WO2014021163A1 - Procédé de production d'éthanol faisant appel à une levure recombinée - Google Patents

Procédé de production d'éthanol faisant appel à une levure recombinée Download PDF

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WO2014021163A1
WO2014021163A1 PCT/JP2013/070036 JP2013070036W WO2014021163A1 WO 2014021163 A1 WO2014021163 A1 WO 2014021163A1 JP 2013070036 W JP2013070036 W JP 2013070036W WO 2014021163 A1 WO2014021163 A1 WO 2014021163A1
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gene
yeast
strain
ethanol
pcr
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大西　徹
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トヨタ自動車株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; 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
    • 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)
    • 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 invention relates to a method for producing ethanol using recombinant yeast.
  • acetic acid is known as a fermentation inhibitor contained in a reaction system in an ethanol production method utilizing ethanol fermentation. That is, in the reaction system containing acetic acid, the efficiency of ethanol fermentation is reduced, and the ethanol yield is known to be lower than that in the reaction system not containing acetic acid.
  • acetic acid is contained in a large amount in the hydrolysis product of cellulosic biomass and inhibits ethanol fermentation of yeast.
  • yeast introduced with a xylose utilization gene significantly inhibits ethanol fermentation using xylose as a sugar source (Non-patent Documents 1 and 2).
  • moromi fermented saccharified cellulosic biomass with cellulase mainly contains unfermented residues, difficult-to-ferment residues, enzymes and fermentation microorganisms.
  • the reaction liquid containing moromi for the subsequent fermentation the fermentation microorganisms can be reused, the amount of new input microorganisms for fermentation can be reduced, and the cost can be reduced.
  • acetic acid contained in the moromi is also brought in at the same time. As a result, the concentration of acetic acid contained in the fermentation medium increases, which may inhibit ethanol fermentation.
  • acetic acid As another method for avoiding fermentation inhibition by acetic acid, it is conceivable to metabolize acetic acid in the medium simultaneously with ethanol fermentation.
  • the metabolism of acetic acid is an aerobic reaction and overlaps with the metabolic pathway of ethanol. Therefore, when fermentation is performed under aerobic conditions, acetic acid may be metabolized, but at the same time, the target substance ethanol is also metabolized.
  • yeast has an acetic acid biosynthesis pathway, and the ALD4 gene, ALD5 gene and ALD6 gene, which are acetaldehyde dehydrogenase genes, are involved in acetic acid biosynthesis. Therefore, by destroying those genes, it is considered that acetic acid production decreases (Non-patent Document 5), and fermentation inhibition may be avoided.
  • ALD6 gene-disrupted strains do not change or decrease the ethanol productivity in a medium using glucose as a sugar source (Non-patent Document 6).
  • the present invention provides a method for producing ethanol using a recombinant yeast that can improve ethanol fermentation efficiency in the presence of acetic acid and can realize excellent ethanol productivity, in view of the above-described circumstances. For the purpose.
  • the present invention includes the following.
  • the ethanol production method according to the present invention can achieve excellent ethanol productivity without reducing the efficiency of ethanol fermentation even when acetic acid is contained in the medium. Therefore, the ethanol production method according to the present invention is particularly preferably applied to a system (for example, simultaneous saccharification and fermentation) in which a sugar component obtained by saccharifying cellulosic biomass is used as a raw material for ethanol fermentation.
  • a system for example, simultaneous saccharification and fermentation
  • the method for producing ethanol according to the present invention is a method for synthesizing ethanol from a medium containing acetic acid using a mutant yeast strain lacking a specific acetaldehyde dehydrogenase gene.
  • the yeast mutant used in the method for producing ethanol according to the present invention is one lacking a specific acetaldehyde dehydrogenase gene.
  • the specific acetaldehyde dehydrogenase gene is an acetaldehyde dehydrogenase gene called ALD4 gene, ALD5 gene, and ALD6 gene in Saccharomyces cerevisiae. That is, when the yeast mutant used in the method for producing ethanol according to the present invention is prepared from a wild Saccharomyces cerevisiae strain, at least one acetaldehyde dehydration selected from the group consisting of these ALD4 gene, ALD5 gene and ALD6 gene is used. Deletion of elementary enzyme gene.
  • a yeast mutant used in the method for producing ethanol according to the present invention is produced using a yeast other than Saccharomyces cerevisiae
  • at least one of genes corresponding to the ALD4 gene, ALD5 gene and ALD6 gene in the yeast is used.
  • One acetaldehyde dehydrogenase gene is deleted.
  • the base sequence of the ALD4 gene of Saccharomyces cerevisiae and the amino acid sequence of the protein encoded by the ALD4 gene are shown in SEQ ID NOs: 1 and 2, respectively.
  • the nucleotide sequence of the ALD5 gene of Saccharomyces cerevisiae and the amino acid sequence of the protein encoded by the ALD5 gene are shown in SEQ ID NOs: 3 and 4, respectively.
  • the nucleotide sequence of the ALD6 gene of Saccharomyces cerevisiae and the amino acid sequence of the protein encoded by the ALD6 gene are shown in SEQ ID NOs: 5 and 6, respectively.
  • the acetaldehyde dehydrogenase gene to be deleted is not limited to those specified in SEQ ID NOs: 1 to 6, but is a gene that has a different base sequence or amino acid sequence, but has a paralogous relationship or a narrowly defined homologous relationship. There may be.
  • the acetaldehyde dehydrogenase gene is not limited to those specified by these SEQ ID NOs: 1 to 6, and for example, 70% or more, preferably 80% or more with respect to the amino acid sequence of SEQ ID NO: 2, 4 or 6 More preferably, it may encode a protein having an amino acid sequence having a sequence similarity or identity of 90% or more, most preferably 95% or more, and having acetaldehyde dehydrogenase activity. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX program that implements the BLAST algorithm (default setting).
  • sequence similarity was calculated by comparing the total of amino acid residues that completely matched when paired amino acid sequences were subjected to pair-wise alignment analysis and amino acid residues having similar physicochemical functions. Calculated as a percentage of the total number of all amino acid residues.
  • identity value is calculated as the ratio of the number of amino acid residues in all amino acid residues compared by calculating the amino acid residues that completely match when a pair of amino acid sequences are subjected to pair-wise alignment analysis. .
  • the acetaldehyde dehydrogenase gene is not limited to those specified in SEQ ID NOs: 1 to 6, and for example, one or several amino acids are substituted for the amino acid sequence of SEQ ID NOs: 2, 4 or 6. It may be a protein having a deleted, inserted or added amino acid sequence and encoding a protein having acetoacetaldehyde dehydrogenase activity.
  • everal means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • the acetaldehyde dehydrogenase gene is not limited to those specified in these SEQ ID NOs: 1 to 6, and for example, all or part of the complementary strand of DNA consisting of the base sequence of SEQ ID NO: 1, 3 or 5
  • it may be one that hybridizes under stringent conditions and encodes a protein having acetoacetaldehyde dehydrogenase activity.
  • stringent conditions means the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition) can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, it is 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate), and the temperature is 42 ° C.
  • a gene consisting of a base sequence different from SEQ ID NO: 1, 3 or 5 or a gene encoding an amino acid sequence different from SEQ ID NO: 2, 4 or 6 functions as an acetaldehyde dehydrogenase gene
  • an expression vector in which the gene is inserted between an appropriate promoter and terminator, etc. transform a host such as yeast using this expression vector, and measure the acetaldehyde dehydrogenase activity of the expressed protein. That's fine.
  • Acetaldehyde dehydrogenase activity can be measured by preparing a solution containing acetaldehyde or NADP + as a substrate, allowing the protein to be tested to act at an appropriate temperature, and spectroscopically measuring the produced NADH or NADPH. .
  • the yeast mutant strain used in the method for producing ethanol according to the present invention is a yeast having xylose metabolic ability, in addition to lacking a specific acetaldehyde dehydrogenase gene as described above.
  • having xylose metabolism ability means that, when xylose metabolism-related gene is imparted by introducing xylose metabolism-related gene, it inherently has xylose metabolism-related gene and inherently has xylose metabolism ability. It is meant to include any case. That is, in the case of yeast having xylose metabolism ability, it means that both recombinant yeast introduced with a xylose metabolism-related gene and yeast originally having a xylose metabolism-related gene are included.
  • Yeast having the ability to metabolize xylose can assimilate xylose contained in the medium to produce ethanol.
  • the xylose contained in the medium may be obtained by a process of saccharifying xylan, hemicellulose or the like containing xylose as a constituent sugar, or xylan or hemicellulose or the like contained in the medium is saccharified by a saccharifying enzyme. May be supplied to the medium. In the latter case, it means a so-called simultaneous saccharification and fermentation system.
  • Genes related to xylose metabolism are xylose reductase gene encoding xylose reductase that converts xylose to xylitol, xylitol dehydrogenase gene encoding xylitol dehydrogenase that converts xylitol to xylulose, and xylulose is phosphorylated to produce xylulose 5-phosphate It is meant to include a xylulokinase gene encoding xylulokinase. Xylulose 5-phosphate produced by xylulokinase enters the pentose phosphate pathway and is metabolized.
  • genes related to xylose metabolism include, but are not limited to, xylose reductase gene and xylitol dehydrogenase gene derived from Pichia stipitis, and xylulokinase gene derived from Saccharomyces cerevisiae (Eliasson A. et al., Appl. Environ. Microbiol 66: 3381-3386 and Toivari MN et al., Metab. Eng. 3: 236-249).
  • a xylose reductase gene derived from Candida ⁇ ⁇ ⁇ tropicalis or Candida prapsilosis can be used as the xylose reductase gene.
  • xylitol dehydrogenase gene a xylitol dehydrogenase gene derived from Candida tropicalis or Candida prapsilosis can be used.
  • xylulokinase gene a xylulokinase gene derived from Pichia stipitis can also be used.
  • a xylose isomerase gene derived from Streptomyces murinus cluster or the like can also be used.
  • yeasts that inherently have the ability to metabolize xylose include, but are not limited to, Pichia stipitis, Candida tropicalis, Candida prapsilosis, and the like.
  • the acetaldehyde dehydrogenase gene to be introduced into yeast having xylose metabolic ability is not particularly limited, and any organism-derived gene may be used.
  • the acetaldehyde dehydrogenase gene is modified in accordance with the codon usage in the yeast to be introduced when using genes derived from organisms other than fungi such as yeast, such as bacteria, animals, plants, insects, and algae. It is preferable to use the gene obtained.
  • the yeast mutant used in the method for producing ethanol according to the present invention may be a yeast further introduced with another gene.
  • another gene for example, the gene involved in sugar metabolisms, such as glucose, can be mentioned.
  • a yeast mutant modified to have ⁇ -glucosidase activity by introducing a ⁇ -glucosidase gene can also be used.
  • ⁇ -glucosidase activity means an activity catalyzing a reaction of hydrolyzing a ⁇ -glycoside bond of a sugar. That is, ⁇ -glucosidase can decompose cellooligosaccharides such as cellobiose into glucose.
  • the ⁇ -glucosidase gene can also be introduced as a cell surface display type gene.
  • the cell surface display type gene is a gene modified so that a protein encoded by the gene is expressed so as to be displayed on the surface layer of the cell.
  • the cell surface-presenting ⁇ -glucosidase gene is a gene obtained by fusing a ⁇ -glucosidase gene and a cell surface localized protein gene.
  • the cell surface localized protein refers to a protein that is fixed on the cell surface layer of yeast and is present on the cell surface layer.
  • ⁇ - or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned.
  • a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal on the C-terminal side. Although it has the same secretory protein in that it has a secretion signal, the cell surface localized protein is different from the secreted protein in that it is transported by being immobilized on the cell membrane via a GPI anchor.
  • the GPI anchor attachment recognition signal sequence is selectively cleaved, and is bound to the GPI anchor at the newly protruding C-terminal portion and fixed to the cell membrane. Thereafter, the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Next, the protein cut off from the cell membrane is incorporated into the cell wall, fixed to the cell surface layer, and localized on the cell surface layer (see, for example, JP-A-2006-174767).
  • PI-PLC phosphatidylinositol-dependent phospholipase C
  • the ⁇ -glucosidase gene is not particularly limited, and examples thereof include ⁇ -glucosidase genes derived from Aspergillus aculeatus (Murai et al., Appl. Environ. Microbiol. 64: 4857-4861).
  • ⁇ -glucosidase gene a ⁇ -glucosidase gene derived from Aspergillus oryzae, a ⁇ -glucosidase gene derived from Clostridium cellulovorans, a ⁇ -glucosidase gene derived from Saccharomycopsis fibligera, and the like can be used.
  • the recombinant yeast used in the method for producing ethanol according to the present invention may be one in which a gene encoding another enzyme constituting cellulase is introduced in addition to or in addition to the ⁇ -glucosidase gene.
  • cellulase is composed of exo-type cellobiohydrolase (CBH1 and CBH2) that releases cellobiose from the end of crystalline cellulose, and amorphous cellulose (amorphous cellulose) chains are randomized, although crystalline cellulose cannot be degraded.
  • CBH1 and CBH2 exo-type cellobiohydrolase
  • amorphous cellulose (amorphous cellulose) chains are randomized, although crystalline cellulose cannot be degraded.
  • An endo-type endoglucanase (EG) that cleaves can be mentioned.
  • a yeast mutant strain that can be used in the present invention can be prepared.
  • the deficiency of the above-mentioned acetaldehyde dehydrogenase gene is synonymous with suppressing the gene, suppressing or reducing the expression level of the gene, and inhibiting the function of the protein encoded by the gene. It means to include.
  • Deletion of a gene means that a region including a part or all of the coding region of the gene is deleted from the chromosome, and that the gene is destroyed by incorporating a transposon or the like into the coding region of the gene.
  • the method for reducing the expression level of the gene is not particularly limited, and examples thereof include a method for reducing the transcription amount by modifying the expression control region of the gene, a method for selectively degrading the transcription product of the gene, and the like. be able to.
  • transposon method as a technique for suppressing a gene, the so-called transposon method, transgene method, post-transcriptional gene silencing method, RNAi method, nonsense-mediated decay (NNM) method, ribozyme method, antisense method, miRNA (Micro-RNA) method, siRNA (small interfering RNA) method and the like.
  • the above-mentioned acetaldehyde dehydrogenase gene may be deleted in a recombinant yeast in which a gene related to xylose metabolism is introduced into a yeast that does not have xylose metabolism ability, or in yeast that originally has xylose metabolism ability.
  • the gene may be introduced even if it is deficient, or a gene related to xylose metabolism may be introduced into yeast having no ability to metabolize xylose and the gene may be deficient.
  • the acetaldehyde dehydrogenase gene may be deleted in yeast that does not have xylose metabolism, and then xylose metabolism may be imparted by introducing a gene related to xylose metabolism into the recombinant yeast.
  • yeasts such as Candida Shehatae, Pichia stipitis, Pachysolen tannophilus, Saccharomyces cerevisiae and Schizosaccaromyces pombe, and Saccharomyces cerevisiae is particularly preferable.
  • the yeast may be an experimental strain used for experimental convenience, or an industrial strain (practical strain) used for practical utility. Examples of industrial strains include yeast strains used for wine, sake and shochu making.
  • yeast having homothallic properties is synonymous with homothallic yeast.
  • the yeast having homothallic properties is not particularly limited, and any yeast can be used. Examples of yeast having homothallic properties include, but are not limited to, Saccharomyces cerevisiae ⁇ OC-2 strain (NBRC2260).
  • yeasts with homothallic properties include alcoholic yeasts (Taken 396, NBRC0216) (Source: “Characteristics of Alcoholic Yeast”, Sakekenkai Bulletin, No37, p18-22 (1998.8)), isolated in Brazil and Okinawa Ethanol producing yeast (Source: “Genetic properties of wild strains of Saccharomyces cerevisiae isolated in Brazil and Okinawa” Journal of Japanese Agricultural Chemical Society, Vol.65, No.4, p759-762 (1991.4)) and 180 (Source: Alcohol The screening of yeast having a strong fermenting ability ”, Journal of Japan Brewing Association, Vol.82, No.6, p439-443 (1987.6)).
  • yeast having homothallic properties can be used as a yeast having homothallic properties by introducing the HO gene so that it can be expressed. That is, in the present invention, the yeast having homothallic properties is meant to include yeast into which the HO gene has been introduced so as to be expressed.
  • the Saccharomyces cerevisiae OC-2 strain is preferable because it is a strain that has been confirmed to be safe and has been used in the winemaking scene. Further, the Saccharomyces cerevisiae OC-2 strain is preferable because it has excellent promoter activity under high sugar concentration conditions, as shown in the Examples described later. In particular, the Saccharomyces cerevisiae OC-2 strain is preferable because it has an excellent promoter activity of the pyruvate decarboxylase gene (PDC1) under high sugar concentration conditions.
  • PDC1 pyruvate decarboxylase gene
  • the promoter of the gene to be introduced is not particularly limited.
  • the promoter of glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), the promoter of 3-phosphoglycerate kinase gene (PGK1), the hyperosmotic response 7 gene ( HOR7) promoters can be used.
  • the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene.
  • the above-described gene may be introduced into the yeast genome together with a promoter controlling expression and other expression control regions.
  • the above-described gene may be introduced so that the expression is controlled by a promoter of a gene originally present in the genome of yeast as a host or other expression control regions.
  • any conventionally known method known as a yeast transformation method can be applied. Specifically, for example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”, acetic acid Lithium Method “J. Bacteriology, 153, p163 (1983)”, Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual The method can be implemented, but is not limited thereto.
  • the medium for ethanol fermentation is not particularly limited.
  • the yeast mutant described above has the characteristic that the efficiency of ethanol fermentation does not decrease even if acetic acid is contained in the medium, or the efficiency of ethanol fermentation is improved when acetic acid is contained in the medium. ing. Therefore, the yeast mutant described above is more preferably used in a system in which ethanol fermentation is performed using a medium containing acetic acid.
  • the medium containing acetic acid is not particularly limited, and examples thereof include a medium containing a carbon source obtained by saccharifying cellulosic biomass with a saccharifying enzyme.
  • a medium containing a carbon source obtained by saccharifying cellulosic biomass with a saccharifying enzyme When cellulosic biomass is saccharified with a saccharification enzyme, fermentation inhibitors such as acetic acid are produced. Therefore, when ethanol is produced using a medium obtained by saccharifying cellulosic biomass with a saccharifying enzyme, acetic acid is contained in the medium.
  • the medium obtained by saccharifying cellulosic biomass with a saccharifying enzyme may be a medium after saccharifying cellulosic biomass with a saccharifying enzyme, or may be a medium before saccharification treatment containing cellulosic biomass and saccharifying enzyme. . In the medium before saccharification treatment containing cellulosic biomass and saccharification enzyme, saccharification treatment by saccharification enzyme and ethanol fermentation by the
  • yeast mutant having xylose metabolism ability as the yeast mutant described above.
  • ethanol fermentation can be performed using xylose derived from hemicellulose as a carbon source in addition to glucose derived from cellulose contained in cellulosic biomass.
  • the cellulosic biomass may be one that has been subjected to conventionally known pretreatment.
  • pre-processing For example, the process which decomposes
  • a treatment such as a treatment in which the pulverized cellulose biomass is immersed in a dilute sulfuric acid solution, an alkali solution or an ionic liquid, a hydrothermal treatment, or a fine pulverization treatment may be applied.
  • the saccharification rate of biomass can be improved.
  • the saccharification method is not particularly limited, and examples thereof include an enzyme method using a cellulase preparation such as cellulase or hemicellulase.
  • the cellulase preparation contains a plurality of enzymes involved in the degradation of cellulose chains and hemicellulose chains, and exhibits a plurality of activities such as endoglucanase activity, endoxylanase activity, cellobiohydrolase activity, glucosidase activity, and xylosidase activity.
  • the cellulase preparation is not particularly limited, and examples thereof include cellulase produced by Trichoderma reesei, Acremonium cerulolyticus, and the like. As the cellulase preparation, a commercially available product may be used.
  • the method for producing ethanol according to the present invention includes a step of saccharifying cellulosic biomass contained in a medium with a saccharification enzyme, and a step of ethanol fermentation using a carbon source such as glucose or xylose produced by saccharification. It is preferable to use a so-called simultaneous saccharification and fermentation treatment in which the saponification proceeds simultaneously.
  • the simultaneous saccharification and fermentation treatment means a treatment that is carried out simultaneously without distinguishing between the step of saccharifying cellulosic biomass and the ethanol fermentation step.
  • the cellulase preparation and the above-described yeast mutant are added to a medium containing cellulosic biomass (may be after pretreatment), and the yeast mutant is cultured in a predetermined temperature range.
  • the culture temperature is not particularly limited, but can be 25 to 45 ° C., preferably 30 to 40 ° C. in consideration of the efficiency of ethanol fermentation.
  • the pH of the culture solution is preferably 4-6. Moreover, you may stir and shake in the case of culture
  • ethanol is recovered from the medium after ethanol fermentation.
  • the method for recovering ethanol is not particularly limited, and any conventionally known method can be applied.
  • a liquid layer containing ethanol and a solid layer containing yeast mutants and solid components are separated by solid-liquid separation operation.
  • ethanol contained in the liquid layer is separated and purified by a distillation method, whereby high purity ethanol can be recovered.
  • the purity of ethanol can be adjusted as appropriate according to the intended use of ethanol.
  • acetic acid is known to inhibit the growth and proliferation of yeast and to reduce the efficiency of ethanol fermentation using xylose as a sugar source.
  • a yeast mutant lacking a predetermined acetaldehyde dehydrogenase gene is used, ethanol fermentation efficiency does not decrease even in a medium containing acetic acid, or ethanol fermentation efficiency is improved. To do. Therefore, the ethanol production method according to the present invention can achieve an excellent ethanol yield as compared with the case of using yeast that does not lack the acetaldehyde dehydrogenase gene.
  • Example 1 ⁇ Test stock>
  • xylose metabolism gene S. stipitis derived xylose reductase gene (XYL1) and xylitol dehydrogenase gene (XYL2) host-derived XKS1 gene and Strain Uz326 introduced into the GRE3 locus (see reference examples below)
  • Uz644 strain that gave histidine requirement to Uz326 strain
  • Uz644 strain as the parent strain Uz675 strain that disrupted ALD4 gene
  • Uz677 that disrupted ALD5 gene Uracil-requiring mutant Uz632 of Uz679 strain with disrupted ALD6 gene and tryptophan-requiring OC2-T strain (Saitoh, S.
  • a DNA fragment containing the ALD4 gene and its 5 ′ upstream and 3 ′ downstream untranslated regions was amplified by PCR using the genomic DNA of yeast BY4742 (manufactured by Open Biosystems) as a template.
  • TB3248 and TB3249 were used as PCR primers (primer base sequences are summarized in Table 2. Hereinafter, the base sequences of other primers are also the same).
  • the PCR primer design for DNA sequence amplification of the yeast BY4742 strain containing TB3248 and TB3249 was based on the DNA sequence data of the Saccharomyces Genome Database.
  • the amplified DNA fragment was cloned into a plasmid using Zero Blunt TOPO PCR Cloning Kit (manufactured by Life Technologies) and named pCR-5U_ALD4-ALD4-3U_ALD4.
  • pCR-5U_ALD4-ALD4-3U_ALD4 as a template, 5 ′ upstream and 3 ′ downstream untranslated regions of the ALD4 gene and a region containing pCR-BluntII-TOPO, and genomic DNA of yeast BY4742 strain as a template
  • a DNA fragment containing the TRP1 gene and its promoter and terminator region was amplified by PCR.
  • the PCR primers used were TB3486 and TB3460, TB2800 and TB2799 designed so that the DNA fragments obtained by amplification overlap each other by about 15 bp.
  • the above two DNA fragments were cloned using In-Fusion® HD® Cloning® Kit (manufactured by Takara Bio Inc.) and named pCR-5U_ALD4-TRP1-3U_ALD4.
  • a DNA fragment containing the ALD5 gene and its 5 ′ upstream and 3 ′ downstream untranslated regions was amplified by PCR using the genomic DNA of yeast BY4742 strain as a template.
  • TB3469 and TB3470 were used as PCR primers.
  • the amplified DNA fragment was cloned into a plasmid using Zero Blunt TOPO PCR Cloning Kit and named pCR-5U_ALD5-ALD5-3U_ALD5.
  • DNA fragments of the 5 ′ upstream and 3 ′ downstream untranslated regions of the ALD5 gene and the region containing pCR-BluntII-TOPO were amplified by PCR.
  • a DNA fragment containing the TRP1 gene, its promoter and terminator region was amplified by PCR using the genomic DNA of the yeast BY4742 strain as a template.
  • TB3631 designed so that the DNA fragment obtained by amplification overlaps with a DNA fragment amplified by PCR using genomic DNA of yeast BY4742 as a template. And used TB3599.
  • the DNA fragment obtained by each PCR was cloned using In-Fusion HD Cloning Kit and named pCR-5U_ALD5-TRP1-3U_ALD5.
  • a DNA fragment containing the ALD6 gene and its 5 ′ upstream and 3 ′ downstream untranslated regions was amplified by PCR using the genomic DNA of yeast BY4742 strain as a template.
  • TB3406 and TB3408 were used as PCR primers, respectively.
  • the amplified DNA fragment was cloned into a plasmid using Zero Blunt TOPO PCR Cloning Kit and named pCR-5U_ALD6-ALD6-3U_ALD6.
  • DNA fragments of the 5 ′ upstream and 3 ′ downstream untranslated regions of the ALD6 gene and the region containing pCR-BluntII-TOPO were amplified by PCR.
  • a DNA fragment containing the TRP1 gene, its promoter and terminator region was amplified by PCR using the genomic DNA of the yeast BY4742 strain as a template.
  • TB3710 designed so that the DNA fragment obtained by amplification overlaps by about 15 bp with the DNA fragment amplified by PCR using genomic DNA of yeast BY4742 strain as a template. And used TB3600.
  • the DNA fragment obtained by each PCR was cloned using In-Fusion® HD® Cloning® Kit and named pCR-5U_ALD6-TRP1-3U_ALD6.
  • ⁇ DNA fragment for HIS3 gene disruption A DNA fragment containing the HIS3 gene and its 5 ′ upstream and 3 ′ downstream untranslated regions was amplified by PCR using the genomic DNA of yeast BY4742 strain as a template. TB3164 and TB3165 were used as PCR primers. The amplified DNA fragment was cloned into a plasmid using Zero Blunt TOPO PCR Cloning Kit and named pCR-5U_HIS3-HIS3-3U_HIS3.
  • pCR-5U_HIS3-HIS3-3U_HIS3 As a template, 5 'upstream and 3' downstream untranslated regions of the HIS3 gene and a region containing pCR-BluntII-TOPO, and pAUR101 (manufactured by Takara Bio Inc.) as a template
  • the AUR1-C gene, its promoter, and a DNA fragment containing a terminator region were amplified by PCR.
  • the PCR primers used were TB3458 and TB3459, TB2885 and TB2859 designed so that the DNA fragments obtained by amplification overlap each other by about 15 bp.
  • the DNA fragment obtained by each PCR was cloned using In-Fusion® HD® Cloning® Kit and named pCR-5U_HIS3-AUR1-C-3U_HIS3.
  • ⁇ HIS3 gene disruption strain Using the pCR-5U_HIS3-AUR1-C-3U_HIS3 prepared as described above as a template, DNA fragments other than pCR-Blunt II TOPO in the vector portion were amplified by PCR. TB3164 and TB3165 were used as PCR primers. Using the DNA fragment amplified by PCR, the Uz326 strain overexpressed xylose-utilizing genes (XYL1, XYL2, XKS1) in the diploid homothallic yeast Saccharomyces cerevisiaeOC2 was transformed into the Frozen-EZ Yeast Transformation II kit (ZYMO RESEARCH The product was transformed according to the protocol attached to the kit.
  • the ALD5 gene disruption DNA fragment and the ALD6 gene disruption DNA fragment were amplified by PCR (primers were TB3469, TB3470, TB3406, respectively). And TB3408), ALD5 gene and ALD6 gene disruption strains were obtained by transformation and doubling.
  • the ALD5 gene disruption strain derived from the Uz644 strain was the Uz677 strain
  • the ALD6 gene disruption strain was the Uz679 strain.
  • the ALD5 gene disruption strain derived from the Uz632 strain was designated as the Uz830 strain
  • the ALD6 gene disruption strain was designated as the Uz832 strain.
  • Ethanol and glucose in the fermentation broth were quantified using HPLC (LC-10A; Shimadzu Corporation). Specifically, in HPLC, AminexHPX-87H was used as a column, 0.01N H 2 SO 4 was used as a mobile phase, the flow rate was 0.6 ml / min, and the temperature was 30 ° C.
  • the detector used was a differential refractive index detector: RID-10A.
  • a medium containing cellulose and a saccharifying enzyme (cellulase preparation: Cellic TM CTec2) is a so-called simultaneous saccharification in which the saccharification step by saccharifying enzyme and ethanol fermentation by the test strain proceed simultaneously. It corresponds to the fermentation system.
  • Tables 4 and 5 show the results of quantification of ethanol and glucose in the fermentation broth of XR / XDH non-introduced strains (Uz632 strain and Ald gene-disrupted strain derived from Uz632 strain). As can be seen from Tables 4 and 5, the ethanol yield in the medium containing acetic acid decreased in the control strain Uz632 in both the simultaneous saccharification fermentation system and the normal ethanol fermentation system not containing saccharification treatment.
  • the ethanol yield in the medium containing acetic acid was superior to that of the Uz632 strain in both the simultaneous saccharification fermentation system and the normal ethanol fermentation system that did not include saccharification treatment. It was revealed that an ethanol yield equivalent to that obtained when a medium containing no acetic acid was used can be achieved.
  • Table 6 shows the results of quantifying ethanol and glucose in the fermentation broth of the XR / XDH-introduced strain (Uz644 strain and Ald gene-disrupted strain derived from the Uz644 strain).
  • the ethanol strain of the control strain Uz644 decreased in the medium containing acetic acid in the simultaneous saccharification and fermentation system.
  • the Uz644 strain had no significant difference in ethanol yield between a medium containing acetic acid and a medium not containing acetic acid in a normal ethanol fermentation system without saccharification treatment. That is, in yeast having xylose metabolism ability, ethanol fermentation inhibition by acetic acid was confirmed only in the simultaneous saccharification and fermentation system.
  • XYL1, XYL2, and XKS1 gene overexpression in the homothallic diploid yeast Saccharomyces cerevisiae OC2-T strain (Saitoh, S. et al., J. Ferment. Bioeng. 1996, 81, 98-103) A procedure for producing a strain into which the GRE3 gene has been heterogeneously introduced will be described.
  • RNA fragment containing the GRE3 gene and its 5 ′ upstream and 3 ′ downstream untranslated regions was amplified by PCR.
  • TB2358 (5′-TGGGAATATTACCGCTCGAAG-3 ′: SEQ ID NO: 27) and TB2359 (5′-AAGGGGGAAGGTGTGGAATC-3 ′: SEQ ID NO: 28) were used as PCR primers, respectively.
  • PCR primer design for the DNA sequence amplification of yeast BY4742 strain was referred to the DNA sequence data of Saccharomyces Genome Database.
  • plasmid pUC19 as a template, a linear DNA fragment containing the full length of pUC19 that was cleaved at the multicloning site of pUC19 was amplified by PCR.
  • TB2373 (5′-CACACCTTCCCCCTTGATCCTCTAGAGTCGACC-3 ′: SEQ ID NO: 29) and TB2374 (5′-GCGGTAATATTCCCAGATCCCCGGGTACCGAGCTC-3 ′: SEQ ID NO: 30) were used as PCR primers, respectively.
  • the above two DNA fragments were cloned using In-Fusion TM Advantage PCR Cloning Kit (Takara Bio) and named pUC19-5U_GRE3-GRE3-3U_GRE3.
  • the primers for PCR are TB3018 (5'-AACGAGGCGCGCTCTTCCAGCCAGTAAAATCCA-3 ': SEQ ID NO: 31) and TB3017 (5'-GCTATGGTGTGTGGGCTTTAAAAAATTTCCAATTTTCCTTTACG-3': SEQ ID NO: 32), TB2210 (5'-CCCACACACCATAGCTTCAAAATG-3 ': SEQ ID NO: 33) TB2269 (5'-TCTTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGCAAG-3 ': SEQ ID NO: 34), TB2345 (5'-AATCTAATCTAAAGAATGTTGTGTTCAGTAATTCAGAGAC-3': SEQ ID NO: 35) and TB2346 (5'-CTGCGGCCGGCCGCATTAGATGAGAGTCTTTTCCAGTTC-3 ': 140 TGCGGCCGGCCGCAGC-3 ′: SEQ ID NO: 37) and TB2683 (5′-GCGCCTCGTTCAGAAT
  • pUC19-5U_GRE3-P_TEF1-XKS1-T_HIS3-3U_GRE3 as a template, a linear DNA fragment containing the entire length of the plasmid as cleaved between the HIS3 terminator region and the 3 ′ downstream untranslated region of the GRE3 gene, and the yeast BY4742 strain
  • the XYL1 gene was amplified by PCR using a DNA fragment containing the TDH2 promoter region, a DNA fragment containing the ILV3 terminator region, and the genomic DNA of Pichia stipitis as a template.
  • the primers for PCR are TB9020 (5'-TCCAGCCAGTAAAATCCATAC-3 ': SEQ ID NO: 39), TB2457 (5'-CCGTCAAGAGAGCGCGCCTCGTTCAG-3': SEQ ID NO: 40), TB2844 (5'-GCGCTCTCTTGACGGGTATTCTGAGCATCTTAC-3 ': SEQ ID NO: 41), respectively.
  • TB2595 (5'-TTTGTTTTGTTTGTTTGTGTGATGAATTTAATTTG-3 ': SEQ ID NO: 42), TB2314 (5'-AACAAACAAAACAAAATGCCTTCTATTAAGTTGAAC-3': SEQ ID NO: 43) and TB2455 (5'-GGGGCCTATAATGCATTAGACGAAGATAGGAATCTTG-3 ': SEQ ID NO: 44, TB-4)
  • AACAAACAAAACAAAATGCCTTCTATTAAGTTGAAC-3 ′: SEQ ID NO: 45) and TB3019 (5′-ATTTTACTGGCTGGAATTTCGTAGATTATAATTAAGGCGAC-3 ′: SEQ ID NO: 46) were used.
  • PCR primer design for XYL1 gene sequence amplification referred to the Pichia stipitis XYL1 gene (registration number XM_001385144) registered in GeneBank.
  • the above four DNA fragments were cloned using In-Fusion TM Advantage PCR Cloning Kit and named pUC19-5U_GRE3-P_TEF1-XKS1-T_HIS3-P_TDH2-XYL1-T_ILV3-3U_GRE3.
  • pUC19-5U_GRE3-P_TEF1-XKS1-T_HIS3-P_TDH2-XYL1-T_ILV3-3U_GRE3 as a template, linear DNA containing the entire length of the plasmid as cleaved between the ILV3 terminator region and the 3 'downstream untranslated region of the GRE3 gene
  • the XYL2 gene was amplified by PCR using the fragment, genomic DNA of yeast BY4742 strain as a template, DNA fragment containing PDC1 promoter region and DNA fragment containing ILV6 terminator region, and genomic DNA of Pichia stipitis.
  • the primers for PCR were TB2375 (5'-AGTTGCTTGACACGGTGGAAGAAGGTCCAGCCAGTAAAATCCATA-3 ': SEQ ID NO: 47) and TB3021 (5'-ATTTCGTAGATTATAATTAAGGCGAC-3': SEQ ID NO: 48), TB2010 (5'-TTTGATTGATTTGACTGTGTTATTTTGC-3 ': SEQ ID NO: 49), respectively.
  • TB2261 (5'-CCGTGTCAAGCAACTATGGG-3 ': SEQ ID NO: 50), TB3022 (5'-TATAATCTACGAAATTAATAAGAAAGGTGACCGTG-3': SEQ ID NO: 51) and TB2347 (5'-GTTAGTCTCTCGGCCTTGCG-3 ': SEQ ID NO: 52), TB2351 (5'- GGCCGAGAGACTAACTTACTCAGGGCCGTCAAT-3 ′: SEQ ID NO: 53) and TB2352 (5′-GTCAAATCAATCAAAATGACTGCTAACCCTTCC-3 ′: SEQ ID NO: 54) were used.
  • the PCR primer design for XYL2 gene sequence amplification was based on the Pichia stipitis XYL2 gene (registration number AF127801 or X55392) registered in GeneBank.
  • the above four DNA fragments were cloned using In-Fusion TM Advantage PCR Cloning Kit and named pUC19-5U_GRE3-P_TEF1-XKS1-T_HIS3-P_TDH2-XYL1-T_ILV3-ILV6_T-XYL2-PDC1_P-3U_GRE3.
  • OC2-T strain was transformed with Frozen-EZ Yeast Transformation II kit (ZYMO RESEARCH) according to the protocol attached to the kit. After transformation, it was spread on a plate using xylose as a sole carbon source and cultured at 30 ° C. for 7 days to obtain a transformant.
  • ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit
  • Genomic DNA was prepared from the transformant, and TB2356 (5'-TGGGGCTAAACGAGATTTGG-3 ': SEQ ID NO: 55) and TB592 (5'-GAAATTTAGTATGCTGTGCTTGGG-3': primers outside the inserted DNA fragment and inside the vector were prepared by PCR. Using SEQ ID NO: 56), it was confirmed that one copy was normally integrated into the chromosome.

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Abstract

L'objet de la présente invention est d'améliorer l'efficacité de la fermentation de l'éthanol en présence d'acide acétique et d'obtenir une excellente productivité d'éthanol. Pour ce faire, une souche mutante de levure, qui présente un déficit en gène de l'acétoaldéhyde déshydrogénase correspondant au moins à un gène d'acétoaldéhyde déshydrogénase choisi dans le groupe constitué par le gène ALD4, le gène ALD5 et le gène ALD6 chez Saccharomyces cerevisiae, est cultivée dans un milieu de culture contenant de l'acide acétique, et l'éthanol dans le milieu de culture est ensuite collecté.
PCT/JP2013/070036 2012-08-01 2013-07-24 Procédé de production d'éthanol faisant appel à une levure recombinée WO2014021163A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010239925A (ja) * 2009-04-08 2010-10-28 Toyota Central R&D Labs Inc キシロースを利用して有用物質を生産する方法
WO2011065539A1 (fr) * 2009-11-30 2011-06-03 国立大学法人神戸大学 Procédé pour la production d'éthanol à partir de biomasse
US20120122172A1 (en) * 2010-11-16 2012-05-17 Snu R&Db Foundation Method for producing ethanol from xylose using recombinant saccharomyces cerevisiae transformed to eliminate functions of genes involved in tor signal transduction pathway

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010239925A (ja) * 2009-04-08 2010-10-28 Toyota Central R&D Labs Inc キシロースを利用して有用物質を生産する方法
WO2011065539A1 (fr) * 2009-11-30 2011-06-03 国立大学法人神戸大学 Procédé pour la production d'éthanol à partir de biomasse
US20120122172A1 (en) * 2010-11-16 2012-05-17 Snu R&Db Foundation Method for producing ethanol from xylose using recombinant saccharomyces cerevisiae transformed to eliminate functions of genes involved in tor signal transduction pathway

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EGLINTON, J. M. ET AL.: "Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene.", YEAST, vol. 19, 2002, pages 295 - 301 *
REMIZE, F ET AL.: "Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: role of the cytosolic Mg2+ and mitochondrial K+ acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation.", APPL. ENVIRON. MICROBIOL., vol. 66, no. 8, 2000, pages 3151 - 3159 *
SAINT-PRIX, F. ET AL.: "Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP'-dependent Ald6p and Ald5p isoforms play a major role in acetate formation.", MICROBIOLOGY, vol. 150, 2004, pages 2209 - 2220 *
SONDEREGGER, M ET AL.: "Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae.", APPL. ENVIRON. MICROBIOL., vol. 70, no. 5, 2004, pages 2892 - 2897 *
WANG, X. ET AL.: "Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehyde dehydrogenases in ethanol metabolism in Saccharomyces cerevisiae.", J. BACTERIOL., vol. 180, no. 4, 1998, pages 822 - 830 *

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