US20110236941A1 - Recombinant microorganism and methods of production thereof - Google Patents

Recombinant microorganism and methods of production thereof Download PDF

Info

Publication number
US20110236941A1
US20110236941A1 US13/049,263 US201113049263A US2011236941A1 US 20110236941 A1 US20110236941 A1 US 20110236941A1 US 201113049263 A US201113049263 A US 201113049263A US 2011236941 A1 US2011236941 A1 US 2011236941A1
Authority
US
United States
Prior art keywords
butanol
dehydrogenase
microorganism
seq
clostridium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/049,263
Other languages
English (en)
Inventor
Michael Koepke
Fungmin Liew
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Venture lending and Leasing VI Inc
Lanzatech NZ Inc
Original Assignee
Lanzatech New Zealand Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lanzatech New Zealand Ltd filed Critical Lanzatech New Zealand Ltd
Priority to US13/049,263 priority Critical patent/US20110236941A1/en
Assigned to LANZATECH NEW ZEALAND LIMITED reassignment LANZATECH NEW ZEALAND LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LIEW, FUNGMING, KOEPKE, MICHAEL
Priority to CN201180062114.3A priority patent/CN103339261B/zh
Priority to KR1020137012815A priority patent/KR20130079599A/ko
Priority to EA201390444A priority patent/EA028760B1/ru
Priority to NZ609150A priority patent/NZ609150A/en
Priority to AU2011318676A priority patent/AU2011318676B2/en
Priority to BR112013009552-0A priority patent/BR112013009552B1/pt
Priority to MYPI2013700647A priority patent/MY157111A/en
Priority to ES11834686.5T priority patent/ES2626448T3/es
Priority to PCT/NZ2011/000203 priority patent/WO2012053905A1/en
Publication of US20110236941A1 publication Critical patent/US20110236941A1/en
Priority to JP2013534846A priority patent/JP6014042B2/ja
Priority to EP11834686.5A priority patent/EP2630247B1/en
Priority to CA2813431A priority patent/CA2813431C/en
Priority to DK11834686.5T priority patent/DK2630247T3/en
Priority to KR1020147000771A priority patent/KR101640325B1/ko
Assigned to VENTURE LENDING & LEASING VI, INC. reassignment VENTURE LENDING & LEASING VI, INC. SECURITY AGREEMENT Assignors: LANZATECH FREEDOM PINES BIOREFINERY LLC, LANZATECH HONG KONG LIMITED, LANZATECH NEW ZEALAND LIMITED, LANZATECH PRIVATE LIMITED, LANZATECH, INC.
Assigned to LANZATECH NEW ZEALAND LIMITED reassignment LANZATECH NEW ZEALAND LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SIMPSON, SEAN DENNIS
Priority to ZA2013/02898A priority patent/ZA201302898B/en
Priority to US14/180,423 priority patent/US9359611B2/en
Assigned to LANZATECH, INC., LANZATECH FREEDOM PINES BIOREFINERY LLC, LANZATECH NEW ZEALAND, LANZATECH PRIVATE LIMITED, LANZATECH HONG KONG LIMITED reassignment LANZATECH, INC. RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: VENTURE LENDING & LEASING VI, INC.
Assigned to LANZATECH NZ, INC. reassignment LANZATECH NZ, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: LANZATECH NEW ZEALAND LIMITED
Assigned to LANZATECH, INC., LANZATECH HONG KONG LIMITED, LANZATECH NEW ZEALAND, LANZATECH FREEDOM PINES BIOREFINERY LLC, LANZATECH PRIVATE LIMITED reassignment LANZATECH, INC. CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: VENTURE LENDING & LEASING VI, INC.
Assigned to VENTURE LENDING & LEASING VI, INC. reassignment VENTURE LENDING & LEASING VI, INC. CORRECTIVE ASSIGNMENT TO CORRECT THE THE U.S. APPLICATION NUMBER 13497,969 PREVIOUSLY RECORD PREVIOUSLY RECORDED AT REEL: 028991 FRAME: 0471. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: LANZATECH PRIVATE LIMITED, LANZATECH FREEDOM PINES BIOREFINERY LLC, LANZATECH HONG KONG LIMITED, LANZATECH NEW ZEALAND LIMITED, LANZATECH, INC.
Assigned to LANZATECH NZ, INC. reassignment LANZATECH NZ, INC. CORRECTIVE ASSIGNMENT TO CORRECT THE U.S. PATENT NUMBER 8,979,228 PREVIOUSLY RECORDED AT REEL: 059911 FRAME: 0400. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: LANZATECH NEW ZEALAND LIMITED
Assigned to LANZATECH NZ INC. reassignment LANZATECH NZ INC. CORRECTIVE ASSIGNMENT TO CORRECT THE THE PATENT NUMBE 9,5348,20 PREVIOUSLY RECORDED AT REEL: 059911 FRAME: 0400. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: LANZATECH NEW ZEALAND LIMITED
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • 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
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • the present disclosure relates to methods for the production of biofuels by microbial fermentation and genetically modified micro-organisms suitable for use in such methods.
  • Butanol is an important bulk chemical with a wide range of industrial uses that has worldwide production of 4.5-5.5 million tonnes per annum. It is used as a precursor for the production of acrylate and methacrylate esters (used in coatings, plastics, textiles, adhesives, etc), glycol ethers (coatings, electronics) and butyl acetate (paints, ink, coatings, synthetic fruit flavoring) as well as butylamines (production of pesticides and pharmaceuticals) and amine resins. It also has direct use as a solvent (in ink, dyes, etc), an extractant (for the production of drugs and natural substances such as alkaloids, antibiotics, hormones, and vitamins), and in deicing fluids, cosmetics and chromatography.
  • solvent in ink, dyes, etc
  • an extractant for the production of drugs and natural substances such as alkaloids, antibiotics, hormones, and vitamins
  • Butanol also has potential as a second generation biofuel, and in this context is referred to as Biobutanol (Köpke & Dürre, 2010). It has similar properties to gasoline and superior properties to ethanol. Specifically, it has increased mileage due to higher energy density, it can be mixed with gasoline in any concentration (while ethanol can only be blended up to 85%) and is not hygroscopic or corrosive.
  • Biofuels for transportation are attractive replacements for gasoline and are rapidly penetrating fuel markets as low concentration blends.
  • Biofuels derived from natural plant sources, are more environmentally sustainable than those derived from fossil resources (such as gasoline), their use allowing a reduction in the levels of so-called fossil carbon dioxide (CO 2 ) gas that is released into the atmosphere as a result of fuel combustion.
  • CO 2 fossil carbon dioxide
  • biofuels can be produced locally in many geographies, and can act to reduce dependence on imported fossil energy resources.
  • first generation biofuels The vast majority of biofuels are produced via traditional yeast-based fermentation processes that use crop derived carbohydrates as the main carbon source and are known as first generation biofuels. However, these crops are required for food and many crops also require high agricultural inputs in the form of fertilizers. These limitations mean that first generation biofuels are considered unsustainable and the greenhouse gas reductions that can be achieved are limited.
  • the aim of second generation biofuels is the sustainable use of non-food parts of current crops or other industrial waste to reduce greenhouse gas emissions and reduce dependency on fossil fuels.
  • Recent 1-butanol production has been mainly by oxo synthesis (Wei ⁇ ermel & Arpe, 2003).
  • Petrochemicals including crude oil are cracked to form propylene which is used during oxo synthesis.
  • the synthesis process requires use of non-renewable resources as well as suffering from being expensive and non-specific in the products formed.
  • Butanol can also be produced through biological production methods, the most common being the Acetone-Butanol-Ethanol (ABE) fermentation which has been used industrially since 1913 (Köpke & Dürre, 2010).
  • ABE Acetone-Butanol-Ethanol
  • This method has the unwanted by-product of acetone which is usually produced at about half the volume of butanol which therefore substantially reduces the yield.
  • this method of fermentation is limited by the toxicity of butanol to the micro-organism which results in growth being almost completely inhibited at such low butanol concentrations as 1.5% (Köpke and Dürre 2010).
  • ABE fermentation uses sugar from corn, starch, cassaya and sugar cane as a feedstock. This results in the undesirable use of arable land to produce fuel rather than food. It can also exacerbate problems related to deforestation and desertification.
  • Clostridia A number of organisms have been genetically modified to produce 1-butanol including E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Pseudomonas putida , or Lactobacillus brevis . However all of these organisms still rely on sugar as feedstock (Köpke & Dürre, 2010). Despite over 250 Clostridium species being known, only a few are genetically accessible. There is no natural competence (uptake of extracellular DNA from the cell's environment) known in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues present significant difficulties in effectively transforming Clostridia species. Most Clostridia have one or more restriction/methylation systems to protect against foreign and phage DNA which means that transformation is particularly difficult and unpredictable.
  • a genetically modified microorganism is capable of using CO to produce 1-butanol or a precursor thereof as the main fermentation product.
  • the invention provides an acetogenic recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides an acetogenic recombinant microorganism which is capable of producing 1-butanol and/or a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 g/l per litre of fermentation broth.
  • the microorganism comprises exogenous nucleic acids adapted to express one or more enzymes in the butanol biosynthesis pathway.
  • the one or more enzymes are chosen from the group consisting of: Thiolase 3-hydroxybutyryl-CoA dehydrogenase, Crotonase/crotonyl-CoA hydratase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, and Electron Transfer Flavoprotein B
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes.
  • the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.
  • the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
  • the microorganism comprises a plasmid encoding one or more of, or preferably each of, Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
  • the microorganism comprises one or more exogenous nucleic acids encoding each of the enzymes thiolase 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase and butyryl-CoA dehydrogenase.
  • the microorganism further comprises an exogenous phosphotransacetylase/acetate kinase promoter.
  • the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
  • the promoter is contained on a construct encoding one or more of the enzymes referred to herein before.
  • the microorganism comprises exogenous nucleic acids adapted to express one or more of the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase; butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase and butanol dehydrogenase.
  • the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase; butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase and butanol dehydrogenase.
  • the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the microorganism comprises exogenous nucleic acids adapted to express one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism comprises exogenous nucleic acids adapted to express each of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids outlined in tables 7 to 10 herein after and functionally equivalent variants thereof.
  • the microorganism comprises one or more nucleic acid adapted to express at least two of the enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.
  • the microorganism is selected from the group of acetogenic bacteria.
  • the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii , and Thermoanaerobacter kiuvi.
  • the microorganism is Clostridium autoethanogenum DSM23693.
  • the recombinant microorganism of the invention has the defining characteristics of the microorganism deposited at the DSMZ (Deutsche Sammlung für Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) under the accession number DSM24138.
  • the invention provides a recombinant methyltransferase gene according to nucleotide SEQ_ID NO 27 or a functionally equivalent variant thereof.
  • the invention provides a methyltransferase according to SEQ_ID NO 28 or a functionally equivalent amino acid variant thereof.
  • the invention provides a recombinant microorganism comprising a methyltransferase gene according to the second aspect.
  • the methyltransferase gene may be present on a nucleic acid construct or integrated into the genome of the microorganism.
  • the invention provides a nucleic acid comprising SEQ_ID No 1 to 6, or functionally equivalent variants thereof, in any order.
  • the nucleic acid comprises SEQ_ID No 1 to 6 in the order shown in FIG. 2 .
  • the nucleic acid further comprises a phosphotransacetylase/acetate kinase promoter.
  • the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
  • the invention provides an expression construct comprising one or more nucleic acid sequences wherein the construct, when expressed in an acetogenic microorganism, results in 1-butanol and/or a precursor thereof being produced as the main fermentation product.
  • the one or more nucleic acid sequences encode one or more enzymes that are part of the 1-butanol biosynthesis pathway.
  • the nucleic acids are selected from nucleic acids encoding thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, electron transfer flavoprotein A and/or electron transfer flavoprotein B.
  • the one or more nucleic acid sequences are selected from SEQ_ID NO. 1 to SEQ_ID NO. 6 or functionally equivalent variants thereof.
  • the nucleic acids are further selected from nucleic acids encoding Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the nucleic acids are selected from the group of nucleic acids outlined in tables 7 to 10 herein after and functionally equivalent variants thereof.
  • the expression construct encodes at least 2 enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 of the enzymes.
  • the expression construct further comprises a phosphotransacetylase/acetate kinase operon promoter.
  • the expression construct comprises another highly active promoter such as the promoter of the pyruvate:ferredoxin oxidoreductase (SEQ_ID No. 48), the Wood-Ljungdahl gene cluster (SEQ_ID No 47), Rnf operon (SEQ_ID No 49) or the ATP synthase operon (SEQ_ID No 50).
  • the phosphotransacetylase/acetate kinase operon promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
  • the invention provides a methylation construct comprising a methyltransferase gene as described herein.
  • the invention provides a composition comprising the expression construct of the fifth aspect and the methylation construct of the sixth aspect.
  • the composition is able to produce a recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
  • both the methylation construct and the expression construct are isolated in step C.
  • the expression construct is isolated in step C.
  • only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
  • the expression construct is as defined in the fifth aspect.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • the expression construct is as defined in the fifth aspect.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the methyltransferase is produced by expressing a methyltransferase gene, preferably according to SEQ_ID No 27 or a functionally equivalent variant thereof, in a microorganism and isolating the methyltransferase enzyme.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • the expression construct is as defined in the fifth aspect.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • the methyltransferase is encoded by a methyltransferase gene as defined in the second aspect or a methyltransferase as defined in the third aspect.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
  • both the methylation construct and the expression construct are isolated in step C.
  • the expression construct is isolated in step C.
  • only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism that produces 1-butanol or a precursor thereof as the main fermentation product comprising:
  • expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
  • both the methylation construct and the expression construct are isolated in step C.
  • the expression construct is isolated in step C.
  • only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
  • the expression construct is as defined in the fifth aspect.
  • the methylation construct is as defined in the sixth aspect.
  • the invention provides a method of production of 1-butanol and/or a precursor thereof by microbial fermentation comprising fermenting a substrate using a recombinant microorganism.
  • 1-butanol and/or a precursor thereof is the main fermentation product.
  • the recombinant microorganism is as described in any one of the eighth to the tenth aspects.
  • 1-butanol and/or a precursor thereof is produced in a yield of from approximately 0.075 grams per litre of fermentation broth (g/l) to approximately 20 g/l. In one embodiment, the yield is from approximately 0.15 g/l to approximately 1.54 g11. In other embodiments, the yield is approximately 10 g/l, approximately 5 g/l, or approximately 2 g/l. Preferably, the yield of 1-butanol is up to the limit at which butanol becomes toxic to the surrounding media.
  • the substrate comprises CO.
  • the substrate is a gaseous substrate comprising CO.
  • the substrate comprises an industrial waste gas.
  • the gas is steel mill waste gas or syngas.
  • the substrate will typically contain a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume.
  • the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
  • the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H2:CO.
  • the substrate comprises about 30% or less H 2 by volume, 20% or less H 2 by volume, about 15% or less H 2 by volume or about 10% or less H 2 by volume.
  • the substrate stream comprises low concentrations of H2, for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free.
  • the substrate may also contain some CO 2 for example, such as about 1% to about 80% CO 2 by volume, or 1% to about 30% CO 2 by volume.
  • the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
  • the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the microorganism produces butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehyrogenase/butanol deydrogenase before and after introduction of an exogenous nucleic acid.
  • the invention provides 1-butanol or a precursor thereof when produced by the method of the eleventh aspect.
  • the invention provides a shuttle microorganism comprising a methylation construct as defined herein.
  • the shuttle microorganism further comprises an expression construct as defined herein.
  • the shuttle microorganism is E. coli or Bacillus subtillis.
  • the methylation construct of any of the previous aspects comprises a lac promoter and the methyltransferase gene and is induced by Isopropyl- ⁇ -D-thio-galactoside (IPTG).
  • IPTG Isopropyl- ⁇ -D-thio-galactoside
  • Expression of the methyltransferase could also be controlled by other inducible promoter systems such as ara, tet, or T7.
  • the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 8 to 13.
  • the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 16 to 23.
  • the invention provides a nucleic acid comprising at least the nucleic acid sequence of SEQ ID No. 7 or a functionally equivalent variant thereof, a nucleic acid construct or vector comprising same, and microorganisms comprising said nucleic acid or nucleic acid construct or vector.
  • the invention provides a nucleic acid which encodes a methyltransferase according to SEQ_ID No 28.
  • the invention provides a nucleic acid comprising a nucleic acid encoding a polypeptide having the amino acid sequence of a polypeptide chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
  • the invention provides a nucleic acid comprising a nucleic acid chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
  • the invention provides constructs and microorganisms comprising a nucleic acid of the eighteenth or nineteenth aspects of the invention.
  • the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 32 to 38 and 123 to 135.
  • the invention provides a polypeptide comprising the amino acid sequence of a polypeptide chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • FIG. 1 shows the butanol biosynthesis pathway from CO.
  • FIG. 2 shows an exemplary expression plasmid encoding genes involved in 1-butanol biosynthesis.
  • FIG. 3 shows sequencing results of pMTL85245-thlA-crt-hbd which demonstrate that the 1-butanol biosynthesis genes found on the expression plasmid were free of mutations.
  • FIGS. 4 a , 4 b and 4 c show a nucleotide alignment of the C. autoethanogenum (CAU), C. ljungdahlii (CLJ), C. ragsdalei (CRA) and the designed methyltransferase (DMT) genes.
  • CAU C. autoethanogenum
  • CLJ C. ljungdahlii
  • CRA C. ragsdalei
  • DMT methyltransferase
  • FIG. 4 d shows an amino acid alignment of the methyltransferases from C. autoethanogenum (CAU1+2), C. ljungdahlii (CLJ), C. ragsdalei (CRA1+2) and the designed methyltransferase (DMT).
  • FIG. 5 shows an exemplary methylation plasmid of the invention
  • FIG. 6 shows an agarose gel electrophoresis image of isolated plasmid DNA.
  • Lane 1, 6, 11, 16, 21 and 26 show 100 by Plus DNA Ladder.
  • Lane 2-5 shows PCR with original methylated plasmid mix as template in the following order: ermB, ColE1, thlA, crt.
  • Lane 7-10, 12-15, 17-20, 22-25 and 27-30 show PCR with isolated plasmids from 4 different clones as template, each in the following order ermB, ColE1, thlA, crt.
  • Lane 32-35 shows plasmid prep from 4 different clones.
  • Lane 36 shows plasmid prep from original C. autoethanogenum DSM23693.
  • FIG. 7 shows HPLC results showing 1-butanol production with C. autoethanogenum harboring butanol plasmid pMTL85245-thlA-crt-hbd.
  • FIG. 8 shows an analysis of expression of over 200 genes during a typical fermentation with Clostridium autoethanogenum at standard conditions using real-time PCR to identify appropriate promoter regions for the expression of heterologous genes.
  • FIG. 9 shows the sequence for SEQ_ID No 1, 2 and 3.
  • FIG. 10 shows the sequence for SEQ_ID No 4, 5 and 6.
  • FIG. 11 shows the sequence for promoter regions encoded by SEQ_ID No 7, 47, 48, 49 and 50.
  • FIG. 12 shows the sequence for SEQ_ID No 14
  • FIG. 13 shows the sequence for SEQ_ID No 15
  • FIG. 14 shows the sequence for SEQ_ID No 24 and 25
  • FIG. 15 shows the sequence for SEQ_ID No 26
  • FIG. 16 shows the sequence for SEQ_ID No 27
  • FIG. 17 shows the sequence for SEQ_ID No 28
  • FIG. 18 shows the sequence for SEQ_ID No 29
  • FIG. 19 shows the 16s rRNA gene of C. autoethanogenum (Y18178, GI:7271109)
  • FIGS. 20 and 21 show the sequence for SEQ_ID No 31
  • FIG. 22 shows Seq. ID 39: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. autoethanogenum
  • FIG. 23 shows Seq. ID 40: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. autoethanogenum
  • FIG. 24 shows Seq. ID 41: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum ; and, Seq. ID 42: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
  • FIG. 25 shows Seq. ID 43: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum ; and, Seq. ID 44: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
  • FIG. 26 shows Seq. ID 45: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
  • FIG. 27 shows Seq. ID 46: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum ; and, Seq. ID 119: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
  • FIG. 28 shows Seq. ID 120: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and Seq. ID 121: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
  • FIG. 29 shows Seq. ID 122: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and, Seq. ID 51: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
  • FIG. 30 shows Seq. ID 52: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and, Seq. ID 53: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
  • FIG. 31 shows Seq. ID 54: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and, Seq. ID 55: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
  • FIG. 32 shows Seq. ID 56: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and, Seq. ID 57: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
  • FIG. 33 shows Seq. ID 58: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum ; and Seq. ID 59: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum ; and Seq. ID 60: Amino acid sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum.
  • FIG. 34 shows Seq. ID 61: Nucleotide sequence of acetate/butyrate kinase from C. autoethanogenum ; and Seq. ID 62: Amino acid sequence of acetate/butyrate kinase from C. autoethanogenum.
  • FIG. 35 shows Seq. ID 63: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum ; and Seq. ID 64: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum.
  • FIG. 36 shows Seq. ID 65: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum ; and Seq. ID 66: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum.
  • FIG. 37 shows Seq. ID 67: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
  • FIG. 38 shows Seq. ID 68: Amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
  • FIG. 39 shows Seq. ID 69: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
  • FIG. 40 shows Seq. ID 70: Amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii ; and Seq. ID 71: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii.
  • FIG. 41 shows Seq. ID 72: Amino acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii ; and Seq. ID 73: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii ; and Seq. ID 74: Amino acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii.
  • FIG. 42 shows Seq. ID 75: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 76: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 77: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii.
  • FIG. 43 shows Seq. ID 78: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 79: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 80: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii.
  • FIG. 44 shows Seq. ID 81: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 82: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 83: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii.
  • FIG. 45 shows Seq. ID 84: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii ; and Seq. ID 85: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. ljungdahlii ; and Seq. ID 86: Amino acid sequence of phosphate acetyl/butyryl transferase from C. ljungdahlii ; and Seq. ID 87: Nucleotide sequence of acetate/butyrate kinase from C. ljungdahlii.
  • FIG. 46 shows Seq. ID 88: Amino acid sequence of acetate/butyrate kinase from C. ljungdahlii ; and Seq. ID 89: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii ; and Seq. ID 90: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii.
  • FIG. 47 shows Seq. ID 91: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii ; and Seq. ID 92: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii.
  • FIG. 48 shows Seq. ID 93: Nucleotide Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei
  • FIG. 49 shows Seq. ID 94: Amino Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei
  • FIG. 50 shows Seq. ID 95: Nucleotide Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei.
  • FIG. 51 shows Seq. ID 96: Amino Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei ; and Seq. ID 97: Nucleotide Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei.
  • FIG. 52 shows Seq. ID 98: Amino Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei ; Seq. ID 99: Nucleotide Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei ; and Seq. ID 100: Amino Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei.
  • FIG. 53 shows Seq. ID 101: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 102: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 103: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei.
  • FIG. 54 shows Seq. ID 104: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 105: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 106: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei:
  • FIG. 55 shows Seq. ID 107: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 108: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 109: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei.
  • FIG. 56 shows Seq. ID 110: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei ; and Seq. ID 111: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. ragsdalei ; and Seq. ID 112: Amino acid sequence of phosphate acetyl/butyryl transferase from C. ragsdalei ; and Seq. ID 113: Nucleotide sequence of acetate/butyrate kinase from C. ragsdalei.
  • FIG. 57 shows Seq. ID 114: Amino acid sequence of acetate/butyrate kinase from C. ragsdalei ; and Seq. ID 115: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei ; and Seq. ID 116: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei.
  • FIG. 58 shows Seq. ID 117: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei ; and Seq. ID 118: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei.
  • the inventors have found that when particular genes encoding proteins in the 1-butanol biosynthesis pathway ( FIG. 1 ) were introduced into acetogenic microorganisms, such microorganisms were able to use a gaseous substrate to produce 1-butanol or a precursor thereof as the main fermentation product. Although some unmodified microorganisms are known to produce 1-butanol, the yield of 1-butanol from CO produced by such unmodified microorganisms is very low. As a result, their utility for production of biofuels from gaseous substrates is extremely limited due to their low efficiency and a subsequent lack of commercial viability. Clostridium autoethanogenum naturally produces ethanol, acetate, 2,3-butandiol and lactic acid but is not known to produce 1-butanol.
  • the Wood-Ljungdahl pathway converts CO to acetyl-CoA.
  • This compound may be further converted to 1-butanol in acetogenic microorganisms by the action of the enzymes thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase and butanol dehydrogenase.
  • the microorganism expresses the first four enzymes which may be encoded by the nucleic acid SEQ_ID Nos 1 to 4 or functionally equivalent variants thereof.
  • the present invention provides a microorganism that facilitates the conversion of acetyl-CoA to 1-butanol by the action of enzymes encoded by recombinant nucleic acids as well as naturally occurring enzymes.
  • the invention also provides for the use of microorganisms expressing other recombinant nucleic acid sequences which encode enzymes at other stages in the Wood-Ljungdahl or butanol biosynthesis pathways.
  • the inventors have also identified a number of novel enzymes and nucleic acids.
  • Clostridia Since there is no natural competence (uptake of extracellular DNA from the cell's environment) known in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues present significant difficulties in effectively transforming Clostridium species. Additionally, the restriction/methylation systems found in Clostridia protect against foreign and phage DNA and result in their genetic transformation being particularly troublesome. Transformation of several Clostridium strains ( C. acetobutylicum ATCC824 , C. cellulolyticum ATCC35319, C. botulinum ATCC25765, and C. difficile CD3 and CD6) was shown to be only possible if DNA is methylated in vivo in E.
  • a novel methylation system comprising a novel methyltransferase gene was developed to circumvent the naturally occurring restriction barriers present in native acetogenic microorganisms. Accordingly, the methylation method and methyltransferase gene of the present invention may be applied to a number of compatible microorganisms that have restriction barriers preventing effective introduction and expression of desirable recombinant nucleic acids in microorganisms.
  • precursors of 1-butanol include butyryl CoA, butyryl-phosphate, butyrate, and butyraldehyde.
  • a “fermentation broth” is a culture medium comprising at least a nutrient media and bacterial cells.
  • a “shuttle microorganism” is a microorganism in which a methyltransferase enzyme is expressed and is distinct from the destination microorganism.
  • a “destination microorganism” is a microorganism in which the genes included on the expression construct are expressed and is distinct from the shuttle microorganism.
  • main fermentation product is intended to mean the one fermentation product which is produced in the highest concentration and/or yield.
  • increasing the efficiency when used in relation to a fermentation process, include, but are not limited to, increasing one or more of the rate of growth of microorganisms catalysing the fermentation, the volume of desired product (such as alcohols) produced per volume of substrate (such as sugar) consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.
  • desired product such as alcohols
  • substrate such as sugar
  • substrate comprising carbon monoxide and like terms should be understood to include any substrate in which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.
  • gaseous substrate comprising carbon monoxide includes any gas which contains a level of carbon monoxide.
  • the substrate contains at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume.
  • the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
  • the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H2:CO.
  • the substrate comprises about 30% or less H 2 by volume, 20% or less H 2 by volume, about 15% or less H 2 by volume or about 10% or less H 2 by volume.
  • the substrate stream comprises low concentrations of H2, for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free.
  • the substrate may also contain some CO 2 for example, such as about 1% to about 80% CO 2 by volume, or 1% to about 30% CO 2 by volume. In one embodiment the substrate comprises less than or equal to about 20% CO 2 by volume. In particular embodiments the substrate comprises less than or equal to about 15% CO 2 by volume, less than or equal to about 10% CO 2 by volume, less than or equal to about 5% CO 2 by volume or substantially no CO 2 .
  • the gaseous substrate may be provided in alternative forms.
  • the gaseous substrate containing CO may be provided dissolved in a liquid.
  • a liquid is saturated with a carbon monoxide containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology.
  • a microbubble dispersion generator Hensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002
  • the gaseous substrate containing CO may be adsorbed onto a solid support.
  • Such alternative methods are encompassed by use of the term “substrate containing CO” and the like.
  • the CO-containing gaseous substrate is an industrial off or waste gas.
  • “Industrial waste or off gases” should be taken broadly to include any gases comprising CO produced by an industrial process and include gases produced as a result of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, and coke manufacturing. Further examples may be provided elsewhere herein.
  • the phrases “fermenting”, “fermentation process” or “fermentation reaction” and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process.
  • the bioreactor may comprise a first growth reactor and a second fermentation reactor.
  • the addition of metals or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.
  • bioreactor includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.
  • CSTR Continuous Stirred Tank Reactor
  • ICR Immobilized Cell Reactor
  • TBR Trickle Bed Reactor
  • Bubble Column Gas Lift Fermenter
  • Static Mixer Static Mixer
  • Exogenous nucleic acids are nucleic acids which originate outside of the microorganism to which they are introduced. Exogenous nucleic acids may be derived from any appropriate source, including, but not limited to, the microorganism to which they are to be introduced, strains or species of microorganisms which differ from the organism to which they are to be introduced, or they may be artificially or recombinantly created. In one embodiment, the exogenous nucleic acids represent nucleic acid sequences naturally present within the microorganism to which they are to be introduced, and they are introduced to increase expression of or over-express a particular gene (for example, by increasing the copy number of the sequence (for example a gene)).
  • the exogenous nucleic acids represent nucleic acid sequences not naturally present within the microorganism to which they are to be introduced and allow for the expression of a product not naturally present within the microorganism or increased expression of a gene native to the microorganism (for example in the case of introduction of a regulatory element such as a promoter).
  • the exogenous nucleic acid may be adapted to integrate into the genome of the microorganism to which it is to be introduced or to remain in an extra-chromosomal state.
  • nucleic acids whose sequence varies from the sequences specifically exemplified herein provided they perform substantially the same function.
  • nucleic acid sequences that encode a protein or peptide this means that the encoded protein or peptide has substantially the same function.
  • nucleic acid sequences that represent promoter sequences the variant sequence will have the ability to promote expression of one or more genes.
  • Such nucleic acids may be referred to herein as “functionally equivalent variants”.
  • functionally equivalent variants of a nucleic acid include allelic variants, fragments of a gene, genes which include mutations (deletion, insertion, nucleotide substitutions and the like) and/or polymorphisms and the like.
  • Homologous genes from other bacteria capable of butyric acid or butanol fermentation may also be considered as examples of functionally equivalent variants of the sequences specifically exemplified herein. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium tetani, Clostridium pasteurianum, Clostridium kluyveri, Clostridium cellulovorans, Clostridium perfringens, Clostridium botulinum, Clostridium butyricum strain DSM10702, Clostridium tyrobutyricum strain ATCC 25755 , Anaerococcus prevotii DSM 20548, Thermoanaerobacter tengcongensis, Brachyspira pilosicoli, Bacillus megaterium, Streptococcus pyogenes and Clostridium saccharoperbutylacetonicum details of which are publicly
  • “functionally equivalent variants” should also be taken to include nucleic acids whose sequence varies as a result of codon optimisation for a particular organism. “Functionally equivalent variants” of a nucleic acid herein will preferably have at least approximately 70%, preferably approximately 80%, more preferably approximately 85%, preferably approximately 90%, preferably approximately 95% or greater nucleic acid sequence identity with the nucleic acid identified.
  • a functionally equivalent variant of a protein or a peptide includes those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or greater amino acid identity with the protein or peptide identified and has substantially the same function as the peptide or protein of interest.
  • variants include within their scope fragments of a protein or peptide wherein the fragment comprises a truncated form of the polypeptide wherein deletions may be from Ito 5, to 10, to 15, to 20, to 25 amino acids, and may extend from residue 1 through 25 at either terminus of the polypeptide, and wherein deletions may be of any length within the region; or may be at an internal location.
  • Functionally equivalent variants of the specific polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria, for example as exemplified in the previous paragraph.
  • “Substantially the same function” as used herein is intended to mean that the nucleic acid or polypeptide is able to perform the function of the nucleic acid or polypeptide of which it is a variant.
  • a variant of an enzyme of the invention will be able to catalyse the same reaction as that enzyme.
  • the variant has the same level of activity as the polypeptide or nucleic acid of which it is a variant.
  • “Over-express”, “over expression” and like terms and phrases when used in relation to the invention should be taken broadly to include any increase in expression of one or more protein as compared to the expression level of the protein of a parental microorganism under the same conditions. It should not be taken to mean that the protein is expressed at any particular level.
  • a “parental microorganism” is a microorganism used to generate a recombinant microorganism of the invention.
  • the parental microorganism may be one that occurs in nature (ie a wild type microorganism) or one that has been previously modified but which does not express or over-express one or more of the enzymes the subject of the present invention. Accordingly, the recombinant microorganisms of the invention have been modified to express or over-express one or more enzymes that were not expressed or over-expressed in the parental microorganism.
  • nucleic acid “constructs” or “vectors” and like terms should be taken broadly to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle to transfer genetic material into a cell.
  • the terms should be taken to include plasmids, viruses (including bacteriophage), cosmids and artificial chromosomes.
  • Constructs or vectors may include one or more regulatory elements, an origin of replication, a multicloning site and/or a selectable marker, among other elements, sites and markers.
  • the constructs or vectors are adapted to allow expression of one or more genes encoded by the construct or vector.
  • Nucleic acid constructs or vectors include naked nucleic acids as well as nucleic acids formulated with one or more agents to facilitate delivery to a cell (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).
  • nucleic acids of the invention may be in any appropriate form, including RNA, DNA, or cDNA, including double-stranded and single-stranded nucleic acids.
  • the invention provides genetically modified microorganisms capable of using CO to produce 1-butanol and/or a precursor thereof as the main fermentation product.
  • the microorganism is preferably an acetogenic recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the acetogenic recombinant microorganism is capable of producing 1-butanol or a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 g/l of butanol per litre of fermentation broth.
  • the microorganism comprises one or more exogenous nucleic acid adapted to express or over-express one or more enzymes in the butanol biosynthesis pathway.
  • the microorganism is adapted to express one or more enzyme in the butanol biosynthesis pathway which is not naturally present in the parental microorganism from which it is derived, or to over-express one or more enzyme in the butanol biosynthesis pathway which are naturally present in the parental microorganism.
  • the microorganism may be adapted to express or over-express the one or more enzymes by any number of recombinant methods including, for example, increasing expression of native genes within the microorganism (for example, by introducing a stronger or constitutive promoter to drive expression of a gene), increasing the copy number of a gene encoding a particular enzyme by introducing exogenous nucleic acids encoding and adapted to express the enzyme, introducing an exogenous nucleic acid encoding and adapted to express an enzyme not naturally present within the parental microorganism.
  • the parental microorganism may be transformed to provide a combination of increased or over-expression of one or more genes native to the parental microorganism and introduction of one or more genes not native to the parental microorganism.
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes chosen from the group consisting: Thiolase; 3-hydroxybutyryl-CoA dehydrogenase; Crotonase/crotonyl-CoA hydratase; Butyryl-CoA dehydrogenase; Electron Transfer Flavoprotein A; and, Electron Transfer Flavoprotein B.
  • the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.
  • the recombinant microorganism is adapted to express one or more of the genes which encode the enzymes thiolase (IUBMB enzyme nomenclature EC:2.3.1.9) (thlA), 3-hydroxybutyryl-CoA dehydrogenase (EC:1.1.1.157) (hbd), crotonase/crotonyl-CoA hydratase (EC:1.1.1.157) (crt or cch) and/or butyryl-CoA dehydrogenase (EC4.2.1.55) (bcd).
  • the microorganism is adapted to express all of these enzymes.
  • the genes correspond to one or more of the nucleic acid sequences selected from SEQ_ID Nos 1 to 4 or functionally equivalent variants thereof.
  • the recombinant microorganism of the invention may also contain two electron transferring proteins.
  • the electron transferring proteins are electron transferring flavoproteins (EC1.3.99.2) (etfAB) encoded by SEQ_ID Nos 5 and 6, or functionally equivalent variants thereof. The use of these electron-transferring flavoproteins enhances the efficiency of the microorganism in producing 1-butanol.
  • the flavoproteins provide a stable complex that is required for the activity of Bcd.
  • the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
  • the microorganism comprises a plasmid encoding one or more of, or preferably each of, Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
  • the microorganism alternatively or further comprises exogenous nucleic acids adapted to express one or more of the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase (or in other words aledhyde:ferredoxin oxidoreductase); butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase (or in other words aledhyde:ferredoxin oxidoreductase); butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional buty
  • the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • the microorganism comprises exogenous nucleic acids adapted to express one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism comprises exogenous nucleic acids adapted to express each of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
  • the microorganism comprises one or more nucleic acid adapted to express at least two of the enzymes in the 1-butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.
  • the microorganism further comprises an exogenous phosphotransacetylase/acetate kinase promoter, although other promoters may be used.
  • the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
  • the promoter is contained on a construct encoding one or more of the enzymes referred to herein before.
  • the parental microorganism is selected from the group of acetogenic bacteria.
  • the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii , and Thermoanaerobacter kiuvi.
  • the parental microorganism is selected from the cluster of ethanologenic, acetogenic Clostridia comprising the species C. autoethanogenum, C. ljungdahlii , and C. ragsdalei and related isolates.
  • These include but are not limited to strains C. autoethanogenum JAI-1 T (DSM10061) [Abrini J, Naria H, Nyns E-J: Clostridium autoethanogenum , sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351] , C.
  • rhamnose, arabinose e.g. gluconate, citrate
  • amino acids e.g. arginine, histidine
  • substrates e.g. betaine, butanol.
  • auxotroph to certain vitamins (e.g. thiamine, biotin) while others were not.
  • the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
  • the microorganism produces butyraldehyde dehydrogenase and/or butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
  • the microorganism is Clostridium autoethanogenum DSM23693.
  • the recombinant microorganism of the invention has the defining characteristics of the microorganism deposited at the DSMZ (Deutsche Sammlung für Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) under the accession number DSM24138.
  • the one or more exogenous nucleic acids may be delivered to a parental microorganism as naked nucleic acids or may be formulated with one or more agents to facilitate the tranformation process (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).
  • the one or more nucleic acids may be DNA, RNA, or combinations thereof, as is appropriate.
  • microorganisms of the invention may be prepared from a parental microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms.
  • transformation including transduction or transfection
  • transformation techniques are described for example in Sambrook et al, 1989.
  • methylate the nucleic acid to be introduced into the microorganism due to the restriction systems which are active in the microorganism to be transformed, it is necessary to methylate the nucleic acid to be introduced into the microorganism. This can be done using a variety of techniques, including those described below, and further exemplified in the Examples section herein after.
  • the invention provides a method of producing a recombinant microorganism comprising the following steps:
  • the methyltransferase gene of step B is expressed constitutively. In another embodiment, expression of the methyltransferase gene of step B is induced.
  • the shuttle microorganism is a microorganism, preferably a restriction negative microorganism, that facilitates the methylation of the nucleic acid sequences that make up the expression construct.
  • the shuttle microorganism is a restriction negative E. coli or Bacillus subtillis.
  • the methyltransferase gene present on the methylation construct is expressed.
  • induction may be by any suitable promoter system although in one particular embodiment of the invention, the methylation construct comprises an inducible lac promoter (preferably encoded by SEQ_ID NO 28) and is induced by addition of lactose or an analogue thereof, more preferably isopropyl- ⁇ -D-thio-galactoside (IPTG).
  • suitable promoters include the ara, tet, or T7 system.
  • the methylation construct promoter is a constitutive promoter.
  • the expression construct promoter is a constitutive promoter that is preferably highly active under appropriate fermentation conditions.
  • an inducible promoter could be used.
  • the expression construct promoter is selected from the group comprising phosphotransacetylase/acetate kinase operon promoter, pyruvate:ferredoxin oxidoreductase (SEQ_ID No. 48), the Wood-Ljungdahl gene cluster (SEQ_ID No 47), Rnf operon (SEQ_ID No 49) or the ATP synthase operon ((SEQ_ID No 50).
  • the phosphotransacetylase/acetate kinase operon promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof
  • FIG. 8 shows that expression of genes operably linked to these promoters have a high level of expression in Clostridium autoethanogenum under standard conditions.
  • the methylation construct has an origin of replication specific to the identity of the shuttle microorganism so that any genes present on the methylation construct are expressed in the shuttle microorganism.
  • the expression construct has an origin of replication specific to the identity of the destination microorganism so that any genes present on the expression construct are expressed in the destination microorganism.
  • the expression construct may then be isolated from the shuttle microorganism according to any one of a number of known methods. By way of example only, the methodology described in the Examples section described hereinafter may be used to isolate the expression construct.
  • both constructs are concurrently isolated.
  • the expression construct may be introduced into the destination microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section hereinafter may be used. Since the expression construct is methylated, the nucleic acid sequences present on the expression construct are able to be incorporated into the destination microorganism and successfully expressed.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • a methyltransferase gene of the invention may be introduced into a shuttle microorganism and over-expressed.
  • the resulting methyltransferase enzyme may be collected using known methods and used in vitro to methylate an expression construct, preferably, the expression construct is as defined in the fifth aspect.
  • the expression construct may then be introduced into the destination microorganism for expression.
  • the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention provides a method of producing a recombinant microorganism comprising:
  • Standard methods are used for the introduction of a methyltransferase gene, preferably according to SEQ_ID No 27, into the genome of the shuttle microorganism.
  • the methyltransferase may be constitutively expressed by the microorganism and result in the production of a methyltransferase enzyme, preferably according to SEQ_ID No 28 or a functionally equivalent variant thereof.
  • An expression construct is methylated, isolated and introduced into the destination microorganism which preferably, produces 1-butanol and/or a precursor thereof as the main fermentation product.
  • the invention also includes microorganisms comprising a recombinant methyltransferase gene or methylation construct as herein described.
  • the present invention also provides a hybrid methyltransferase gene (SEQ_ID NO 28) developed following analysis of methyltransferase nucleic acid sequences and restriction barrier systems from C. autoethanogenum, C. ljungdahlii , and C. ragsdalei.
  • SEQ_ID NO 28 hybrid methyltransferase gene developed following analysis of methyltransferase nucleic acid sequences and restriction barrier systems from C. autoethanogenum, C. ljungdahlii , and C. ragsdalei.
  • the methyltransferase gene is expressed in a shuttle microorganism which results in the production of a methyltransferase enzyme which methylates the sequence of the expression construct.
  • the methyltransferase gene may be present on a construct or integrated into the genome of the shuttle microorganism.
  • the hybrid methyltransferase gene is codon optimised for E. coli and may be incorporated into a methylation construct ( FIG. 5 ).
  • the methyltransferase gene may be codon optimised for use in another species of microorganism where appropriate, for example Bacillus subtillus . Methods for codon optimisation are standard and would be known to one of skill in the art (Carbone et al, 2003).
  • methyltransferase genes that have at least 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or greater nucleic acid sequence identity to SEQ_ID NO 28 and express a polypeptide which is able to methylate DNA.
  • the methylation method and methyltransferase gene will have utility across a range of microorganisms.
  • the destination microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi .
  • the destination microorganism is selected from the group consisting Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei . In one particular embodiment the destination microorganism is Clostridium autoethanogenum DSM23693.
  • the invention also provides various nucleic acids or nucleic acid constructs as outlined in aspects 4, 5, 14, 15, 16, 18, 19 and 21 of the invention herein before described.
  • an expression construct comprising one or more nucleic acids encoding one or more enzymes chosen from Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase and an electron transfer protein or a functionally equivalent variant thereof.
  • the electron transfer protein is Electron Transfer Flavoprotein A or Electron Transfer Flavoprotein B.
  • both Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B are included on the expression construct.
  • sequence information for each gene and equivalent enzyme is provided on GenBank as detailed in Table 1 herein after. Skilled persons will readily appreciate alternative genes and enzymes which may be used.
  • the enzymes are encoded by the nucleic acid SEQ_ID No 1 to 6 which may be present in any order on the construct or in the order shown in FIG. 2 .
  • SEQ_ID Nos 8 to 13 and SEQ_ID Nos 16 to 23 are novel sequences used to clone and sequence the genes referred to in the immediately preceding paragraph.
  • butyraldehyde dehydrogenase EC1.2.1.10
  • alcohol dehydrogenase EC 1.1.1.1
  • phosphotransbutyrylase EC 2.3.1.19
  • butyrate kinase EC 2.7.2.7
  • aldehyde:ferredoxin oxidoreductase EC1.2.7.5
  • alcohol dehydrogenase EC 1.1.1.1
  • the alcohol dehydrogenase of the invention is a butanol dehydrogenase.
  • the butyraldehyde dehydrogenase and butanol dehydrogenase activity is supplied by a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
  • butanol biosynthesis pathway depicted in FIG. 1 .
  • butyraldehyde dehydrogenase, butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase, and/or aldehyde:ferredoxin oxidoreductase are naturally expressed by the microorganism and therefore catalyse the conversion of butyryl-CoA to 1-butanol.
  • the expression construct comprises nucleic acids encoding one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase in addition to or in the alternative to one or more of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase and an electron transfer protein.
  • Examples of appropriate enzymes and amino acid and nucleic acid sequence information include, but are not limited to: butyraldehyde dehydrogenase, such as Ald from C. beijerinckii (ABR35947, GI:149905114), C. saccharobutylicum (CAQ57983, GI:189310620), or Clostridium saccharoperbutylacetonium (AAP42563, GI:31075383); butanol dehydrogenase, such as BdhB from C. acetobutylicum (NP — 349891, GI:15896542); bifunctional butyraldehyde/butanol dehydrogenase enzyme, such as AdhE1 from C.
  • butyraldehyde dehydrogenase such as Ald from C. beijerinckii (ABR35947, GI:149905114), C. saccharobutylicum (CAQ57983, GI:189310620
  • acetobutylicum NP — 149325, GI:15004865 or AdhE2 from C. acetobutylicum (NP — 149199, GI:15004739), C. beijerinckii . YP — 001307449, GI:150015195); a phosphotransbutyrylase such as Ptb from C. acetobutylicum (NP — 348368); butyrate kinase such as Buk from C. acetobutylicum (AAK81015.1); aldehyde:ferredoxin oxidoreductase AOR from C. acetobutylicum (NP — 348637).
  • the inclusion of one or more of these genes may help avoid co-production of butyrate completely, increasing the efficiency of 1-butanol production.
  • the invention also provides recombinant microorganisms comprising one or more nucleic acids adapted to express or increase expression of one or more of these enzymes.
  • the nucleic acid(s) encode an enzyme chosen from the group of enzymes listed in tables 7 to 10 herein after and functional equivalents of any one or more thereof.
  • the nucleic acids are chosen from the group of nucleic acids listed in tables 7 to 10 herein after and functional equivalents of any one or more thereof.
  • the expression construct encodes at least 2 enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 of the enzymes.
  • the expression construct further comprises a suitable promoter as hereinbefore described.
  • the promoter is a phosphotransacetylase/acetate kinase promoter.
  • the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
  • the expression construct comprises a nucleic acid encoding all of said enzymes. It will be appreciated by one of skill in the art that the expression construct may comprise nucleic acids encoding alternative electron transferring proteins.
  • the genes to be expressed in the recombinant microorganism may be assembled in the expression construct under the control of any appropriate promoter.
  • the promoter allows for substantially constitutive expression of the genes under its control.
  • the promoter is a phosphotransacetylase/acetate kinase (SEQ_ID NO 7) promoter.
  • Other promoters which may find use in the invention include those from C. autoethanogenum (or C. ljungdahlii ).
  • the inventors have also identified a number of other promoters that are operably linked to genes that were highly expressed under typical fermentation conditions in Clostridium autoethanogenum ( FIG. 8 ).
  • the invention comprises a construct, recombinant microorganism or a nucleic acid sequence comprising nucleic acid SEQ_ID NOs 1 to 6 in the order shown in FIG. 2 .
  • the invention may still have the desired utility when the nucleic acid sequences are presented in any order and with one or more of the sequences absent.
  • the invention comprises a nucleic acid comprising the promoter sequence represented by Seq ID No. 7, or a functionally equivalent variant thereof, construct comprising said promoter and recombinant microorganisms comprising same.
  • an expression construct of the present invention may contain any number of regulatory elements in addition to the promoter as well as additional genes suitable for expression of further proteins if desired.
  • the construct includes one promoter.
  • the construct includes two or more promoters.
  • the construct includes one promoter for with each gene to be expressed.
  • the construct includes one or more ribosomal binding sites, preferably a ribosomal binding site for each gene to be expressed.
  • nucleic acid sequences and construct sequences defined herein may contain standard linker nucleotides such as those required for ribosome binding sites and/or restriction sites. Such linker sequences should not be interpreted as being required and do not provide a limitation on the sequences defined.
  • the microorganism When the expression construct of the invention is expressed in an acetogenic microorganism, the microorganism produces 1-butanol or a precursor thereof as the main fermentation product. It is envisaged that other genes which encode enzymes catalyzing different steps of the Wood-Ljungdahl or butanol biosynthesis pathways may also be incorporated in the expression construct in order to produce 1-butanol as the main fermentation product.
  • the expression construct and the methylation construct as defined above may be combined to provide a composition of matter.
  • a composition has particular utility in circumventing restriction barrier mechanisms in a wide variety of microorganisms but in a preferred embodiment, the recombinant microorganism produced by use of the composition produces 1-butanol or a precursor thereof as the main fermentation product.
  • Nucleic acids and nucleic acid constructs may be constructed using any number of techniques standard in the art. For example, chemical synthesis or recombinant techniques may be used. Such techniques are described, for example, in Sambrook et al (1989). Further exemplary techniques are described in the Examples section herein after. Essentially, the individual genes and regulatory elements will be operably linked to one another such that the genes can be expressed to form the desired proteins. Suitable vectors for use in the invention will be appreciated by those of ordinary skill in the art. However, by way of example, the following vectors may be suitable: pMTL80000 shuttle vectors, pIMP1, pJIR750 and the plasmids exemplified in the Examples section herein after.
  • nucleic acids which are capable of hybridising to at least a portion of a nucleic acid herein described, a nucleic acid complementary to any one thereof, or a functionally equivalent variant of any one thereof.
  • Such nucleic acids will preferably hybridise to such nucleic acids, a nucleic acid complementary to any one thereof, or a functionally equivalent variant of any one thereof, under stringent hybridisation conditions.
  • Stringent hybridisation conditions means that the nucleic acid is capable of hybridising to a target template under standard hybridisation conditions such as those described in Sambrook et al (1989).
  • the minimal size of such nucleic acids is a size which is capable of forming a stable hybrid between a given nucleic acid and the complementary sequence to which it is designed to hybridise. Accordingly, the size is dependent on the nucleic acid composition and percent homology between the nucleic acid and its complementary sequence, as well as the hybridisation conditions which are utilised (for example, temperature and salt concentrations).
  • the nucleic acid is at least 10 nucleotides in length, at least 15 nucleotides in length, at least, 20 nucleotides in length, at least 25 nucleotides in length, or at least 30 nucleotides in length.
  • nucleic acids of the invention may be in any appropriate form, including RNA, DNA, or cDNA, including double-stranded and single-stranded nucleic acids.
  • the invention also provides host organisms, particularly microorganisms, and including viruses, bacteria, and yeast, comprising any one or more of the nucleic acids described herein.
  • the invention provides a method of production of 1-butanol and/or a precursor thereof by microbial fermentation comprising fermenting a gaseous substrate comprising CO using a recombinant microorganism.
  • 1-butanol or a precursor thereof is co-produced with another fermentation product (for example, ethanol).
  • the 1-butanol or a precursor thereof is the main fermentation product.
  • the recombinant microorganism is as herein before described.
  • 1-butanol and/or a precursor thereof is produced in a yield of from approximately 0.075 grams per litre of fermentation broth (g/l) to approximately 20 g/l. In one embodiment, the yield is from approximately 0.15 g/l to approximately 1.54 g11. In other embodiments, the yield is approximately 10 g/l, approximately 5 g/l, or approximately 2 g/l. Preferably, the yield of 1-butanol is up to the limit at which butanol becomes toxic to the bacteria.
  • the fermentation comprises the steps of anaerobically fermenting a substrate in a bioreactor to produce 1-butanol and/or a precursor thereof using recombinant microorganisms as described herein.
  • the precursor of 1-butanol is referred to herein it is envisaged that it may be optionally converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase, a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase, and/or ferredoxin dependent aldehyde oxidoreductase.
  • the microorganism produces one or more of these enzymes both before and after introduction of a recombinant nucleic acid.
  • the gaseous substrate fermented by the microorganism is a gaseous substrate containing CO.
  • the gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
  • the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.
  • the CO may be a component of syngas (gas comprising carbon monoxide and hydrogen).
  • syngas gas comprising carbon monoxide and hydrogen.
  • the CO produced from industrial processes is normally flared off to produce CO 2 and therefore the invention has particular utility in reducing CO 2 greenhouse gas emissions and producing butanol for use as a biofuel.
  • the gaseous substrate may be filtered or scrubbed using known methods.
  • a suitable liquid nutrient medium will need to be fed to the bioreactor.
  • a nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used.
  • Anaerobic media suitable for fermentation to produce butanol using CO are known in the art.
  • suitable media are described Biebel (2001). In one embodiment of the invention the media is as described in the Examples section herein after.
  • the fermentation should desirably be carried out under appropriate conditions for the CO-to-butanol fermentation to occur.
  • Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
  • reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
  • WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively.
  • example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.
  • the composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction.
  • O 2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.
  • a culture of a bacterium of the invention is maintained in an aqueous culture medium.
  • the aqueous culture medium is a minimal anaerobic microbial growth medium.
  • Suitable media are known in the art and described for example in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438, and as described in the Examples section herein after.
  • Butanol, or a mixed alcohol stream containing butanol and one or more other alcohols may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, and extractive fermentation, including for example, liquid-liquid extraction.
  • By-products such as acids including butyrate may also be recovered from the fermentation broth using methods known in the art.
  • an adsorption system involving an activated charcoal filter or electrodialysis may be used.
  • continuous gas stripping may also be used.
  • butanol and by-products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering butanol and optionally acid from the broth.
  • Alcohols may conveniently be recovered for example by distillation, and acids may be recovered for example by adsorption on activated charcoal.
  • the separated microbial cells are preferably returned to the fermentation bioreactor.
  • the cell free permeate remaining after the alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.
  • the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • butanol is recovered from the fermentation reaction using extractive fermentation procedures in which butanol is recovered into an oil phase in the reactor. Skilled persons would readily appreciate techniques for achieving this
  • Genomic DNA from Clostridium acetobutylicum ATCC824 and Clostridum autoethanogenum DSM 10061 was isolated using a modified method by Bertram and Dürre (1989). A 100-ml overnight culture was harvested (6,000 ⁇ g, 15 min, 4° C.), washed with potassium phosphate buffer (10 mM, pH 7.5) and suspended in 1.9 ml STE buffer (50 mM Tris-HCl, 1 mM EDTA, 200 mM sucrose; pH 8.0). 300 ⁇ l lysozyme (400,000 U) were added and the mixture was incubated at 37° C.
  • Butanol biosynthesis genes and the phosphotransacetylase/acetate kinase promoter were amplified by PCR with oligonucleotides in table 2 using iProof High Fidelity DNA Polymerase (Bio-Rad Laboratories) and the following program: initial denaturation at 98° C. for 30 seconds, followed by 32 cycles of denaturation (98° C. for 10 seconds), annealing (50-62° C. for 30-120 seconds) and elongation (72° C. for 45 seconds), before a final extension step (72° C. for 10 minutes).
  • the amplified 498 by promoter region of the phosphotransacetylase/acetate kinase operon was cloned into the E. coli —Clostridium shuttle vector pMTL 85141 (Seq. ID 14; FJ797651.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using NotI and NdeI restriction sites and strain DH5 ⁇ -T1 R (Invitrogen).
  • the created plasmid pMTL85145 and the 1,194 by PCR product of the thiolase gene were both cut with NdeI and EcoRI. A ligation was transformed into E.
  • a hybrid methyltransferase gene fused to an inducible lac promoter was designed (Seq. ID 28), by alignment of methyltransferase genes from C. autoethanogenum (SEQ_ID No. 24), C. ljungdahlii (SEQ_ID No. 25), and C. ragsdalei (SEQ_ID No. 26) ( FIGS. 4 a , 4 b and 4 c ). Expression of the methyltransferase gene resulted in production of a methyltransferase enzyme according to SEQ_ID No. 28. Methyltransferase amino acid sequence alignment data is shown in FIG. 4 d .
  • the hybrid methyltransferase gene (SEQ_ID No.
  • C. autoethanogenum DSM23693 was grown in PETC media (Tab. 4) with 10 g/l fructose and 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N 2 , 22% CO 2 , 2% H 2 ) as carbon source at 37° C. using standard anaerobic techniques described by Hungate (1969) and Wolfe (1971).
  • a 50 ml culture of C. autoethanogenum DSM23693 was subcultured to fresh media for 3 consecutive days. These cells were used to inoculate 50 ml PETC media containing 40 mM DL-threonine at an OD 600nm of 0.05. When the culture reached an OD 600nm of 0.4, the cells were transferred into an anaerobic chamber and harvested at 4,700 ⁇ g and 4° C. The culture was twice washed with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl 2 , 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 ⁇ l fresh electroporation buffer.
  • ice-cold electroporation buffer 270 mM sucrose, 1 mM MgCl 2 , 7 mM sodium phosphate, pH 7.4
  • This mixture was transferred into a pre-cooled electroporation cuvette with a 0.4 cm electrode gap containing 1 ⁇ g of the methylated plasmid mix and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 ⁇ l, and 25 ⁇ F. Time constants of 3.7-4.0 ms were achieved.
  • the culture was transferred into 5 ml fresh media. Regeneration of the cells was monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, the cells start growing again.
  • the cells were harvested, suspended in 200 ⁇ l fresh media and plated on selective PETC plates (containing 1.2% BactoTM Agar (BD)) with 4 ⁇ g/ ⁇ l Clarithromycin. After 4-5 days of inoculation with 30 psi steel mill gas at 37° C., 15-80 colonies per plate were clearly visible.
  • selective PETC plates containing 1.2% BactoTM Agar (BD)
  • the colonies were used to inoculate 2 ml PETC media containing 4 ⁇ g/ ⁇ l Clarithromycin. When growth occurred, the culture was upscaled into 5 ml and later 50 ml PETC media containing 4 ⁇ g/ ⁇ 1 Clarithromycin and 30 psi steel mill gas as sole carbon source.
  • a plasmid mini prep was performed from 10 ml culture volume using the QIAprep Spin Miniprep Kit (Qiagen). Due to Clostridial exonuclease activity (Burchhardt and Dürre, 1990), the isolated plasmid DNA from 4 analyzed clones were partly degraded and only resulted in a smear on an agarose gel, while a plasmid isolation from the original C. autoethanogenum DSM23693 strain didn't result in a signal at all ( FIG. 6 ). However, the quality of the isolated plasmid DNA was sufficient to run a control PCR using 4 sets of primers, covering all relevant different regions of the plasmid (Table 5).
  • PCR was performed with illustra PuReTaq Ready-To-GoTM PCR Beads (GE Healthcare) using a standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min).
  • PCR of all 4 analyzed transformants resulted in the same signals as with the original methylated plasmid mix as template ( FIG. 6 ).
  • 1 ⁇ l of each of the partly degraded isolated plasmids were re-transformed in E. coli XL1-Blue MRF′ Kan (Stratagene), from where the plasmids could be isolated cleanly and verified by restriction digests.
  • genomic DNA was isolated (see above) from 40 ml of each culture and a PCR was performed against the 16s rRNA gene (Tab. 5; Weisberg et al., 1991) using illustra PuReTaq Ready-To-GoTM PCR Beads (GE Healthcare) and standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min). The respective PCR products were purified and sequenced. Sequences of all clones showed at least 99.9% identity against the 16s rRNA gene of C. autoethanogenum (Seq. ID 30; Y18178, GI:7271109).
  • PETC media without yeast extract and fructose were prepared and inoculated with the novel C. autoethanogenum strain harboring butanol plasmid pMTL85245-thlA-crt-hbd.
  • Bottles were pressurized with 30 psi of a CO containing gas stream from two industrial sources, steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N 2 , 22% CO 2 , 2% H 2 ) and syngas (Range Fuels Inc., Broomfield, Colo.; composition: 29% CO, 45% H 2 , 13% CH 4 , 12% CO 2 , 1% N 2 ).
  • 1-Butanol production could be demonstrated on both gas mixes over several subculturing periods and co-production of butyrate was observed as well.
  • the expression plasmid only contains the genes necessary for production of butyryl-CoA from acetyl-CoA. Butyryl-CoA can then be converted directly to butanol by action of a butyraldehyde dehydrogenase and butanol dehydrogenase ( FIG. 1 ).
  • a second possibility is that butyryl-CoA is converted to butyrate via a phosphotransbutyrylase and butyrate kinase ( FIG. 1 ), in which case ATP is gained via substrate level phosphorylation (SLP).
  • Respective genes/enzymes with butyraldehyde dehydrogenase, butanol dehydrogenase, phophotransbutyrylase, butyrate kinase, and aldehyde:ferredoxin oxidoreductase activity have been identified by the inventors in C. autoethanogenum, C. ljungdahlii , and C. ragsdalei (Tab. 7-10).
  • Potential genes and enzymes were predicted by comparison with characterized genes and enzymes using BLAST (Altschul et al, 1990), COG (Tatusov et al, 2003), and TIGRFAM (Haft et al, 2002) databases.
  • Genomes of C. autoethanogenum, C. ljungdahlii , and C. ragsdalei contain several genes encoding enzymes with alcohol and aldehyde dehydrogenase activity. As indicated in tables 7 to 10, some of these were found to have high homology of over 70% to characterized butyraldehyde and butanol dehydrogenases from C. acetobutylicum, C. beijerinckii , or C. saccharobutylicum , while others have at least in some 40% identity to these enzymes.
  • RT-PCR reactions were performed in MyiQ Single Colour Real-Time PCR Detection System (Bio-Rad Labratories) in a reaction volume of 15 ⁇ L with 25 ng of cDNA template, 67 nM of each primer (Tab. 11), and 1 ⁇ iQ SYBR Green Supermix (Bio-Rad Labratories, Hercules, Calif. 94547, USA). Guanylate kinase and formate tetrahydrofolate ligase were used as housekeeping gene and non-template controls were included. The reaction conditions were 95° C. for 3 min, followed by 40 cycles of 95° C. for 15 s, 55° C. for 15 s and 72° C. for 30 s.
  • a melting-curve analysis was performed immediately after completion of the RT PCR (38 cycles of 58° C. to 95° C. at 1° C./s), for detection of primer dimerisation or other artifacts of amplification. mRNA from housekeeping and all target genes were successfully detected.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US13/049,263 2010-10-22 2011-03-16 Recombinant microorganism and methods of production thereof Abandoned US20110236941A1 (en)

Priority Applications (17)

Application Number Priority Date Filing Date Title
US13/049,263 US20110236941A1 (en) 2010-10-22 2011-03-16 Recombinant microorganism and methods of production thereof
KR1020147000771A KR101640325B1 (ko) 2010-10-22 2011-09-29 재조합 미생물에 의한 일산화탄소로부터 부탄올의 제조
EP11834686.5A EP2630247B1 (en) 2010-10-22 2011-09-29 Production of butanol from carbon monoxide by a recombinant microorganism
DK11834686.5T DK2630247T3 (en) 2010-10-22 2011-09-29 PREPARATION OF BUTANOL FROM CARBON MONOXIDE BY A RECOMBINANT MICROORGANISM
EA201390444A EA028760B1 (ru) 2010-10-22 2011-09-29 Производство бутанола из монооксида углерода рекомбинантным микроорганизмом
NZ609150A NZ609150A (en) 2010-10-22 2011-09-29 Production of butanol from carbon monoxide by a recombinant microorganism
AU2011318676A AU2011318676B2 (en) 2010-10-22 2011-09-29 Production of butanol from carbon monoxide by a recombinant microorganism
BR112013009552-0A BR112013009552B1 (pt) 2010-10-22 2011-09-29 Microorganismo recombinante que produz 1-butanol e método para a produção de 1-butanol
MYPI2013700647A MY157111A (en) 2010-10-22 2011-09-29 Recombinant microorganisms and methods of production thereof
ES11834686.5T ES2626448T3 (es) 2010-10-22 2011-09-29 Producción de butanol a partir de monóxido de carbono mediante un microorganismo recombinante
PCT/NZ2011/000203 WO2012053905A1 (en) 2010-10-22 2011-09-29 Production of butanol from carbon monoxide by a recombinant microorganism
KR1020137012815A KR20130079599A (ko) 2010-10-22 2011-09-29 재조합 미생물에 의한 일산화탄소로부터 부탄올의 제조
JP2013534846A JP6014042B2 (ja) 2010-10-22 2011-09-29 組換え微生物による一酸化炭素からのブタノールの産生
CN201180062114.3A CN103339261B (zh) 2010-10-22 2011-09-29 通过重组微生物从一氧化碳产生丁醇
CA2813431A CA2813431C (en) 2010-10-22 2011-09-29 Production of butanol from carbon monoxide by a recombinant microorganism
ZA2013/02898A ZA201302898B (en) 2010-10-22 2013-04-22 Production of butanol from carbon monoxide by a recombinant microorganism
US14/180,423 US9359611B2 (en) 2010-10-22 2014-02-14 Recombinant microorganism and methods of production thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US40587110P 2010-10-22 2010-10-22
US13/049,263 US20110236941A1 (en) 2010-10-22 2011-03-16 Recombinant microorganism and methods of production thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/180,423 Continuation US9359611B2 (en) 2010-10-22 2014-02-14 Recombinant microorganism and methods of production thereof

Publications (1)

Publication Number Publication Date
US20110236941A1 true US20110236941A1 (en) 2011-09-29

Family

ID=44656929

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/049,263 Abandoned US20110236941A1 (en) 2010-10-22 2011-03-16 Recombinant microorganism and methods of production thereof
US14/180,423 Active 2031-03-28 US9359611B2 (en) 2010-10-22 2014-02-14 Recombinant microorganism and methods of production thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/180,423 Active 2031-03-28 US9359611B2 (en) 2010-10-22 2014-02-14 Recombinant microorganism and methods of production thereof

Country Status (14)

Country Link
US (2) US20110236941A1 (enExample)
EP (1) EP2630247B1 (enExample)
JP (1) JP6014042B2 (enExample)
KR (2) KR101640325B1 (enExample)
CN (1) CN103339261B (enExample)
AU (1) AU2011318676B2 (enExample)
CA (1) CA2813431C (enExample)
DK (1) DK2630247T3 (enExample)
EA (1) EA028760B1 (enExample)
ES (1) ES2626448T3 (enExample)
MY (1) MY157111A (enExample)
NZ (1) NZ609150A (enExample)
WO (1) WO2012053905A1 (enExample)
ZA (1) ZA201302898B (enExample)

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110256600A1 (en) * 2011-02-02 2011-10-20 Lanzatech New Zealand Limited Recombinant Microorganisms and Methods of Use Thereof
WO2013152236A1 (en) * 2012-04-05 2013-10-10 Lanzatech New Zealand Limited Enzyme-altered metabolite activity
WO2013180584A1 (en) 2012-06-01 2013-12-05 Lanzatech New Zealand Limited Recombinant microorganisms and uses therefor
WO2014012055A1 (en) 2012-07-13 2014-01-16 Calysta Energy, Llc Biorefinery system, methods and compositions thereof
WO2014113209A1 (en) * 2013-01-18 2014-07-24 Coskata, Inc. Method for production of n-butanol from syngas using syntrophic co-cultures of anaerobic microorganisms
EP2774986A1 (en) * 2013-03-06 2014-09-10 Clariant Produkte (Deutschland) GmbH Improvement of clostridial butanol production by gene overexpression
US20140273236A1 (en) * 2013-03-13 2014-09-18 Coskata, Inc. Use of Clostridial Methyltransferases for Generating Novel Strains
EP2816096A1 (de) 2013-06-18 2014-12-24 Evonik Industries AG Verfahren zur Speicherung von Überschussenergie
US20150210977A1 (en) * 2014-01-28 2015-07-30 Lanzatech New Zealand Limited Bacterium with increased tolerance to butyric acids
CN104955938A (zh) * 2013-01-30 2015-09-30 朗泽科技新西兰有限公司 包含nadph依赖性酶的重组微生物及其生产方法
WO2015138977A3 (en) * 2014-03-14 2015-11-26 Ohio State Innovation Foudation Metabolic engineering of clostridium for biofuel production
US20150376654A1 (en) * 2011-02-02 2015-12-31 Lanzatech New Zealand Limited Recombinant microorganisms with increased tolerance to ethanol
WO2017066498A1 (en) 2015-10-13 2017-04-20 Lanzatech New Zealand Limited Genetically engineered bacterium comprising energy-generating fermentation pathway
WO2017096324A1 (en) 2015-12-03 2017-06-08 Lanzatech New Zealand Limited Arginine supplementation to improve efficiency in gas fermenting acetogens
US9790522B2 (en) * 2015-04-07 2017-10-17 Synata Bio Compositions and methods for the conversion of short-chained carboxylic acids to alcohols using clostridial enzymes
WO2017191483A1 (en) 2016-05-05 2017-11-09 Newpek S.A. De C.V. Enzymatic methods for butanol production
US9816111B2 (en) 2012-09-18 2017-11-14 Calysta, Inc. Propylene synthesis using engineered enzymes
WO2020188033A1 (en) 2019-03-20 2020-09-24 Global Bioenergies Improved means and methods for producing isobutene from acetyl-coa
WO2021165229A1 (en) 2020-02-17 2021-08-26 Scientist Of Fortune S.A. Method for the incorporation of formaldehyde into biomass
US11142751B2 (en) 2019-03-07 2021-10-12 Auburn University CRISPR-cas system for Clostridium genome engineering and recombinant strains produced thereof
EP4144838A1 (en) 2021-09-06 2023-03-08 Global Bioenergies Organisms producing less crotonic acid
WO2023031482A1 (en) 2021-09-06 2023-03-09 Global Bioenergies Organisms producing less crotonic acid
WO2023077101A1 (en) 2021-10-29 2023-05-04 Synata Bio, Inc. Method of dewatering
WO2023077103A1 (en) 2021-10-29 2023-05-04 Synata Bio, Inc. Green methods of making product from hydrogen enriched synthesis gas
US11680216B2 (en) 2019-01-29 2023-06-20 Lanzatech, Inc. Production of bio-based liquefied petroleum gas
US20230193325A1 (en) * 2017-08-29 2023-06-22 Gwangju Institute Of Science And Technology Method for converting non-ethanol producing, acetogenic strain to ethanol-producing strain and method for producing ethanol from same ethanol-producing strain by using carbon monoxide
US12291734B2 (en) 2022-06-21 2025-05-06 Lanzatech, Inc. Microorganisms and methods for the continuous co-production of high-value, specialized proteins and chemical products from C1-substrates
US12371727B2 (en) 2022-06-21 2025-07-29 Lanzatech, Inc. Microorganisms and methods for the continuous co-production of tandem repeat proteins and chemical products from C1-substrates

Families Citing this family (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6381525B2 (ja) * 2012-06-08 2018-08-29 ランザテク・ニュージーランド・リミテッド 組換え微生物およびその使用
US9347076B2 (en) 2012-06-21 2016-05-24 Lanzatech New Zealand Limited Recombinant microorganisms that make biodiesel
KR102121888B1 (ko) 2012-08-28 2020-06-12 란자테크 뉴질랜드 리미티드 재조합 미생물 및 이에 대한 용도
BR112015007638B1 (pt) 2012-10-08 2020-05-05 Calysta Inc sistemas de fermentação alimentados por gás
JP2014161252A (ja) * 2013-02-22 2014-09-08 Sekisui Chem Co Ltd 組換え細胞
AU2014274838A1 (en) * 2013-06-05 2015-12-24 Lanzatech New Zealand Limited Recombinant microorganisms exhibiting increased flux through a fermentation pathway
US9745566B2 (en) 2013-09-12 2017-08-29 Lanzatech New Zealand Limited Recombinant microorganisms and methods of use thereof
KR102320074B1 (ko) 2014-01-28 2021-11-03 란자테크 뉴질랜드 리미티드 재조합 미생물의 생산 방법
EP3099783A4 (en) 2014-01-30 2017-09-20 Lanzatech New Zealand Limited Recombinant microorganisms and methods of use thereof
WO2015144447A1 (en) 2014-03-26 2015-10-01 Scientist Of Fortune S.A. Enzymatic production of acetyl phosphate from formaldehyde
JP6662868B2 (ja) 2014-10-22 2020-03-11 ランザテク・ニュージーランド・リミテッド 複数ステージバイオリアクタープロセス
EP3210012B1 (en) 2014-10-22 2023-06-07 LanzaTech NZ, Inc. Gas testing unit and method
HUE052810T2 (hu) 2014-12-08 2021-05-28 Lanzatech New Zealand Ltd Rekombináns mikroorganizmusok, amelyek megnövekedett fluxust mutatnak fermetációs útvonalon keresztül
US10131884B2 (en) 2015-02-23 2018-11-20 Lanzatech New Zealand Limited Recombinant acetogenic bacterium for the conversion of methane to products
CN105368863B (zh) * 2015-12-14 2019-03-08 中国科学院微生物研究所 一种构建高产丁醇菌株的方法与应用
US10358661B2 (en) 2015-12-28 2019-07-23 Lanzatech New Zealand Limited Microorganism with modified hydrogenase activity
CN108603201A (zh) 2016-02-01 2018-09-28 朗泽科技新西兰有限公司 整合发酵和电解工艺
SG11201807025SA (en) 2016-02-26 2018-09-27 Lanzatech New Zealand Ltd Crispr/cas systems for c-1 fixing bacteria
KR102514023B1 (ko) 2016-05-14 2023-03-23 란자테크, 인크. 알데히드:페레독신 옥시도리덕타제 활성이 변경된 미생물 및 관련 방법
CN107460148B (zh) * 2017-09-01 2020-12-25 湖州至美生物科技有限公司 一种枯草芽孢杆菌菌粉的制备方法及应用
ES2926336T3 (es) 2017-09-08 2022-10-25 Lanzatech Inc Proceso para la producción de metabolitos utilizando sustratos que contienen C1 ricos en hidrógeno
WO2019126400A1 (en) 2017-12-19 2019-06-27 Lanzatech, Inc. Microorganisms and methods for the biological production of ethylene glycol
KR101985307B1 (ko) 2018-01-23 2019-06-04 한국과학기술연구원 헥사노익산의 생산 수율이 우수한 클로스트리디움 속 신규 균주 및 이를 이용한 헥사노익산의 생산 방법
CN111683731B (zh) 2018-02-12 2022-09-02 朗泽科技有限公司 用于提高碳转化效率的工艺
ES2960999T3 (es) 2018-04-20 2024-03-07 Lanzatech Inc Proceso de fermentación para producir etanol
EP3884060A4 (en) 2018-11-19 2022-08-10 Lanzatech, Inc. FERMENTATION AND GASIFICATION INTEGRATION
KR20240135053A (ko) 2019-02-08 2024-09-10 란자테크, 인크. 유사한 비등점 생성물의 회수 방법
CN111621510B (zh) * 2019-02-27 2024-01-05 南京食气生化科技有限公司 一种优化的扬氏梭菌外源dna电转化方法
KR20240158377A (ko) 2019-07-11 2024-11-04 란자테크, 인크. 가스 이용률을 최적화하는 방법
CN111088193B (zh) * 2020-01-10 2022-09-09 南京工业大学 一种提高食甲基丁酸杆菌电转频率的方法
CN111154683B (zh) * 2020-01-19 2022-05-13 南京工业大学 一种食甲基丁酸杆菌的优化培养方法及其应用
US11932818B2 (en) 2020-03-16 2024-03-19 Lanzatech, Inc. Tail gas of gas fermentation to dry gasification feedstock
JP2023518045A (ja) 2020-03-18 2023-04-27 ランザテク，インコーポレイテッド ガス基質からの２－フェニルエタノールの発酵産生
US12134794B2 (en) 2020-04-29 2024-11-05 Lanzatech, Inc. Fermentative production of B-ketoadipate from gaseous substrates
KR20230156819A (ko) 2020-06-06 2023-11-14 란자테크, 인크. 아세토락테이트 탈카복실화효소 유전자위에 녹인이 있는 미생물
US11788092B2 (en) 2021-02-08 2023-10-17 Lanzatech, Inc. Recombinant microorganisms and uses therefor
CN116615549A (zh) * 2021-03-08 2023-08-18 朗泽科技有限公司 重组微生物及其用途
JP2024513395A (ja) 2021-04-09 2024-03-25 ランザテク，インコーポレイテッド 供給原料を提供するためのプロセス及び装置
WO2023004293A1 (en) 2021-07-20 2023-01-26 Lanzatech, Inc. Recombinant microorganisms and uses therefor
TW202307202A (zh) 2021-08-06 2023-02-16 美商朗澤科技有限公司 用於改良乙二醇之生物產生的微生物及方法
US12091648B2 (en) 2021-11-03 2024-09-17 Lanzatech, Inc. System and method for generating bubbles in a vessel
US12280331B2 (en) 2022-04-29 2025-04-22 Lanzatech, Inc. Low residence time gas separator
US12077800B2 (en) 2022-06-16 2024-09-03 Lanzatech, Inc. Liquid distributor system and process of liquid distribution
WO2024253883A1 (en) 2023-06-05 2024-12-12 Lanzatech, Inc. Integrated gas fermentation and carbon black processes
US12281344B2 (en) 2023-06-05 2025-04-22 Lanzatech, Inc. Integrated gas fermentation

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173429A (en) * 1990-11-09 1992-12-22 The Board Of Trustees Of The University Of Arkansas Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism
US5593886A (en) * 1992-10-30 1997-01-14 Gaddy; James L. Clostridium stain which produces acetic acid from waste gases
US5807722A (en) * 1992-10-30 1998-09-15 Bioengineering Resources, Inc. Biological production of acetic acid from waste gases with Clostridium ljungdahlii
US5821111A (en) * 1994-03-31 1998-10-13 Bioengineering Resources, Inc. Bioconversion of waste biomass to useful products
US6136577A (en) * 1992-10-30 2000-10-24 Bioengineering Resources, Inc. Biological production of ethanol from waste gases with Clostridium ljungdahlii
US6340581B1 (en) * 1992-10-30 2002-01-22 Bioengineering Resources, Inc. Biological production of products from waste gases
US6368819B1 (en) * 1998-09-08 2002-04-09 Bioengineering Resources, Inc. Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth
US7285402B2 (en) * 2000-07-25 2007-10-23 Emmaus Foundation, Inc. Methods for increasing the production of ethanol from microbial fermentation
US20070275447A1 (en) * 2006-05-25 2007-11-29 Lewis Randy S Indirect or direct fermentation of biomass to fuel alcohol
US20100151543A1 (en) * 2008-12-16 2010-06-17 Andrew Reeves Recombinant microorganisms having modified production of alcohols and acids

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001288360A1 (en) * 2000-08-23 2002-03-04 Genome Therapeutics Corporation Genomics-assisted rapid identification of targets
JP4762481B2 (ja) * 2000-08-30 2011-08-31 株式会社セルフリーサイエンス 無細胞タンパク質合成用転写鋳型の設計および構築、並びにこれを用いる希釈バッチ方式コムギ胚芽無細胞タンパク質合成法
MY139496A (en) * 2004-03-18 2009-10-30 Univ Putra Malaysia Novel lipase gene from bacillus sphaericus 205y
AU2005335216A1 (en) * 2004-10-05 2007-02-15 Wyeth Probe arrays for detecting multiple strains of different species
GB0612301D0 (en) * 2006-06-21 2006-08-02 Morvus Technology Ltd DNA molecules and methods
US7704723B2 (en) 2006-08-31 2010-04-27 The Board Of Regents For Oklahoma State University Isolation and characterization of novel clostridial species
CA2715092A1 (en) 2006-12-21 2008-07-03 Gevo, Inc. Butanol production by metabolically engineered yeast
JP2011510611A (ja) * 2007-02-09 2011-04-07 ザ レジェンツ オブ ザ ユニヴァースティ オブ カリフォルニア 組換え微生物によるバイオ燃料の生成
US20090111154A1 (en) * 2007-04-04 2009-04-30 The Regents Of The University Of California Butanol production by recombinant microorganisms
CN101743302B (zh) 2007-06-15 2013-11-13 塞卢升生物技术有限公司 改进的柔性的生物反应器
US20090035848A1 (en) 2007-08-03 2009-02-05 Robert Hickey Moving bed biofilm reactor (mbbr) system for conversion of syngas components to liquid products
CN102876609A (zh) 2007-11-13 2013-01-16 兰扎泰克新西兰有限公司 新型菌及其使用方法
US7803589B2 (en) * 2008-01-22 2010-09-28 Genomatica, Inc. Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol
US8586335B2 (en) * 2008-03-11 2013-11-19 Ineos Bio Sa Process for the production of ethanol and butanol
BRPI0909334B1 (pt) * 2008-03-12 2019-10-29 Lanzatech New Zealand Ltd método para a conversão de ácido em álcool correspondente
GB0806093D0 (en) * 2008-04-03 2008-05-14 Green Biologics Ltd Production of butanol
AU2010214147B2 (en) * 2009-01-29 2013-01-10 Lanzatech Nz, Inc. Alcohol production process
CN113528417A (zh) 2009-06-04 2021-10-22 基因组股份公司 生产1,4-丁二醇的微生物和相关方法

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173429A (en) * 1990-11-09 1992-12-22 The Board Of Trustees Of The University Of Arkansas Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism
US5593886A (en) * 1992-10-30 1997-01-14 Gaddy; James L. Clostridium stain which produces acetic acid from waste gases
US5807722A (en) * 1992-10-30 1998-09-15 Bioengineering Resources, Inc. Biological production of acetic acid from waste gases with Clostridium ljungdahlii
US6136577A (en) * 1992-10-30 2000-10-24 Bioengineering Resources, Inc. Biological production of ethanol from waste gases with Clostridium ljungdahlii
US6340581B1 (en) * 1992-10-30 2002-01-22 Bioengineering Resources, Inc. Biological production of products from waste gases
US5821111A (en) * 1994-03-31 1998-10-13 Bioengineering Resources, Inc. Bioconversion of waste biomass to useful products
US6368819B1 (en) * 1998-09-08 2002-04-09 Bioengineering Resources, Inc. Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth
US6753170B2 (en) * 1998-09-08 2004-06-22 Bioengineering Resources, Inc. Microbial process for the preparation of acetic acid, as well as solvent for its extraction from the fermentation broth
USRE39175E1 (en) * 1998-09-08 2006-07-11 Bioengineering Resources, Inc. Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth
US7196218B2 (en) * 1998-09-08 2007-03-27 Celanese International Corporation Microbial process for the preparation of acetic acid, as well as solvent for its extraction from the fermentation broth
US7285402B2 (en) * 2000-07-25 2007-10-23 Emmaus Foundation, Inc. Methods for increasing the production of ethanol from microbial fermentation
US20070275447A1 (en) * 2006-05-25 2007-11-29 Lewis Randy S Indirect or direct fermentation of biomass to fuel alcohol
US20100151543A1 (en) * 2008-12-16 2010-06-17 Andrew Reeves Recombinant microorganisms having modified production of alcohols and acids

Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110256600A1 (en) * 2011-02-02 2011-10-20 Lanzatech New Zealand Limited Recombinant Microorganisms and Methods of Use Thereof
WO2012105853A1 (en) * 2011-02-02 2012-08-09 Lanzatech New Zealand Limited Recombinant microorganisms with increased tolerance to ethanol
US20150376654A1 (en) * 2011-02-02 2015-12-31 Lanzatech New Zealand Limited Recombinant microorganisms with increased tolerance to ethanol
WO2013152236A1 (en) * 2012-04-05 2013-10-10 Lanzatech New Zealand Limited Enzyme-altered metabolite activity
US9550979B2 (en) 2012-04-05 2017-01-24 Lanza Tech New Zealand Limited Enzyme-altered metabolite activity
CN104822823A (zh) * 2012-06-01 2015-08-05 郎泽科技新西兰有限公司 重组代谢微生物及其用途
US10913958B2 (en) 2012-06-01 2021-02-09 Lanzatech New Zealand Limited Microbial fermentation for the production of terpenes
CN111304260A (zh) * 2012-06-01 2020-06-19 朗泽科技新西兰有限公司 重组代谢微生物及其用途
CN111218418A (zh) * 2012-06-01 2020-06-02 朗泽科技新西兰有限公司 重组代谢微生物及其用途
EP3346008A1 (en) 2012-06-01 2018-07-11 LanzaTech New Zealand Limited Recombinant microorganisms and uses therefor
EP3795680A1 (en) 2012-06-01 2021-03-24 LanzaTech New Zealand Limited Recombinant microorganisms and uses therefor
WO2013180584A1 (en) 2012-06-01 2013-12-05 Lanzatech New Zealand Limited Recombinant microorganisms and uses therefor
EA028892B1 (ru) * 2012-06-01 2018-01-31 Ланцатек Нью Зилэнд Лимитед Способ получения терпенов с помощью рекомбинантных микроорганизмов
EP3305907A1 (en) 2012-07-13 2018-04-11 Calysta, Inc. Biorefinery system, methods and compositions thereof
WO2014012055A1 (en) 2012-07-13 2014-01-16 Calysta Energy, Llc Biorefinery system, methods and compositions thereof
US9816111B2 (en) 2012-09-18 2017-11-14 Calysta, Inc. Propylene synthesis using engineered enzymes
US9469860B2 (en) * 2013-01-18 2016-10-18 Synata Bio, Inc. Method for production of n-butanol from syngas using syntrophic co-cultures of anaerobic microorganisms
CN104968793A (zh) * 2013-01-18 2015-10-07 科斯卡塔公司 使用厌氧微生物的互养共培养物从合成气生产n-丁醇的方法
WO2014113209A1 (en) * 2013-01-18 2014-07-24 Coskata, Inc. Method for production of n-butanol from syngas using syntrophic co-cultures of anaerobic microorganisms
CN104955938A (zh) * 2013-01-30 2015-09-30 朗泽科技新西兰有限公司 包含nadph依赖性酶的重组微生物及其生产方法
KR102290728B1 (ko) * 2013-01-30 2021-08-19 란자테크 뉴질랜드 리미티드 Nadph 의존적 효소를 포함하는 재조합 미생물 및 이의 생산방법
EP2951286A4 (en) * 2013-01-30 2016-08-10 Lanzatech New Zealand Ltd RECOMBINANT MICROORGANISMS WITH NADPH-DEPENDENT ENZYMES AND METHOD FOR THE PRODUCTION THEREOF
CN104955938B (zh) * 2013-01-30 2018-03-02 朗泽科技新西兰有限公司 包含nadph依赖性酶的重组微生物及其生产方法
KR20150110806A (ko) * 2013-01-30 2015-10-02 란자테크 뉴질랜드 리미티드 Nadph 의존적 효소를 포함하는 재조합 미생물 및 이의 생산방법
WO2014135633A1 (en) * 2013-03-06 2014-09-12 Clariant Produkte (Deutschland) Gmbh Improvement of clostridial butanol production by gene overexpression
EP2774986A1 (en) * 2013-03-06 2014-09-10 Clariant Produkte (Deutschland) GmbH Improvement of clostridial butanol production by gene overexpression
US9428755B2 (en) 2013-03-13 2016-08-30 Coskata, Inc. Use of clostridial methyltransferases for generating novel strains
US9045758B2 (en) * 2013-03-13 2015-06-02 Coskata, Inc. Use of clostridial methyltransferases for generating novel strains
US20140273236A1 (en) * 2013-03-13 2014-09-18 Coskata, Inc. Use of Clostridial Methyltransferases for Generating Novel Strains
EP2816096A1 (de) 2013-06-18 2014-12-24 Evonik Industries AG Verfahren zur Speicherung von Überschussenergie
US20150210977A1 (en) * 2014-01-28 2015-07-30 Lanzatech New Zealand Limited Bacterium with increased tolerance to butyric acids
WO2015138977A3 (en) * 2014-03-14 2015-11-26 Ohio State Innovation Foudation Metabolic engineering of clostridium for biofuel production
US9790522B2 (en) * 2015-04-07 2017-10-17 Synata Bio Compositions and methods for the conversion of short-chained carboxylic acids to alcohols using clostridial enzymes
WO2017066498A1 (en) 2015-10-13 2017-04-20 Lanzatech New Zealand Limited Genetically engineered bacterium comprising energy-generating fermentation pathway
EP3901267A1 (en) 2015-10-13 2021-10-27 Lanzatech New Zealand Limited Genetically engineered bacterium comprising energy-generating fermentation pathway
EP3981869A1 (en) 2015-12-03 2022-04-13 LanzaTech NZ, Inc. Arginine as sole nitrogen source for c1-fixing microorganism
WO2017096324A1 (en) 2015-12-03 2017-06-08 Lanzatech New Zealand Limited Arginine supplementation to improve efficiency in gas fermenting acetogens
WO2017191483A1 (en) 2016-05-05 2017-11-09 Newpek S.A. De C.V. Enzymatic methods for butanol production
US20230193325A1 (en) * 2017-08-29 2023-06-22 Gwangju Institute Of Science And Technology Method for converting non-ethanol producing, acetogenic strain to ethanol-producing strain and method for producing ethanol from same ethanol-producing strain by using carbon monoxide
US11976314B2 (en) * 2017-08-29 2024-05-07 Gwangju Institute Of Science And Technology Method for converting non-ethanol producing, acetogenic strain to ethanol-producing strain and method for producing ethanol from same ethanol-producing strain by using carbon monoxide
US11680216B2 (en) 2019-01-29 2023-06-20 Lanzatech, Inc. Production of bio-based liquefied petroleum gas
US11142751B2 (en) 2019-03-07 2021-10-12 Auburn University CRISPR-cas system for Clostridium genome engineering and recombinant strains produced thereof
US12385023B2 (en) 2019-03-07 2025-08-12 Auburn University CRISPR-Cas system for clostridium genome engineering and recombinant strains produced thereof
WO2020188033A1 (en) 2019-03-20 2020-09-24 Global Bioenergies Improved means and methods for producing isobutene from acetyl-coa
WO2021165229A1 (en) 2020-02-17 2021-08-26 Scientist Of Fortune S.A. Method for the incorporation of formaldehyde into biomass
EP4144838A1 (en) 2021-09-06 2023-03-08 Global Bioenergies Organisms producing less crotonic acid
WO2023031482A1 (en) 2021-09-06 2023-03-09 Global Bioenergies Organisms producing less crotonic acid
WO2023077101A1 (en) 2021-10-29 2023-05-04 Synata Bio, Inc. Method of dewatering
WO2023077103A1 (en) 2021-10-29 2023-05-04 Synata Bio, Inc. Green methods of making product from hydrogen enriched synthesis gas
US12466752B2 (en) 2021-10-29 2025-11-11 Synata Bio, Inc. Method of dewatering
US12291734B2 (en) 2022-06-21 2025-05-06 Lanzatech, Inc. Microorganisms and methods for the continuous co-production of high-value, specialized proteins and chemical products from C1-substrates
US12371727B2 (en) 2022-06-21 2025-07-29 Lanzatech, Inc. Microorganisms and methods for the continuous co-production of tandem repeat proteins and chemical products from C1-substrates

Also Published As

Publication number Publication date
KR20140012212A (ko) 2014-01-29
DK2630247T3 (en) 2017-06-26
JP6014042B2 (ja) 2016-10-25
EA201390444A1 (ru) 2013-11-29
KR101640325B1 (ko) 2016-07-15
CA2813431C (en) 2014-09-09
EP2630247A1 (en) 2013-08-28
AU2011318676A1 (en) 2013-05-02
KR20130079599A (ko) 2013-07-10
BR112013009552A2 (pt) 2016-07-19
US9359611B2 (en) 2016-06-07
US20140186928A1 (en) 2014-07-03
MY157111A (en) 2016-05-13
JP2013543724A (ja) 2013-12-09
CN103339261B (zh) 2017-03-29
ES2626448T3 (es) 2017-07-25
CA2813431A1 (en) 2012-04-26
EA028760B1 (ru) 2017-12-29
ZA201302898B (en) 2014-12-23
EP2630247A4 (en) 2014-05-07
AU2011318676B2 (en) 2014-07-03
WO2012053905A1 (en) 2012-04-26
EP2630247B1 (en) 2017-03-15
NZ609150A (en) 2015-02-27
CN103339261A (zh) 2013-10-02

Similar Documents

Publication Publication Date Title
US9359611B2 (en) Recombinant microorganism and methods of production thereof
US9834795B2 (en) Recombinant microorganisms and uses therefor
CA2995872C (en) Recombinant clostridium bacterium and uses thereof in acetone production
US9994878B2 (en) Recombinant microorganisms and uses therefor
JP6302471B2 (ja) 組換え微生物およびその使用
US9890384B2 (en) Recombinant microorganisms and uses therefor
US9365868B2 (en) Fermentation process for producing isopropanol using a recombinant microorganism
US20130323809A1 (en) Metabolic engineering of clostridium tyrobutyricum for butanol production
US20130224839A1 (en) Recombinant Microorganism and Methods of Production Thereof
JP2015512646A (ja) 代謝物活性が改変された酵素
BR112013009552B1 (pt) Microorganismo recombinante que produz 1-butanol e método para a produção de 1-butanol
NZ614459B2 (en) Recombinant microorganisms and uses therefor

Legal Events

Date Code Title Description
AS Assignment

Owner name: LANZATECH NEW ZEALAND LIMITED, NEW ZEALAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KOEPKE, MICHAEL;LIEW, FUNGMING;SIGNING DATES FROM 20110404 TO 20110415;REEL/FRAME:026323/0152

AS Assignment

Owner name: VENTURE LENDING & LEASING VI, INC., CALIFORNIA

Free format text: SECURITY AGREEMENT;ASSIGNORS:LANZATECH NEW ZEALAND LIMITED;LANZATECH, INC.;LANZATECH FREEDOM PINES BIOREFINERY LLC;AND OTHERS;REEL/FRAME:028991/0471

Effective date: 20120914

AS Assignment

Owner name: LANZATECH NEW ZEALAND LIMITED, NEW ZEALAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SIMPSON, SEAN DENNIS;REEL/FRAME:030218/0618

Effective date: 20130402

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: LANZATECH, INC., ILLINOIS

Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:042051/0377

Effective date: 20170320

Owner name: LANZATECH FREEDOM PINES BIOREFINERY LLC, GEORGIA

Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:042051/0377

Effective date: 20170320

Owner name: LANZATECH PRIVATE LIMITED, INDIA

Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:042051/0377

Effective date: 20170320

Owner name: LANZATECH HONG KONG LIMITED, HONG KONG

Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:042051/0377

Effective date: 20170320

Owner name: LANZATECH NEW ZEALAND, NEW ZEALAND

Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:042051/0377

Effective date: 20170320

AS Assignment

Owner name: LANZATECH NZ, INC., ILLINOIS

Free format text: CHANGE OF NAME;ASSIGNOR:LANZATECH NEW ZEALAND LIMITED;REEL/FRAME:059911/0400

Effective date: 20211028

AS Assignment

Owner name: LANZATECH PRIVATE LIMITED, INDIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:061053/0481

Effective date: 20170320

Owner name: LANZATECH HONG KONG LIMITED, HONG KONG

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:061053/0481

Effective date: 20170320

Owner name: LANZATECH FREEDOM PINES BIOREFINERY LLC, GEORGIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:061053/0481

Effective date: 20170320

Owner name: LANZATECH, INC., ILLINOIS

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:061053/0481

Effective date: 20170320

Owner name: LANZATECH NEW ZEALAND, NEW ZEALAND

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE APPPLICATION 13467969 PREVIOUSLY RECORDED AT REEL: 042051 FRAME: 0377. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:VENTURE LENDING & LEASING VI, INC.;REEL/FRAME:061053/0481

Effective date: 20170320

Owner name: VENTURE LENDING & LEASING VI, INC., CALIFORNIA

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE U.S. APPLICATION NUMBER 13497,969 PREVIOUSLY RECORD PREVIOUSLY RECORDED AT REEL: 028991 FRAME: 0471. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNORS:LANZATECH NEW ZEALAND LIMITED;LANZATECH, INC.;LANZATECH FREEDOM PINES BIOREFINERY LLC;AND OTHERS;SIGNING DATES FROM 20120914 TO 20210914;REEL/FRAME:061053/0365

AS Assignment

Owner name: LANZATECH NZ, INC., ILLINOIS

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE U.S. PATENT NUMBER 8,979,228 PREVIOUSLY RECORDED AT REEL: 059911 FRAME: 0400. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:LANZATECH NEW ZEALAND LIMITED;REEL/FRAME:061058/0076

Effective date: 20211028

AS Assignment

Owner name: LANZATECH NZ INC., ILLINOIS

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE PATENT NUMBE 9,5348,20 PREVIOUSLY RECORDED AT REEL: 059911 FRAME: 0400. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:LANZATECH NEW ZEALAND LIMITED;REEL/FRAME:061084/0646

Effective date: 20211028

Owner name: INC., LANZATECH N, ILLINOIS

Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE THE PATENT NUMBE 9,5348,20 PREVIOUSLY RECORDED AT REEL: 059911 FRAME: 0400. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNOR:LANZATECH NEW ZEALAND LIMITED;REEL/FRAME:061084/0646

Effective date: 20211028