aoshima and Igarashi, J. Bacteriol. 190:2050-2055 (2008)). Based on sequence homology, gene candidates have also been found in Thiobacillus denitrificans and Thermocrinis albus. Protein GenBank ID GI Number Organism cfiA BAF34932.1 116234991 Hydrogenobacter thermophilus cifB BAF34931.1 116234990 Hydrogenobacter thermophilus Icd BAD02487.1 38602676 Hydrogenobacter thermophilus Tbd_1556 YP_315314 74317574 Thiobacillus denitrificans Tbd_1555 YP_315313 74317573 Thiobacillus denitrificans Tbd_0854 YP_314612 74316872 Thiobacillus denitrificans Thal_0268 YP_003473030 289548042 Thermocrinis albus Thal0267 YP_003473029 289548041 Thermocrinis albus Thal_0646 YP_003473406 289548418 Thermocrinis albus Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate cis-aconitate. Two aconitase enzymes 180 are encoded in the E. coli genome by acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active under conditions of oxidative or acid stress (Cunningham et al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in Salmonella typhimurium are encoded by acnA and acnB (Horswill and Escalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiae aconitase, encoded by AC01, is localized to the mitochondria where it participates in the TCA cycle (Gangloff et al., Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (Regev Rudzki et al., Mol. Biol. Cell. 16:4163-4171 (2005)). Protein GenBank ID GI Number Organism acnA AAC7438.1 1787531 Escherichia coli acnB AAC73229.1 2367097 Escherichia coli HP0779 NP_207572.1 15645398 Helicobacterpylori 26695 H16_B0568 CAJ95365.1 113529018 Ralstonia eutropha DesfrDRAFT 3783 ZP_07335307.1 303249064 Desulfovibriofructosovorans JJ Suden_1040 ABB44318.1 78497778 Sulfurimonas denitrificans (acnB) Hydth_0755 AD045152.1 308751669 Hydrogenobacter thermophilus CT0543 (acn) AAM71785.1 21646475 Chlorobium tepidum Clim_2436 YP_001944436.1 189347907 Chlorobium limicola Clim_0515 ACD89607.1 189340204 Chlorobium limicola acnA NP_460671.1 16765056 Salmonella typhimurium acnB NP_459163.1 16763548 Salmonella typhimurium ACO1 AAA34389.1 170982 Saccharomyces cerevisiae Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation of pyruvate to form acetyl-CoA. The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al,, J. Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. Two cysteine residues in this enzyme form a disulfide bond that prtotects it against inactivation in the form of oxygen. This disulfide bond and 181 the stability in the presence of oxygen has been found in other Desulfovibrio species also (Vita et al., Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also well characterized (Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, encoding a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)). PFORs have also been described in other organisms, including Rhodobacter capsulatas (Yakunin and Hallenbeck, Biochimica et Biophysica Acta 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002)). The five subunit PFOR from H. thermophilus, encoded by porEDABG, was cloned into E. coli and shown to function in both the decarboxylating and C0 2 -assimilating directions (Ikeda et al. 2006; Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs also exist in C. carboxidivorans P7. Several additional PFOR enzymes are described in the following review (Ragsdale, S.W., Chem. Rev. 103:2333-2346 (2003)). Finally, flayodoxin reductases (e.g.,fqrB from Helicobacter pylori or Campylobacterjejuni) (St Maurice et al., J. Bacteriol. 189:4764-4773 (2007)) or Rnf type proteins (Seedorf et al., Proc. NatI. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below. Protein GenBank ID GI Number Organism DesfrDRAFT_0121 ZP_07331646.1 303245362 Desulfovibriofructosovorans JJ Por CAA70873.1 1770208 Desulfovibrio africanus por YP_012236.1 46581428 Desulfovibrio vulgaris str. Hildenborough Dde_3237 ABB40031.1 78220682 Desulfo Vibrio desulfuricans G20 Ddes_0298 YP_002478891.1 220903579 Desulfovibrio desulfuricans subsp. desulfuricans str. A TCC 27774 Por YP_428946.1 83588937 Moorella thermoacetica 182 YdbK NP_415896.1 16129339 Escherichia coli nifJ (CT1628) NP_662511.1 21674446 Chlorobium tepidum CJE 1649 YP_179630.1 57238499 Campylobacterjeuni nifi ADE85473.1 294476085 Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacter thermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porA BAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891 Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacter thermophilus FqrB YP_001482096.1 157414840 Campylobacterjejuni HP1164 NP_207955.1 15645778 Helicobacterpylori RnfC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK333 11.1 146346775 Clostridium kluyveri The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes or their combinations thereof. For example, pyruvate dehydrogenase can transform pyruvate into acetyl CoA with the concomitant reduction of a molecule of NAD into NADH. It is a multi-enzyme complex that catalyzes a series of partial reactions which results in acylating oxidative decarboxylation of pyruvate. The enzyme comprises of three subunits: the pyruvate decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae. 183 In the E. coli enzyme, specific residues in the El component are responsible for substrate specificity (Bisswanger, H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem. 8:535-540 (1969); Gong et al., J. Biol, Chem. 275:13645-13653 (2000)). Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (5). Crystal structures of the enzyme complex from bovine kidney (18) and the E2 catalytic domain from Azotobacter vinelandii are available (4). Yet another enzyme that can catalyze this conversion is pyruvate formate lyase. This enzyme catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate. Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli encoded bypflB (Knappe and Sawers, FEMS.Microbiol Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al., Oral.Microbiol Immunol. 18:293-297 (2003)). E. coli possesses an additional pyruvate formate lyase, encoded by tdcE, that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Both pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA. Further, a short protein encoded by yfiD in E. coli can associate with and restore activity to oxygen-cleaved pyruvate formate lyase (Vey et al., Proc.Natl. Acad. Sci. U.S.A. 105:16137-16141 (2008). Note that pfA and pfB from E. coli were expressed in S. cerevisiae as a means to increase cytosolic acetyl-CoA for butanol production as described in WO/2008/080124]. Additional pyruvate formate lyase and activating enzyme candidates, encoded by pfl and act, respectively, are found in Clostridium pasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)). Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde; the latter is oxidized to acetate by acetaldehyde dehydrogenase and subsequently activated to form acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is 184 a native enzyme in several other organisms including E. coli (Kumari et al., J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology 151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica (described already). Alternatively, acetate can be activated to form acetyl-CoA by acetate kinase and phosphotransacetylase. Acetate kinase first converts acetate into acetyl-phosphate with the accompanying use of an ATP molecule. Acetyl-phosphate and CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase. Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several Clostridia and Methanosarcina thermophila. Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor. In E. coli, this activity is encoded by poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiae and Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor (Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then be converted into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, as described earlier. Some of these enzymes can also catalyze the reverse reaction from acetyl-CoA to pyruvate. For enzymes that use reducing equivalents in the form of NADH or NADPH, these reduced carriers can be generated by transferring electrons from reduced ferredoxin. Two enzymes catalyze the reversible transfer of electrons from reduced ferredoxins to NAD(P)*, ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982); Fujii et al., 1977). The Helicobacterpylori FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacterjejuni (St et al. 2007). A ferredoxin:NADP+ oxidoreductase enzyme is encoded in the E. coli genome byfpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase utilizes 185 reduced ferredoxin to generate NADH from NAD*. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD* oxidoreductase of E. coli, encoded by hcaD, is a component of the 3 phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus strain TK-6, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7. Protein GenBank ID GI Number Organism HPJ 164 NP_207955.1 15645778 Helicobacter pylori RPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris fpr BAH29712.1 225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736 Bacillus subtilis CJE0663 AAW35824.1 57167045 Campylobacterjejuni fpr P28861.4 399486 Escherichia coli hcaD AAC75595.1 1788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea mays RnJC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnJG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfR EDK333 11.1 146346775 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707 Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706 Clostridium carboxidivorans P7 186 CcarbDRAFT_2636 ZP_05392636.1 255525704 Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241 Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514 Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124 Clostridium carboxidivorans P7 Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP* oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H. thennophilus genefdx1 encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe 4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacterpylori (Mukhopadhyay et al. 2003) and Campylobacterjejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7 and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed in the table below. Protein GenBank ID GI Number Organism fdxI BAE02673.1 68163284 Hydrogenobacter thernophilus 187 M 11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius Fdx AAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367 Helicobacterpylori fdxA CAL34484.1 112359698 Campylobacterjejuni Moth_0061 ABC 18400.1 83571848 Moorella thermoacetica Moth_1200 ABC19514.1 83572962 Moorella thennoacetica Moth_1888 ABC20188.1 83573636 Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorella thennoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoacetica CcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7 CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7 CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7 CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7 CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7 CcarbDRAFT _1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN CAA35699.1 46143 Rhodobacter capsulatus RruA2264 ABC23064.1 83576513 Rhodospirillum rubrum RruA1916 ABC22716.1 83576165 Rhodospirillum rubrum RruA2026 ABC22826.1 83576275 Rhodospirillum rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605 Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatium vinosum DSM 180 fdx YP_002801146.1 226946073 Azotobacter vinelandii DJ 188 CKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 ferl NP_949965.1 39937689 Rhodopseudomonaspalustris CGA009 fdx CAA12251.1 3724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermus hydrogenoformans fer YP_359966.1 78045103 Carboxydothermus hydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis fdx1 NP_249053.1 15595559 Pseudomonas aeruginosa PA 01 yfhL AP_003148.1 89109368 Escherichia coliK-12 Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule, Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2 methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3 mercaptopropionate, propionate, vinylacetate, and butyrate, among others. The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl CoA:Acetyl-CoA transferase. The gene product of cat] of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004). The succinyl-CoA:acetate CoA transferase from Acetobacter aceti, encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al. 2008). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl CoA transferase encoded by pcal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are identified below. 189 Protein GenBank ID GI Number Organism cat] P38946.1 729048 Clostridium kluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3 Tb1.02.0290 XP_828352 71754875 Trypanosoma brucei pcal AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida aarC ACD85596.1 189233555 Acetobacter aceti An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism HPAGI_0676 YP_627417 108563101 Helicobacterpylori HPAG_0677 YP_627418 108563102 Helicobacterpyori ScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT NP_000427 4557817 Homo sapiens 1 OXCT NP_071403 11545841 Homo sapiens 2 Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA:acetate:CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa. Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814 7818 (2007), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechno 77:1219 190 1224 (2008), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)) are shown below. Protein GenBank ID GI Number Organism AtoA NP_416726.1 2492994 Escherichia coli AtoD NP_416725.1 2492990 Escherichia coli CtfA NP_149326.1 15004866 Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridium acetobutylicum CtfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)-Benzylsuccinate CoA Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)). Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbN1, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism bbsE AAF89840 9622535 Thauera aromatica Bbsf AAF89841 9622536 Thauera aromatica bbsE AAU45405.1 52421824 Azoarcus sp. T bbsF AAU45406.1 52421825 Azoarcus sp. T bbsE YP_158075.1 56476486 Aromatoleum aromaticum EbNJ bbsF YP_158074.1 56476485 Aromatoleum aromaticum EbNJ Gmet_1521 YP_384480.1 78222733 Geobacter metallireducens GS-15 Gmet_1522 YP_384481.1 78222734 Geobacter metallireducens GS-15 Additionally, ygfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below. 191 Protein GenBank ID GI Number Organism ygfH NP_417395.1 16130821 Escherichia coli str. K-12 substr. MGI655 CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae A TCC 29220 SARI_04582 YP_001573497.1 161506385 Salmonella enterica subsp. arizonae serovar yinte0001_14430 ZP_04635364.1 238791727 Yersinia intermedia A TCC 29909 Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting in the cleavage of citrate to acetate and oxaloacetate. The enzyme is active under anaerobic conditions and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and a acyl lyase (beta). Enzyme activation uses covalent binding and acetylation of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3-'-dephospho-CoA, which is similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme into the active holo enzyme (Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD and the citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman, Biochemistry 22:4657 4663 (1983)). The Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in E. coli (Bekal et al., J. Bacterial. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and Klebsiella pneumoniae (Bott, Arch. Microbial. 167: 78-88 (1997); Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below. Protein GenBank ID GI Number Or2anism citF AAC73716.1 1786832 Escherichia coli Cite AAC73717.2 87081764 Escherichia coli citD AAC73718. 1 1786834 Escherichia coli citC AAC73719.2 87081765 Escherichia coli 192 Protein GenBank ID GI Number Organism citG AAC73714.1 1786830 Escherichia coli citX AAC73715.1 1786831 Escherichia coli citF CAA71633.1 2842397 Leuconostoc mesenteroides Cite CAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998 Salmonella typhimurium cite AAL19573.1 16419133 Salmonella typhimurium citD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.1 16764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonella typhimurium citX NP_459612.1 16763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citC BAH6654 1.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependent phosphorylation of acetate to acetylphosphate. Exemplary acetate kinase enzymes have been characterized in many organisms including E. coli, Clostridium acetobutylicum and Methanosarcina thermophila 193 (Ingram-Smith et al., J. Bacterial. 187:2386-2394 (2005); Fox and Roseman, J. Biol. Chem. 261:13487-13497 (1986); Winzer et al, Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity has also been demonstrated in the gene product of E. colipurT (Marolewski et al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example buki and buk2 from Clostridium acetobutylicum, also accept acetate as a substrate (Hartmanis, M.G., J. Biol. Chem. 262:617-621 (1987)). Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231 Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum Ack AAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155 Escherichia coli buki NP_349675 15896326 Clostridium acetobutylicum buk2 Q97111 20137415 Clostridium acetobutylicum The formation of acetyl-CoA from acetylphosphate is catalyzed by phosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)). Additional acetyltransferase enzymes have been characterized in Bacillus subtilis (Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973), Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955), and Thermotoga maritima (Bock et al., J. Bacteriol. 181:1861-1867 (1999)). This reaction is also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19) including the ptb gene products from Clostridium acetobutylicum (Wiesenborn et al., App. Environ. Microbiol. 55:317-322 (1989); Walter et al., Gene 134:107-111 (1993)). Additional ptb genes are found in butyrate-producing bacterium L2-50 (Louis et al., J Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al., Curr. Microbiol. 42:345-349 (2001). Protein GenBank ID GI Number Organism Pta NP_416800.1 71152910 Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801 146346896 Clostridium kluyveri Pta Q9XOL4 6685776 Thermotoga maritima 194 Protein GenBank ID GI Number Or2anism Ptb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.1 butyrate-producing bacterium 38425288 L2-50 Ptb CAC07932.1 10046659 Bacillus megaterium The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase activity. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431 (2004)). ADP-forming acetyl-CoA synthetases are reversible enzymes with a generally broad substrate range (Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetases -are encoded in the Archaeoglobusfulgidus genome by are encoded by AF1211 and AF 1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and reversibility of the enzyme was demonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate, isobutyryl CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)). Additional candidates include the succinyl-CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Femandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154 (1993)). The aforementioned proteins are tabulated below. 195 Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha aesI ABC87079.1 86169671 Methanothermobacter thermautotrophicus acsl AAL23099.1 16422835 Salmonella enterica ACSJ Q01574.2 257050994 Saccharomyces cerevisiae AF1211 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax, are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from synthesis gas components such as CO and H 2 using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes, respectively. 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 and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway and reductive TCA cycle enzymes. Herein below the enzymes and the corresponding genes used for extracting redox from synags components are described. CODH is a reversible enzyme that interconverts CO and CO 2 at the expense or gain of electrons. The natural physiological role of the CODH in ACS/CODH complexes is to convert CO 2 to CO for incorporation into acetyl-CoA by acetyl-CoA synthase. 196 Nevertheless, such CODH enzymes are suitable for the extraction of reducing equivalents from CO due to the reversible nature of such enzymes. Expressing such CODH enzymes in the absence of ACS allows them to operate in the direction opposite to their natural physiological role (i.e., CO oxidation). In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The M. thermoacetica gene (Genbank Accession Number: YP_430813) is expressed by itself in an operon and is believed to transfer electrons from CO to an external mediator like ferredoxin in a "Ping-pong" reaction. The reduced mediator then couples to other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH II is also available (Dobbek et al., Science 293:1281-1285 (2001)). Similar ACS-free CODH enzymes can be found in a diverse array of organisms including Geobacter metallireducens GS 15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H 10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, and Campylobacter curvus 525.92. Protein GenBank ID GI Number Organism CODH (putative) YP_430813 83590804 Moorella thermoacetica CODH-II (CooS- YP_358957 78044574 Carboxydothermus II) hydrogenoformans CooF YP_358958 78045112 Carboxydothermus hydrogenoformans CODH (putative) ZP_05390164.1 255523193 Clostridium carboxidivorans P7 CcarbDPAFT_0341 ZP_05390341.1 255523371 Clostridium carboxidivorans P7 197 CcarbDRAFT_1756 ZP_05391756.1 255524806 Clostridium carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1 255526020 Clostridium carboxidivorans P7 CODH YP_384856.1 78223109 Geobacter metallireducens GS 15 Cpha266_0148 YP_910642.1 119355998 Chlorobium cytochromee c) phaeobacteroides DSM 266 Cpha266_0149 YP_910643.1 119355999 Chlorobium (CODH) phaeobacteroides DSM 266 Ccel_0438 YP_002504800.1 220927891 Clostridium cellulolyticum H10 Ddes_0382 YP_002478973.1 220903661 Desulfovibrio desulfuricans (CODH) subsp. desulfuricans str. A TCC 27774 Ddes_0381 YP_002478972.1 220903660 Desulfovibrio desulfuricans (CooC) subsp. desulfuricans str. A TCC 27774 Pcar_0057 YP_355490.1 7791767 Pelobacter carbinolicus DSM (CODH) 2380 Pcar_0058 YP_35549 1.1 7791766 Pelobacter carbinolicus DSM (CooC) 2380 Pcar_0058 YP_355492.1 7791765 Pelobacter carbinolicus DSM (HypA) 2380 CooS (CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92 In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H 2 0 to CO 2 and H 2 (Fox et al., JBacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and CO 2 reduction activities when linked to an electrode (Parkin et al., JAm.Chem.Soc. 129:10328-10329 (2007)), The protein sequences of 198 exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers. Protein GenBank ID GI Number Organism CODH-I YP_360644 78043418 Carboxydothermus (CooS-1) hydrogenofornans CooF YP_360645 78044791 Carboxydothermus hydrogenoformans HypA YP_360646 78044340 Carboxydothermus hydrogenoformans CooH YP_360647 78043871 Carboxydothermus hydrogenoformans CooU YP_360648 78044023 Carboxydothermus hydrogenoformans CooX YP_360649 78043124 Carboxydothermus hydrogenoformans CooL YP_360650 78043938 Carboxydothermus hydrogenoformans CooK YP_360651 78044700 Carboxydothermus hydrogenoformans CooM YP_360652 78043942 Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296 Carboxydothernus hydrogenoformans CooA-1 YP_360655.1 78044021 Carboxydothermus hydrogenoformans CooL AAC451 18 1515468 Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooU AAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746 Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749 Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJ AAC45126 1498751 Rhodospirillum rubrum 199 Native to E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al, JBacterial. 164:1324 1331 (1985); Sawers and Boxer, Eur.JBiochem. 156:265-275 (1986); Sawers et al., JBacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities, E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1 and Hyd 2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters, respectively (Lukey et al., How E. coli is equipped to oxidize hydrogen under different redox conditions, JBiol Chem published online Nov 16, 2009). Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinone reduction via the hyaC cytochrome. Hyd-2 is sensitive to 02, reversible, and transfers electrons to the periplasmic ferredoxin hybA which, in turn, reduces a quinone via the hybB integral membrane protein. Reduced quinones can serve as the source of electrons for fumarate reductase in the reductive branch of the TCA cycle. Reduced ferredoxins can be used by enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPH or NADH. They can alternatively be used as the electron donor for reactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxin oxidoreductase, and 5,10-methylene-H4folate reductase. Protein GenBank ID GI Number Organism HyaA AAC74057.1 1787206 Escherichia coli HyaB AAC74058.1 1787207 Escherichia coli HyaC AAC74059.1 1787208 Escherichia coli HyaD AAC74060.1 1787209 Escherichia coli HyaE AAC74061.1 1.787210 Escherichia coli HyaF AAC74062.1 1787211 Escherichia coli Protein GenBank ID GI Number Organism HybO AAC76033.1 1789371 Escherichia coli HybA AAC76032.1 1789370 Escherichia coli HybB AAC76031.1 2367183 Escherichia coli HybC AAC76030.1 1789368 Escherichia coli HybD AAC76029.1 1789367 Escherichia coli HybE AAC76028,1 1789366 Escherichia coli 200 HybF AAC76027.1 1789365 Escherichia coli HybG AAC76026.1 1789364 Escherichia coli The hydrogen-lyase systems of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyfgene clusters, respectively. Hydrogenase 3 has been shown to be a reversible enzyme (Maeda et al., Appl Microbiol Biotechnol 76(5):1035 42 (2007)). Hydrogenase activity in E., coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al., J. Bacteriol. 190:1447 1458 (2008)). Protein GenBank ID GI Number Organism HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichia coli HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624 Escherichia coli Protein GenBank ID GI Number Organism HyfA NP_416976 90111444 Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfD NP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichia coli HyfF NP_416981 16130411 Escherichia coli 201 HyfG NP_416982 16130412 Escherichia coli HyfH NP_416983 16130413 Escherichia coli Hyfl NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfR NP_416986 90111447 Escherichia coli Protein GenBank ID GI Number Organism HypA NP_417206 16130633 Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_417208 16130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypE NP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichia coli The M. thermoacetica hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica can grow with CO 2 as the exclusive carbon source indicating that reducing equivalents are extracted from H2 to enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res. Microbiol 155:869-883 (2004); Kellum and Drake, J. Bacteriol. 160:466-469 (1984)) (see Figure 21). M. thennoacetica has homologs to several hyp, hyc, and hyfgenes from E. coli. The protein sequences encoded for by these genes are identified by the following GenBank accession numbers. Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below. Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998 Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorella thermoacetica Moth_2177 YP_431009 83591000 Moorella thermoacetica Moth_2178 YP_431010 83591001 Moorella thermoacetica 202 Moth_2179 YP_431011 83591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorella thermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica Proteins in M. thermoacetica that are homologous to the E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table. Protein GenBank ID GI Number Organism Moth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorella thermoacetica Moth_2185 YP_431017 83591008 Moorella thermoacetica Moth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorella thermoacetica Moth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorella thennoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica In addition, several gene clusters encoding hydrogenase functionality are present in M, thermoacetica and their corresponding protein sequences are provided below. Protein GenBank ID GI Number Organism Moth_0439 YP_429313 83589304 Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_429315 83589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_429670 83589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorella thermoacetica 203 Moth_0811 YP_429672 83589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorella thermoacetica Moth_0814 YP_429674 83589665 Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_429676 83589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194 YP_430051 83590042 Moorella thennoacetica Moth__1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP 430053 83590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorella thermoacetica Moth_1718 YP_430563 83590554 Moorella thermoacetica Moth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_430726 83590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorella thermoacetica Moth_1885 YP_430728 83590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_430730 83590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorella thennoaceica Moth_1452 YP_430305 83590296 Moorella thermoacetica Moth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_430307 83590298 Moorella thermoacetica Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an "02-tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0 2 -tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biachim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122 3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms 204 including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, AppL. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J. Bio. Chem. 284(52), 36462-36472 (2009)). Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753 Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16 HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.1 38637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstonia eutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxE NP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815 Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobacter sulfurreducens HoxY NP_953764.1 39997813 Geobacter suifurreducens HoxH NP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.1 39997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690 Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803 Unknown function NP_441416.1 16330688 Synechocystis str. PCC 6803 HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxY NP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown function NP_441413.1 16330685 Synechocystis str. PCC 6803 Unknown function NP_441412.1 16330684 Synechocystis str. PCC 6803 HoxH NP_441411.1 16330683 Synechocystis str. PCC 6803 HypF NP484737.1 17228189 Nostoc sp. PCC 7120 205 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.1 17228191 Nostoc sp. PCC 7120 Unknown function NP_484740.1 17228192 Nostoc sp. PCC 7120 HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypA NP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195 Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicina Hox1F AAP50520.1 37787352 Thiocapsa roseopersicina HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina Several enzymes and the corresponding genes used for fixing carbon dioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycle intermediates, oxaloacetate or malate are described below. Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by phosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys. 414:170-179 (2003), ppcA in Methylobacterium extorquens AM] (Arps et al., J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacterium glutamicum (Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989). Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichia coli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB53270 80973080 Corynebacterium glutamicum An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP 206 at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCKJ, serves a gluconeogenic role (Valdes-Hevia et al., FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher Km for bicarbonate of PEP carboxykinase (Kim et al., Appl. Environ. Microbiol. 70:1238 1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated inppc mutants of E. coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHCO 3 concentrations. Mutant strains of E. coli can adopt Pck as the dominant C02-fixing enzyme following adaptive evolution (Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum succiniciproducens (Laivenieks et al., AppL. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP. Protein GenBank ID GI Number Organism PCKI NP_013023 6322950 Saccharonyces cerevisiae pek NP_417862.1 16131280 Eseherichia coli pckA YP_089485.1 52426348 Mannheimia succiniciproducens pckA 009460.1 3122621 Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571 Actinobacillus succinogenes pckA P43923.1 1172573 Haenophilus influenza Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate to oxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes are encoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun. 176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomyces 207 cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475:191-206 (2000)). Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium smegmatis Malic enzyme can be applied to convert CO 2 and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent). For example, one of the E. coli malic enzymes (Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion of pyruvate and CO 2 to palate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport. Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E. coli while restoring the lethal Apfl-AldhA phenotype under anaerobic conditions by operating in the carbon-fixing direction (Stols and Donnelly, AppL. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from Ascaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355 65 (1979)). Protein GenBank ID GI Number Organism maeA NP_415996 90111281 Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443 126732 Ascaris suum 208 The enzymes used for converting oxaloacetate (formed from, for example, PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate (formed from, for example, malic enzyme or malate dehydrogenase) to succinyl-CoA via the reductive branch of the TCA cycle are malate dehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, and succinyl CoA transferase. The genes for each of the enzymes are described herein above. Enzymes, genes and methods for engineering pathways from succinyl-CoA to various products into a microorganism are now known in the art. The additional reducing equivalents obtained from CO and/or H 2 , as disclosed herein, improve the yields of a primary alcohol, a fatty acyl CoA, a fatty ester, or a wax when utilizing carbohydrate-based feedstock. For example, a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax can be produced from succinyl-CoA via the reverse TCA cycle for production of acytyl-CoA, which is then converted to acyl-CoA by a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and and enoyl-CoA reductase. Exemplary enzymes for the conversion acyl-CoA to a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax include an acyl-CoA reductase, an alcohol dehydrogenase, a wax ester synthase and an alcohol acetyltransferase. Enzymes, genes and methods for engineering pathways from glycolysis intermediates to various products into a microorganism are known in the art. The additional reducing equivalents obtained from CO and H 2 , as described herein, improve the yields of all these products on carbohydrates. EXAMPLE V Methods for Handling CO and Anaerobic Cultures This example describes methods used in handling CO and anaerobic cultures. A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures and assays that utilized CO required special handling. Several assays, including CO oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization in small batch cultures, called for small quantities of the CO gas that were dispensed and handled within a fume hood. Biochemical assays called for saturating very small quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing the assay. All of the CO handling steps were 209 performed in a fume hood with the sash set at the proper height and blower turned on; CO was dispensed from a compressed gas cylinder and the regulator connected to a Schlenk line. The latter ensures that equal concentrations of CO were dispensed to each of several possible cuvettes or vials. The Schlenk line was set up containing an oxygen scrubber on the input side and an oil pressure release bubbler and vent on the other side. Assay cuvettes were both anaerobic and CO containing. Threfore, the assay cuvettes were tightly sealed with a rubber stopper and reagents were added or removed using gas-tight needles and syringes. Secondly, small (~50 mL) cultures were grown with saturating CO in tightly stoppered serum bottles. As with the biochemical assays, the CO-saturated microbial cultures were equilibrated in the fume hood using the Schlenk line setup. Both the biochemical assays and microbial cultures were in portable, sealed containers and in small volumes making for safe handling outside of the fume hood. The compressed CO tank was adjacent to the fume hood. Typically, a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber. The glass or quartz spectrophotometer cuvettes have a circular hole on top into which a Kontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned proximal to the fume hood. B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and H 2 to simulate syngas as a feedstock in fermentative production. Therefore, quantities of cells ranging from 1 liter to several liters can include the addition of CO gas to increase the dissolved concentration of CO in the medium. In these circumstances, fairly large and continuously administered quantities of CO gas are added to the cultures. At different points, the cultures are harvested or samples removed. Alternatively, cells are harvested with an integrated continuous flow centrifuge that is part of the fermenter. The fermentative processes are carried out under anaerobic conditions. In some cases, it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen saturation to provide a respiratory environment. In addition, the reducing power generated during anaerobic fermentation may be needed in product formation rather than respiration. Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying degrees. Classic acetogens 210 such as M. thennoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive to irreversible inactivation by molecular oxygen. While there are oxygen-tolerant acetogens, the repertoire of enzymes in the Wood-Ljungdahl pathway might be incompatible in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins, and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize energy acquisition. At the same time, cells in culture act as oxygen scavengers that moderate the need for extreme measures in the presence of large cell growth. C. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an 02 concentration of 1 ppm or less and 1 atm pure

N

2 . In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 02 electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N 2 prior to introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels. The chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve. The anaerobic chambers achieved levels of 02 that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based 02 monitoring, test strips can be used instead. D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with 211 nitrogen for -30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Belico 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCI is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B 12 (10 gM cyanocobalamin), nickel chloride (NiC1 2 , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 pM-made as 100 1 000x stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pA l-lacOl promoter in the vectors was performed by addition of isopropyl p-D 1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs. Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once 212 all medium components and cells are added, the bottles are incubated in an incubator in room air but with- continuous nitrogen sparging into the bottles. EXAMPLE VI CO oxidation (CODH) Assay This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH). The 7 gene CODH/ACS operon of Moorella thennoacetica was cloned into E. coli expression vectors. The intact ~10 kbp DNA fragment was cloned, and it is likely that some of the genes in this region are expressed from their own endogenous promoters and all contain endogenous ribosomal binding sites. These clones were assayed for CO oxidation, using an assay that quantitatively measures CODH activity. Antisera to the M. thermoacetica gene products was used for Western blots to estimate specific activity. M thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in E. coli. This activity, described below in more detail, was estimated to be -1/50th of the M. thermoacetica specific activity. It is possible that CODH activity of recombinant E. coli cells could be limited by the fact that M. thermoacetica enzymes have temperature optima around 55'C. Therefore, a mesophilic CODH/ACS pathway could be advantageous such as the close relative of Moorella that is mesophilic and does have an apparently intact CODH/ACS operon and a Wood-Ljungdahl pathway, Desulfitobacterium hafniense. Acetogens as potential host organisms include, but are not limited to, Rhodospirillum rubrumn, Moorella thermoacetica and Desulfitobacterium hafniense. CO oxidation is both the most sensitive and most robust of the CODH/ACS assays. It is likely that an E. coli-based syngas using system will ultimately need to be about as anaerobic as Clostridial (i.e., Moorella) systems, especially for maximal activity. Improvement in CODH should be possible but will ultimately be limited by the solubility of CO gas in water. Initially, each of the genes was cloned individually into expression vectors. Combined expression units for multiple subunits/I complex were generated. Expression in E. coli at the 213 protein level was determined. Both combined M thermoacetica CODH/ACS operons and individual expression clones were made. CO oxidation assay, This assay is one of the simpler, reliable, and more versatile assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955 (2004)). A typical activity of M. thermoacetica CODH specific activity is 500 U at 55"C or -60U at 25*C. This assay employs reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes. In more detail, glass rubber stoppered cuvettes were prepared after first washing the cuvette four times in deionized water and one time with acetone. A small amount of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen (CH 3 viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration. When methyl viologen was added, an 18 Ga needle (partial) was used as a jacket to facilitate use of a Hamilton syringe to withdraw the CH 3 viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up the CH 3 viologen stock to slightly reduce the CH 3 viologen. The temperature was equilibrated to 55"C in a heated Olis spectrophotometer (Bogart GA). A blank reaction (CH 3 viologen + buffer) was run first to measure the base rate of CH 3 viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91 (CODH-ACS operon of M thermoacetica with and without, respectively, the first cooC). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH 3 viologen turns purple. The results of an assay are shown in Table I. 214 Table . Crude extract CO Oxidation Activities. ACS90 17.7_mg/mi AC91 11.8 mg/mi Mta98 9.8 mg/mI Mta99 11.2 mg/mI Extract Vol1 OQD U/m... -- Zngm ACS90 10 microliters 0.073 0.376 0.049 ACS91 10 microliters 0.096 0.494 0.042 Mta99 10 microliters 0.0031 0.016 0.0014 ACS90 10 microliters 0.099 0.51 0.066 Mta99 25 microliters 0.012 0.025 0.0022 ACS91 25 microliters 0.215 0.443 0.037 Mta98 25 microliters 0.019 0.039 0.004 ACS91 10 microliters 0.129 066 0.056 A C590 0.057 U/mg ACS91 0.045 U/mg Mta99 00 018 U/rn Mta98/Mta99 are E. coli MG 1655 strains that express methanol methyltransferase genes from M. thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M thermoacetica CODH operons. If~' 1% of the cellular protein is CODH, then these figures would be approximately 100OX less than the 500 U/mg activity of pure M. thermoacetica CODH. Actual estimates based on Western blots are 0.5% of the cellular protein, so the activity is about 50X less than for M thermoacetica CODH. Nevertheless, this experiment demonstrates CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation

(CH

3 viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce CH 3 viologen. To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe-S assays. The antisera used were polyclonal to purified M thermoacetica CODH-ACS and Mtr proteins and were visualized using an alkaline phosphatase 215 linked goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown in Figure 23. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes. Expression of CODH-ACS operon genes including 2 CODH subunits and the methyltransferase were confirmed via Western blot analysis. Therefore, the recombinant E. coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase and corrinoid iron sulfur protein were active in the same recombinant E. coli cells. These proteins are part of the same operon cloned into the same cells. The CO oxidation assays were repeated using extracts of Moorella thermoacetica cells for the positive controls. Though CODH activity in E. coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the M. thermoacetica control. The results of the assay are shown in Figure 24. Briefly, cells (M. thermoacetica or E. coli with the CODHIACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared as described herein. Assays were performed as described above at 55"C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. These results describe the CO oxidation (CODH) assay and results. Recombinant E. coli cells expressed CO oxidation activity as measured by the methyl viologen reduction assay. EXAMPLE VII E. coli CO Tolerance Experiment and CO Concentration Assay (myoglobin assay) This example describes the tolerance of E. coli for high concentrations of CO. To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing solution, NiC 2 , Fe(II)NH 4

SO

4 , cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology in small volumes. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33) was used as a control, and cultures containing the pZA33 empty vector as well as both ACS90 and ACS91 were tested with both N 2 and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37"C. At the end of the 36 hour period, 216 examination of the flasks showed high amounts of growth in all. The bulk of the observed growth occurred overnight with a long lag. Given that all cultures appeared to grow well in the presence of CO, the final CO concentrations were confirmed. This was performed using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20 0 C and 1 atm. For the myoglobin test of CO concentration, cuvettes were washed 1OX with water, IX with acetone, and then stoppered as with the CODH assay. N 2 was blown into the cuvettes for ~10 min. A volume of 1 ml of anaerobic buffer (HEPES, pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton syringe. A volume of 10 microliter myoglobin (~1 mM -can be varied, just need a fairly large amount) and I microliter dithionite (20 mM stock) were added. A CO standard curve was made Vsing CO saturated buffer added at 1 microliter increments. Peak height and shift was recorded for each increment. The cultures tested were pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in I microliter increments to the same cuvette. Midway through the experiment a second cuvette was set up and used. The results are shown in Table II. Table II. Carbon Monoxide Concentrations, 36 hrs. Strain and Growth Conditions Final CO concentration (micromolari. 494 734 ave 687 SD 164 ACS91-CO 728 812 760 611 217 The results shown in Table II indicate that the cultures grew whether or not a strain was cultured in the presence of CO or not. These results indicate that E. coli can tolerate exposure to CO under anaerobic conditions and that E. coli cells expressing the CODH-ACS operon can metabolize some of the CO. These results demonstrate that E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in place of CO. This experiment demonstrated that laboratory strains of E. coli are insensitive to CO at the levels achievable in a syngas project performed at normal atmospheric pressure. In addition, preliminary experiments indicated that the recombinant E. coli cells expressing CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide. EXAMPLE VIII The following example provides various gene candidates for enzymes disclosed herein for convertion of acyl-CoA to fatty alcohols and other related compounds. Acetyl-CoA carboxylase Gene Organism Accession number GI Number Escherichia coli K accA 12 AAC73296.1 1786382 Escherichia coli K accB 12 AAC76287.1 1789653 Escherichia coli K accC 12 AAC76288.1 1789654 Escherichia coli K accD 12 AAC75376.1 1788655 accA Salmonella enterica CAD08690.1 16501513 accB Salmonella enterica CAD07894.1 16504441 accC Salmonella enterica CAD07895.1 16504442 accD Salmonella enterica CAD07598.1 16503590 Saccharomyces YMR207C cerevisiae NP 013934.1 6323863 YNRO16C Saccharomyces NP 014413.1 6324343 218 cerevisiae Saccharomyces YGR037C cerevisiae NP_011551.1 6321474 Saccharomyces YKL182W cerevisiae NP_012739.1 6322666 Saccharomyces YPL231W cerevisiae NP 015093.1 6325025 Fatty acid synthase Multiple genes are required for that step: 1. Acetyl-CoA: ACP transcylase (2.3.1.38)- for converting acetyl-CoA to acetyl-ACP, 2. malonyl-CoA:ACP transacylase that converts malonyl CoA into malonyl-ACP (2.3.1.39), 3. acetyl[acp]:malonyl-[acp] C-acyl transferase (2.3.1.41) and others in fatty acid elongation Accession Gene number GI number Organism fabH AP_001717.1 89107937 Escherichia coli K12 fabB NP 416826.1 16130258 Escherichia coli K12 fabF NP_415613.1 16129058 Escherichia coli K12 fabD NP_415610.1 16129055 Escherichia coli K12 fabi NP_415804.1 16129249 Escherichia coli K12 fabA NP_415474.1 16128921 Escherichia coliK12 fabZ NP_414722.1 16128173 Escherichia coliK12 fabG NP 415611.1 16129056 Escherichia coli K12 Fasil (fatty acid synthase, alpha Saccahromyces subunit) AAA34601.1 171502 cerevisiae Fasl (fatty acid synthase, beta Saccahromyces subunit) AAA34602.1 171506 cerevisiae Mycobacterium fas NP_217040.1 15609661 tuberculosis H37Rv Bifidobacterium fas AAN25329.1 23326820 longum NCC2705 Bifidobacterium fas YP_003971698.1 311064972 bifidum Corynebacterium fas AEG82252.1 334697455 ulcerans Acyl acp thioesterase 219 Gene Accession number GI number Organism fatA AEE76980 332643459 Arabidopsis thaliana fatA ACC41415 183176305 Mycobacterium marinum M fatA AAX54527 61741120 Helianthus annuus fatA CAC14164 10944734 Brassicajuncea Mycobacterium kansasii A TCC fatA ZP_04749108 240170449 12478 fatA ZP_04384386.1 229490548 Rhodococcus erythropolis SK121 Mycobacterium smegmatis str. fatA YP 885312.1 118472377 MC2 155 fatB AAQ08202.1 33325193 Helianthus annuus fatB AEE28300,1 332190179 Arabidopsis thaliana fatB ABI18986.1 112455672 Brassicajuncea tesA NP 415027.1 16128478 Escherichia coli K12 Acyl CoA synthetase and acyl CoA ligase Gene Accession number GI number Organism scs YP_135572.1 55377722 Haloarcula marismortui sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSCI NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP 011760 6321683 Saccharomyces cerevisiae paaF AAC24333.2 22711873 Pseudomonasputida matB AAC83455.1 3982573 Rhizobium leguminosarum bioW NP_390902.2 50812281 Bacillus subtilis bioW CAA10043.1 3850837 Pseudomonas mendocina bioW P22822.1 115012 Bacillus sphaericus phl CAJ15517.1 77019264 Penicillium chrysogenum phiB ABS19624.1 152002983 Penicillium chrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bio W NP_390902.2 50812281 Bacillus subtilis AA CS NP_084486.1 21313520 Mus musculus AA CS NP_076417.2 31982927 Homo sapiens acs AAC77039.1 1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha 220 Methanothermobacter acs] ABC87079.1 86169671 thermautotrophicus acs] AAL23099.1 16422835 Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiae AF1211 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus EXAMPLE IX Exemplary Carboxylic Acid Reductases This example describes the use of carboxylic acid reductases to carry out the conversion of a caroboxylic acid to an aldehyde. In particular, conversion of a fatty acid to a fatty aldehyde (see Figure 25 and Figure 26) can be carried out by these enzymes. 1.2.1.e Acid reductase. The conversion of unactivated acids to aldehydes can be carried out by an acid reductase. Examples of such conversions include, but are not limited, the conversion of 4-hydroxybutyrate, succinate, alpha-ketoglutarate, and 4-aminobutyrate to 4-hydroxybutanal, succinate semialdehyde, 2,5-dioxopentanoate, and 4-aminobutanal, respectively. One notable carboxylic acid reductase can be found in Nocardia iowensis which catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). This enzyme is encoded by the car gene and was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL. (2006)). Gene Accession No. GI No. Organism car AAR91681.1 40796035 Nocardia iowensis (sp. NRRL 221 5646) Nocardia iowensis (sp. NRRL npt AB183656.1 114848891 5646) Additional car and npt genes can be identified based on sequence homology. Gene Accession No. GI No. Organism fadD9 YP 978699.1 121638475 Mycobacterium bovis BCG BCG 2812c YP 978898.1 121638674 Mycobacterium bovis BCG nfa20150 YP 118225.1 54023983 Nocardiafarcinica IFM 10152 nfa40540 YP 120266.1 54026024 Nocardia farcinica IFM 10152 Streptomyces griseus subsp. griseus SGR 6790 YP 001828302.1 182440583 NBRC 13350 Streptomyces griseus subsp. griseus SGR 665 YP 001822177.1 182434458 NBRC 13350 An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4 hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3 acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et aL, J. Antibiot. 60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial. Gene Accession No. GI No. Organism griC 182438036 YP-00182575 Streptomyces griseus subsp. 5.1 griseus NBRC 13350 griD 182438037 -YP00182575 Streptomyces griseus subsp. 6.1 griseusNBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacterium smegmatis _ _ MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis MSMEG_2648 YP886985.1 118471293 Mycacterium smegmatis MAP1040c NP_959974.1 41407138 Mycobacteriun -avium subsp. paratuberculosis K- 10 MAP2899c NP_961833.1 41408997 Mycobacterium avium _____________subsp. paratuberculosis K- 10 MMDAR_2117 YP_001850422. 183982131 Mycobacterium marinum M MM-AR_2936 YP001851230. 183982939 Mycobacterium marinum M 222 IMMAR_1916 YP001850220. 183981929 Mycobacterium marinum M 1 TpauDRAFT_33060 ZP_04027864.1 227980601 Tsarurella paurometabola TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamure a paurometabola CPCC7001 1320 ZP 05045132.1 254431429 Cyanobium PCC7001 DDBDRAFT_01877 XP636931.1 66806417 Dictyostelium discoideum 29 AX4 An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date. Gene Accession No. GI No. Organism LYS2 AAA34747.1 171867 Saccharomyces cerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiae LYS2 AAC02241.1 2853226 Candida albicans LYS5 AA026020.1 28136195 Candida albicans Lysip P40976.3 13124791 Schizosaccharomyces pombe Lys 7p Q10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium chrysogenum Cloning and Expression of Carboxylic Acid Reductase, Escherichia coli is used as a target organism to engineer the pathway for primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax or a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax or a primary alcohol, a fatty acyl-CoA, a fatty ester, or a 223 wax. E. coli is amenable to genetic manipulation and is known to be capable of producing various intermediates and products effectively under various oxygenation conditions. To generate a microbial organism strain such as an E. coli strain engineered to produce primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax or a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax, nucleic acids encoding a carboxylic acid reductase and phosphopantetheine transferase are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, car genes from Nocardia iowensis (designated 720), Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K-10 (designated 891) and Mycobacterium marinum M (designated 892) were cloned into pZS* 13 vectors (Expressys, Ruelzheim, Germany) under control of PAl/lacO promoters. The npt (ABI83656.1) gene (i.e., 721) was cloned into the pKJL33S vector, a derivative of the original mini-F plasmid vector PML31 under control of promoters and ribosomal binding sites similar to those used in pZS* 13. The car gene (GNM 720) was cloned by PCR from Nocardia genomic DNA. Its nucleic acid and protein sequences are shown in Figures 27A and 27B, respectively. A codon-optimized version of the npt gene (GNM_721) was synthesized by GeneArt (Regensburg, Germany). Its nucleic acid and protein sequences are shown in Figures 28A and 28B, respectively. The nucleic acid and protein sequences for the Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K- 10 (designated 891) and Mycobacterium marinum M (designated 892) genes and enzymes can be found in Figures 29, 30, and 31, respectively. The plasmids are transformed into a host cell to express the proteins and enzymes required for primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax or a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax production. Additional CAR variants were generated. A codon optimized version of CAR 891 was generated and designated 891GA. The nucleic acid and amino acid sequences of CAR 891GA are shown in Figures 32A and 32B, respectively. Over 2000 CAR variants were generated. In particular, all 20 amino acid combinations were made at positions V295, M296, G297, G391, G421, D413, G414, Y415, G416, and S417, and additional variants were tested as well. Exemplary CAR variants include: El6K; Q95L; L100M; A1011T; K823E; T941S; H15Q; 224 D198E; G446C; S392N; F699L; V8831; F467S; T987S; R12H; V295G; V295A; V295S; V295T; V295C; V295V; V295L; V2951; V295M; V295P; V295F; V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K; V295R; M296G; M296A; M296S; M296T; M296C; M296V; M296L; M2961; M296M; M296P; M296F; M296Y; M296W; M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G; G297A; G297S; G297T; G297C; G297V; G297L; G2971; G297M; G297P; G297F; G297Y; G297W; G297D; G297E; G297N; G297Q; G297H; G297K; G297R; G391G; G391A; G391S; G391T; G391C; G391V; G391L; G3911; G391M; G391P; G391F; G391Y; G391W; G391D; G391E; G391N; G391Q; G391H; G391K; G391R; G421G; G421A; G421S; G421T; G421C; G421V; G421L; G4211 G421M; G421P; G421F; G421Y; G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R; D413G; D413A; D413S; D413T; D413C; D413V; D413L; D4131; D413M; D413P; D413F; D413Y; D413W; D413D; D413E; D413N; D413Q; D413H; D413K; D413R; G414G; G414A; G414S; G414T; G414C; G414V; G414L; G4141; G414M; G414P; G414F; G414Y; G414W; G414D; G414E; G414N; G414Q; G414H; G414K; G414R; Y415G; Y415A; Y415S; Y415T; Y415C; Y415V; Y415L; Y4151; Y415M; Y415P; Y415F; Y415Y; Y415W; Y415D; Y415E; Y415N; Y415Q; Y415H; Y415K; Y415R; G416G; G416A; G416S; G416T; G416C; G416V; G416L; G4161; G416M; G416P; G416F; G416Y; G416W; G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G; S417A; S417S; S417T; S417C; S417V S417L; S4171; S417M; S417P; S417F; S417Y; S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and S417R. The CAR variants were screened for activity, and numerous CAR variants were found to exhibit CAR activity. This example describes the use of CAR for converting carboxylic acids to aldehydes. Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. 225 Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated feature but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 226

Claims (47)

1. A non-naturally occurring microbial organism, comprising a microbial organism having a primary alcohol pathway comprising at least one exogenous nucleic acid encoding a primary alcohol pathway enzyme expressed in a sufficient amount to produce a primary alcohol; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof; wherein said primary alcohol pathway comprises: (a) a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl CoA reductase; and an acyl-reduction pathway comprising an acyl-CoA reductase and an alcohol dehydrogenase.

2. A non-naturally occurring microbial organism, comprising a microbial organism having a fatty acyl-CoA pathway comprising at least one exogenous nucleic acid encoding a fatty acyl-CoA pathway enzyme expressed in a sufficient amount to produce a fatty acyl-CoA; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected 227 from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof; wherein said fatty acyl-CoA pathway comprises: (a) a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl CoAreductase.

3. A non-naturally occurring microbial organism, comprising a microbial organism having a fatty ester pathway comprising at least one exogenous nucleic acid encoding a fatty ester pathway enzyme expressed in a sufficient amount to produce a fatty ester; said non naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof; 228 wherein said fatty ester pathway comprises a pathway selected from: (a) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl CoA acyltransferase or ketoacyl- CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase.

4. A non-naturally occurring microbial organism, comprising a microbial organism having a wax pathway comprising at least one exogenous nucleic acid encoding a wax pathway enzyme expressed in a sufficient amount to produce a wax; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof; wherein said wax pathway comprises a pathway selected from: (a) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl-CoA 229 acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and (b) a malonyl-CoA-independent fatty acid synthesis (FAS) pathway and an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway comprises ketoacyl CoA acyltransferase or ketoacyl- CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase.

5. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

6. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

7. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol, a fatty acyl-CoA, a fatty ester or a wax pathway enzyme.

8. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises: (A) four exogenous nucleic acids encoding malonyl-CoA-independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoA reductase; and two exogenous nucleic acids encoding acyl-reduction pathway enzymes comprising an acyl-CoA reductase and an alcohol dehydrogenase. 230

9. The non-naturally occurring microbial organism of claim 2, wherein said microbial organism comprises: (A) four exogenous nucleic acids encoding malonyl-CoA-independent FAS pathway enzymes comprising ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase, 3-hydroxyacyl CoA dehydrogenase, enoyl-CoA hydratase and enoyl-CoAreductase.

10. The non-naturally occurring microbial organism of claim 3, wherein said microbial organism comprises: (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one exogenous nucleic acid encoding a wax ester synthase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl CoA hydratase and an enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one exogenous nucleic acid encoding an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl- CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase.

11. The non-naturally occurring microbial organism of claim 4, wherein said microbial organism comprises: (A) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one nucleic acid encoding a wax ester synthase, wherein said malonyt-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and and enoyl-CoA reductase; or (B) four exogenous nucleic acids encoding malonyl-CoA-independent fatty acid synthesis (FAS) pathway enzymes and one nucleic acid encoding an alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA 231 acyltransferase or a ketoacyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase and an enoyl-CoA reductase.

12. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

13. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

14. The non-naturally occurring microbial organism of claims 1-4, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP citrate lyase or a citrate lyase; a fumarate reductase; and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.

15. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

16. The non-naturally occurring microbial organism of any one of claims 1-4, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

17. A method for producing a primary alcohol, comprising culturing the non-naturally occurring microbial organism of any one of claims 1, 5-8 and 12-15 under conditions and for a sufficient period of time to produce a primary alcohol. 232

18. A method for producing a fatty acyl-CoA, comprising culturing the non-naturally occurring microbial organism of any one of claims 2, 5-7, 9 and 12-15 under conditions and for a sufficient period of time to produce a fatty acyl-CoA.

19. A method for producing a fatty ester, comprising culturing the non-naturally occurring microbial organism of any one of claims 3, 5-7, 10 and 12-15 under conditions and for a sufficient period of time to produce a fatty ester.

20. A method for producing a wax, comprising culturing the non-naturally occurring microbial organism of any one of claims 4-7 and 11-15 under conditions and for a sufficient period of time to produce a wax.

21. The method of any one of claims 17-20, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

22. The non-naturally occurring microbial organism of claim 1, wherein said exogenous nucleic acid encoding an acyl-reduction pathway enzyme comprises an enzyme having acyl-CoA reductase and alcohol dehydrogenase activity.

23. The non-naturally occurring microbial organism of claim 22, wherein said enzyme having acyl-CoA reductase and alcohol dehydrogenase activity comprises fatty alcohol forming acyl-CoA reductase (FAR).

24. The non-naturally occurring microbial organism of claim 1, wherein said primary alcohol is produced in amounts at least 10% greater levels compared to a microbial organism lacking said exogenous nucleic acid encoding a malonyl-CoA-independent FAS pathway enzyme.

25. The non-naturally occurring microbial organism of claim 1, wherein said primary alcohol comprises an alcohol having between 4-24 carbon atoms.

26. The non-naturally occurring microbial organism of claim 25, wherein said alcohol having between 4-24 carbon atoms is selected from butanol, hexanol, heptanol, octanol, nonanol, decanol, dodecanol, tetradecanol or hexadecanol. 233

27. A non-naturally occurring microbial organism, comprising a microbial organism having an acyl-ACP pathway comprising at least one exogenous nucleic acid encoding a acyl ACP pathway enzyme expressed in a sufficient amount to produce acyl-ACP; said non-naturally occurring microbial organism further comprising: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof; wherein said acyl-ACP pathway comprises a pathway selected from: (A) acetyl-CoA carboxylase and a fatty acid synthase; and (B) a fatty acid synthase.

28. The non-naturally occurring microbial organism of claim 27, further comprising a fatty acid pathway comprising an exogenous nucleic acid encoding a thioesterase expressed in a sufficient amount to produce fatty acid.

29. The non-naturally occurring microbial organism of claim 28, further comprising a acyl-CoA pathway comprising an exogenous nucleic acid encoding an acyl-CoA synthetase or a acyl-CoA ligase expressed in a sufficient amount to produce acyl-CoA.

30. The non-naturally occurring microbial organism of claim 29, further comprising a fatty aldehyde pathway comprising an exogenous nucleic acid enconding an acyl-CoA reductase 234 (aldehyde forming) or carboxylic acid reductase expressed in a sufficient amount to produce a fatty aldehyde.

31. The non-naturally occurring microbial organism of claim 30, further comprising a fatty alcohol pathway comprising an exogenous nucleic acid encoding an aldehyde reductase expressed in a sufficient amount to produce a fatty alcohol.

32. The non-naturally occurring microbial organism of claim 29, further comprising a fatty alcohol pathway comprising an exogenous nucleic acid encoding an acyl-CoA reductase (alcohol forming) expressed in a sufficient amount to produce a fatty alcohol.

33. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

34. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

35. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a acyl-ACP, fatty acid, acyl-CoA, fatty adehyde or fatty alcohol pathway enzyme.

36. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

37. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme. 235

38. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

39. The non-naturally occurring microbial organism of claims 27-32, wherein said microbial organism comprising (i) comprises three exogenous nucleic acids encoding an ATP citrate lyase or a citrate lyase; a fumarate reductase; and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises four exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase; a phosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase; a CO dehydrogenase; and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.

40. The non-naturally occurring microbial organism of any one of claims 27-32, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

41. A method for producing acyl-ACP, comprising culturing the non-naturally occurring microbial organism of claim 27 under conditions and for a sufficient period of time to produce acyl-ACP.

42. A method for producing fatty acid, comprising culturing the non-naturally occurring microbial organism of claim 28 under conditions and for a sufficient period of time to produce fatty acid.

43. A method for producing acyl-CoA, comprising culturing the non-naturally occurring microbial organism of claim 29 under conditions and for a sufficient period of time to produce acyl-CoA.

44. A method for producing fatty aldehyde, comprising culturing the non-naturally occurring microbial organism of claim 31 under conditions and for a sufficient period of time to produce fatty aldehyde. 236

45. A method for producing fatty alcohol, comprising culturing the non-naturally occurring microbial organism of claims 31 or 32 under conditions and for a sufficient period of time to produce fatty alcohol.

46. The method of any one of claims 41-45, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

47. The non-naturally occurring microbial organism of claim 1, where said microbial organism further comprises an acyl-reduction pathway comprising an acyl-CoA hydrolase, an acyl-CoA transferase or an acyl-CoA ligase; a carboxylic acid reductase and an alcohol dehydrogenase. 237