WO2012099934A2 - Production d'alcool butylique par microorganismes ayant un couplage nadh - Google Patents

Production d'alcool butylique par microorganismes ayant un couplage nadh Download PDF

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WO2012099934A2
WO2012099934A2 PCT/US2012/021679 US2012021679W WO2012099934A2 WO 2012099934 A2 WO2012099934 A2 WO 2012099934A2 US 2012021679 W US2012021679 W US 2012021679W WO 2012099934 A2 WO2012099934 A2 WO 2012099934A2
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coa
recombinant microorganism
microorganism
gene
acetyl
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WO2012099934A3 (fr
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James C. Liao
Roa Pu Claire Shen
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The Regents Of The University Of California
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01038—Trans-2-enoyl-CoA reductase (NADPH) (1.3.1.38)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Metabolically-modified microorganisms and methods of producing such organisms are provided. Also provided are methods of producing biofuels by contacting a suitable substrate with a metabolically-modified microorganism and enzymatic preparations there from.
  • n-Butanol is generally preferred because of its hydrophobicity, lower vapor pressure, and higher energy content.
  • microorganisms that include recombinant biochemical pathways useful for producing n-butanol via fermentation of a suitable substrate. Also provided are methods of producing biofuels using
  • the disclosure provides a recombinant microorganism for producing 1-butanol comprising a recombinant pathway including enzymes selected from the group consisting of keto-thiolase, acetyl—CoA acetyltransferase, hydroxybutyryl-CoA dehydrogenase, crotonase, crotonyl-CoA reductase, butyryl-CoA dehydrogenase, and an alcohol dehydrogenase, wherein NADH use is tightly coupled to the butanol production pathway.
  • the NADH utilization can be tightly coupled through selection in various organisms or through mutagenesis and screening.
  • the NADH utilization is controlled by knocking out competing pathways for NADH utilization or increase production of NADH.
  • the competing pathway utilizes a phosphate acetyltransferase (e.g., a pta) .
  • a phosphate acetyltransferase e.g., a pta
  • the microorganism comprises one or more knockouts selected from the group consisting of frdBc, ldhA, adhE and pta.
  • the microorganism comprises a heterologous trans-2-enoyl-CoA reductase (ter) .
  • the ter is derived from a Treponema denticola or F. succinogenes and comprises an M11K substitution.
  • the disclosure provides a recombinant microorganism provided herein comprising elevated expression of a keto-thiolase as compared to a parental microorganism.
  • the recombinant microorganism provided herein comprising elevated expression of a keto-thiolase as compared to a parental microorganism.
  • microorganism produces a metabolite that includes acetoacetyl-CoA from a substrate that includes acetyl-CoA.
  • the keto-thiolase can be encoded by an atoB gene or homolog thereof, or a fadA gene or homolog thereof.
  • the atoB gene or fadA gene can be derived from the genus Escherichia.
  • the microorganism is also engineered to produce an intracellular pool of NADH using a heterologous or over' expressed endogenous formate dehydrogenase (fdh) gene, while simultaneously reducing NADH consumption in pathways that compete for the NADH pool for production of 1-butanol.
  • fdh heterologous or over' expressed endogenous formate dehydrogenase
  • the microorganism comprises a heterologous trans-2-enoyl-CoA reductase (ter) .
  • the ter is derived from a Treponema denticola or F. succinogenes and comprises an M11K substitution .
  • a recombinant microorganism provided herein includes elevated expression of hydroxybutyryl-CoA dehydrogenase as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes a 3- hydroxybutyryl-CoA from a substrate that includes acetoacetyl-CoA .
  • the hydroxybutyryl CoA dehydrogenase can be encoded by an hbd gene or homolog thereof.
  • the hbd gene can be derived from various microorganisms including Clostridium acetobutylicum, Clostridium difficile, Dastricha ruminatium, Butyrivibrio fibrisolvens ,
  • a recombinant microorganism provided herein includes elevated expression of crotonase as compared to a parental microorganism.
  • the recombinant microorganism includes elevated expression of crotonase as compared to a parental microorganism.
  • microorganism produces a metabolite that includes crotonyl-CoA from a substrate that includes 3-hydroxybutyryl-CoA .
  • the crotonase can be encoded by a crt gene or homolog thereof.
  • the crt gene can be derived from various microorganisms including Clostridium
  • a recombinant microorganism provided herein includes elevated expression of a crotonyl-CoA reductase as compared to a parental microorganism.
  • microorganism produces a metabolite that includes butyryl-CoA from a substrate that includes crotonyl-CoA .
  • the crotonyl-CoA reductase can be encoded by a ccr gene or homolog thereof.
  • the ccr gene can be derived from the genus Streptomyces .
  • a recombinant microorganism provided herein includes elevated expression of a butyryl-CoA dehydrogenase (e.g., Bed) as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes butyryl-CoA from a substrate that includes crotonyl-CoA .
  • the butyryl-CoA dehydrogenase can comprise an electron transfer flavoprotein (etfAB) (e.g., Bcd-EtfAB) .
  • etfAB electron transfer flavoprotein
  • dehydrogenase and electron transfer flavoprotein can be encoded by a bed gene and an etfAB or homologs thereof.
  • the bed gene and etfAB can be derived from Clostridium acetobutylicum, Mycobacterium tuberculosis, or Megasphaera elsdenii.
  • the butyryl-CoA reductase and flavoprotein are encoded by one or more genes selected from the group consisting of bed, etfA, etfB and may be tightly coupled to Ter, and TDE0597.
  • a recombinant microorganism provided herein includes elevated expression of an alcohol dehydrogenase (ADHE2) as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes butanol from a substrate that includes butyryl-CoA.
  • the alcohol dehydrogenase can be encoded by an aad gene or homolog thereof, or an adhE2 gene or homolog thereof.
  • These enzymes are members of a class of enzymes that possess alcohol/aldehyde dehydrogenase activity.
  • the E. coli adhE enzyme converts acetyl-CoA to ethanol .
  • the aad gene or adhE2 gene can be derived from
  • a recombinant microorganism including a recombinant biochemical pathway to produce n-butanol from fermentation of a suitable carbon substrate.
  • the recombinant biochemical pathway includes elevated expression of: a) a keto-thiolase as compared to a parental microorganism or an acetyl-CoA acetyltransferase as compared to a parental
  • microorganism b) a hydroxybutyryl-CoA dehydrogenase as compared to a parental microorganism; c) a crotonase as compared to a parental microorganism; d) a crotonyl-CoA reductase as compared to a parental microorganism or a butyryl-CoA dehydrogenase eletron transfer flavoprotein as compared to a parental microorganism; e) an alcohol dehydrogenase (ADH) as compared to a parental
  • microorganism and f) a formate dehydrogenease (FDH) compared to a parental organism.
  • FDH formate dehydrogenease
  • one or more genes selected from the group consisting of frdB, frdC, frdBC operon, ldhA, adhE and pta are knockedout or their expression or activity reduced .
  • a method of producing a recombinant microorganism that converts a suitable carbon substrate to n-butanol includes transforming a microorganism with one or more recombinant polynucleotides encoding polypeptides that include keto thiolase or acetyl-CoA
  • acetyltransferase activity hydroxybutyryl-CoA dehydrogenase activity, crotonase activity, crotonyl-CoA reductase or butyryl-CoA dehydrogenase electron transfer flavoprotien activity, alcohol dehydrogenase activity and formate dehydrogenase activity.
  • the method includes knocking out or reducing activity of an enzyme selected from fumarate reductase (e.g., frd) , phosphate acetyltransferase (e.g., pta), lactate dehydrogenase (e.g., ldhA) and ethanol dehydrogenase (e.g., adhE) .
  • fumarate reductase e.g., frd
  • phosphate acetyltransferase e.g., pta
  • lactate dehydrogenase e.g., ldhA
  • ethanol dehydrogenase e.g., adhE
  • a method for producing n-butanol includes: a) providing a recombinant microorganism as provided herein; b) culturing the microorganism in the presence of a suitable carbon substrate and under conditions suitable for the conversion of the substrate to n-butanol; and c) detecting the production of n-butanol and/or isolate the n-butanol.
  • Figure 1A shows 1-butanol production pathway engineered in E. coli from C. acetobutylicum (boxed) .
  • Acetyl-CoA initiates the NADH-consuming reactions (thick gray arrows) .
  • a total of four NADH is needed to make one 1-butanol.
  • Figure 1B-H shows various types of driving force with example productions given below, (b) decarboxylation (c)
  • Figure 2 shows a comparison of anaerobic 1-butanol production using different Ter homologues and mutants. Production level with C. acetobutylicum Bcd-EtfAB is also shown. Successful Ter mutants (dashed box) as a result of first-round growth
  • Figure 3A shows the effect of Fdh over-expression
  • Solid gray lines represent JCL166 transformed with plasmids pELll, pIM8, and pCS138.
  • Dashed gray lines (Aldh Aadh Afrd / Fdh-) refer to JCL166 transformed with plasmids pELll and pIM8.
  • Time indicates time since inoculation.
  • JCL299 BW AldhA AadhE AfrdBC Apta .
  • FIG. 3B shows 1-butanol productions in the pH- controlled fed batch fermentor with gas-stripping. Quite similar performances of T. denticola (Tde) Ter and the F. succinogenes
  • Figure 4 is a comparison of intracellular NADH levels and anaerobic 1-butanol production titers in the wild type and the engineered strains. All strains contained plasmids pELll and pIM8. Strains indicated as "Fdh over-expressed" also carry plasmid pCS138. A concentration of 20 mM formate was fed to the culture at the time of anaerobic switch where noted. The intracellular NADH level was measured using crude extracts prepared from the
  • JCL16 wild type
  • JCL166 BW ldhA adhE frdBC
  • JCL299 BW ldhA adhE frdBC pta .
  • Figure 5 depicts SEQ ID NO : 1 and 3, a nucleic acid sequence of fadA and fadB, respectively.
  • Figure 6 depicts SEQ ID NO: 5, a nucleic acid sequence derived from an atoB gene encoding a polypeptide having keto thiolase activity.
  • Figure 7 depicts SEQ ID NO: 7, a nucleic acid sequence derived from a thlA gene encoding a polypeptide having acetyl-CoA acetyltransferase activity.
  • Figure 8 depicts SEQ ID NO: 9, a nucleic acid sequence derived from a crt gene encoding a polypeptide having crotonase activity .
  • Figure 9 depicts SEQ ID NO: 11, a nucleic acid sequence derived from a hbd gene encoding a polypeptide having
  • Figure 10 depicts SEQ ID NO: 13, a nucleic acid sequence derived from a bed gene encoding a polypeptide having butyryl-CoA dehydrogenase activity.
  • Figure 11 depicts SEQ ID NO: 15, a nucleic acid sequence derived from an etfA gene encoding an ETF polypeptide.
  • Figure 12 depicts SEQ ID NO: 17, a nucleic acid sequence derived from an etfB gene encoding an ETF polypeptide.
  • Figure 13 depicts SEQ ID NO: 19, a nucleic acid sequence derived from a bed gene encoding a polypeptide having butyryl-CoA dehydrogenase activity.
  • Figure 14 depicts SEQ ID NO: 21, a nucleic acid sequence derived from an etfA gene encoding an ETF polypeptide.
  • Figure 15 depicts SEQ ID NO: 23, a nucleic acid sequence derived from an etfB gene encoding an ETF polypeptide.
  • Figure 16 depicts SEQ ID NO: 25, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 17 depicts SEQ ID NO: 27, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 18 depicts SEQ ID NO: 29, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 19 depicts SEQ ID NO: 31, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 20 depicts SEQ ID NO: 33, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 21 depicts SEQ ID NO: 35, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 22 depicts SEQ ID NO: 37, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having crotonyl CoA reductase activity.
  • Figure 23 depicts SEQ ID NO: 39, a nucleic acid sequence derived from a ccr gene encoding a polypeptide having alcohol dehydrogenase activity.
  • Figure 24 shows Multiple sequence alignments of Ter homologues from various organisms using ClustalW.
  • the M11K amino acid substitution found in the F. succinogenes Ter mutants is shaded. Fully conserved residues are noted with asterisks.
  • TD T. denticola (SEQ ID NO:41)
  • TV T. vincentii (SEQ ID NO:42)
  • FS F. succinogenes (SEQ ID NO: 43)
  • FJ F. johnsonia (SEQ ID NO: 44).
  • Figure 25A shows the effect of aeration level on 1- butanol production. Fermentations of strain JCL166 harboring plasmids pELll and pIM8 were carried out under three different oxygen condition as listed below. Detail procedure of each condition is described in Supplementary Methods. Samples were taken after 24 hours. Cell densities are listed on the right y- axis .
  • Figure 25B shows the effect of pH adjustment on 1- butanol production. Fermentations of strain JCL166 harboring plasmids pELll and pIM8 were carried out with or without pH adjustments. Samples were taken after 24 hours. Time indicates time since inoculation.
  • Figure 26 shows media analysis on 1-butanol production.
  • Fermentations of strain JCL299 harboring plasmids pELll, pIM8 and pCS138 were carried out under different media compositions as indicated on the x-axis (- indicates the absence and + indicates the presence of that particular component) . Effect of every element in the TB media was analyzed by subtraction of each component one by one. Production of 1-butanol in glucose minimal media with 0.5% yeast extract (YE) was also shown. Samples were taken after 30 hours. Cell densities are listed on the right y-axis.
  • Butanol is hydrophobic and less volatile than ethanol .
  • butanol has an energy density closer to gasoline. Butanol at 85 percent strength can be used in cars without any change to the engine (unlike ethanol) and it produces more power than ethanol and almost as much power as gasoline. Butanol is also used as a solvent in chemical and textile processes, organic synthesis and as a chemical intermediate. Butanol also is used as a component of hydraulic and brake fluids and as a base for perfumes.
  • microorganisms are relatively difficult to manipulate. Genetic manipulation tools for these organisms are not as efficient as those for user-friendly hosts such as E. coli and Sarcomyces sp . and physiology and their metabolic regulation are much less understood, prohibiting rapid progress towards high-efficiency production.
  • the disclosure provides a recombinant microorganism comprising elevated expression of at least one target enzyme as compared to a parental microorganism or encodes an enzyme not found in the parental organism.
  • the microorganism comprises a reduction, disruption or knockout of at least one gene encoding an enzyme that competes with a metabolite necessary for the production of a desired higher alcohol product or which produces an unwanted product.
  • the recombinant microorganism produces at least one metabolite involved in a biosynthetic pathway for the production of 1-butanol.
  • the recombinant microorganisms comprises at least one recombinant metabolic pathway that comprises a target enzyme and may further include a reduction in activity or expression of an enzyme in a competitive biosynthetic pathway.
  • the pathway acts to modify a substrate or metabolic intermediate in the production of 1-butanol.
  • the target enzyme is encoded by, and expressed from, a polynucleotide derived from a suitable biological source.
  • the polynucleotide comprises a gene derived from a bacterial or yeast source and recombinantly engineered into the microorganism of the disclosure.
  • metabolic engineering involves rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired metabolite, such as an acetoacetyl-CoA or higher alcohol, in a microorganism.
  • a desired metabolite such as an acetoacetyl-CoA or higher alcohol
  • a biosynthetic gene can be heterologous to the host microorganism, either by virtue of being foreign to the host, or being modified by mutagenesis, recombination, and/or association with a heterologous expression control sequence in an endogenous host cell.
  • the polynucleotide can be codon optimized .
  • biosynthetic pathway also referred to as
  • metabolic pathway refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another.
  • Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
  • substrate refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
  • the term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof.
  • substrate encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein.
  • a “biomass derived sugar” includes, but is not limited to, molecules such as glucose, sucrose, mannose, xylose, and arabinose .
  • biomass derived sugar encompasses suitable carbon substrates ordinarily used by microorganisms, such as 6 carbon sugars, including, but not limited to, glucose, lactose, sorbose, fructose, idose, galactose and mannose in either D or L form, or a combination of 6 carbon sugars, such as glucose and fructose, and/or 6 carbon sugar acids
  • 2-keto-L-gulonic acid including, but not limited to, 2-keto-L-gulonic acid, idonic acid (IA), gluconic acid (GA) , 6-phosphogluconate , 2-keto-D-gluconic acid (2 KDG) , 5-keto-D-gluconic acid, 2-ketogluconatephosphate, 2 , 5-diketo-L-gulonic acid, 2 , 3-L-diketogulonic acid,
  • dehydroascorbic acid dehydroascorbic acid, erythorbic acid (EA) and D-mannonic acid.
  • the term "1-butanol” or “n-butanol” generally refers to a straight chain isomer with the alcohol functional group at the terminal carbon.
  • the straight chain isomer with the alcohol at an internal carbon is sec-butanol or 2-butanol.
  • the branched isomer with the alcohol at a terminal carbon is isobutanol, and the branched isomer with the alcohol at the internal carbon is tert- butanol .
  • Recombinant microorganisms provided herein can express a plurality of target enzymes involved in pathways for the production of 1-butanol from a suitable carbon substrate.
  • metabolically "engineered” or “modified” microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism.
  • the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, an
  • the introduction of genetic material into a parental microorganism results in a new or modified ability to produce 1-butanol.
  • the genetic material introduced into the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of 1- butanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences .
  • An engineered or modified microorganism can also include in the alternative or in addition to the introduction of a genetic material into a host or parental micoorganism, the disruption, deletion or knocking out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the reduction, disruption or knocking out of a gene or
  • polynucleotide the microorganism acquires new or improved
  • properties e.g., the ability to produced a new or greater quantities of an interacellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of undesireable by-products.
  • Microorganisms provided herein are modified to produce metabolites in quantities not available in the parental
  • a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., acetyl-coA) in, or an end product (e.g., 1- butanol) , of metabolism.
  • Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones.
  • Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
  • the disclosure demonstrates that the expression of one or more heterologous polynucleotide (s) and/or over-expression of one or more polynucleotide (s) encoding; (i) a polypeptide that catalyzes the production of acetoacetyl-coA from two molecules of acetyl-coA; (ii) a polypeptide that catalyzes the conversion of acetoacetyl-coA to 3-hydroxybutyryl-CoA; (iii) a polypeptide the catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA;
  • a polypeptide (or polypeptide combination) that catalyzes the reduction of crotonyl-CoA to butyryl-CoA; and (v) a polypeptide that preferentially catalyzes the conversion of butyryl-CoA to butyraldehyde and butyraldehyde to 1-butanol, and a coupling of NADH consumption/use to butanol production can provide production of at least 0.1 g/liter/H (e.g., 0.12, 0.15, 0.18 or 0.2 g/L/H, and any number therebetween), titers of at least 10 g/L (e.g., 11, 12, 13, 14, 15 g/L) in flasks and at least 15 g/liter (e.g., 15, 20, 25, 30 g/L) in the fermentor and yields of approximately 88% of the theoretical in flasks and 70% of the theoretical in the fermentor.
  • g/liter/H e.g. 0.12, 0.15, 0.
  • the organism is a not a Clostridium sp.
  • the disclosure demonstrates that with over-expression of the heterologous atoB or thl , hbd, crt, bed, etfAB, and adhE2 genes in E. coli with reduction in NADH use by competing pathways and coupling of NADH use to the 1-butanol production pathway that the production of 1-butanol can be obtained at about 20-30 g/L in a fermentor .
  • the disclosure provides a recombinant microorganisms that produce 1-butanol and include the expression or elevated expression of target enzymes such as a acetyl-coA acetyl transferase (e.g., atoB) , an acetoacetyl-coA thiolase (e.g., thl), a 3-hydroxybutryl-coA dehydrogenase (e.g., hbd), a crotonase (e.g., crt), a butyryl-CoA dehydrogeanse (e.g., bed), and electron transfer flavoprotein (e.g., etf) , and an aldehyde/alcohol dehydrogenase (e.g., adhE2) , or any combination thereof, as compared to a parental microorganism.
  • target enzymes such as a acetyl-coA acetyl transferase (e.g.,
  • microorganism may include a disruption, deletion or knockout of expression of an alcohol/acetoaldehyde dehydrogenase that
  • acetyl-coA as a substrate (e.g. adhE gene), as compared to a parental microorganism.
  • Other disruptions, deletions or knockouts can include one or more genes encoding a polypeptide or protein selected from the group consisting of: (i) an enzyme that catalyzes the NADH-dependent conversion of pyruvate to D- lactate (e.g., ldhA) ; (ii) an enzyme that promotes catalysis of fumarate and succinate interconversion (e.g., frdBC) ; (iii) an oxygen transcription regulator; and (iv) an enzyme that catalyzes the conversion of acetyl-coA to acetyl-phosphate (e.g., pta) .
  • the microorganism comprises a disruption, deletion or knockout of a combination of an alcohol/acetoaldehyde
  • acetoacetyl-CoA can be produced by a recombinant microorganism metabolically engineered to express or over-express keto thiolase or acetyl-CoA
  • 3-hydroxybutyryl-CoA can be produced by a recombinant microorganism metabolically engineered to express or over-express hydroxybutyryl CoA dehydrogenase and crotonyl-CoA can be produced by a recombinant microorganism metabolically engineered to express or over-express crotonase .
  • the metabolite butyryl-CoA can be produced by a
  • a recombinant microorganism metabolically engineered to express or over-express crotonyl-CoA reductase or butyryl-CoA dehydrogenase The metabolites buteraldehyde and n-butanol can be produced by a recombinant microorganism metabolically engineered to express or over-express alcohol dehydrogenase (AdhE2) .
  • AdhE2 alcohol dehydrogenase
  • a recombinant microorganism provided herein includes the elevated expression of at least one target enzyme, such as keto thiolase.
  • a recombinant microorganism can express a plurality of target enzymes involved in pathway to produce n-butanol as depicted in Figure la from fermentation of a suitable substrate.
  • the plurality of enzymes can include keto thiolase, acetyl-CoA acetyltransferase, hydroxybutyryl CoA dehydrogenase, crotonase, crotonyl-CoA reductase, butyryl-CoA dehydrogenase, and alcohol dehydrogenase (AdhE2) , or any combination thereof.
  • the target enzymes described throughout this disclosure generally produce metabolites.
  • a keto thiolase produces acetoacetyl-CoA from a substrate that includes acetyl-CoA.
  • the target enzymes described throughout this disclosure are encoded by polynucleotide.
  • a keto thiolase can be encoded by an atoB gene, polynucleotide or homolog thereof, or an fadA gene, polynucleotide or homolog thereof (see, e.g., EC 2.3.1.16, which includes a number of genes encoding a keto thiolase) .
  • the atoB gene or fadA gene can be derived from any biologic source that provides a suitable nucleic acid sequence encoding a suitable enzyme.
  • atoB gene or fadA gene can be derived from E. coli or
  • a recombinant microorganism provided herein includes expression or elevated expression of an acetyl-CoA acetyltransferase as compared to a parental
  • the microorganism produces a metabolite that includes acetoacetyl-CoA from a substrate that includes acetyl-CoA.
  • the acetyl-CoA acetyltransferase can be encoded by a thlA gene, polynucleotide or homolog thereof.
  • the thlA gene or polynucleotide can be derived from the genus Clostridium.
  • a recombinant microorganism in another embodiment, includes expression or elevated expression of a hydroxybutyryl CoA dehydrogenase as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes a 3-hydroxybutyryl-CoA from a substrate that includes acetoacetyl-CoA .
  • the hydroxybutyryl CoA dehydrogenase can be encoded by an hbd gene, polynucleotide or homolog thereof.
  • the hbd gene can be derived from various microorganisms including
  • Clostridium acetobutylicum Clostridium difficile, Dastricha ruminatium, Butyrivibrio fibrisolvens, Treponema phagedemes ,
  • Acidaminococcus fermentans Clostridium kluyveri , Syntrophospora bryanti , and Thermoanaerobacterium thermosaccharolyticum.
  • a recombinant microorganism provided herein includes expression or elevated expression of crotonase as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes crotonyl-CoA from a substrate that includes 3-hydroxybutyryl-CoA .
  • the crotonase can be encoded by a crt gene, polyncleotide or homolog thereof.
  • the crt gene or polynucleotide can be derived from various microorganisms including Clostridium acetobutylicum, Butyrivibrio fibrisolvens , Thermoanaerobacterium thermosaccharolyticum, and Clostridium difficile .
  • a recombinant microorganism includes expression or elevated expression of a crotonyl-CoA reductase as compared to a parental microorganism.
  • the microorganism produces a metabolite that includes butyryl-CoA from a substrate that includes crotonyl-CoA .
  • the crotonyl-CoA reductase can be encoded by a ccr gene, polynucleotide or homolog thereof.
  • the ccr gene or polynucleotide can be derived from the genus Streptomyces .
  • a recombinant microorganism includes expression or elevated expression of a butyryl-CoA dehydrogenase as compared to a parental microorganism.
  • the recombinant microorganism produces a metabolite that includes butyryl-CoA from a substrate that includes crotonyl-CoA .
  • the butyryl-CoA dehydrogenase can be encoded by a bed gene,
  • the bed gene, polynucleotide can be derived from Clostridium acetobutylicum, Mycobacterium tuberculosis, or Megasphaera elsdenii.
  • a recombinant microorganism provided herein includes expression or elevated expression of an alcohol dehydrogenase (ADHE2) as compared to a parental
  • the recombinant microorganism produces a metabolite that includes butanol from a substrate that includes butyryl-CoA.
  • the alcohol dehydrogenase can be encoded by bdhA/bdhB
  • polynucleotide or homolog thereof an aad gene, polynucleotide or homolog thereof, or an adhE2 gene, polynucleotide or homolog thereof.
  • the aad gene or adhE2 gene or polynucleotide can be derived from Clostridium acetobutylicum.
  • the recombinant microorganism comprises a NADH utilization pathway that improves butanol production.
  • the recombinant microorganism may be selected using methods described herein.
  • the NADH driving force created by a cell's inability to regenerate NAD+ anaerobically without an external electron acceptor can be used as a selection platform to improve enzymes and pathways that consume NADH.
  • the microorganism comprises a heterologous trans-2-enoyl-CoA reductase (ter) .
  • Trans-2-enoyl-CoA reductase or TER is a protein that is capable of catalyzing the conversion of crotonyl-CoA to butyryl-CoA.
  • the recombinant microorganism expresses a TER which catalyzes the same reaction as Bcd/EtfA/EtfB from Clostridia and other bacterial species.
  • Mitochondrial TER from E. gracilis has been described, and many TER proteins and proteins with TER activity derived from a number of species have been identified forming a TER protein family
  • a truncated cDNA of the E. gracilis gene has been functionally expressed in E. coli.
  • This cDNA or the genes of homologues from other microorganisms can be expressed together with the n-butanol pathway genes thl, crt, adhE2, and hbd to produce n-butanol in E. coli, S. cerevisiae or other hosts.
  • TER proteins can also be identified by generally well known bioinformatics methods, such as BLAST. Examples of TER proteins include, but are not limited to, TERs from species such as: Euglena spp. including, but not limited to, E. gracilis,
  • Aeromonas spp. including, but not limited, to A. hydrophila,
  • Psychromonas spp. including, but not limited to, P. ingrahamii, Photobacterium spp. including, but not limited, to P. profundum, Vibrio spp. including, but not limited, to V angustum, V. cholerae, V alginolyticus , V parahaemolyticus , V vulnificus , V fischeri, V spectacularus , Shewanella spp. including, but not limited to, S.
  • Pseudoalteromonas spp. including, but not limited to, P. atlantica, Alteromonas spp., Saccharophagus spp. including, but not limited to, S. degradans , S. marine gamma proteobacterium, S. alpha proteobacterium, Pseudomonas spp. including, but not limited to, P. aeruginosa, P. putida, P. fluorescens , Burkholderia spp. including, but not limited to, B. phytofirmans , B. cenocepacia, B. cepacia, B. ambifaria, B.
  • M. flageliatus including, but not limited to, S. maltophilia, Congregibacter spp. including, but not limited to, C. litoralis, Serratia spp. including, but not limited to, S. proteamaculans , Marinomonas spp. , Xytella spp. including, but not limited to, X fastidiosa, Reinekea spp. , Colweffia spp. including, but not limited to, C.
  • Yersinia spp. including, but not limited to, Y. pestis , Y. pseudotuberculosis, Methylobacillus spp. including, but not limited to, M flageliatus, Cytophaga spp. including, but not limited to, C. hutchinsonii , Flavobacterium spp. including, but not limited to, F. j ohnsoniae, Microscilla spp. including, but not limited to, M marina, Polaribacter spp. including, but not limited to, P. irgensii, Clostridium spp. including, but not limited to, C. acetobutylicum, C. beijerenckii , C. cellulolyticum, Coxiella spp. including, but not limited to, C. burnetii.
  • Yersinia spp. including, but not limited to, Y. pestis , Y. pseudotuberculosis,
  • the ter is derived from a Treponema denticola or F. succinogenes .
  • the ter is a mutant ter comprising an M11K substitution.
  • the microorganism comprises expression or over expression or one or more or all of the
  • the microorganism comprises one or more knockouts selected from the group consisting of frdBc, ldhA, adhE and pta.
  • genes useful in the methods, compositions and organisms of the disclosure are not necessary.
  • changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutation and silent mutations.
  • modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme activity using methods known in the art .
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non- optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E.
  • DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure.
  • the native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as they modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
  • homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
  • the term "homologs" used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR.
  • a protein has "homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences) .
  • two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology”) .
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al . , 1994, hereby incorporated herein by reference) .
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryp
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) , Threonine (T) ; 2) Aspartic Acid (D) , Glutamic Acid (E) ; 3) Asparagine (N) , Glutamine (Q) ; 4) Arginine (R) , Lysine (K) ; 5) Isoleucine (I) , Leucine (L) , Methionine (M) , Alanine (A) , Valine (V) , and 6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) .
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG) , University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.
  • BLAST Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997) .
  • Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
  • polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference) .
  • percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.
  • polypeptide is reduced or knocked-out.
  • knockout or a reduction in expression are optional in the synthesis of the product, however, such knockouts increase various substrate intermediates and improve yield.
  • Exemplary yield data for E.coli comprising overexpression of atoB (EC) , hbd (CA) , crt (CA) , bed (CA) , etfAB (CA) , and adhE2 (CA)
  • the disclosure provides recombinant microorganism comprising a biosynthetic pathway that provides a yield of greater than 0.02 grams of n-butanol per gram of glucose.
  • the recombinant microorganism can produce about 0.02 to about 0.06 grams of n-butanol per gram of glucose (e.g., greater than about 0.025, about 0.028 to about 0.03, about 0.033 to 0.035 to about 0.06, and any ranges or values therebetween) .
  • the parental microorganism does not produce n-butanol.
  • the parental microorganism produced only trace amounts of n-butanol (e.g., less than 0.010 grams of n-butanol per gram of glucose) .
  • the recombinant microorganism can produce about 0.02 to about 0.06 grams of n-butanol per gram of glucose (e.g., greater than about 0.025, about 0.028 to about 0.03, about 0.033 to 0.035 to about 0.06, and any ranges or values therebetween) .
  • the parental microorganism does not produce n-butanol.
  • the parental microorganism
  • microorganism produces at least about 0.14 g/L of 1-butanol in 24 hours (e.g., 0.15 g/L, 0.16 g/L, 0.17 g/L, 0.18 g/L, 0.19 g/L, 0.20 g/L, 0.21 g/L, 0.22 g/L, 0.23 g/L , 0.24 g/L , 0.25 g/L , 0.26 g/L , 0.27 g/L , 0.28 g/L, 0.29 g/L, 0.30 g/L, 0.31 g/L, 0.32 g/L, 0.33 g/L, 0.34 g/L, 0.35 g/L, 0.36 g/L, 0.37 g/L, 0.38 g/L or more in 24 hours) .
  • microorganisms are described that are capable of metabolizing a carbon source for producing n-butanol at a yield of at least 4% of theoretical, and, in some cases, a yield of over 50% of theoretical.
  • yield refers to the molar yield. For example, the yield equals 100% when one mole of glucose is converted to one mole of n-butanol.
  • yield is defined as the mole of product obtained per mole of carbon source monomer and may be expressed as percent. Unless otherwise noted, yield is expressed as a percentage of the theoretical yield.
  • Theoretical yield is defined as the maximum moles of product that can be generated per a given mole of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to n-butanol is 100%. As such, a yield of n-butanol from glucose of 95% would be expressed as 95% of theoretical or 95% theoretical yield. In one embodiment, the yield is at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or more. For example, the yield of a recombinant E.
  • the coli of the disclosure can generate a yield of 4-15% (e.g., 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%).
  • the yield of a recombinant yeast cell can be from 5% to 50%.
  • the microorganism is an E.coli.
  • the microorganism is a yeast.
  • a culture comprises a population microorganism that is substantially homogenous (e.g., from about 70-100% homogenous) .
  • a culture can comprise a combination of micoorganism each having distinct biosynthetic pathways that produced metabolites that can be used by at least one other microorganism in culture leading to the production of n-butanol .
  • homologs and variants described herein are exemplary and non- limiting. Additional homologs, variants and sequences are available to those of skill in the art using various databases including, for example, the National Center for Biotechnology Information (NCBI) access to which is available on the World-Wide- Web. Furthermore, the disclosure demonstrates that by reducing oxidation of NADH by competitive pathways, effective n-butanol production and/or coupling NADH utilization more closely to the n- butanol production pathway described herein provides an increase in n-butanol production. Identifying competing (oxidative) pathways in various organism is within the skill in the art and various enzymes in such pathways can be reduced by knocking out the polynucleotide encoding such enzyme or reducing expression.
  • NCBI National Center for Biotechnology Information
  • Trans-2-enoyl-CoA reductase is encoded in T. denticola
  • T. vincentii or F. johnsoniae ter gene F. succinogens, T. vincentii or F. johnsoniae ter gene.
  • denticoloa TER has the accession number Q73Q47 (see also Figure 24) .
  • the F. succinogens TER comprises the sequence set forth in Figure 24 and has a MetllLys mutation.
  • Other TER polypeptides are set forth in Figure 24.
  • TER proteins can also be identified by generally well known bioinformatics methods, such as BLAST. Examples of TER proteins include, but are not limited to, TERs from species such as: Euglena spp. including, but not limited to, E. gracilis, Aeromonas spp. including, but not limited, to A. hydrophila, Psychromonas spp. including, but not limited to, P.
  • Photobacterium spp. including, but not limited, to P. profundum, Vibrio spp. including, but not limited, to V. angustum, V. cholerae, V. alginolyticus, V. parahaemolyticus, V. vulnificus, V. fischeri , V. spectacularus,
  • Shewanella spp. including, but not limited to, S. amazonensis, S. woodyi , S. frigidimarina, S. paeleana, S. baltica, S.
  • denitrificans Oceanospirillum spp.
  • Xanthomonas spp. including, but not limited to, X. oryzae, X. campestris, Chromohalobacter spp. including, but not limited, to C. salexigens, Idiomarina spp.
  • Pseudoalteromonas spp. including, but not limited to, P. atlantica, Alteromonas spp., Saccharophagus spp. including, but not limited to, S. degradans, S. marine gamma proteobacterium, S. alpha proteobacterium, Pseudomonas spp. including, but not limited to, P. aeruginosa, P. putida, P. fluorescens, Burkholderia spp. including, but not limited to, B. phytofirmans, B. cenocepacia, B. cepacia, B. ambifaria, B.
  • M. flageliatus including, but not limited to, S. maltophilia, Congregibacter spp. including, but not limited to, C. litoralis, Serratia spp. including, but not limited to, S. proteamaculans, Marinomonas spp., Xytella spp. including, but not limited to, X. fastidiosa, Reinekea spp., Colweffia spp. including, but not limited to, C.
  • Yersinia spp. including, but not limited to, Y. pestis, Y. pseudotuberculosis, Methylobacillus spp. including, but not limited to, M. flageliatus, Cytophaga spp. including, but not limited to, C. hutchinsonii , Flavobacterium spp. including, but not limited to, F. j ohnsoniae, Microscilla spp. including, but not limited to, M. marina, Polaribacter spp. including, but not limited to, P. irgensii , Clostridium spp. including, but not limited to, C. acetobutylicum, C. beijerenckii , C. cellulolyticum, Coxiella spp. including, but not limited to, C. burnetii.
  • Yersinia spp. including, but not limited to, Y. pestis, Y. pseudotuberculosis
  • the Ter is derived from a Treponema denticola or F. succinogenes .
  • the ter is a mutant ter comprising an M11K substitution.
  • Ethanol Dehydrogenase (also referred to as Aldehyde- alcohol dehydrogenase) is encoded in E.coli by adhE.
  • adhE comprises three activities: alcohol dehydrogenase (ADH) ; acetaldehyde/acetyl- CoA dehydrogenase (ACDH) ; pyruvate-formate-lyase deactivase (PFL deactivase) ; PFL deactivase activity catalyzes the quenching of the pyruvate-formate-lyase catalyst in an iron, NAD, and CoA dependent reaction. Homologs are known in the art (see, e.g., aldehyde- alcohol dehydrogenase (Polytomella sp . Pringsheim 198.80)
  • aldehyde-alcohol dehydrogenase includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating) (EC 1.2.1.10) (acdh) ; pyruvate-formate- lyase deactivase (pfl deactivase)) ⁇ Clostridium botulinum A str. ATCC 3502) gi I 148287832 I em I CAL81898.1 I (148287832) ; aldehyde- alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH);
  • Acetaldehyde dehydrogenase (acetylating) (ACDH) ; Pyruvate-formate- lyase deactivase (PFL deactivase) )
  • aldehyde-alcohol dehydrogenase includes: alcohol dehydrogenase and acetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase ⁇ Erwinia carotovora subsp. atroseptica SCRI1043
  • dehydrogenase includes: alcohol dehydrogenase and acetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase (Erwinia carotovora subsp. atroseptica SCRI1043)
  • dehydrogenase Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase (acetylating) (ACDH) )
  • Aldehyde-alcohol dehydrogenase Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase (acetylating) (ACDH) ; Pyruvate-formate-lyase
  • PFL deactivase PFL deactivase
  • aldehyde-alcohol dehydrogenase includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating) ; pyruvate-formate-lyase deactivase (Clostridium difficile 630)
  • aldehyde-alcohol dehydrogenase includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating) ; pyruvate-formate-lyase deactivase (Clostridium difficile 630)
  • dehydrogenase includes alcohol dehydrogenase (ADH) and
  • acetaldehyde dehydrogenase acetylating (ACDH)
  • ACDH acetylating
  • PFL deactivase pyruvate-formate- lyase deactivase
  • Photorhabdus luminescens subsp. laumondii TTOl gi
  • aldehyde- alcohol dehydrogenase 2 includes: alcohol dehydrogenase;
  • acetaldehyde dehydrogenase (Streptococcus pyogenes str. Manfredo) gi I 134271169 I em I CAM29381.1 I (134271169); Aldehyde-alcohol
  • dehydrogenase includes alcohol dehydrogenase (ADH) and
  • acetaldehyde dehydrogenase acetylating (ACDH)
  • ACDH acetylating
  • PFL deactivase pyruvate-formate- lyase deactivase
  • Photorhabdus luminescens subsp. laumondii TTOl gi
  • aldehyde- alcohol dehydrogenase includes: alcohol dehydrogenase and
  • pyruvate-formate-lyase deactivase (Clostridium difficile 630) gi I 126700586 I ref I YP_001089483.1 I (126700586); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase and pyruvate- formate-lyase deactivase (Clostridium difficile 630)
  • dehydrogenase 2 (Streptococcus pyogenes str. Manfredo)
  • ADHE1 Distridium acetobutylicum ATCC 8244
  • ADHE1 Distridium acetobutylicum ATCC 824) gi I 14994351
  • ACDH acetaldehyde/acetyl-CoA dehydrogenase
  • Lactate Dehydrogenase (also referred to as D-lactate dehydrogenase and fermentive dehydrognase) is encoded in E.coli by IdhA and catalyzes the NADH-dependent conversion of pyruvate to D- lactate . IdhA homologs and variants are known. In fact there are currently 1664 bacterial lactate dehydrogenases available through NCBI .
  • such homologs and variants include, for example, D-lactate dehydrogenase (D-LDH) (Fermentative lactate dehydrogenase) gi
  • Fumarate reductase comprises multiple subunits (e.g., frdA, B, and C in E.coli) . Modification of any one of the subunits can result in the desired activity herein. For example, a knockout of frdB, frdC or frdBC is useful in the methods of the disclosure.
  • frdB, frdC or frdBC is useful in the methods of the disclosure.
  • frd homologs and variants are known.
  • homologs and variants includes, for example, Fumarate reductase subunit D
  • flavoprotein subunit precursor (Flavocytochrome c) (FL cyt) gi I 25452947
  • FRDA_SHEON 25452947
  • fumarate reductase iron-sulfur subunit (Wolinella succinogenes)
  • Phosphate acetyltransferase is encoded in E.coli by pta .
  • PTA is involved in conversion of acetate to acetyl-CoA.
  • PTA catalyzes the conversion of acetyl-coA to acetyl- phosphate .
  • PTA homologs and variants are known. There are approximately 1075 bacterial phosphate acetyltransferases available on NCBI . For example, such homologs and variants include phosphate acetyltransferase Pta (Rickettsia felis URRWXCal2)
  • acetyltransferase (pta) (Treponema pallidum subsp. pallidum str. Nichols) gi I 3322356 I gb I AAC65090.1 I (3322356) , each sequence associated with the accession number is incorporated herein by reference in its entirety.
  • Pyruvate-formate lyase (Formate acetlytransferase) is an enzyme that catalyzes the conversion of pyruvate to acetly-coA and formate. It is induced by pf1-activating enzyme under anaerobic conditions by generation of an organic free radical and decreases significantly during phosphate limitation. Formate acetlytransferase is encoded in E.coli by pflB. PFLB homologs and variants are known.
  • such homologs and variants include, for example, Formate acetyltransferase 1 (Pyruvate formate-lyase 1) gi
  • acetyltransferase 1 ⁇ Yersinia pseudotuberculosis IP 32953) gi I 51589030 I emb I CAH20648.1 I (51589030) ; formate acetyltransferase 1
  • acetyltransferase 1 ⁇ Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150) gi
  • acetyltransferase (Staphylococcus aureus subsp. aureus Mu3) gi 11567206911 dbj
  • acetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03) gi I 165976461 I ref I YP_001652054.1 I (165976461) ; formate acetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03) gi I 165876562 I gb I ABY69610.1 I (165876562) ; formate
  • acetyltransferase (Staphylococcus aureus subsp. aureus MW2) gi I 21203365 I dbj
  • gi 86556286 gb ABD01243.1 (86556286); formate acetyltransferase (Synechococcus sp . JA-3-3Ab) gi
  • FNR transcriptional dual regulators are transcription requlators responsive to oxygen contenct .
  • FNR is an anaerobic regulator that represses the expression of PDHc . Accordingly, reducing FNR will result in an increase in PDHc expression.
  • FNR homologs and variants are known.
  • such homologs and variants include, for example, DNA-binding transcriptional dual regulator, global regulator of anaerobic growth (Escherichia coli W3110) gi 1742191 dbj BAA14927.1 (1742191) ; DNA-binding
  • An acetoacetyl-coA thiolase (also sometimes referred to as an acetyl-coA acetyltransferase) catalyzes the production of acetoacetyl-coA from two molecules of acetyl-coA.
  • a heterologous acetoacetyl-coA thiolase (acetyl- coA acetyltransferase) can be engineered for expression in the organism.
  • a native acetoacetyl-coA thiolase E.C. 2.3.1.19
  • acetyl-coA acetyltransferase can be overexpressed .
  • Acetoacetyl-coA thiolase is encoded in E.coli by thl .
  • Acetyl-coA acetyltransferase is encoded in C. acetobutylicum by atoB. THL and AtoB homologs and variants are known.
  • Homologous enzymes have also been identified, and may be identified by performing a BLAST search against above protein sequence. These homologs can also serve as suitable thiolases in a heterologously expressed n-butanol pathway. Just to name a few, these homologous enzymes include, but are not limited to, those from C. acetobutylicum sp . (e.g., protein ID AAC26026.1), C. pasteurianum (e.g., protein ID ABA18857.1), C.
  • beijerinckii sp. e.g., protein ID EAP59904.1 or EAP59331.1
  • Clostridium perfringens sp . e.g., protein ID ABG86544.1
  • Clostridium difficile sp . e.g., protein ID CAJ67900.1 or ZP . sub . --01231975.1
  • Clostridium difficile sp . e.g., protein ID CAJ67900.1 or ZP . sub . --01231975.1
  • thermosaccharolyticum e.g., protein ID CAB07500.1
  • Thermoanaerobacter tengcongensis (e.g., AAM23825.1),
  • Carboxydothermus hydrogenoformans e.g., protein ID ABB13995.1
  • Desulfotomaculum reducens MI-1 e.g., protein ID EAR45123.1
  • Candida tropicalis e.g., protein ID BAA02716.1 or BAA02715.1
  • Saccharomyces cerevisiae e.g., protein ID AAA62378.1 or
  • S. cerevisiae thiolase could also be active in a hetorologously expressed n-butanol pathway (ScERGlO) .
  • homologs and variants include, for example, acetyl-coa acetyltransferase (thiolase) (Streptomyces coelicolor A3 (2) ) gi
  • acetyltransferase (thiolase) ⁇ Alcanivorax borkumensis SK2) gi I 110647539 I em I CAL17015.1 I (110647539) ; acetyl CoA acetyltransferase (thiolase) (Saccharopolyspora erythraea NRRL 2338) gi I 133915420 I emb I CAM05533.1 I (133915420) ; acetyl-coa acetyltransferase (thiolase) (Saccharopolyspora erythraea NRRL 2338) gi I 134098403 I ref I YP_001104064.1 I (134098403) ; acetyl-coa acetyltransferase (thiolase) (Saccharopolyspora erythraea NRRL 2338) gi I
  • acetyltransferase (thiolase) (Ralstonia metallidurans CH34) gi I 93358270 I gb I ABF12358.1 I (93358270); acetyl-CoA acetyltransferase (thiolase) (Ralstonia metallidurans CH34)
  • 3 hydroxy-butyryl-coA-dehydrogenase catalyzes the conversion of acetoacetyl-coA to 3-hydroxybutyryl-CoA.
  • a heterologous 3-hydroxy-butyryl-coA- dehydrogenase can be engineered for expression in the organism. Alternatlively a native 3-hydroxy-butyryl-coA-dehydrogenase can be overexpressed .
  • 3-hydroxy-butyryl-coA-dehydrogenase is encoded in C. acetobuylicum by hbd. HBD homologs and variants are known.
  • such homologs and variants include, for example, 3- hydroxybutyryl-CoA dehydrogenase (Clostridium acetobutylicum ATCC 824) gi I 15895965 I ref
  • Crotonase catalyzes the conversion of 3-hydroxybutyryl- CoA to crotonyl-CoA .
  • Crotonase catalyzes the conversion of 3-hydroxybutyryl- CoA to crotonyl-CoA .
  • heterologous Crotonase can be engineered for expression in the organism. Alternatlively a native Crotonase can be overexpressed . Crotonase is encoded in C. acetobuylicum by crt . CRT homologs and variants are known. For examples, such homologs and variants include, for example, crotonase (butyrate-producing bacterium L2- 50) gi 119370267 gb ABL68062.1 (119370267) ; crotonase
  • gi 149203066 ref ZP_01880037.1 (149203066); crotonase (Roseovarius sp. TM1035) gi 149143612 gb EDM31648.1 (149143612) ; crotonase; 3- hydroxbutyryl-CoA dehydratase (Mesorhizobium loti MAFF303099) gi 14027492 dbj BAB53761.1 (14027492) ; crotonase (Roseobacter sp.
  • SK209-2-6) gi 126738922 ref ZP_01754618.1 (126738922); crotonase (Roseobacter sp. SK209-2-6) gi
  • Butyryl-coA dehydrogenase is an enzyme in the protein pathway that catalyzes the reduction of crotonyl-CoA to butyryl- CoA.
  • a butyryl-CoA dehydrogenase complex (Bcd/EtfAB) couples the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin.
  • a heterologous butyryl-CoA dehydrogenase can be engineered for expression in the organism.
  • a native butyryl-CoA dehydrogenase can be overexpressed .
  • Butyryl-coA dehydrognase is encoded in
  • BCD homologs and variants are known.
  • such homologs and variants include, for example, butyryl-CoA dehydrogenase (Clostridium acetobutylicum ATCC 824) gi I 15895968 I ref
  • BCD can be expressed in combination with a
  • flavoprotien electron transfer protein Useful flavoprotein electron transfer protein subunits are expressed in
  • etfA and etfB or the operon etfAB
  • ETFA, B, and AB homologs and variants are known.
  • such homologs and variants include, for example, putative a-subunit of electron-transfer flavoprotein gi I 1055221 I g I AAA95970.1 I (1055221) ; putative b-subunit of electron- transfer flavoprotein gi
  • ( 1055220 ) each sequence associated with the accession number is incorporated herein by reference in its entirety.
  • Aldehyde/alcohol dehydrogenase catalyzes the conversion of butyryl-CoA to butyraldehyde and butyraldehyde to 1-butanol. In one embodiment, the aldehyde/alcohol dehydrogenase preferentially catalyzes the conversion of butyryl-CoA to butyraldehyde and butyraldehyde to 1-butanol.
  • a heterologous aldehyde/alcohol dehydrogenase can be engineered for expression in the organism.
  • a native amino acid dehydrogenase can be engineered for expression in the organism.
  • a native amino acid dehydrogenase can be engineered for expression in the organism.
  • aldehyde/alcohol dehydrogenase can be overexpressed .
  • aldehyde/alcohol dehydrogenase is encoded in C. acetobuylicum by adhE (e.g., an adhE2) .
  • adhE e.g., an adhE2
  • ADHE e.g., ADHE2
  • homologs and variants are known.
  • such homologs and variants include, for example, aldehyde-alcohol dehydrogenase ⁇ Clostridium
  • NAD+ (Clostridium acetobutylicum ATCC 824) gi I 15004865 I ref I NP_149325.1 I (15004865) ; alcohol dehydrogenase E (Clostridium acetobutylicum) gi
  • ADHE1 Clostridium acetobutylicum ATCC 824) gi I 14994351
  • Crotonyl-coA reductase catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Depending upon the organism used a heterologous Crotonyl-coA reductase can be engineered for
  • Crotonyl-coA reductase is encoded in S.coelicolor by ccr.
  • CCR homologs and variants are known.
  • such homologs and variants include, for example, crotonyl CoA reductase (Streptomyces coelicolor A3 (2))
  • culture conditions useful in producing a 1-butanol product comprise conditions of culture medium pH, ionic strength, nutritive content, etc.;
  • microorganism Appropriate culture conditions are well known for microorganisms that can serve as host cells.
  • microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of n-butanol . It is also understood that various microorganisms can act as "sources" for genetic material encoding target enzymes suitable for use in a recombinant microorganism provided herein.
  • microorganism includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • yeast can be used in the methods and compositions of the disclosure (see, e.g., US Patent Publ . No.
  • yeast If yeast are used, NADH oxidation will be reduced in pathways that compete with NADH utilization in the recombinant n- butanon production pathway.
  • prokaryotes is art recognized and refers to cells which contain no nucleus or other cell organelles.
  • the prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on
  • the term "Archaea” refers to a categorization of organisms of the division Mendosicutes , typically found in unusual environments and distinguished from the rest of the procaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls.
  • the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota .
  • Crenarchaeota Crenarchaeota
  • Euryarchaeota On the basis of their physiology, the Archaea can be organized into three types:
  • methanogens prokaryotes that produce methane
  • extreme halophiles prokaryotes that produce methane
  • thermophilus prokaryotes that live at very high temperatures.
  • Bacteria i.e., no murein in cell wall, ester-linked membrane lipids, etc.
  • prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats.
  • the Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes and the
  • Euryarchaeota contains the methanogens and extreme halophiles.
  • Bacteria refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes ,
  • Mycobacteria Mycobacteria, Micrococcus, others
  • low G+C group Bollus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas
  • Proteobacteria e.g., Purple photosynthetic +non- photosynthetic Gram-negative bacteria (includes most "common” Gram- negative bacteria)
  • Cyanobacteria e.g., oxygenic phototrophs
  • Spirochetes and related species (5) Planctomyces ; (6)
  • Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium .
  • Gram positive bacteria include cocci, nonsporulating rods, and sporulating rods.
  • the genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium,
  • Mycobacterium Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces .
  • microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express non- endogenous polynucleotide, such as those included in a vector.
  • the polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite as described above, but may also include protein factors necessary for
  • recombinant microorganisms described herein have been genetically engineered to express or over-express target enzymes not previously expressed or over-expressed by a parental microorganism. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. [ 00117 ] A "parental microorganism” refers to a cell used to generate a recombinant microorganism. The term "parental microorganism"
  • microorganism describes a cell that occurs in nature, i.e. a "wild-type” cell that has not been genetically modified.
  • parental microorganism also describes a cell that has been genetically modified but which does not express or over-express a target enzyme e.g., an enzyme involved in the biosynthetic pathway for the production of a desired metabolite such as n-butanol .
  • a wild-type microorganism can be any suitable microorganism.
  • a wild-type microorganism can be any suitable microorganism.
  • This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or over-express a second target enzyme e.g.,
  • microorganism modified to express or over express e.g., thiolase and
  • hydroxybutyryl-CoA dehydrogenase can be modified to express or over express a third target enzyme e.g., crotonase.
  • a parental microorganism functions as a reference cell for successive genetic modification events.
  • Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction
  • the term “facilitates” encompasses the activation or reduction of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental
  • the method includes transforming a microorganism with one or more recombinant polynucleotides encoding polypeptides that includes keto thiolase or acetyl-CoA
  • acetyltransferase activity hydroxybutyryl CoA dehydrogenase activity, crotonase activity, crotonyl-CoA reductase or butyryl-CoA dehydrogenase activity, and alcohol dehydrogenase activity.
  • metabolites e.g., keto thiolase, acetyl-CoA
  • FIG. 8 through 25 provide exemplary polynucleotide sequences encoding polypeptides useful in the methods described herein. It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid.
  • the "activity" of an enzyme is a measure of its ability to catalyze a reaction resulting in a metabolite, i.e., to
  • enzyme activity can be represented as the amount of metabolite produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
  • a "protein” or “polypeptide”, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
  • An “enzyme” means any substance, typically composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.
  • the term “enzyme” can also refer to a catalytic polynucleotide (e.g., RNA or DNA) .
  • polynucleotide, gene, or cell means a protein, enzyme,
  • a polynucleotide described above include “genes” and that the nucleic acid molecules described above include “vectors” or “plasmids . "
  • a polynucleotide encoding a keto thiolase can comprise an atoB gene or homolog thereof, or an fadA gene or homolog thereof.
  • the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular polypeptide comprising a sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA
  • transcribed region of the gene may include untranslated regions, including introns, 5 ' -untranslated region (UTR) , and 3'-UTR, as well as the coding sequence.
  • UTR 5 ' -untranslated region
  • 3'-UTR 3'-UTR
  • polynucleotide refers to polynucleotides such as deoxyribonucleic acid (DNA) , and, where appropriate, ribonucleic acid (RNA) .
  • expression with respect to a gene or polynucleotide refers to transcription of the gene or polynucleotide and, as appropriate, translation of the resulting mRNA transcript to a protein or polypeptide.
  • expression of a protein or polypeptide results from transcription and translation of the open reading frame.
  • a "vector” generally refers to a polynucleotide that can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include viruses, bacteriophage, pro- viruses, plasmids, phagemids, transposons, and artificial
  • chromosomes such as YACs (yeast artificial chromosomes) , BACs
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a peptide-conj ugated DNA or RNA, a liposome-conj ugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.
  • Transformation refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection) , can be achieved by any one of a number of means including electroporation, microinjection, biolistics (or particle bombardment-mediated delivery) , or agrobacterium mediated
  • the disclosure provides polynucleotides in the form of recombinant DNA expression vectors or plasmids, as described in more detail elsewhere herein, that encode one or more target enzymes.
  • such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the
  • the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions) .
  • the disclosure provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.
  • the disclosure provides methods for the heterologous expression of one or more of the biosynthetic genes or
  • expression vector refers to a polynucleotide that can be introduced into a host microorganism or cell-free transcription and translation system. An expression vector can be maintained permanently or transiently in a
  • An expression vector also comprises a promoter that drives expression of an RNA, which typically is translated into a polypeptide in the microorganism or cell extract.
  • the expression vector also typically contains a ribosome-binding site sequence positioned upstream of the start codon of the coding sequence of the gene to be expressed.
  • Other elements such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes by which host
  • microorganisms containing the vector can be identified and/or selected, may also be present in an expression vector.
  • Selectable markers i.e., genes that confer antibiotic resistance or
  • sensitivity are preferred and confer a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • an expression vector can vary widely, depending on the intended use of the vector and the host cell (s) in which the vector is intended to replicate or drive expression.
  • Expression vector components suitable for the expression vector can vary widely, depending on the intended use of the vector and the host cell (s) in which the vector is intended to replicate or drive expression.
  • suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.
  • suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.
  • promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac) , maltose, tryptophan (trp) , beta- lactamase (bla) , bacteriophage lambda PL, and T5 promoters.
  • synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433, which is incorporated herein by reference in its entirety), can also be used.
  • E. coli expression vectors it is useful to include an E. coli origin of replication, such as from pUC, plP, pi, and pBR.
  • recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of PKS and/or other biosynthetic gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells.
  • the host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.
  • a nucleic acid of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and those procedures described in the Examples section below.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • nucleic acid molecule encoding a polypeptide homologous to the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the
  • polynucleotide by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a non-conservative amino acid substitutions (see above) , in some positions it is preferable to make conservative amino acid substitutions.
  • a method for producing n-butanol includes culturing a recombinant
  • microorganism as provided herein in the presence of a suitable carbon substrate and under conditions suitable for the conversion of the substrate to n-butanol.
  • the butanol produced by a microorganism provided herein can be detected by any method known to the skilled artisan. Such methods include mass spectrometry.
  • RNA polymerase mediated techniques e.g., NASBA
  • Clostridium has been challenging because of the complex physiology of this organism.
  • Various attempts have been reported to transfer the CoA-dependent 1-butanol pathway to more tractable organisms, such as Escherichia coli, Saccharomyces cerevisiae, Lactococcus brevis, Pseudomonas putida and Bacillus subtilis.
  • these efforts achieved only low titers (2.5 mg/L to about 1 g/L) .
  • the CoA-dependent 1-butanol pathway lacks such a driving force.
  • the pathway consists of five reversible steps starting from the condensation of acetyl-CoA and does not possess any decarboxylation reaction to drive the carbon flux (Fig. la) .
  • thermodynamic driving force is necessary to channel high metabolic flux to the desired pathway.
  • a driving force may exist in several different forms: 1) release of a gaseous molecule (e.g., C0 2 ) , which escapes out of the cell through diffusion and becomes diluted in the gas phase (Fig. lb); 2) irreversible reaction or polymerization (e.g., glycogen and polyhydroxyalkanoate ) where synthesis and degradation reactions are catalyzed by completely different sets of enzymes (Fig.
  • a weak acid e.g., lactate and succinate
  • ATP-generation coupled pathways e.g. acetate and butyrate
  • concentration gradient they all function by enlarging the entropic or energetic potential difference which drives the target pathway. It appears that presence of both a pushing source and a pulling sink is essential, regardless of which one being the driving force and which one being the desired reaction.
  • the driving forces can include build-in irreversible reactions (such as decarboxylation and polymerization) or artificial driving forces created by pushing or pulling essential co-factors and
  • M9 medium (6 g Na 2 HP0 4 , 3g KH 2 P0 4 , 0.5 g NaCl, 1 g NH 4 C1, ImM MgS0 4 , lmg vitamin Bl and 0.1 mM CaCl 2 per liter of water) containing 2% glucose and 0.5% yeast extract, appropriate antibiotics, and lOOOx Trace Metal Mix A5 (2.86g H 3 B0 3 , 1.81g MnCl 2 ⁇ 4 ⁇ 2 0, 0.222g ZnS0 4 -7H 2 0, 0.39 g Na 2 Mo0 4 ⁇ 2 ⁇ 2 0, 0.079 g CuS0 4 -5H 2 0, 49.4 mg Co (N0 3 ) 2 ⁇ 6H 2 0 per liter of water) .
  • Oxygen in the headspace and media was then evacuated through the needle by repeated vacuuming and refilling of nitrogen and hydrogen in the anaerobic transfer chamber.
  • the needles were taken off from the caps inside the anaerobic chamber.
  • the sealed tubes were then taken outside and wrapped with parafilm and tape to prevent bursting of the caps (when cells grow, pressure built up due to C0 2 released) .
  • the cultures were then incubated at 37 °C in a rotary shaker (250rpm) for a few days and growths were examined.
  • the 5ml induced cultures were transferred from test tubes to the 10ml BD Vacutainer sealed tubes. Oxygen was evacuated with the same method as described under anaerobic growth rescue. Cultures were then incubated at 37°C in a rotary shaker (250rpm) and samples were taken everyday inside the anaerobic chamber to maintain anaerobicity. If a time course was taken, 1.5% glucose in IX TB medium was fed to the cultures everyday. Unless noted otherwise, culture pH was adjusted using 10M NaOH on a daily basis.
  • Strain JCL299 bearing plasmids pELll, pIM8, and pCS138 was used in this fermentation. Overnight pre-culture was inoculated in LB containing the appropriate antibiotics and allowed to grow at 37°C in a rotary shaker (250 rpm) .
  • fermentation was allowed to proceed in batch mode until the anaerobic switch (around 17 hours) , and then intermittent lineal feeding of glucose solution (500 g/L) was initiated to avoid glucose depletion.
  • the pH was controlled at 6.8 by automatic addition of 2M NaOH solution.
  • Dissolved oxygen (DO) was maintained above 20% with respect to air saturation during aerobic stage by raising stirrer speed (from 200 to 600 rpm) . Fermentation samples were collected to determine cell growth, 1-butanol production, organic acids, and glucose concentration.
  • the cells were harvested by centrifugation at 15,000 rpm under room temperature. The pellets were then resuspended with 0.2 ml of the lysis buffer (50mM Tris-HCl at pH 7.5, 10X bugbuster and 1000X Lysonase) . Lysis was allowed to go for 10 to 20 minutes until the cell resuspension turned clear. The lysate was then centrifuged at 13,200 rpm for 20 minutes at 4°C. The supernatant was retrieved for the subsequent enzyme assays.
  • the lysis buffer 50mM Tris-HCl at pH 7.5, 10X bugbuster and 1000X Lysonase
  • AtoB assay All spectrophotometric assays were done using the Biotek microplate reader (model Powerwave XS) at 30°C. Reaction volume was 0.2 ml. Protein concentrations were determined using Bradford assay or Nanodrop 2000C spectrophotometer from Thermo Scientific.
  • AtoB activity was measured by monitoring the disappearance of acetoacetyl-CoA, corresponding to the thiolysis direction of the enzymatic reaction. The disappearance of
  • acetoacetyl-CoA was monitored by the decrease in absorbance at 303 nm which is the characteristic absorption band of an enolate complex formed by acetoacetyl-CoA with Mg 2+ .
  • the reaction mixture contained 100 mM Tris-HCl pH 8.0, 10 mM MgS0 4 , 200 ⁇ acetoacetyl- CoA, 200 ⁇ CoA, and cell extract prepared as described before. Standard curve was constructed by measuring absorbance of
  • Hbd assay The Hbd activity was measured by monitoring the decrease of absorption at 340 nm, corresponding to consumption of NADH.
  • the reaction mixture contained 100 mM 3- (N- morpholino) propanesulfonic acid (MOPS) pH 7.0, 200 ⁇ NADH, 200 ⁇ acetoacetyl-CoA, and the crude cell extract. The reaction was initiated by the addition of the cell extracts.
  • MOPS propanesulfonic acid
  • [00161] Crt assay The Crt activity was measured by the decrease of absorption at 263nm, corresponding to disruption of the ⁇ - ⁇ unsaturation of crotonyl-CoA .
  • the assay mixture contained 100 mM Tris-HCl pH 7.6, 100 ⁇ crotonyl-CoA, and the crude extract. The reaction was initiated by addition of the cell extracts.
  • the standard curve of crotonyl-CoA and 3-hydroxybutyryl-CoA was constructed by measuring the absorbance of the two compounds at 263 nm with different concentrations.
  • the Ter activity was measured at 340 nm.
  • the reaction mixture contained 100 mM potassium phosphate buffer pH 6.2, 200 ⁇ NADH, 200 ⁇ crotonyl-CoA and crude extract. The reaction was initiated by the addition of the extract.
  • AdhE2 assay The aldehyde and alcohol dehydrogenase activities of AdhE2 were measured by monitoring the decrease of absorbance at 340 nm corresponding to the consumption of NADH or NADPH.
  • the reaction mixture contained 100 mM Tris-HCl, pH 7.5, 5 mM dithiothreitol (DTT) , 300 ⁇ NADH, and 1 mM butyryl-CoA for the butyraldehyde dehydrogenase (BYDH) reaction and 50 mM butyraldehyde for the butanol dehydrogenase (BDH) reaction.
  • the reaction was initiated by the addition of the extract.
  • Lysis was allowed to go for 10 to 20 minutes at 60°C until the cell resuspension turned clear. The lysate was then centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was retrieved for subsequent NADH assays.
  • the cell lysates were mixed with the enzyme and the fluorescent detection reagent provided in the kit. The reaction was allowed to go for 1 - 1.5 hour under room temperature in dark. Then readings were taken with excitation at 530-570 nm and emission at 590-600nm.
  • Escherichia coli BW25113 (rrnB T14 AlacZ MJ16 hsdR514 AaraBAD AH33 ArhaBAD LD78 ) was designated as the wild- type.
  • XL-1 Blue (Stratagene, La Jolla, CA) was used to propagate all plasmids .
  • Construction of JCL16 (BW25113 with lacl q provided on F' ) , JCL166, and JCL299 was described previously (Atsumi, S. et al . Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10, 305-311 (2008)).
  • Plasmid construction Plasmid construction.
  • plasmid constructs were sequenced for verification by Genewiz.
  • a list of plasmids and primers used is shown in Table below.
  • Fdh from C. boidinii was amplified with primer fdhfacc65 and fdhrsal by PCR from genomic DNA, digested and cloned into an empty medium copy plasmid cut with the same restriction enzymes, resulting in pCS102.
  • pCS102 was digested by Avrll and Aatll and the piece containing the pl5A origin and Kan R was removed by gel purification and replaced by the pSClOl origin and Cm R cut with the same enzymes.
  • the resulting plasmid is pCS138.
  • Plasmid pELll was constructed by inserting the crt -hbd fragment into pJCL17.
  • the crt-hbd fragment was created by
  • crtfxba and crtrSOE individually amplifying crt with primers crtfxba and crtrSOE, and hbd with hbdfSOE and hbdrxba using pJCL60 as a template.
  • the two pieces were then connected together by SOE and further amplified by crtfxba and hbdrxba.
  • the resulting crt -hbd fragment and pJCL17 were both digested with Xba l and ligated to give pELll.
  • Fjo ter r sal acgcagtcgacTTATTTGATAGATTCGATATTAACAACTTCGT (62) pCS128 [00169]
  • the ter gene was amplified with PCR from the corresponding genomic DNA purchased from ATCC .
  • the resulting fragment of ter amplified with Tdeterfacc65 and Tdeterrsal was digested and ligated into pCS106 cut with Acc65I and Sail to yield pIM8.
  • Treponema vincentii which the genomic DNA was not available, the ter was amplified by colony PCR using commercially available polymerase kit ImmoMix-Red (Bioline) and the
  • acetobutylicum Bcd- EtfAB was cloned into pCS106 by amplification of the operon using primers CacBcdfacc65 and CacEtfBrsal from genomic DNA followed by digestion with Acc65I and Sail.
  • PCR-based gene synthesis The ter from Fibrobacter succinogenes and Flavobacterium j ohnsoniae were synthesized by PCR assembly of 50bp oligonucleotides. Oligo sequences were obtained by using automated oligonucleotide design from Helix Systems (NIH: http : ( / / ) helixweb . nih . gov/dnaworks/ ) . The protein sequences of the two ter genes were entered into Helix systems. The parameters were left as default. The codon frequency was selected for E.coli standard, and Acc65I and Sail restriction sites were added to the sequence for subsequent insertion into a vector plasmid. The oligos received were then mixed together into a super-mix and used in PCR reactions .
  • mutant libraries were mini-preped and co- transformed with pELll into JCL166.
  • the resulting cells were plated on LB 1% glucose with appropriate antibiotics and incubated at 37°C anaerobically for the next few days. Colonies were picked based on size, re-streaked on fresh LB glucose plates, then incubated anaerobically to isolate single colonies. Plasmids were retrieved from the potential positive cells, and Xbal was used to cut pELll, leaving behind only the plasmid containing the mutant ter. The digested mixture was again transformed into XL-1 Blue to obtain the pure mutant plasmid. Production and growth rescue tests were then repeated to confirm the validity of ter mutants.
  • Glucose level was detected with the YSI glucose analyzer 2700.
  • filtered supernatant was applied (0.02 ml) to an Agilent 1200 HPLC equipped with an auto- sampler (Agilent Technologies) and a BioRad (Biorad Laboratories, Hercules, CA) Aminex HPX87 column (0.5mM H 2 S0 4 , 0.6 mL/min, column temperature at 65 °C) .
  • Organic acids were detected using a
  • the NADH driving force could be established by deleting the mixed-acid fermentation reactions (ethanol, lactate, and succinate) in E. coli.
  • the resulting strain, JCL166 ⁇ /XadhE /XldhA IXfrd) lost its ability to grow anaerobically due to the lack of NADH-consuming pathways as an electron sink (Fig. lh) .
  • Such a strain is unable to recycle NADH, thereby creating a driving force for reactions that consume NADH. Since the Clostridial CoA- dependent pathway for 1-butanol synthesis is extremely reversible on both thermodynamic and enzymatic grounds and lacks the intrinsic driving forces found in other fermentation pathways, presence of an artificial driving force could be much more significant.
  • Ter trans-enoyl-CoA reductase
  • Treponema denticola which has been shown to possess significant specific activity towards crotonyl-CoA reduction using NADH as a direct reducing equivalent without the help of electron transfer flavoproteins or ferredoxin.
  • This NADH-dependent enzyme could potentially facilitate tighter coupling of 1-butanol production with the NADH driving force (Fig. lh) .
  • T. denticola Ter was utilized in the 1-butanol production pathway in E. coli as shown in a not-yet granted patent application 27 , only limited success was achieved with less than 0.25 g/L of 1-butanol accumulated in 72 hours. This result suggests that additional factors need to be considered.
  • acetyltransferase encoded by pta was also eliminated to help build an acetyl-CoA driving force coupled with the use of E. coli AtoB to provide a better link to the 1-butanol pathway.
  • NADH driving force created by a cell's inability to regenerate NAD + anaerobically without an external electron acceptor can also be used as a selection platform to improve enzymes and pathways that consume NADH.
  • NAD + anaerobically without an external electron acceptor can also be used as a selection platform to improve enzymes and pathways that consume NADH.
  • succinogenes supported minimal growth of JCL166 under anaerobic condition and yielded around 0.3 g/L of 1-butanol when grown aerobically followed by anaerobic fermentation (Fig 2) .
  • Fig 2 To demonstrate that the NADH surplus created in the host can be used as a selection pressure for optimizing the 1-butanol pathway, the three Ter homologues from T. vincentii, F. succinogenes, and F. johnsonia were subjected to error-prone PCR mutagenesis followed by co-transformation with plasmid pELll into JCL166. Anaerobic growth was then examined on LB with glucose plates incubated at 37°C for the next 1-3 days.
  • succinogenes Ter carry the identical amino acid substitution MetllLys in addition to other silent mutations that may have contributed to the optimization of protein expression and stability in the heterologous system. Importance of the single MetllLys substitution was confirmed by re-introducing this mutation using site-directed mutagenesis to the wild type F. succinogenes ter gene followed by anaerobic 1-butanol production using the resulting Ter variant. The success in the mutation and selection of Ter
  • homologues established the basis for strain and/or enzyme evolution of NADH-consuming pathways using this platform, even under incomplete NADH balance conditions .
  • the 1-butanol flux in E. coli has exceeded or achieved a comparable level to the Clostridium species in flasks and batch fermentors: productivity (0.2 g/L/hr) , titer (15 g/L in flasks, 30 g/L in fermentor) , and yield (approximately 88% of theoretical in flasks and 70% in fermentor) in un-optimized cultures of E. coli.
  • productivity 0.2 g/L/hr
  • titer 15 g/L in flasks, 30 g/L in fermentor
  • yield approximately 88% of theoretical in flasks and 70% in fermentor

Abstract

L'invention concerne des microorganismes qui catalysent la synthèse de biocarburants à partir d'un substrat approprié tel que le glucose. L'invention concerne également des procédés de génération de tels organismes et des procédés de synthèse de biocarburants à l'aide de tels organismes. L'invention concerne des microorganismes comportant une voie métabolique d'origine non naturelle pour la production d'alcools supérieurs.
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US9777297B2 (en) 2011-11-03 2017-10-03 Easel Biotechnologies, Llc Microbial production of n-butyraldehyde
EP2773761B1 (fr) * 2011-11-03 2019-03-06 Easel Biotechnologies, LLC Production microbienne de n-butyraldéhyde
EP3187590A4 (fr) * 2014-08-04 2018-01-24 Tokyo Institute of Technology Procédé de production d'un copolymère de polyhydroxyalcanoate à partir d'une matière première saccharidique
US10538791B2 (en) 2014-08-04 2020-01-21 Tokyo Institute Of Technology Method for producing polyhydroxyalkanoate copolymer from saccharide raw material
WO2016137976A1 (fr) * 2015-02-23 2016-09-01 Biocheminsights, Inc. Module de bioréacteur électrochimique et voies métaboliques modifiées pour la production de 1-butanol avec un rendement carbone élevé
CN107849584A (zh) * 2015-02-23 2018-03-27 百奥堪引赛股份有限公司 以高碳效率生产1‑丁醇的电化学生物反应器模块和工程代谢路径
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WO2018056794A1 (fr) * 2016-09-26 2018-03-29 한국생명공학연구원 Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone
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TWI622648B (zh) * 2016-12-27 2018-05-01 國立清華大學 正丁醇表現匣、重組質體及正丁醇生產相關基因的表現方法
CN108641973A (zh) * 2018-04-03 2018-10-12 中海石油(中国)有限公司湛江分公司 一株产聚羟基烷酸酯的海洋细菌及其应用
CN114933997A (zh) * 2022-06-13 2022-08-23 中国热带农业科学院热带生物技术研究所 一株珊瑚来源的共生链霉菌sh001及其应用
CN114933997B (zh) * 2022-06-13 2023-06-23 中国热带农业科学院热带生物技术研究所 一株珊瑚来源的共生链霉菌sh001及其应用

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