WO2012109578A2 - Souches de clostridium thermocellum pour production améliorée d'éthanol et leur procédé d'utilisation - Google Patents

Souches de clostridium thermocellum pour production améliorée d'éthanol et leur procédé d'utilisation Download PDF

Info

Publication number
WO2012109578A2
WO2012109578A2 PCT/US2012/024713 US2012024713W WO2012109578A2 WO 2012109578 A2 WO2012109578 A2 WO 2012109578A2 US 2012024713 W US2012024713 W US 2012024713W WO 2012109578 A2 WO2012109578 A2 WO 2012109578A2
Authority
WO
WIPO (PCT)
Prior art keywords
organism
gene
protein
thermocellum
inactivated
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.)
Ceased
Application number
PCT/US2012/024713
Other languages
English (en)
Other versions
WO2012109578A3 (fr
Inventor
Adam Guss
Lee R. Lynd
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.)
Dartmouth College
Original Assignee
Dartmouth College
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 Dartmouth College filed Critical Dartmouth College
Publication of WO2012109578A2 publication Critical patent/WO2012109578A2/fr
Publication of WO2012109578A3 publication Critical patent/WO2012109578A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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; 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0067—Oxidoreductases (1.) acting on hydrogen as donor (1.12)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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

Definitions

  • the present disclosure pertains to the field of biomass processing to produce ethanol.
  • new thermophilic organisms that can use a variety of biomass derived substrates and produce ethanol in high yield are disclosed.
  • Lignocellulosic biomass represents one of the most abundant renewable resources on Earth. It is formed of three major components - cellulose, hemicellulose, and lignin - and includes, for example, agricultural and forestry residues, municipal solid waste (MSW), fiber resulting from grain operations, waste cellulosics (e.g., paper and pulp operations), and energy crops.
  • MSW municipal solid waste
  • the cellulose and hemicellulose polymers of biomass may be hydrolyzed into their component sugars, such as glucose and xylose, which can then be fermented by microorganisms to produce ethanol. Conversion of even a small portion of the available biomass into ethanol could substantially reduce current gasoline consumption and dependence on petroleum.
  • SSF simultaneous saccharification and fermentation
  • SSCF simultaneous saccharification and co-fermentation
  • co-fermentation processes may also provide improved product yield because certain compounds that would otherwise accrue at levels that inhibit metabolysis or hydrolysis are consumed by the co-fermenting organism(s).
  • ⁇ - glucosidase ceases to hydrolyze cellobiose in the presence of glucose and, in turn, the build-up of cellobiose impedes cellulose degradation.
  • An SSCF process involving co- fermentation of cellulose and hemicellulose hydrolysis products may alleviate this problem by converting glucose into one or more products that do not inhibit the hydrolytic activity of ⁇ -glucosidase.
  • Consolidated bioprocessing involves four biologically-mediated events: (1) enzyme production, (2) substrate hydrolysis, (3) hexose fermentation and (4) pentose fermentation. In contrast to conventional approaches, which perform each step independently, all four events may be performed simultaneously in a CBP configuration. This strategy requires a microorganism that utilizes both cellulose and hemicellulose. Otherwise, a CBP process that utilizes more than one organism to accomplish the four biologically-mediated events is referred to as a consolidated bioprocessing co-culture fermentation.
  • Acetyl-CoA is further converted to acetate by phosphotransacetylase ("pta”) and acetate kinase (“ack”), or reduced to ethanol by acetaldehyde dehydrogenase (“AcDH”) and alcohol
  • Carbohydrate metabolic pathways such as those described above, may be altered by directing the flow of carbon to a desired end product, such as ethanol. See generally, Lynd, L. R., P. J. Weimer, W. H. van Zyl, and I. S. Pretorius (2002) Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66: 506.
  • a "carbon-centered" approach to metabolic engineering involves inactivating enzymatic pathways that direct carbon containing molecules away from ethanol or otherwise promoting the flow of carbon towards ethanol. For instance, Desai, S. G., M. L. Guerinot, L. R.
  • the present instrumentalities advance the art by providing methods for manipulating branched end-product metabolism of fermentative microorganisms.
  • the end-product ratio between ethanol and organic acids is altered by eliminating one or more enzymatic activities associated with the formation of hydrogen.
  • one or more mutations in a maturation protein implicated in post-translational modification of hydrogenases are introduced into a organism to modify the hydrogenase activities within the organism.
  • the maturation proteins that facilitates the maturation of other proteins may be referred to as “maturase” or "maturation protein,” and those maturases that facilitate maturation of hydrogenase may be referred to as "hydrogenase maturase, " or “hydrogenase maturation protein.”
  • one or more genes encoding enzymes implicated in the production of organic acids are mutated to reduce production of these organic acids and to direct the carbon flow towards ethanol.
  • Example of organic acids may include but are not limited to lactic acid, acetic acid, formic acid or salts thereof.
  • the resulting organism may utilize a variety substrates derived from biomass to generate ethanol in higher yield as compared to ethanol yield by the parental strain.
  • the gene encoding the pyruvate-formate lyase (pfl) which is required for the production of formate may be disrupted in order to obtain an modified organism with higher ethanol yield. Methods for generating such organisms by genetic engineering are also disclosed.
  • mutations may be generated in various genes either singly or in combination, where such genes encode proteins that play some roles in the formation of hydrogen and/or organic acids in the native (host) organism.
  • genes may include but are not limited to: (a) hydrogenase genes, (b) genes encoding one or more maturation proteins that modify hydrogenase post-translationally, such as HydE, HydF and HydG, (c) any other genes encoding proteins that modulate the activities of hydrogenase, (d) pyruvate-formate lyase (pfl) gene, (e) pfl and/or one or more of HydE, HydF and HydG genes, (f) acetate kinase (ack) gene, (g) phosphotransacetylase (pta) gene, and (h) lactate dehydrogenase (Idh) gene; or (i) combination of two or more genes from (a)-(h) listed above.
  • a mutation may include but not limited to knockout, deletion, insertion, substitution and so on. Mutations may affect the coding sequence which determine the sequence of the encoded proteins. Mutations may also occur in the non-coding regions of the target genes.
  • enzymatic activities may be eliminated by rendering non-expression of a target gene through methods other than mutations.
  • expression of a gene can be eliminated by RNA interference, gene silencing, among others.
  • saccharification product of a substrate derived from a biomass is created to obtain a modified organism capable of producing ethanol in higher yield as compared to the ethanol yield of the unmodified organism.
  • a first gene endogenous to the organism which encodes a post-translational modifying protein is inactivated through genetic engineering, which, in turn, leads to a decrease in hydrogenase activities in the organism.
  • the saccharification product is derived from a carbohydrate-rich biomass substrate.
  • the post-translational modifying protein is a protein capable of facilitating maturation of at least one hydrogenase synthesized by said organism in its native state.
  • the post-translational modifying protein may be a protein that facilitates the assembly of protein complexes having hydrogenase activities.
  • the first gene may be a gene selected from the group consisting of HydE (SEQ ID NO. 1), HydF (SEQ ID NO. 2) and HydG (SEQ ID NO. 3).
  • the first gene may encode a protein having at least 80%, 90%, 95%, 99% or more preferably, 100%) sequence identity with a protein encoded by a
  • polynucleotide sequence selected from the group consisting of SEQ ID Nos. 1-3.
  • a first gene endogenous to the organism which encodes a protein required for the generation of formate or formic acid is inactivated through genetic engineering, which, in turn, leads to a decrease in formate production in the organism.
  • the first gene may encode a pyruvate-formate lyase (pfl).
  • the first gene may encode a protein having at least 80%, 90%, 95%, 99%, or more preferably, 100%) sequence identity with a protein encoded by the pflA gene (SEQ ID No. 4) or pflB gene (SEQ ID No. 5) of C. thermocellum. Note that in C.
  • thermocellum the the pflA and pflB genes are so close to each other that the pflA locus and the pflB locus may be referred to as the pflAB locus.
  • the organism is a bacterium, preferably, a thermophilic, anaerobic bacterium, more preferably, a Clostridium thermocellum.
  • the organism is a bacterium strain deposited under Patent Deposit Designation No. PTA-11763 or PTA-11764.
  • an isolated polynucleotide comprising a polynucleotide sequence having at least 90%, 95%, 99%, or more preferably, 100%) sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID Nos. 1-5 is described.
  • a method for producing ethanol includes generating a modified organism with a first gene encoding a post-translational modifying protein inactivated, and incubating the organism in a medium containing at least one substrate.
  • the substrate may include but are not limited to glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, cellulose, pectin or combinations thereof.
  • the post-translational modifying protein is capable of facilitating maturation of at least one hydrogenase synthesized by the organism in its native state. By inactivating the first gene encoding such a post-translational modifying protein, the modified organism has decreased hydrogenase activities which, in turn, leads to enhanced ethanol production.
  • a method for producing ethanol includes generating a modified organism with a first gene inactivated which encodes a protein required for the formation of formate or formic acid, and incubating the organism in a medium containing at least one substrate.
  • the substrate may include but are not limited to glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, cellulose, pectin or combinations thereof.
  • a method for producing ethanol includes providing within a reaction vessel, a reaction mixture comprising a carbohydrate-rich biomass substrate, a cellulolytic material, and a fermentation agent.
  • the fermentation agent contains a bacterium that has been genetically modified to inactivate a first gene encoding a post-translational modifying protein.
  • the post- translational modifying protein is a maturation protein (or maturase) that facilitates the hydrogenase in the bacterium.
  • the reaction mixture may be incubated under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the carbohydrate-rich biomass substrate.
  • the fermentation agent contains a bacterium that has been genetically modified to inactivate a first gene encoding a protein required for the production of formate or formic acid by the bacterium.
  • the reaction mixture may be incubated under a suitable condition wherein the temperature is at least 50°C.
  • one or more genes in addition to the pfl gene and/or the maturation genes may be inactivated in an organism.
  • these one or more genes encode one or more proteins selected from the group consisting of lactate dehydrogenase (ldh), acetate kinase (ack), phosphotransacetylase ipta), hydrogenase, and combination thereof.
  • a modified organism may be created wherein both the pfl gene and at least one gene encoding a hydrogenase maturase are inactivated.
  • a modified organism may be created wherein the pfl gene, the ldh gene and at least one gene encoding a hydrogenase maturase are inactivated.
  • a modified organism may be created wherein the pfl gene, the ldh gene, the ack gene, the pta gene, and at least one gene encoding a hydrogenase maturase are inactivated.
  • Fig. 1 shows a modified glycolytic pathway after hydrogenase inactivation, according to an embodiment.
  • Fig. 2 shows the sequences of the hydrogenase maturase genes HydE
  • Fig. 3 showns a diagram of the plasmid pAMG258, along with its full sequence.
  • Fig. 4 showns a diagram of the plasmid pAMG278.
  • Fig. 5 showns a diagram of the plasmid pAMG281.
  • Fig. 6 panels A and B are eletrophoresis pictures showing deletion of the HydG gene.
  • Fig. 7 shows the end-product profile in the mutant hydG strain and mutant pfl strain.
  • thermophilic, anaerobic, Gram-positive bacteria in the conversion of biomass to ethanol.
  • an organism is "in a native state" if it has not been genetically engineered or otherwise manipulated by the hand of man in a manner that alters the genotype and/or phenotype of the organism.
  • a wild-type organism may be considered to be in a native state.
  • Identity refers to a comparison between sequences of polynucleotide or polypeptide molecules. Methods for determining sequence identity are commonly known. Computer programs typically employed for performing an identity comparison include, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), which uses the algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489.
  • “Lignocellulosic substrate” generally refers to any lignocellulosic biomass suitable for use as a substrate to be converted into ethanol.
  • a “Lignocellulosic substrate” may be referred to as a “substrate” in this disclosure.
  • Saccharification refers to the process of breaking a complex carbohydrate, such as starch or cellulose, into its monosaccharide or oligosaccharide components.
  • a complex carbohydrate is preferably processed into its monosaccharide components during a saccharification process.
  • endogenous is used to describe a molecule that exists naturally in an organism.
  • a molecule that is introduced into an organism using molecular biology tools, such as transgenic techniques, is not endogenous to that organism.
  • inactivated means more than seventy percent.
  • a gene is considered inactivated if its expression or its function has been reduced by more than seventy percent.
  • Techniques for inactivation of a target gene may include, but are not limited to, deletion, insertion, substitution in the coding or non-coding regulatory sequences of the target gene, as well as the use of RNA interference to suppress gene expression.
  • the process of inactivating a gene is also referred to as “knocking out” a gene.
  • an organism that has one or more of its genes inactivated may be called a “knockout” (KO) strain.
  • an organism that possesses the necessary biological and chemical components including polynucleotides, polypeptides, carbohydrates, lipids and other molecules, as well as cellular or subcellular structures that may be required for performing or facilitating certain biological and/or chemical processes is deemed to be capable of performing said processes.
  • an organism that contains certain inducible genes may be considered capable of performing the function attributable to the proteins encoded by those genes.
  • genetic engineering is used to refer to a process by which genetic materials, including DNA and/or RNA, are manipulated in a cell or introduced into a cell to affect expression of certain proteins in the cell. Manipulation may include introduction of a foreign (or “exogenous”) gene into the cell or inactivation or
  • Such a modified cell may be called a "genetically engineered cell” or a “genetically modified cell”. If the original cell to be genetically engineered is a bacterial cell, said genetically engineered cell may be said to have been derived from a bacterial cell.
  • a molecule that is introduced into a cell to genetically modify the cell may be called a genetic construct.
  • a genetic construct typically carries one or more DNA or RNA sequences on a single molecule.
  • the expression of a protein is generally regulated by the non-coding region of a gene termed promoter.
  • promoter controls the transcription of a gene, it can also be said that the expression of the gene (or the encoded protein) is driven by the promoter.
  • a promoter is placed in proximity of a coding sequence, such that transcription of the coding sequence is under control of the promoter, it can be said that the coding sequence is operably linked to the promoter.
  • a promoter that is not normally associated with a gene is called a heterologous promoter.
  • a "cellulolytic material” is a material that may facilitate the breakdown of cellulose into its component oligosaccharides or monosaccharides.
  • cellulolytic material may comprise a cellulase or hemicellulase.
  • microorganism may be altered by directing the flow of carbon to a desired end product, such as ethanol, using a "carbon-centered" approach to metabolic engineering.
  • a desired end product such as ethanol
  • An alternative, "electron-centered” approach is disclosed herein where ethanol yield may be increased by inactivation of an enzymatic pathway that produces hydrogen.
  • Fig. 1 illustrates a portion of the glycolytic pathway, where a cross indicates blocking of hydrogenase activity that leads to hydrogen production. Based on stoichiometric equations, hydrogen production is related to acetic acid production. Therefore, disrupting the ability of an organism to produce hydrogen may result in decreased production of acetic acid and increased ethanol production.
  • thermophilic bacterium Clostridium thermocellum
  • Clostridium thermocellum is used by way of example to illustrate how maturases in an organism may be manipulated to affect hydrogenase activities and to increase ethanol production.
  • the methods and materials disclosed herein may however apply to members of the Clostridium,
  • Clostridium genus may include but are not limited to, Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium thermocellulaseum.
  • Thermoanaerobacter and Thermoanaerobacterium genera may include, for example, Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum,
  • thermohydrosulfuricus Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus,
  • Thermoanaerobacter brockii variants thereof, and/or progeny thereof. Both the carbon- centered and the electron-centered approaches for maximizing ethanol production from biomass may be applicable in metabolic engineering of other microorganisms, such as yeast or fungi.
  • Major groups of bacteria include eubacteria and archaebacteria.
  • Thermophilic eubacteria include: phototropic bacteria, such as cyanobacteria, purple bacteria and green bacteria; Gram-positive bacteria, such as Bacillus, Clostridium, lactic acid bacteria and Actinomyces; and other eubacteria, such as Thiobacillus, Spirochete, Desulfotomaculum, Gram-negative aerobes, Gram-negative anaerobes and Thermotoga.
  • the present instrumentalities relate to Gram-negative
  • thermophiles of the genus Thermus Gram-positive eubacteria, such as Clostridium, which comprise both rods and cocci; eubacteria, such as Thermosipho and Thermotoga; archaebacteria, such as Thermococcus, Thermoproteus (rod-shaped), Thermofilum (rod-shaped), Pyrodictium, Acidianus, Sulfolobus, Pyrobaculum,
  • thermophilic or mesophilic organisms including bacteria, prokaryotic microorganisms and fungi
  • Clostridium thermosulfurogenes Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium
  • thermosaccharolyticum Clostridium tartarivorum, Clostridium thermocellulaseum, Anaerocellum sp., Thermoanaerobacterium thermosaccharolyticum,
  • Thermoanaerobium brockii Methanobacterium thermoautotrophicum, Pyrodictium occultum, Thermoproteus neutrophilus, Thermofllum librum, Thermothrix thioparus, Desulfovibrio thermophilus, Thermoplasma acidophilum, Hydrogenomonas
  • thermophilus Thermomicrobium roseum, Thermus flavas, Thermus ruber, Pyrococcus furiosus, Thermus aquaticus, Thermus thermophilus, Chloroflexus aurantiacus,
  • Lactobacillus thermophilus Lactobacillus bulgaricus, Bifidobacterium thermophilum, Streptomyces fragmentosporus, Streptomyces thermonitrificans, Streptomyces
  • thermovulgaris Pseudonocardia thermophila, Thermoactinomyces vulgaris,
  • thermophilic bacteria for use with the disclosed instrumentalities may be selected from the group consisting of
  • Fervidobacterium gondwanense Clostridium thermolacticum, Moorella sp. and
  • Rhodothermus marinus Rhodothermus marinus.
  • the disclosed instrumentalities relate to microorganisms of the genera Geobacillus, Saccharococcus, Paenibacillus, Bacillus and Anoxybacillus, including but not limited to species selected from the group consisting of: Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof, and/or progeny thereof.
  • Geobacillus thermoglucosidasius Geobacillus stearothermophilus
  • Saccharococcus caldoxylosilyticus Saccharoccus thermophilus
  • Paenibacillus campinasensis Bacillus flavothermus
  • Anoxybacillus kamchatkensis Anoxybacillus gonensis, variants thereof, and/or progen
  • the disclosed instrumentalities relate to mesophilic bacteria selected from the group consisting of Saccharophagus degradans; Flavobacterium johnsoniae; Fibrobacter succinogenes; Clostridium hungatei;
  • Clostridium phytofermentans Clostridium cellulolyticum; Clostridium aldrichii;
  • Acetivibrio multivorans Bacteroides cellulosolvens; and Alkalibacter
  • saccharofomentans variants thereof, and/or progeny thereof.
  • the disclosed instrumentalities relate to organisms having a ferredoxin- linked hydrogenase (EC subclass 1.12.7.2), including but not limited to organisms selected from the groups of eubacteria and achaebacteria, phototropic bacteria (such as cyanobacteria, purple bacteria and green bacteria), Gram- positive bacteria and lactic acid bacteria and Gram-negative anaerobes, as well as organisms selected from the genera including, but not limited to: Bacillus, Clostridium, Thermotoga, Pyrococcus and Saccharococcus.
  • EC subclass 1.12.7.2 ferredoxin- linked hydrogenase
  • Such organisms include those selected from the group consisting of: Thermotoga maritima, Clostridium acetobutylicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium
  • thermohydrosulfuricum Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium thermocellulaseum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium saccharolyticum, Thermobacteroides acetoethylicus,
  • Figure 1 shows the general pathways of sugar fermentation.
  • hexoses may be converted to pyruvate through the glycolytic pathway.
  • Pyruvate is a branch point from which carbon and electron may flow to various fermentation end products. Examples of these end products include but are not limited to lactate, acetate, formate, carbon dioxide, hydrogen, and ethanol.
  • thermocellum thermocellum
  • thermocellum contains two putative pyruvate-formate lyase (pfl) genes which are predicted to be involved in formate production during pyruvate conversion to acetyl-CoA.
  • a polynucleotide having 70%, 80%, 90%, 99% sequence identity with the polynucleotide of SEQ ID No. 4 or SEQ ID No. 5 may encode a functional putative pyruvate-formate lyase. It is disclosed here that disruption of one or both of the pfl genes in C. thermocellum reduces formate formation and increases ethanol production.
  • one or both of the pfl genes as well as one or all of the hydrogenase maturation protein genes may be disrupted in one organism.
  • one or more of the genes involved in the production of lactate, acetate, formate, or hydrogen may be disrupted in an organism in order to direct carbon flow to ethanol.
  • lactate dehydrogenase ⁇ Idh the gene that confers the ability to produce lactic acid
  • acetate kinase (ack) and/or phosphotransacetylase ipta) the genes that confer the ability to produce acetic acid
  • lactate dehydrogenase ⁇ Idh the gene that confers the ability to produce lactic acid
  • ack acetate kinase
  • phosphotransacetylase ipta phosphotransacetylase
  • an organism may be generated in which all hydrogenase activities leading to synthesis of hydrogen are disrupted in order to maximize ethanol production. For instance, maturation proteins required for enzymatic activities of Fe-Fe hydrogenase and Ni-Fe hydrogenase may all be inactivated to remove any residual hydrogen production.
  • optimal cellulase activity parameters include a pH between 4-5 and temperature between 40-50°C, which are substantially similar to the optimal growth conditions of thermophilic bacteria.
  • optimal growth temperature for T. saccharolyticum is about 50-60°C.
  • C. thermocellum is capable of rapidly degrading cellulose, but it cannot ferment pentose sugars, which, in the form of xylan and other polysaccharides, may account for up to 30% of total carbohydrates in a typical saccharified biomass.
  • T. saccharolyticum is capable of fermenting and utilizing pentose sugars. A process utilizing both C.
  • thermocellum and a knockout of T. saccharolyticum may therefore be an efficient way to improve cellulosic ethanol production, and reduce process costs. See Lynd, L. R., W. H. van Zyl, J. E. McBride, and M. Laser (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16: 577-583.
  • thermophilic temperatures offer several important benefits over conventional mesophilic fermentation temperatures of 30-37°C.
  • enzyme concentrations necessary to achieve a given amount of conversion may be reduced due to higher enzyme activity at
  • thermophilic temperatures are substantially reduced for thermophilic SSF and SSCF (e.g., 2-fold or more), and are eliminated for CBP. Costs associated with fermentor cooling and heat exchange before and after fermentation are also expected to be reduced for thermophilic SSF, SSCF and CBP. Finally, processes featuring thermophilic biocatalysts may be less susceptible to microbial contamination as compared to processes featuring conventional mesophilic biocatalysts.
  • a method for producing ethanol includes providing within a reaction vessel, a reaction mixture comprising lignocellulosic substrate, a cellulolytic material and a fermentation agent.
  • the fermentation agent comprises an organism that has been transformed to eliminate expression of at least one gene encoding a hydrogenase maturase or the pyruvate-formate lyase.
  • the reaction mixture is reacted under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the lignocellulosic substrate.
  • Appropriate substrates for the production of ethanol include, for example, one or more of glucose, xylose, cellobiose, sucrose, xylan, starch, cellulose, pectin and combinations thereof. These substrates may, in some aspects, be produced during an SSF, SSCF or CBP process to achieve efficient conversion of biomass to ethanol.
  • carbohydrate-rich biomass material that is saccharified to produce one or more of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch cellulose and pectin may be utilized by the disclosed organisms.
  • the biomass may be lignocellulosic biomass that comprises wood, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard, or combinations thereof.
  • a plasmid was constructed using yeast gap repair cloning (Burke, D., D. Dawson, and T. Stearns. 2000. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.) to delete the hydG gene from the C. thermocellum chromosome.
  • a region ca. 1 kilobase (kb) upstream of the C. thermocellum hydG gene was amplified by polymerase chain reaction (PCR) using primers:
  • thermocellum gapD promoter for positive and negative selection in C. thermocellum flanked on one side by a fusion of the upstream and downstream region of C.
  • thermocellum hydG and on the other side by an internal fragment of hydG.
  • the plasmid also contains the Thermoanaerobacterium saccharolyticum tdk gene expressed from the C. thermocellum cbp promoter for a second negative selection in C. thermocellum, the pUC19 origin of replication and bla gene for replication and selection in Escherichia coli, the URA3+ gene and CEN6/ARSH4 origin of replication for selection and propagation of the plasmid in Saccharomyces cerevisiae, and the pNW33N origin of replication for plasmid replication in C. thermocellum.
  • the DNA was transformed into yeast via a modified Lazy Bones protocol (Elble, R. 1992. A simple and efficient procedure for transformation of yeasts. Biotechniques 13:18-20.) and was assembled into a contiguous piece of DNA via yeast homologous recombination. Plasmid DNA was isolated from yeast using Zymoprep Yeast Plasmid Miniprep II kit (Zymo, Orange, CA, USA.) and introduced via
  • E. coli Top 10 Invitrogen, Carlsbad, CA
  • E. coli BL21 DE3
  • All PCR amplified regions were sequenced at the Dartmouth College Molecular Biology Core Facility to verify PCR fidelity.
  • a second plasmid, pAMG281 (Fig. 5), was constructed in an essentially identical manner to delete pflAB from the C. thermocellum genome, except that the upstream region of pflAB was PCR amplified using primers:
  • thermocellum Ahpt strain was obtained from Mascoma Corporation and was transformed via electroporation as described (WO 2010/056450 ELECTRO- TRANSFORMATION OF GRAM-POSITIVE, ANAEROBIC, THERMOPHILIC BACTERIA and Tyurin, M. V., S. G. Desai, and L. R. Lynd. 2004. Electrotransformation of Clostridium thermocellum. Appl Environ Microbiol 70:883-90) with modifications. Briefly, 400 ml C.
  • thermocellum Ahpt strain was grown inside a Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) in modified DSM122 media supplemented with 50 mM MOPS and 10 mM sodium citrate at 51°C. to an OD between 0.8 and 1.0, centrifuged without measures to exclude oxygen at room temperature in a Beckman Coulter Avanti J-25 centrifuge with a JA-10 rotor at 5000 x g, and the supernatant was removed.
  • cell pellets were washed with 400 ml ice cold electroporation buffer prepared without measures to exclude oxygen and consisting of 250 mM sucrose, 10% glycerol, 100 ⁇ MOPS pH 7.0, 0.5 mM MgCl 2 , 0.5 mM MgS0 4 and centrifuged at 4000 x g.
  • the cells were rinsed and centrifuged a second time as above and brought on ice into a Coy anaerobic chamber. Cells were resuspended in an additional 500 ⁇ electroporation buffer and kept on ice until use.
  • Plasmid DNA was diluted to 500 ng/ ⁇ , and 2 ⁇ DNA was mixed with 20 ⁇ washed cells in pre-chilled 1 mm gap electroporation cuvettes. The mixture was then subjected to a 1.2 kV, 1.5 msec square pulse using a BioRad GenePulser XCell. Cells were immediately resuspended in 1 ml room temperature growth medium and serial dilutions were plated with no recovery period (to ensure each colony represents a unique transformant) by mixing with 25 ml molten media + 0.8% agar + thiamphenicol (10 ⁇ g/ml). Once plates had solidified, they were placed in 2.5 L AnaeroPack Rectangular Jars (bioMerieux, Durham, NC, USA) to minimize desiccation and incubated at 51°C for up to one week.
  • thermocellum Ahpt strains containing plasmid pAMG281 or pAMG278 were grown in 5 ml liquid medium supplemented with thiamphenicol.
  • Serial dilutions were plated via pour plating in medium supplemented with 0.8% agar, 10 g/ml thiamphenicol, and 10 g/ml 5-fluoro- 2'-deoxuracil (FUdR) to select for the simultaneous integration of the cat-hpt genes at the hydG or pflAB locus and the loss of the plasmid.
  • Thiamphenicol-resistant, FUdR-resistant colonies were purified via streak plating and isolated colonies were grown in 5 ml liquid medium supplemented with thiamphenicol.
  • Colony PCR was performed to screen for the correct genomic structure of putative deletion mutants (Fig. 6).
  • the gel was loaded with primer sets grouped together and ordered from 1 - 5, flanked by 2-Log DNA ladder from NEB.
  • templates were, in order, (1) No template negative control, (2) C.
  • thermocellum Ahpt parent strain wild type at hydG locus (3)-(5) C. thermocellum Ahpt AhydG clones #1-3.
  • Primer pair #1 targets an internal region of the hydG gene that should generate a ca. 1000 base pair band in strains wild type at this locus and no product in strains deleted for hydG.
  • the PCR results using Primer pair #1 are shown in Fig. 6 A, lanes 2-6. Lane 2: No template negative control; lane 3: C. thermocellum Ahpt parent strain (wild type at hydG locus); lanes 4-6: C. thermocellum Ahpt AhydG clones #1-3.
  • the sequences of Primer pair #1 are:
  • Primer pair #2 targets a ca. 700 base pair fragment of the plasmid backbone and should be absent in all strains.
  • the PCR results using Primer pair #2 are shown in Fig. 6A, lanes 8-12. Lane 8: No template negative control; lane 9: C.
  • thermocellum Ahpt parent strain wild type at hydG locus
  • lanes 10-12 C.
  • Primer pair #3 targets the upstream junction of the deleted region and should produce a ca. 2700 base pair band in the wild type and a ca. 1200 base pair band in the hydG deletion mutant.
  • the PCR results using Primer pair #3 are shown in Fig. 6A, lanes 14-18.
  • Lane 14 No template negative control
  • lane 15 C. thermocellum Ahpt parent strain (wild type at hydG locus)
  • lanes 16-18 C. thermocellum Ahpt AhydG clones #1-3.
  • Primer pair #3 :
  • Primer pair #4 targets the downstream junction of the deleted region and should produce a ca. 2900 base pair band in the wild type and a ca. 1300 base pair band in the hydG deletion mutant.
  • the PCR results using Primer pair #4 are shown in Fig. 6B, lanes 2-6.
  • lanes 4-6 C. thermocellum Ahpt AhydG clones #1-3.
  • Primer pair #4 sequences are:
  • Primer pair #5 flank the upstream and downstream regions, and should result in a band ca. 3600 base pairs in the wild type and a ca. 2200 base pair band in the hydG deletion mutant.
  • the PCR results using Primer pair #5 are shown in Fig. 6B, lanes 10-14.
  • lane 11 C. thermocellum Ahpt parent strain (wild type at hydG locus);
  • lanes 12-14 C. thermocellum Ahpt AhydG clones #1-3.
  • Primer pair #5 :
  • C. thermocellum Ahpt Apfl C. thermocellum Ahpt AhydG
  • parent strain C. thermocellum Ahpt were grown anaerobically in Balch tubes containing 10 ml medium with a ca. 17 ml headspace. After growth was complete, 1 ml samples were removed and centrifuged at 15000 x g to remove cells. Then, 700 ⁇ of the supernatant was combined with 100 ⁇ 10% sulfuric acid and filtered through a 0.22 ⁇ filter.
  • Ethanol, acetic acid, lactic acid, and formic acid were then measured by HPLC using a Bio-Rad Aminex HPX- 87H column with RI detection on a Waters HPLC system at 60°C, as shown in Fig. 7.
  • the parent strain C. thermocellum Ahpt is shown as Dhpt in Fig. 7
  • the mutant strain Ahpt Apfl is indicated as Dpfi
  • the mutant Ahpt AhydG is shown as DhydG in Fig. 7.
  • Additional mutants carrying mutations in more than one genes may be created by genetic engineering. These genes may include, for example, (a) hydrogenase genes, (b) genes encoding one or more maturation proteins that modify hydrogenase post- translationally, such as HydE, HydF and HydG, (c) any other genes encoding proteins that modulate the activities of hydrogenase, (d) pyruvate-formate lyase (pfl) genes, such as pflA and pflB, (e) acetate kinase (ack) gene, (f) phosphotransacetylase (pta) gene, and (g) lactate dehydrogenase (Idh) gene, among others.
  • a hydrogenase genes
  • genes encoding one or more maturation proteins that modify hydrogenase post- translationally, such as HydE, HydF and HydG
  • any other genes encoding proteins that modulate the activities of hydrogenase (d) pyruvate-format
  • Double mutant can be generated wherein two genes that are known to play non-redundant roles in the fermentation pathways are disrupted.
  • the pfl gene may be disrupted in a mutant strain carrying a deletion to one or more of the hydrogenase maturase genes, such as HydE, HydF or HydG.
  • one or more of the hydrogenase maturase genes may be deleted/disrupted in a mutant strain already carrying a pfl gene deletion.
  • Mutations in three or more genes may also be introduced into a strain.
  • the ldh gene may be disrupted in a double mutant strain which already has both the pfl gene and the hydG gene deleted.
  • such a triple mutant may produce much higher levels of ethanol as compared to the pfl- hydG double mutant because the blocking of lactate production may direct the more carbon flow to ethanol.
  • other multiple disruption mutants may be generated to maximize ethanol production.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Selon l'invention, le Clostridium thermocellum est une bactérie anaérobie thermophile qui se spécialise dans la solubilisation et la fermentation rapides de cellulose cristalline en des produits qui comprennent de l'éthanol. Des mutants de délétion de C. thermocellum ont été construits pour éliminer des activités de la lyase pyruvate-formiate (pfl) et de l'hydrogénase. C. thermocellum Apfl ne synthétise plus de formiate. Les mutants pfl produisent moins d'acétate que la souche parentale. Le mutant, qui n'exprime pas l'une des protéines de maturation requises pour la production d'hydrogénases fonctionnelles dans C. thermocellum, présente une production réduite d'acétate par comparaison avec des souches normales.
PCT/US2012/024713 2011-02-11 2012-02-10 Souches de clostridium thermocellum pour production améliorée d'éthanol et leur procédé d'utilisation Ceased WO2012109578A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161442109P 2011-02-11 2011-02-11
US61/442,109 2011-02-11

Publications (2)

Publication Number Publication Date
WO2012109578A2 true WO2012109578A2 (fr) 2012-08-16
WO2012109578A3 WO2012109578A3 (fr) 2012-11-22

Family

ID=46639223

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/024713 Ceased WO2012109578A2 (fr) 2011-02-11 2012-02-10 Souches de clostridium thermocellum pour production améliorée d'éthanol et leur procédé d'utilisation

Country Status (1)

Country Link
WO (1) WO2012109578A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2534880C1 (ru) * 2013-09-23 2014-12-10 Федеральное государственное бюджетное учреждение науки Институт цитологии и генетики Сибирского отделения Российской академии наук (ИЦиг СО РАН) ШТАММ БАКТЕРИИ Geobacillus stearothermophilus - ПРОДУЦЕНТ БИОЭТАНОЛА
US20150210987A1 (en) * 2014-01-30 2015-07-30 Lanzatech New Zealand Limited Recombinant microorganisms and methods of use thereof
CN109055459A (zh) * 2018-08-17 2018-12-21 中国科学院青岛生物能源与过程研究所 用于木质纤维素的全菌糖化方法
CN110540982A (zh) * 2019-09-30 2019-12-06 江南大学 一种提高梭热杆菌纤维素酶产量发酵方法
CN114717174A (zh) * 2022-04-26 2022-07-08 苏州聚维元创生物科技有限公司 一种产高品质还原糖的工程菌株、构建方法及其应用
CN115029264A (zh) * 2022-06-14 2022-09-09 中国科学院青岛生物能源与过程研究所 一株耐酸热纤梭菌及其降解木质纤维素的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770435A (en) * 1995-11-02 1998-06-23 University Of Chicago Mutant E. coli strain with increased succinic acid production
US6280986B1 (en) * 1997-12-01 2001-08-28 The United States Of America As Represented By The Secretary Of Agriculture Stabilization of pet operon plasmids and ethanol production in bacterial strains lacking lactate dehydrogenase and pyruvate formate lyase activities
JP2009513145A (ja) * 2005-10-31 2009-04-02 ザ トラスティーズ オブ ダートマウス カレッジ リグノセルロース系バイオマスをエタノールに変換する好熱性生物
WO2009079584A1 (fr) * 2007-12-17 2009-06-25 The Trustees Of Dartmouth College Modification d'activités d'hydrogénase dans des bactéries thermophiles pour amplifier la production d'éthanol

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2534880C1 (ru) * 2013-09-23 2014-12-10 Федеральное государственное бюджетное учреждение науки Институт цитологии и генетики Сибирского отделения Российской академии наук (ИЦиг СО РАН) ШТАММ БАКТЕРИИ Geobacillus stearothermophilus - ПРОДУЦЕНТ БИОЭТАНОЛА
US20150210987A1 (en) * 2014-01-30 2015-07-30 Lanzatech New Zealand Limited Recombinant microorganisms and methods of use thereof
CN106133132A (zh) * 2014-01-30 2016-11-16 朗泽科技新西兰有限公司 重组微生物和其使用方法
AU2015210892B2 (en) * 2014-01-30 2019-01-17 Lanzatech Nz, Inc. Recombinant microorganisms and methods of use thereof
US11549103B2 (en) 2014-01-30 2023-01-10 Lanzatech Nz, Inc. Recombinant microorganisms and methods of use thereof
CN109055459A (zh) * 2018-08-17 2018-12-21 中国科学院青岛生物能源与过程研究所 用于木质纤维素的全菌糖化方法
CN110540982A (zh) * 2019-09-30 2019-12-06 江南大学 一种提高梭热杆菌纤维素酶产量发酵方法
CN114717174A (zh) * 2022-04-26 2022-07-08 苏州聚维元创生物科技有限公司 一种产高品质还原糖的工程菌株、构建方法及其应用
CN114717174B (zh) * 2022-04-26 2023-10-20 苏州聚维元创生物科技有限公司 一种产高品质还原糖的工程菌株、构建方法及其应用
CN115029264A (zh) * 2022-06-14 2022-09-09 中国科学院青岛生物能源与过程研究所 一株耐酸热纤梭菌及其降解木质纤维素的方法
CN115029264B (zh) * 2022-06-14 2023-06-09 中国科学院青岛生物能源与过程研究所 一株耐酸热纤梭菌及其降解木质纤维素的方法

Also Published As

Publication number Publication date
WO2012109578A3 (fr) 2012-11-22

Similar Documents

Publication Publication Date Title
US8435770B2 (en) Gene knockout mesophilic and thermophilic organisms, and methods of use thereof
US10066217B2 (en) Thermophilic organisms for conversion of lignocellulosic biomass to ethanol
EP3033413B1 (fr) Procédés pour l'amélioration du rendement de production et de la production dans un micro-organisme par recyclage de glycérol
US20120094343A1 (en) Gene Knockout Mesophilic and Thermophilic Organisms, and Methods of Use Thereof
US20120107892A1 (en) Production of ethanol from lignocellulosic biomass
WO2012109578A2 (fr) Souches de clostridium thermocellum pour production améliorée d'éthanol et leur procédé d'utilisation
US20110256601A1 (en) Modification of hydrogenase activities in thermophilic bacteria to enhance ethanol production
US20120295306A1 (en) Modified CIPA Gene From Clostridium Thermocellum for Enhanced Genetic Stability
EP2397556A1 (fr) Organismes thermophiles pour la conversion de biomasse ligno-cellulosique en ethanol
BR112016002359B1 (pt) Microorganismo recombinate, método para diminuir o glicerol celular produzido, e processo para a conversão de biomassa em etanol

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12744792

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12744792

Country of ref document: EP

Kind code of ref document: A2