WO2012177726A1 - Micro-organisme pour produire des alcools primaires et composés associés et procédés associés à ceux-ci - Google Patents

Micro-organisme pour produire des alcools primaires et composés associés et procédés associés à ceux-ci Download PDF

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WO2012177726A1
WO2012177726A1 PCT/US2012/043293 US2012043293W WO2012177726A1 WO 2012177726 A1 WO2012177726 A1 WO 2012177726A1 US 2012043293 W US2012043293 W US 2012043293W WO 2012177726 A1 WO2012177726 A1 WO 2012177726A1
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coa
microbial organism
pathway
acyl
fatty
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Anthony P. Burgard
Robin E. Osterhout
Jun Sun
Priti Pharkya
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Genomatica Inc
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Genomatica Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters

Definitions

  • 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 bio fuels and specialty chemicals. Primary alcohols also can be used to make a large number of additional industrial products including polymers and surfactants. For example, higher primary alcohols (C4-C20) and their ethoxylates are used as surfactants in many consumer detergents, cleaning products and personal care products worldwide such as laundry powders and liquids, dishwashing liquid and hard surface cleaners. They are also used in the manufacture of a variety of industrial chemicals and in lubricating oil additives. Long-chain primary alcohols, such as octanol and hexanol, have useful organoleptic properties and have long been employed as fragrance and flavor materials.
  • C4-C8 higher primary alcohols e.g., butanol
  • Primary alcohols are currently produced from, for example, hydrogenation of fatty acids, hydroformylation of terminal olefins, partial oxidation of n-paraffms and the Al- catalyzed polymerization of ethylene.
  • n-paraffms petroleum-based linear hydrocarbons
  • embodiments disclosed herein relate to a non-naturally occurring microbial 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 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 eno
  • 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 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 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 pathway having ketoacyl-CoA acyltransferase or ketoacyl-CoA thiolase
  • embodiments disclosed herein relate to a non-naturally occurring microbial organism that includes one or more gene disruptions occurring in genes encoding enzymes that couple long-chain alcohol (LCA) production to growth of the non-naturally occurring microbial organism.
  • 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.
  • 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.
  • 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 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.
  • 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 mitochondrion of said organism.
  • embodiments disclosed herein relate to a method for producing long chain alcohols, including culturing these non-naturally occurring eukaryotic organisms.
  • Figure 1 shows the malonyl-CoA-independent fatty acid synthesis and reduction (MI-LCA) 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 (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.
  • 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- 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 V_A (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 V_A 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), XII A (short dashed), and XII B (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 XII B 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 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 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 10a 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 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 10b 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 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 11a shows the formation of dodecanol in the cytosol by relying on the acylating 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 l ib 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- type network
  • the light gray dotted curve shows the production characteristics when acylating acetaldehyde dehydrogenase is added to the network
  • 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.
  • 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.
  • 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 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.
  • 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.
  • Figure 16 shows the formation of dodecanol in the mitochondrion by using the
  • FIG. 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.
  • Figure 18 shows the formation of dodecanol in the mitochondrion by adding 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 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- 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.
  • 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 C0 2 on carbohydrates as substrates.
  • 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 CODH (Moth_1202/1203) or Mtr (Moth l 197) 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
  • 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.
  • FIG 25 panels A and B, show exemplary pathways for fixation of C0 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.
  • Figure 26 panels A and B, show exemplary pathways for fixation of C0 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 27 A 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 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 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 avium subspecies paratuberculosis K-10 (designated 891), and Figure 30B shows the encoded amino acid sequence (SEQ ID NO: 8).
  • Figure 31 A shows the nucleotide sequence (SEQ ID NO: 9) of carboxylic acid reductase from Mycobacterium marinum M (designated 892), and
  • Figure 3 IB shows the encoded amino acid sequence (SEQ ID NO: 10).
  • Figure 32A shows the nucleotide sequence (SEQ ID NO: 11) of carboxylic acid reductase designated 891GA, and Figure 32B shows the encoded amino acid sequence (SEQ ID NO: 12).
  • 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
  • Recombinant microorganisms of the invention can be engineered 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.
  • 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 the use of biosynthetic processes to convert low cost renewable feedstock for the manufacture of chemical products.
  • 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 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 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- 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.
  • non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
  • Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
  • Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
  • Exemplary metabolic polypeptides include enzymes or 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 thereof. Exemplary metabolic modifications are disclosed herein.
  • isolated when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature.
  • the term includes a microbial organism that is removed from some or all components as it is found in its natural environment.
  • the term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
  • Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.
  • microbial As used herein, the terms "microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.
  • 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 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 n H 2n +10H, and include ethanol and methanol.
  • Exemplary primary alcohols include butanol, hexanol, heptanol, octanol, nananol, decanol, dodecanol, tetradecanol, and hexadecanol.
  • 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 transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
  • substantially anaerobic when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
  • the term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.
  • Exogenous as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
  • the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous” refers to a referenced molecule or activity that is present in the host.
  • an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism.
  • heterologous refers to a molecule or activity derived from a source other than the referenced species whereas "homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
  • the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid.
  • a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein.
  • two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism
  • 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.
  • 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.
  • the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.
  • 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 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.
  • metabolic modification is intended to refer to a biochemical reaction that is altered from its naturally occurring state. Metabolic modifications can 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 Table 3.
  • 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 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 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 expressed than under normal culture conditions, or introducing antisense RNA molecules that interact with complementary mRNA molecules of the gene and alter its expression.
  • 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.
  • stable growth-coupled biochemical production will be greater than 10 generations, particularly stable growth-coupled biochemical production will be greater than about 25 generations, and more particularly, stable growth-coupled 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- coupled production of a biochemical can be enhanced through multiple disruptions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.
  • 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.
  • 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 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 mean any eukaryotic organism that exists as a microscopic cell that is included within the domain of eukarya.
  • 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.
  • mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides.
  • Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are
  • Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.
  • Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism.
  • An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species.
  • a specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase.
  • a second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase 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.
  • 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.
  • microsomal epoxide hydrolase epoxide hydrolase I
  • soluble epoxide hydrolase epoxide hydrolase II
  • paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor.
  • Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.
  • a nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species.
  • a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein.
  • Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.
  • Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity.
  • Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined.
  • a computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art.
  • Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related.
  • amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on.
  • Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.
  • 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 generations without loss of the alteration.
  • 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.
  • 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 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.
  • FOS fatty acid synthesis
  • 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 single cell organisms exhibit both algae and protozoan characteristics and, depending on conditions, can utilize either light energy (photosynthesis) or chemical energy (eating) for biochemical processes. Under anaerobic conditions, E.
  • gracilis converts paramylon, the reserve beta-l,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- 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)).
  • 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, 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 malonyl-CoA-independent FAS system involved in anaerobic wax ester fermentation
  • 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.
  • 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.
  • ACP acyl-carrier protein
  • 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 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.
  • ACC acetyl-CoA carboxylase complex
  • the elongation cycle can include (i) ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase, EC 2.3.1.16), (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
  • 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. 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.
  • 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 example, renewable feedstock to acetyl-CoA.
  • acetyl-CoA can be used as both a primer and an extension unit in the elongation cycle described above.
  • 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 invention.
  • acyl-CoA having a desired chain-length is coupled 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.
  • Chain-length of the primary alcohols can be controlled by chain-length specific enoyl-CoA reductase, ketoacyl- CoA thiolase and/or acyl-CoA reductase.
  • microorganisms of the invention having the coupled biosynthetic pathways described above can produce primary alcohols at very high levels.
  • 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- 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. 10/173,547, filed June 14, 2002, and in International Patent Application No.
  • Table 4 Comparison of the maximum theoretical yield of octanol using (1) the malonyl- CoA-independent fatty acid synthesis and acyl-reduction pathway and (2) the ACP-dependent fatty acid synthesis and pathway.
  • a non-naturally occurring microbial organism of the invention employs combinations of metabolic reactions for biosynthetically producing a target primary alcohol or a target 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.
  • 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 product and result in the production of a target primary alcohol or set of alcohols.
  • 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 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 catalyze various enzymatic transformations en route to a primary alcohol, a fatty acyl-CoA, a fatty ester, or a wax.
  • 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.
  • RTCA reductive tricarboxylic acid
  • 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 as CO, C0 2 , and/or H 2 .
  • gaseous carbon sources and/or syngas components such as CO, C0 2 , and/or H 2 .
  • other sources of such gases include, but are not limted 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 C0 2 assimilation which uses reducing equivalents and ATP ( Figure 21).
  • One turn of the RTCA cycle assimilates two moles of C0 2 into one mole of acetyl-CoA, or four moles of C0 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.
  • the reductive TCA cycle coupled with carbon monoxide
  • 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 in 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.
  • 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 full combustion, of organic materials at high temperatures (500-1500°C) to provide syngas as a 0.5: 1-3: 1 H 2 /CO mixture.
  • Carbon dioxide can be provided from the atmosphere or in condensed from, for example, from a tank cylinder, or via sublimation of solid C0 2 .
  • 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 RTCA cycle assimilates two moles of C0 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 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 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.
  • reductive and oxidative (Krebs) TCA cycles are present in the same organism (Hugler et al, supra (2007); Siebers et al, J. Bacteriol. 186:2179-2194 (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
  • TCA cycle reactions are irreversible and can catalyze reactions in the reductive and oxidative directions.
  • TCA cycle reactions are irreversible in 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.
  • citrate is formed from the condensation of oxaloacetate and acetyl-CoA.
  • 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.
  • succinate dehydrogenase The conversion of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse reaction is catalyzed by fumarate reductase.
  • 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) C0 2 and H 2 , 3) CO and C0 2 , 4) synthesis gas 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- ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, acetate kinase, phosphotransacetylase
  • NAD(P)H ferredoxin oxidoreductase, carbon monoxide dehydrogenase, hydrogenase, and 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.
  • the combination of certain carbon gas-utilization pathway components with the pathways for formation of a primary alcohol, a fatty acyl-CoA, 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.
  • 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 (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.
  • 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 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) H 2 , (4) C0 2 and H 2 , (5) CO and C0 2 , (6) CO and H 2 , or (7) CO, C0 2 , and H 2 .
  • 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, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase.
  • such an 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) C0 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.
  • exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin
  • 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 lyase, a fumarate reductase, a pyruvate: ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase.
  • 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 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.
  • the present invention provides a method for enhancing the availability of 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.
  • 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.
  • 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 TCA pathway enzyme.
  • 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.
  • the 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
  • the non-naturally occurring microbial organism includes two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.
  • the non-naturally occurring microbial organisms having a primary 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 thereof.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydr
  • 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.
  • 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) C0 2 , (3) C0 2 and H 2 , (4) CO and H 2 , or (5) CO, C0 2 , and H 2 .
  • 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.
  • 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.
  • 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 nucleic acids encoding an enzyme selected from a pfiosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a pyruvate carboxylase, and a malic enzyme.
  • 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 nucleic acids encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA synthetase, and a succinyl-CoA transferase.
  • 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 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.
  • 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 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,
  • At least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an 3 ⁇ 4 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.
  • 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 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
  • 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.
  • 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.
  • 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.
  • 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 microbial organism is a heterologous nucleic acid. In another specific embodiment, said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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).
  • 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.
  • 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.
  • 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.
  • the invention provides 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 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 exogen
  • fatty acyl-CoA pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA
  • 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 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
  • 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.
  • 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.
  • 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-CoAreductase.
  • 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 microbial organism is a heterologous nucleic acid. In another specific embodiment, said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • the invention provides 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 re
  • 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.
  • 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.
  • 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.
  • 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
  • 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 microbial organism is a heterologous nucleic acid. In another specific embodiment, said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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 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
  • 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
  • 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.
  • 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
  • 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
  • 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.
  • 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 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 alcohol acetyltransferase, wherein said malonyl-CoA-independent FAS pathway enzymes comprise a ketoacyl-CoA acyltransferase
  • 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 microbial organism is a heterologous nucleic acid. In another specific embodiment, said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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 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
  • 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.
  • 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.
  • said non-naturally occurring microbial organism further comprises a 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 sufficient amount to produce a fatty alcohol.
  • 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.
  • 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 enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydr
  • 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.
  • 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.
  • said microbial organism comprising (i) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said said at least one exogenous nucleic acid is a heterologous nucleic acid.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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, 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.
  • a primary alcohol a fatty acyl-CoA, a fatty ester, a wax, acyl-ACP, fatty acid, acyl-CoA, fatty aldehyde or fatty alcohol pathway intermediate.
  • Uptake 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 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
  • the uptake sources can be selected to alter the carbon- 12, carbon- 13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.
  • the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources.
  • An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom.
  • An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction.
  • Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio.
  • 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.
  • 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.
  • a source of carbon for example, can be selected from a fossil fuel- derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as 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 10 12 carbon atoms in the earth's atmosphere and has a half- life of about 5700 years.
  • the stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( 14 N).
  • Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called "Suess effect".
  • Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR).
  • AMS accelerated mass spectrometry
  • SIRMS Stable Isotope Ratio Mass Spectrometry
  • SNIF-NMR Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance
  • 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.
  • ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.
  • the biobased content of a compound is estimated by the ratio of carbon-14 ( 14 C) to carbon- 12 ( 12 C).
  • An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available.
  • the Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933 ⁇ 0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille.
  • ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)).
  • a Fm 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source.
  • a Fm 100%, after correction for the post- 1950 injection of carbon- 14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.
  • the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon- 14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre -bomb" standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by
  • 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.
  • polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30%> (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al, supra, 2000).
  • PPT polypropylene terephthalate
  • 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.
  • 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 least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%o or as much as 100%.
  • the uptake source is C0 2 .
  • 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 petroleum-based carbon uptake source.
  • 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 less than 95%, less than 90%, less than 85%o, less than 80%>, less than 75%, less than 70%>, less than 65%, less than 60%>, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%o, less than 20%>, less than 15%, less than 10%>, less than 5%, less than 2% or less than 1%.
  • 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 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.
  • the present invention relates to the biologically produced 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 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 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 C0 2
  • 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, 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 C0 2 that occurs in the environment, or any of the other ratios disclosed herein.
  • a product can have a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 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 acid, acyl-CoA, fatty aldehyde or fatty alcohol intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product.
  • the invention further provides bio fuels, 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 C0 2 that occurs in the environment, wherein the biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, flavor materials or acrylates are generated directly from or in combination with bioderived 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 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.
  • the invention provides biobased biofuels, chemicals, polymers, surfactants, soaps, detergents, shampoos, lubricating oil additives, fragrances, 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.
  • bioderived means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism.
  • a biological organism in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source.
  • the biological organism can utilize atmospheric carbon.
  • biobased means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention.
  • a biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.
  • 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 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, 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 comprising
  • 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 least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived 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.
  • the invention provides a biobased biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate
  • 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 alcohol or primary alcohol, fatty ester, wax, fatty acid, fatty aldehyde or fatty alcohol intermediate.
  • 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 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 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 intermediate of the invention are well known in the art.
  • 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 protein that converts a substrate to a product selected from the group consisting of ⁇ - ketoacyl-CoA to ⁇ -hydroxyacyl-CoA, ⁇ -hydroxyacyl-CoA to trans-2-enoyl-CoA, trans-2- enoyl-CoA to acyl-CoA, acyl-CoA to ⁇ -ketoacyl-CoA, acyl-CoA to a fatty aldehyde, a fatty aldehyde to a fatty alcohol, acyl-CoA to a fatty acid, a fatty acid pathway
  • the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a 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.
  • the invention additionally provides a non- naturally occurring microbial organism comprising at least one exogenous nucleic acid 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.
  • 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 is exemplified in Figures 1, 9-22, 25 and 26.
  • the invention additionally 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 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 enzyme, where the microbial organism produces a primary alcohol, a fatty acyl-CoA, a fatty ester,
  • 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.
  • a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product.
  • 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.
  • the invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction.
  • reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product.
  • reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.
  • the 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 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.
  • O- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates.
  • Some such O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- or S- carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- 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 fatty acid.
  • Other biosynthetically accessible O-carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations.
  • O-carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an O- or S-carboxylate.
  • S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters.
  • 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.
  • 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.
  • a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression.
  • 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.
  • a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as 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 non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed 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-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.
  • 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 can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
  • exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
  • exogenous expression of all enzymes or proteins in a pathway for production 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 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 .
  • 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, 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.
  • 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. coli.
  • a host organism can be engineered to increase production of a precursor, as disclosed herein.
  • 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. 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.
  • 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.
  • 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 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 aldehyde reductase, or alternatively an alpha-detoglutarate ferredoxin oxidoreductase and a fatty alcohol forming acyl-CoA reductase, and the like.
  • 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.
  • 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 production of the corresponding desired product.
  • 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 desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.
  • a fatty acyl-CoA 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 various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes.
  • 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 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 aldehyde or fatty alcohol.
  • One such procedure includes, for example, the fermentation of a 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.
  • 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 organism that converts 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 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 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 organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
  • 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, acyl-CoA, fatty aldehyde or fatty alcohol.
  • 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.
  • 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 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.
  • 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 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 acid, 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,
  • 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.
  • the non-naturally occurring microbial organisms of the invention 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 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 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 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 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 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.
  • a functionally complete malonyl-CoA independent FAS pathway can be engineererd by introduction of nucleic acids encoding these two enzymes.
  • the non-naturally occurring microbial organisms of the invention will include at least one exogenously 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.
  • 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.
  • the non-naturally occurring microbial organisms also can 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 or more of the malonyl-CoA independent FAS pathway precursors such as acetyl-CoA, ⁇ - ketoacyl-CoA, ⁇ -hydroxyacyl-CoA, tra/?s-2-enoyl-CoA and/or fatty aldehyde.
  • a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize acyl-CoA through 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.
  • 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 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 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.
  • a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the malonyl- CoA independent FAS and/or acyl-reduction biosynthetic pathways.
  • exogenous expression of the encoding nucleic acids is employed.
  • Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
  • 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 of fadB, an E. coli gene having malonyl-CoA independent FAS activity
  • an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
  • an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
  • 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.
  • the ketothiolase, dehydrogenase, and enoyl-CoA hydratase steps are reversible whereas the enoyl-CoA reductase step is primarily oxidative under
  • 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.
  • species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human.
  • Exemplary species for such sources include, for example, Escherichia coli, Acetobacter aceti, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp., Acinetobacter sp.
  • Strain M-l Actinobacillus succinogenes, Aedes aegypti, Aeropyrum pernix ,Alcanivorax borkumensis SK2 ,Alkaliphilus metalliredigens QYMF, Alkaliphilus oremlandii OhILAs, Allochromatium vinosum DSM 180, Anaerobio spirillum succiniciproducens, Anopheles gambiae str.
  • Clostridium saccharoperbutylacetonicum Clostridium tetani E88, Colwellia psychrerythraea 34H, Corynebacterium glutamicum, Corynebacterium ulcerans, Cryptococcus neoformans var, Cyanobium PCC7001, Danio rerio, Desulfatibacillum alkenivorans AK-01,
  • fructosovorans JJ Desulfovibrio vulgaris str. Hildenborough, Dictyostelium discoideum, Dictyostelium discoideum AX4, Escherichia coli K12, Escherichia coli K12 MG1655, Escherichia coli str. K-12 substr.
  • MG1655 Euglena gracilis, Flavobacteria bacterium BAL38, Fragaria x ananassa, Fusarium oxysporum, Fusarium proliferatum, Geobacter metallireducens GS-15, Geobacter sulfurreducens, Haemophilus influenza, Haloarcula marismortui, Helianthus annuus, Helicobacter pylori, Helicobacter pylori 26695, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumonia, Lactococcus lactis,
  • hydrocarbonoclasticus Methanosarcina thermophila, Methanothermobacter
  • thermautotrophicus Methanothermobacter thermautotrophicus, Methylobacterium extorquens, Monosiga brevicollis MX1, Moorella thermoacetica, Mus musculus,
  • Mycobacterium sp. MCS Mycobacterium tuberculosis H37Rv, Myxococcus xanthus DK 1622, Nematostella vectensis, Nocardia farcinica 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. SKA 34, Picea sitchensis, Pseudomonas aeruginosa PA01, Pseudomonas fluorescens,
  • Pseudomonas mendocina Pseudomonas putida, Pseudomonas putida GB-1, Pseudomonas sp, Pseudomonas sp. CF600, Pseudomonas stutzeri A1501, Pyrobaculum aerophilum str. IM2, Ralstonia eutropha, Ralstonia eutropha HI 6, Rattus norvegicus, Reinekea sp.
  • MED297 Rhizobium etli CFN 42, Rhizobium leguminosarum, Rhodobacter capsulatus, Rhodococcus erythropolis SKI 21, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodospirillum rubrum, Rosa hybrid cultivar, Roseovarius sp.
  • HTCC2601 Saccharomyces cerevisiae, Salmonella enteric, Salmonella enterica subsp.
  • arizonae serva Salmonella typhimurium, Schizosaccharomyces pombe, Simmondsia chinensis, Simmondsia chinensis, Sordaria macrospora, Stenotrophomonas maltophilia, Streptomyces griseus subsp. griseus NBRC 13350, Strongylocentrotus 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 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 ATCC 29909, and Zea mays, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes.
  • coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.
  • primary alcohol biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual genes usage between different organisms may differ.
  • teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize the primary alcohol products of the invention.
  • genes fadA and fadB encode a multienzyme complex that exhibits three constituent activities of the malonyl-CoA independent FAS pathway, namely, ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA
  • fadl and fadJ genes encode similar activities which can substitute for the above malonyl-CoA independent FAS conferring genes fadA and fadB. These genes are naturally expressed under anaerobic conditions (Campbell and Cronan, supra, (2002)).
  • the nucleic acid sequences for each of the above fad genes are well known in the art and can be accessed in the public databases such as Genbank using the following accession numbers.
  • fadR NP 415705.1 Escherichia coli
  • 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 of Bacteriology 169:42-52 (1987)), and the two ketothiolases, thiA and thiB, from Clostridium acetobutylicum (Winzer et al., Journal of Molecular Microbiology 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
  • Non-E. coli genes applicable for conferring this activity include AA072312.1 from E. gracilis (Winkler et al, Plant Physiology 131 :1 '53-762 (2003)), paaC from Pseudomonas putida (Olivera et al, PNAS USA 95:6419-6424 (1998)), paaC from Pseudomonas fluorescens (Di Gennaro et al., Archives of Microbiology 188: 117- 125 (2007)), and hbd from C.
  • enoyl-CoA hydratase step Clostridium acetobutylicum
  • 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 of Biochemistry 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol.
  • 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 Hoffmeister et al, supra (2005)).
  • a construct derived from 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.
  • TDE0597 from the prokaryote Treponema denticola represents also can be employed to confer enoyl-CoA reductase activity (Tucci and Martin, FEBS Letters 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 Boynton et al, Journal of Bacteriology 178: 3015-3024 (1996)).
  • 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 chain length preference (Inui et al, European Journal of Biochemistry 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.
  • 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.
  • any or all of the encoding nucleic acids for the malonyl-CoA independent FAS pathway can be readily obtained using methods well 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).
  • TBestDB publically available sequence database
  • 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 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. ketoacyl-CoA acyltransferase (or ketoacyl-CoA thiolase)
  • the resulting acyl-CoA can be reduced to produce a primary alcohol by either a single enzyme or pair of 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 acrl encoding a fatty acyl-CoA reductase (Reiser and Somerville, Journal of Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp.
  • Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol include air A encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol. 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 (Sulzenbacher et al, Journal of Molecular Biology 342:489-502 (2004)). alrA BAB12273.1 Acinetobacter sp. Strain M-l
  • 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
  • the jojoba (Simmondsia chinensis) FAR encodes an 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.
  • 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 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
  • nucleic acids other than those within the malonyl-CoA independent FAS and/or acyl-reduction pathways of the invention also can be introduced into a host organism for further production of primary alcohols.
  • the Ralstonia eutropha BktB and PhbB genes catalyze the condensation of butyryl- CoA and acetyl-CoA to form ⁇ -keto-hexanoyl-CoA and the reduction of ⁇ -keto-hexanoyl- CoA to 3-hydroxy-hexanoyl-CoA (Fukui et al, Biomacromolecules 3:618-624 (2002)).
  • exogenous DNA sequences encoding for these specific enzymes can be expressed in the production host of interest.
  • 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 generate long chain alcohols of a predetermined chain length or lengths.
  • esters of various lengths can be formed between: 1) fatty acyl-CoA and short-chain alcohols such as methanol, ethanol, propanol, etc.; 2) fatty alcohols and 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.
  • 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.
  • Ethanol is an exemplary short chain alcohol that is naturally produced by many
  • 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, 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 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 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.
  • 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. 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. Gene GenBank ID GI Number Organism
  • 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 acetyl-CoA:alcohol O-acetyltransferase (EC 2.3.1.84).
  • Exemplary genes coding for these enzymes include the Acinetobacter sp.
  • ADPl 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 (Lardizabal et al. Plant Physiology 2000, 122: 645-655.); the Alcanivorax borkumensis atfAl and atfA2 encoding bifunctional WS/DGAT enzymes (Kalscheuer et & ⁇ .
  • PON1 has a common polymorphic site at residue 192, glutamine (R) or arginine (Q), that results in qualitative differences.
  • R glutamine
  • Q arginine
  • the R isozyme has a higher esterase activity on GBL than the S isozyme (Billecke et al., Drug Metab Dispos. 28: 1335- 1342 (2000)).
  • PON1 resides on high-density lipoprotein (HDL) particles, and its activity and stability require this environment. Wild type and recombinant PON1 enzymes have been functionally expressed in other organisms (Rochu et al.,
  • 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 Pseudomonas fluorescens, encoded by EstFl, is yet another suitable enzyme
  • esters may also be catalyzed by enzymes in the 3.1.1 family that act on 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 with 4-8 carbon atoms.
  • the gamma lactonase in human blood and rat liver microsomes was purified (Fishbein et al., J Biol Chem 241 :4835-4841 (1966)) and the lactonase activity was activated and stabilized by calcium ions (Fishbein et al., J Biol Chem 241 :4842-4847 (1966)).
  • Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes.
  • Exemplary bacteria include species selected from E. coli, Rhodococcus opacus, Ralstonia eutropha, Klebsiella oxytoca,
  • Anaerobio spirillum succiniciproducens Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum,
  • Gluconobacter oxydans Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens,
  • 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 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).
  • nucleic acids encoding enzymes in the 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.
  • mitochondrial genes will encode an N-terminal targeting signals, which can be removed before transformation into host 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
  • genes can be subjected for codon optimization with techniques well known in the art, to achieve optimal 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.
  • Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunob lotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunob lotting 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.
  • PCR polymerase chain reaction
  • Primary alcohol production can be detected and/or monitored using methods well known to those skilled in the art.
  • 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.
  • 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 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 nucleic acid encoding a malonyl-CoA independent FAS pathway enzyme in sufficient amounts to produce primary alcohol.
  • the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of greater than that which can be synthesized in naturally occurring organisms.
  • the intracellular concentration of, for example, octanol is about 54 ⁇ g/L and decanol is about 148
  • one exemplary growth condition for achieving biosynthesis of primary alcohols includes anaerobic culture or fermentation conditions.
  • the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
  • 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 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 invention further provides a method for the production of primary alcohols.
  • 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 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 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.
  • FOS fatty acid
  • any of the non-naturally occurring microbial organisms described previously can be cultured to produce the biosynthetic products of the invention.
  • the primary alcohol producers can be cultured for the biosynthetic production of its engineered target primary alcohol.
  • the primary alcohol can be isolated or isolated and further utilized in a wide variety of products and procedures.
  • a method for producing a primary alcohol comprising culturing a non-naturally occurring microbial organism described herein as capable of producing a primary alcohol under conditions and for a sufficient period of time to produce a primary alcohol.
  • 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 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 ly
  • At least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; and wherein said primary alcohol pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA
  • the invention provides a 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 oxidoreductase, ferredoxin, and combinations thereof.
  • 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 transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
  • said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a primary alcohol pathway enzyme.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • a method for producing a fatty acyl-CoA comprising culturing a non-naturally occurring microbial organism described herein as capable of producing a fatty acyl-CoA under conditions and for a sufficient period of time to produce a fatty acyl-CoA.
  • 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 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 lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;
  • fatty acyl-CoA pathway comprises a malonyl-CoA-independent FAS pathway comprising ketoacyl-CoA
  • the invention provides a 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 oxidoreductase, ferredoxin, and combinations thereof.
  • 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 synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.
  • said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a fatty acyl-CoA pathway enzyme.
  • 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- CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and enoyl- CoAreductase.
  • 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.
  • said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said at least one exogenous nucleic acid in said non-naturally occurring microbial organism is a heterologous nucleic acid.
  • said non- naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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 ester.
  • 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 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 exogen
  • 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
  • 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.
  • the invention provides a method for producing a fatty ester 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 oxidoreductase, ferredoxin, and combinations thereof.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succiny
  • 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.
  • the invention provides a method for producing a fatty ester using said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a fatty ester enzyme.
  • 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 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.
  • FES malonyl-CoA-independent fatty acid synthesis
  • 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 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 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.
  • a method for producing a wax comprising culturing a non-naturally occurring microbial organism described herein as capable of producing a wax under conditions and for a sufficient period of time to produce a wax.
  • 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 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 fum
  • 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.
  • 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 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.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-Co
  • the invention 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.
  • the 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.
  • 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 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
  • 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.
  • a method for producing acyl-ACP comprising culturing a non-naturally occurring microbial organism described herein as capable of producing acyl-ACP under conditions and for a sufficient period of time to produce acyl- ACP.
  • 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 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
  • acyl-ACP pathway comprises a pathway selected from: (A) acetyl- CoA carboxylase and a fatty acid synthase; and (B) a fatty acid synthase.
  • the invention provides a method for producing acyl-ACP 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 oxidoreductase, ferredoxin, and combinations thereof.
  • 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.
  • the invention provides a method for producing acyl-ACP using said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a acyl-ACP pathway enzyme.
  • 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 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.
  • 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.
  • the invention provides a method for producing a 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.
  • the invention provides a method for producing a fatty acid 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 oxidoreductase, ferredoxin, and combinations thereof.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl
  • the invention provides a method for producing a fatty acid 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
  • the invention provides a method for producing a fatty acid using 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.
  • 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.
  • the invention provides a method for producing a fatty acid using said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said said at least one exogenous nucleic acid is a heterologous nucleic acid.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • 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.
  • the invention provides a method for producing a 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.
  • 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.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl- CoA synthetase, a succin
  • the 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.
  • the invention provides a method for producing acyl-CoA using said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding acyl-CoA pathway enzyme.
  • 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.
  • said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said said at least one exogenous nucleic acid is a heterologous nucleic acid.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • a method for producing a fatty aldehyde 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.
  • 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 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.
  • the invention provides a method for producing a fatty aldehyde 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 oxidoreductase, ferredoxin, and combinations thereof.
  • an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a
  • 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.
  • the invention provides a method 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.
  • 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 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.
  • 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.
  • the invention provides a method for producing a fatty alcohol using said non-naturally occurring microbial organism having a 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.
  • 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 comprising an exogenous nucleic acid encoding an acyl-CoA reductase (alcohol forming) expressed in a sufficient amount to produce a fatty alcohol.
  • 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 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 enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a
  • 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
  • the invention provides a method for 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.
  • 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.
  • said microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.
  • said at least one exogenous nucleic acid is a heterologous nucleic acid.
  • said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
  • the 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 separated from other components in the culture using a variety of methods well known in the art.
  • Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.
  • culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions.
  • Exemplary anaerobic conditions have been described previously and are well known in the art.
  • Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed August 10, 2007.
  • Exemplary anaerobic conditions for fermentation processes are described 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.
  • growth condition 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.
  • the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant.
  • an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress.
  • Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine.
  • the osmoprotectant is glycine betaine.
  • the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used.
  • the amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM 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 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. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of primary alcohols.
  • the continuous and/or near-continuous production of primary alcohols will 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, 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 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.
  • the primary alcohol producing microbial organisms of the invention utilize renewable feedstocks and carbon-containing gas as carbon sources for growth.
  • Renewable feedstocks useful for growth of the primary alcohol producing 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.
  • 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 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.
  • 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, agricultural crop wastes and residues, wood wastes and residues, aquatic plants, and other waste materials including some municipal wastes (see, for example, the URL
  • biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass,
  • biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • the primary alcohol producing microbial organisms of the invention also can be modified for growth on syngas as its source of carbon.
  • one or more proteins or enzymes are 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
  • syngas is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues.
  • Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include C0 2 and other gases in smaller quantities.
  • synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 2 .
  • 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 C0 2 and C0 2 /H 2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.
  • H 2 -dependent conversion of C0 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved.
  • non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 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 branch converts methyl-THF to acetyl-CoA.
  • the reactions in the methyl branch are catalyzed in order by the following enzymes: ferredoxin oxidoreductase, formate
  • cyclodehydratase methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase.
  • the reactions in the carbonyl branch are catalyzed in order by the following enzymes: cobalamide corrinoid/iron- sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase.
  • the invention is also directed, in part, to the design and creation of cells and organisms having growth-coupled production of LCA.
  • 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 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 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 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.
  • 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 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 subset of the gene products marked for disruption.
  • disruptions can be introduced to 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 determined metabolic pathways is confirmed by construction of strains having the designed metabolic genotype.
  • These metabolically engineered cells or organisms can also be subjected to adaptive evolution to further augment growth-coupled product production.
  • the engineered cells or organisms can also incorporate additional copies of beneficial genes to increase flux through a particular metabolic pathway.
  • exogenous gene insertions from another organism can be used to install functionality that is not present in the host organism.
  • 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 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 in an elongation cycle that involves the ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase.
  • 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 acyl-CoA reductase to form the desired primary alcohol.
  • 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.
  • 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:
  • 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 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 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.
  • the invention provides a non-naturally occurring microbial organism, 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.
  • the invention provides a non-naturally occurring eukaryotic 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.
  • enzymes include, for example a cytosolic pyruvate decarboxylase, a mitochondrial pyruvate dehydrogenase, a cytosolic ethanol-specific alcohol dehydrogenase or a mitochondrial ethanol-specific alcohol dehydrogenase.
  • the present invention provides methods of producing such non-naturally microbial organisms having stable growth-coupled production of LCA.
  • the method includes: (a) identifying in silico a set of metabolic modifications requiring LCA production during cell growth, and (b) genetically modifying a microorganism to contain the set of metabolic modifications requiring LCA production.
  • 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.
  • continuous processes are kept in a state of constant exponential growth and, if properly operated, can run for many months at a time.
  • productivities i.e., dilution rate times cell mass
  • OptKnock is a metabolic modeling and simulation program that suggests gene disruption strategies that result in genetically stable microorganisms which overproduce the target product.
  • 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.
  • the OptKnock framework has already been able to identify promising gene disruption strategies for biochemical overproduction, (Burgard, A.P., P. Pharkya, and CD. Maranas, Biotechnol Bioeng, 84(6):647-657 (2003); Pharkya, P., A.P. Burgard, and CD. 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. 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.
  • 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.
  • FBA flux balance analysis
  • OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or 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
  • SimPheny® 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 Application Serial No. 10/173,547, filed June 14, 2002, and in International Patent
  • 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. Analysis methods such as convex analysis, linear
  • 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, 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 VanroUeghem 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 as well as the metabolism of H. influenzae as described in Edwards and Palsson, Proc. Natl. 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).
  • Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny ® and OptKnock.
  • 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.
  • 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 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 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 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 governing physico-chemical constraints, Price et al, Nat Rev Microbiol, 2: 886-97 (2004).
  • constraint-based models and simulations are well known in the art and generally invoke the optimization of a particular cellular objective, subject to network stoichiometry, to suggest a likely flux distribution.
  • V substrate V substrate _uptake Mmol/gDW-hr
  • Vj 0, V j e ⁇ irrev. reactions ⁇
  • Sy is the stoichiometric coefficient of metabolite i in reaction j
  • Vj is the flux of reaction j
  • v substrate t ptake represents the assumed or measured uptake rate(s) of the limiting substrate(s)
  • v a tp_main is the non-growth associated ATP maintenance requirement.
  • the vector v includes both internal and external fluxes.
  • 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 gDW-hr (gram of dry weight times hour) such that biomass formation is expressed as g biomass produced/ gDW -hr or 1/hr.
  • reaction flux Vj is not active ' ⁇ e assume a value of 1 if reaction j is active and a value of 0 if it is inactive.
  • the following constraint, >'/ ⁇ .> ' ; ⁇ » ' ; ⁇ »'; " ⁇ . / ⁇ M ensures that reaction flux v, is set to zero only if variable 3 ⁇ 4 is equal to zero.
  • V j is free to assume any value between a lower vTM 1 " and an upper V j max bound.
  • vf m and vf" are identified by minimizing and maximizing, respectively, every reaction flux subject to the network constraints described above, Mahadevan et al., Metab Eng, 5: 264-76 (2003).
  • this bilevel optimization problem is illustrated in Figure 2.
  • this bilevel optimization problem is expressed as the following bilevel mixed-integer optimization problem: maximize v chemical (OptKnock)
  • 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.
  • 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.
  • one particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene.
  • These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the product coupling are desired or when genetic reversion is less likely to occur.
  • integer cuts an optimization method, termed integer cuts. 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 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
  • 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 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 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.
  • the modified integer cut procedure begins with iteration 'zero' which calculates the 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.
  • the two parameter sets, objstore iter and ystore iter are introduced to store the objective function iy chemical) and reaction on-off information (y ), respectively, at each iteration, iter.
  • the following constraints are then successively added to the OptKnock formulation at each iteration.
  • ⁇ and M are a small and a large numbers, respectively.
  • ⁇ can be set at about 0.01 and M ean 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 ⁇ ensures that adding disruptions to a previously identified strategy must lead to an increase of at least ⁇ 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 growth, of distinct disruption strategies that differ from each other by at least one disruption.
  • 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 alterations.
  • 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 pressures described previously.
  • 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 that are identified by an in silico method selected from OptKnock.
  • modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.
  • metabolic modifications can be selected from the set of metabolic modifications listed in Table 1.
  • the method includes: (a) identifying in silico a 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 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.
  • eukaryotic organisms include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,
  • a microbial organism produced by the methods of the invention is further provided.
  • the invention provides a non-naturally occurring microbial organism
  • the non-naturally occurring microbial organism of the invention includes one or 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 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.
  • 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 . 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.
  • the complete genome sequence available for now more than 550 species 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 organisms is routine and well known in the art.
  • 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.
  • a metabolic alteration exemplified in one organism can be applied equally to other organisms.
  • homologues can include othologs and/or nonorthologous gene displacements.
  • 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.
  • the invention provides non naturally occurring microbial organisms having growth-coupled production of LCA.
  • Product production is obligatorily linked to the exponential growth 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 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.
  • 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 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.
  • 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 reaction of the invention. Such other functions are well known to those skilled in the art.
  • 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.
  • 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 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.
  • 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 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, sucrose, xylose, arabinose, or glycerol. The complete set of growth-coupled LCA
  • D-lactate dehydrogenase LDH D Phosphotransacetylase and/or Acetate kinase PTAr and/or ACKr
  • D-lactate dehydrogenase LDH D Phosphotransacetylase and/or Acetate kinase PTAr and/or ACKr
  • D-lactate dehydrogenase LDH D Phosphotransacetylase and/or Acetate kinase PTAr and/or ACKr
  • Design I 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 (LDH D) 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.
  • PTAr phosphotransacetylase
  • ACKr acetate kinase
  • 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 LDH D 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.
  • Design V_A involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDH D), malate dehydrogenase (MDH), pyruvate formate lyase (PFLi), L-aspartase (ASPT), pyruvate kinase (PYK), glucose 6-phosphate dehydrogenase (G6PDHy), and dihydroxyacetone phosphotransferase (DHAPT).
  • ADHEr Acetaldehyde-CoA dehydrogenase
  • LDH D lactate dehydrogenase
  • MDH malate dehydrogenase
  • PFLi pyruvate formate lyase
  • APT L-aspartase
  • PYK pyruvate kinase
  • G6PDHy glucose 6-phosphate dehydrogenase
  • DHAPT dihydroxyacetone phosphotransferase
  • 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.
  • Design V coli for growth coupled LCA production by first removing genes encoding ADHEr and LDH D activities resulting in Design I.
  • Design V is then constructed by further deleting genes responsible for MDH and PFLi activities.
  • Design V_A is then constructed by deleting genes encoding ASPT, PYK, G6PDHy, and DHAPT activities.
  • several activities i.e., 6-phosphogluconolactonase (PGL), phosphogluconate dehydratase (PGDHY), or 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA)
  • PDL 6-phosphogluconolactonase
  • PGDHY phosphogluconate dehydratase
  • EDA 2-dehydro-3-deoxy-phosphogluconate aldolase
  • Design XII A involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDH D), acetate kinase (ACKr) and/or phosphotransacetylase (PTAr), glutamate dehydrogenase (NADP) (GLUDy), phosphogluconate dehydrogenase (PGDH), and glucose-6-phosphate isomerase (PGI).
  • ADHEr Acetaldehyde-CoA dehydrogenase
  • LDH D lactate dehydrogenase
  • ACKr acetate kinase
  • PTAr phosphotransacetylase
  • NADP glutamate dehydrogenase
  • PGDH phosphogluconate dehydrogenase
  • PGI glucose-6-phosphate isomerase
  • Design XII B involves disruption of Acetaldehyde-CoA dehydrogenase (ADHEr), lactate dehydrogenase (LDH D), 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).
  • ADHEr Acetaldehyde-CoA dehydrogenase
  • LDH D lactate dehydrogenase
  • ACKr acetate kinase
  • PTAr phosphotransacetylase
  • GACpts D-glucose transport via PEP:Pyr PTS
  • an engineered strain lacking the activities specified by Design XII B 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 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.
  • Design 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 XII A is then constructed by deleting the genes responsible for GLUDy, PGDH, and PGI activities.
  • Design XII B is constructed by further deleting a gene essential for GLCpts activity.
  • 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
  • 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),
  • PTAr phosphotransacetylase
  • ACKr acetate kinase
  • FUM fumarate reductase
  • MDH malate dehydrogenase
  • 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.
  • FFD2 fumarate reductase
  • FUM fumarase
  • MDH malate dehydrogenase
  • 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.
  • GLUDy glutamate dehydrogenase
  • 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.
  • NAD(P) transhydrogenase TDD2
  • 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), fumarase (FUM), or malate dehydrogenase (MDH) as exemplified by Designs XIII, XIV, and XV.
  • FGD2 fumarate reductase
  • FUM fumarase
  • MDH malate dehydrogenase
  • 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.
  • ATPS4r ATP synthase
  • the OptKnock algorithm 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides non-naturally occurring eukaryotic microbial organisms that produce LCAs in the cytosol.
  • cytosol herein refers to any compartment outside the mitochondrion.
  • 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.
  • genes endocing these enzymes include, for example, YLR044C, YLR134W, YGR087C, PDC3, YNL071W, YER178W, YBR221C, YGR193C, YFL018C, YBR145W, YGL256W, YOL086C, YMR303, YMR083W, YPL088W, YAL061W, YMR318C, YCR105W, and YDL168W.
  • 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, YDL085W, 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
  • 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
  • cytosolic transhydrogenase a cytosolic transhydrogenase or its gene regulatory region
  • 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.
  • gene regulatory regions include, for example, alternate promoters, inducible promoters, variant promoters or enhancers to enhance 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.
  • 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.
  • 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.
  • MI-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.
  • 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 ACS1 and ACS2 genes.
  • 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.
  • the ACS2 gene is replaced with a mutant ACS 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 ACS1 or ACS2 (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 -> acetyl-CoA + PPi + AMP). Under anaerobic conditions, when energy is available only through substrate-level
  • 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.
  • 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.
  • NADH dehydrogenase 25: 15-37 (2001)
  • 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.
  • the non-naturally occurring eukaryotic organism incorporates an exogenous gene encoding an ADP-forming acetate CoA ligase.
  • 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 Pyrococcus furiosus (Glasemacher et al, Eur. J. Biochem. 244:561-567 (1997)) (Mai and Adams, J.
  • 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 10b 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.
  • 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 11a).
  • 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.
  • 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.
  • 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.
  • each of the strains above can be supplemented with additional disruptions.
  • 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 can be accomplished by disrupting ethanol-specific alcohol dehydrogenase activity.
  • the production envelope is shown in Figure l ib.
  • the wild-type S. cerevisiae (black) network can form only small amounts of dodecanol as an byproduct of growth under anaerobic conditions.
  • 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.
  • (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.
  • the non-naturally occurring eukaryotic organism is engineered to retarget the native mitochondrial pyruvate dehydrogenase to the cytosol.
  • a heterologous cytosolic enzyme is introduced into the organism. The retargeting of an enzyme to 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 l ib. Note that pyruvate decarboxylase is disrupted instead of alcohol dehydrogenase to achieve growth-coupling in the network.
  • 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 et al, Journal of Biochemistry 96:931-934 (1984); Inui et al, Archives of Biochemistry 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.
  • 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)).
  • a non-naturally occurring eukaryotic organism uses a pyruvate formate lyase.
  • a heterologous cytosolic pyruvate formate 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 conversion of pyruvate into acetyl CoA makes the production of dodecanol feasible under anaerobic conditions.
  • 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.
  • 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, YLR134W, YGR087C, PDC3, YBR145W, YGL256W, YOL086C, YMR303, YMR083W, YPL088W, YAL061W, YMR318C, YCR105W, and YDL168W.
  • 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, YOL059W, YIL155C, YMR145C, YDL085W, and
  • 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.
  • an enzyme such as a pyruvate dehydrogenase, a pyruvate: NADP oxidoreductase, a pyruvate formate lyase, an acylating acetaldehyde dehydrogenase, an acetate CoA liga
  • 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.
  • 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)).
  • the enzyme is overexpressed in its native or a heterologous form.
  • the native enzyme can be overexpressed by using a stronger promoter.
  • 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.
  • 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 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.
  • a transhydrogenase 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.
  • 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.
  • 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.
  • a non-naturally occurring eukaryotic organism uses a heterologous acetaldehyde dehydrogenase (acylating).
  • 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., 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.
  • a non-naturally occurring eukaryotic organism uses a mitochondrial acetyl CoA synthetase (AMP-forming).
  • AMP-forming mitochondrial acetyl CoA synthetase
  • the expression of this enzyme requires oxygen for favorable energetics.
  • ACS1 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.
  • pyruvate decarboxylase also can be expressed in the mitochondrion to form acetate.
  • 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.
  • LCA cytosolic malate dehydrogenase
  • glycerol-3-phospate dehydrogenase shuttle the external NADH dehydrogenase
  • the internal mitochondrial NADH the internal mitochondrial NADH
  • 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.
  • 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.
  • 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.
  • an acetate-CoA ligase to convert acetate into acetyl-CoA as shown in Figure 20.
  • 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.
  • 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.
  • 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. Appl. Microbiol. Biotechnol. Sep 6. (2008) [Currently available in online edition], describe a similar non-growth couple production process in a strain of Cory neb acterium 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. Appl.
  • 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, Aspergillus 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 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).
  • 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.
  • the gene disruptions can enhance LCA production in a non- growth dependent manner.
  • 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.
  • 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.
  • 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 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.
  • any quantity, including commercial quantities, can be synthesized using the growth-coupled LCA producers of the invention.
  • 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 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 EvolugatorTM 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.
  • apparatuses such as the EvolugatorTM 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.
  • 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.
  • 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.
  • the gene disruption can include a complete gene deletion.
  • other means to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable.
  • 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.
  • FOS malonyl-CoA-independent fatty acid synthesis
  • 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.
  • the strains 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • 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.
  • OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism.
  • the OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data.
  • OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or 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.
  • OptKnock 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.
  • SimPheny® 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an optimization method termed integer cuts. 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®.
  • 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.
  • 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 resulting network overproduces the biochemical of interest (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)).
  • OptKnock mathematical framework can be applied to pinpoint gene disruptions leading to the growth-coupled production of a desired product.
  • the solution of the bilevel OptKnock problem provides only one set of disruptions.
  • an optimization technique termed integer cuts. 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. O. 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.
  • 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 (i.e., 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 production strains. (Fong, S. S., A. P. Burgard, C.
  • 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 U S.A. 91 :6808-6814 (1994).) in experiments which clearly demonstrated consistent improvement in reproductive rate over period of years.
  • 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.
  • serial dilution is inexpensive and easy to parallelize.
  • EvolugatorTM 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.
  • the cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained.
  • 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.
  • 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.
  • 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%.
  • 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.
  • known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule.
  • 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.
  • Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >104). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol.Eng 22: 11-19 (2005); Huisman and Lalonde, In
  • 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.
  • epRCA Error-prone Rolling Circle Amplification
  • DNA or Family Shuffling 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
  • Nucleases such as Dnase I or EndoV
  • StEP Staggered Extension
  • RPR Random Priming Recombination
  • 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:el8 (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.
  • Random Drift Mutagenesis in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al, Biomol. Eng.
  • 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));
  • CCM Combinatorial Multiple Cassette Mutagenesis
  • LTM Look-Through Mutagenesis
  • 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
  • TGRTM GeneReassemblyTM
  • PDA Silico Protein Design Automation
  • ISM Iterative Saturation Mutagenesis
  • 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.
  • 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.
  • 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).
  • the fadl/fadJ genes (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 971211.1), encoding enoyl-CoA reductase, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter.
  • the acrigene (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 PAl/lacO promoter.
  • the two sets of plasmids are transformed into E. coli strain MG1655 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).
  • 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.
  • 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,
  • 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.
  • 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 H2S04. The growth rate is determined by measuring optical density using a
  • spectrophotometer 600 nm
  • glucose uptake rate by monitoring carbon source depletion over time.
  • Byproducts such as undesirable alcohols, organic acids, and residual glucose can be 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.
  • 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.
  • 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 ⁇ Red recombinase system of Datsenko and Wanner. (Datsenko, K.A. and B.L. Wanner, Proc Natl Acad Sci 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.
  • one exemplary growth condition for achieving biosynthesis of LCA includes anaerobic culture or fermentation conditions.
  • the non-naturally occurring microbial organism of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions.
  • 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
  • Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen.
  • the percent of oxygen can be maintained by, for example, sparging the culture with an N 2 /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.
  • 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, J Bacteriol, 185(21):6400-8 (2003); Ibarra, R.U., IS. 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.
  • 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.
  • EvolugatorTM 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.
  • 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.
  • 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.
  • 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%.
  • 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. EvolugatorTM is a continuous culture device that exhibits significant time and effort savings over traditional evolution techniques, (de Crecy et al, Appl. Microbiol. Biotechnol. 77:489-496 (2007)).
  • 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
  • the growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproduces.
  • 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.
  • 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).
  • URA3 resistance marker
  • 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 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 EcoRI/XhoI enzymes (Vellanki et al., Biotechnol. Lett. 29:313-318 (2007)).
  • the gene is inserted at the EcoRI and Xhol 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 Narl 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.
  • 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
  • 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 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.
  • EvolugatorTM 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.
  • 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.
  • 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.
  • 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
  • the growth-coupled biochemical production concept behind the OptKnock approach should also result in the generation of genetically stable overproduces.
  • 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.
  • 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 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.
  • Table 1 for LCA production.
  • Table 2 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.
  • ADHEr LDH " D ASPT ATPS4r GLUDy MDH TPI 109 ADHEr LDH D ACKr GLUDy PGDH PGI

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Abstract

L'invention concerne un organisme microbien d'origine non naturelle ayant un organisme microbien ayant au moins une insertion de gène exogène et/ou une ou plusieurs ruptures de gène qui confèrent la production d'alcools primaires et au moins un activité exogène qui code pour une enzyme qui augmente les rendements de l'alcool primaire par (i) augmentation de la fixation de carbone via le cycle TCA réducteur, et/ou (ii) accès à des équivalents réducteurs additionnels à partir de sources de carbone gazeuses et/ou des composants de gaz de synthèse tels que CO, CO2, et/ou H2. Un procédé pour produire des alcools à chaîne longue comprend la culture de ces organismes microbiens d'origine non naturelle.
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