WO2017138489A1 - Method for promoting fermentation through loss of function of vacuolar transporter chaperone complex of yeast - Google Patents

Method for promoting fermentation through loss of function of vacuolar transporter chaperone complex of yeast Download PDF

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WO2017138489A1
WO2017138489A1 PCT/JP2017/004212 JP2017004212W WO2017138489A1 WO 2017138489 A1 WO2017138489 A1 WO 2017138489A1 JP 2017004212 W JP2017004212 W JP 2017004212W WO 2017138489 A1 WO2017138489 A1 WO 2017138489A1
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yeast
gene
complex
vtc
fermentation
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French (fr)
Japanese (ja)
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渡辺　大輔
健一 高木
博史 高木
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国立大学法人 奈良先端科学技術大学院大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/023—Preparation of other alcoholic beverages by fermentation of botanical family Solanaceae, e.g. potato
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
    • 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
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/02—Pitching yeast
    • 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 improving fermentability by functional defect of yeast vacuolar transporter chaperone complex.
  • VTC complex a vacuolar transporter chaperone complex
  • the VTC complex is a protein complex composed of Vtc1p, Vtc2p, Vtc3p, and Vtc4p.
  • the VTC complex has been reported to be involved in maintaining the stability of vacuolar proton ATPase, protein transport, microautophagy, and polyphosphate accumulation in the vacuole (Non-Patent Documents 1 and 2). 3, 4).
  • Non-Patent Document 1 a VTC1 gene-disrupted strain that encodes Vtc1p is prepared using a laboratory yeast strain W303, and analysis of the VTC complex is performed.
  • Yeast is used for the production of fermented foods such as bread dough and alcoholic beverages such as sake and bioethanol.
  • an alcohol fermentation process is generally used in which sugars such as glucose are decomposed into ethanol and carbon dioxide by Saccharomyces yeasts.
  • An object of the present invention is to provide a method for improving the fermentability of yeast.
  • this invention consists of the following structures.
  • a method for improving the fermentability of yeast comprising reducing or eliminating the function of a vacuolar transporter chaperone complex (VTC complex) in yeast.
  • VTC complex vacuolar transporter chaperone complex
  • Decreasing or loss of the function of the yeast VTC complex reduces the expression level of at least one gene selected from genes encoding the components of the VTC complex by introducing a genetic mutation in the yeast.
  • At least one gene selected from genes encoding a component of a VTC complex is at least one gene selected from the group consisting of the following (a) to (e): A method for improving the fermentability of yeast according to any one of the following: (A) a gene comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (B) a gene comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (C) a gene comprising a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (D) a gene comprising a polynucleotide comprising the base sequence set forth in any one of SEQ ID NOs: 5 to 8; (E) A gene
  • a method for producing a fermentation product using yeast comprising the method for improving the fermentability of yeast according to any one of 1 to 6 above, wherein the function of the VTC complex is reduced or lost in the yeast.
  • a method for producing a fermented product comprising performing alcoholic fermentation under certain conditions.
  • the method for producing a fermented product according to item 7, wherein the fermented product is a fermented beverage or a fermented food.
  • a method for producing a yeast for alcohol fermentation comprising reducing or losing the function of a VTC complex by introducing a gene mutation in the yeast. 11.
  • a composition for alcoholic fermentation comprising the yeast according to any one of 11 to 13 above.
  • a method for screening a drug for improving the fermentability of yeast comprising a step of allowing a test compound to act on a VTC complex, wherein the test compound decreases or loses the function of the VTC complex.
  • fermentation can be efficiently promoted by performing fermentation in a state where the function of the VTC complex is reduced or lost in yeast. Furthermore, by performing fermentation efficiently according to the present invention, a fermentation product produced through yeast fermentation can be favorably produced. Moreover, according to the present invention, it is possible to systematically produce yeast having high fermentability and ethanol productivity. Yeast with reduced or lost function of the VTC complex produced according to the present invention is useful for producing various fermented foods, fermented beverages, and bioethanol, and is particularly useful in brewing.
  • Example 1 It is a figure which shows the fermentation test result of a VTC1 gene disruption strain, a VTC2 gene disruption strain, a VTC3 gene disruption strain, and a VTC4 gene disruption strain.
  • Example 2 It is a figure which shows the fermentation test result of a VTC1 gene disruption baker's yeast strain.
  • Example 2 It is a figure which shows the fermentation test result of a VTC1 gene disruption bioethanol yeast strain.
  • a fermentation test was performed under laboratory conditions using a yeast gene-disrupted strain library consisting of a yeast gene-disrupted strain, and the influence of each gene disruption on alcohol fermentation was analyzed. It was clarified that the disruption of each gene encoding the component factor significantly promoted alcohol fermentation.
  • the present invention has been achieved based on such findings, and relates to a method for improving the fermentability of yeast by reducing or losing the function of the VTC complex in yeast. Hereinafter, the present invention will be described in detail.
  • VTC complex Vacuolar transporter chaperone complex
  • the VTC complex is a vacuolar transporter chaperone complex present in the vacuolar membrane and was found in the yeast Saccharomyces cerevisiae.
  • the VTC complex is a protein complex formed from a plurality of constituent factors of two or more, and each constituent factor is a membrane permeation protein having a relatively small molecular weight.
  • the Saccharomyces cerevisiae VTC complex is formed by four components, Vtc1p, Vtc2p, Vtc3p, and Vtc4p.
  • Vtc1p the Saccharomyces cerevisiae VTC complex
  • VTC complex examples include those formed by four components of Vtc1p, Vtc2p, Vtc3p, and Vtc4p.
  • Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins function as components of the vacuolar transporter chaperone complex.
  • Each of the components in the VTC complex of the present invention is a protein selected from the proteins shown in the following (A) to (C): (A) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (B) a protein comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4; or (C) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4.
  • SEQ ID NOs: 1 to 4 shown in the sequence listing represent the amino acid sequences of the Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins derived from Saccharomyces cerevisiae, respectively.
  • Vtc1p, Vtc2p, Vtc3p, Vtc4p proteins function as components of the VTC complex.
  • the protein of the above (B) or (C) is a functionally equivalent mutant, derivative, variant, allele, homolog, ortholog, partial peptide, with respect to the protein having the amino acid sequence shown in any of SEQ ID NOs: 1 to 4. Or it is a fusion protein with other protein or peptide, Comprising: The gene which codes the component factor of a vacuolar transporter chaperone complex is intended, The concrete amino acid sequence is not limited.
  • the number of amino acids that may be substituted, deleted, inserted and / or added in (B) is not limited as long as the function is not lost, but site-directed mutagenesis
  • the number which can be substituted, deleted, inserted and / or added by a known mutagenesis method is within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, and most preferably within 5 amino acids (for example, within 5, 4, 3, 2, or 1 amino acid).
  • Whether or not a protein into which a mutation has been introduced imparts a desired character to yeast can be determined by expressing a gene encoding the protein, constructing a VTC complex, and examining whether the VTC complex functions.
  • the “mutation” here means a mutation artificially introduced mainly by site-directed mutagenesis or the like, but may be a similar naturally occurring mutation. “Mutation” includes the destruction of a gene encoding a constituent factor.
  • amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain.
  • amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids having aliphatic side chains (G, A, V, L, I and P), amino acids having hydroxyl group-containing side chains (S, T and Y), sulfur atom-containing side chains Amino acids (C and M) having carboxylic acids and amide-containing side chains (D, N, E and Q), amino acids having base-containing side chains (R, K and H), and aromatic-containing side chains And amino acids (H, F, Y and W) having Furthermore, it is also well known to classify amino acids by, for example, mutational matrix (Taylor 1986, J, Theor.
  • polypeptide having an amino acid sequence modified by deletion, insertion, addition and / or substitution by another amino acid of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. It has been. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.
  • “functionally equivalent” means that the target protein has a biological function and / or biochemical function equivalent to (same and / or similar to) the Vtc1p, Vtc2p, Vtc3p, or Vtc4p protein. Intended to have.
  • examples of the biological function and / or biochemical function of the Vtc1p, Vtc2p, Vtc3p, or Vtc4p protein include, for example, constituting a VTC complex and allowing the VTC complex to function.
  • Biological properties may include the specificity of the site to be expressed and / or the expression level.
  • the homology of the amino acid sequence in (C) above is at least 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% in the entire amino acid sequence (or a region necessary for functional expression). 96%, 97%, 98%, 99% or 99.5% or more of sequence identity. Sequence homology can be determined using the BLASTN (nucleic acid level) or BLASTX (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc.ANatl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci.
  • the term “homology” intends the ratio of the number of amino acid residues having similar properties (homology, positive, etc.), but more preferably the ratio of the number of matched amino acid residues (identity) ).
  • the properties of amino acids are as described above.
  • the gene encoding the constituent factor of the VTC complex may be any gene as long as it encodes any one of the Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins. More specifically, the genes encoding the constituent elements of the VTC complex of the present invention are genes selected from the polynucleotides shown in the following (a) to (e): (A) a gene comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (B) a gene comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (C) a gene comprising a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (D)
  • genes (a) to (c) are genes encoding the proteins (A) to (C).
  • SEQ ID NO: 5 shown in the sequence listing is the nucleotide sequence (ORF) of the VTC1 gene encoding Vtc1p (protein having the amino acid sequence shown in SEQ ID NO: 1) derived from Saccharomyces cerevisiae, No.
  • VTC2 is the base sequence (ORF) of the VTC2 gene encoding Vtc2p derived from Saccharomyces cerevisiae (protein having the amino acid sequence shown in SEQ ID NO: 2)
  • SEQ ID NO: 7 is Vtc3p derived from Saccharomyces cerevisiae (SEQ ID NO: 3
  • SEQ ID NO: 8 is the VTC4 gene encoding the Vtc4p (protein having the amino acid sequence shown in SEQ ID NO: 4) derived from Saccharomyces cerevisiae Base sequence (ORF) Show Re respectively.
  • the ORF means an open reading frame.
  • VTC1 gene, VTC2 gene, VTC3 gene and VTC4 gene are considered to exist widely throughout yeast. That is, the genes according to the present invention include homologous genes of VTC1, VTC2, VTC3, and VTC4 genes that are present in various yeasts.
  • methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, E. M., Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction. (PCR) technology (Saiki, R. K., et al. Science, vol. 230, 1350-1354, 1985, Saiki, R. K. et al. Science, vol.239, 487-491, 1988) It is done.
  • VTC1 gene, VTC2 gene, VTC3 gene and VTC4 gene nucleotide sequences for example, the nucleotide sequences described in SEQ ID NOs: 5 to 8) or a part thereof are used as probes, and the VTC1 gene, VTC2 gene, Isolation of VTC1 gene, VTC2 gene, VTC3 gene, and homologous gene of VTC4 gene from various yeasts using oligonucleotides that specifically hybridize with VTC3 gene or VTC4 gene as primers can be generally performed.
  • the gene of (e) is intended to encode a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide of any of (a) to (d) above. ing.
  • stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed and a non-specific double-stranded polynucleotide is not formed.
  • the hybridization can be carried out in a general hybridization buffer at 68 ° C. for 20 hours.
  • the temperature is 60-68 ° C., preferably 65 ° C., more preferably 68 in a buffer solution consisting of 0.25M Na 2 HPO 4 , pH 7.2, 7% SDS, 1 mM EDTA, 1 ⁇ Denhardt's solution.
  • the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably Can include a condition in which washing is performed twice at 68 ° C. for 15 minutes.
  • Other examples include 25% formamide, 50% formamide under more severe conditions, 4 ⁇ SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10 ⁇ Denhardt's solution, 20 ⁇ g / ml denatured salmon sperm DNA. After prehybridization is performed overnight in a hybridization solution at 42 ° C., a labeled probe is added, and hybridization is performed by incubating overnight at 42 ° C. In the subsequent cleaning, the cleaning solution and temperature conditions are about “1 ⁇ SSC, 0.1% SDS, 37 ° C.”, and more severe conditions are about “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”.
  • the gene of (e) is at least 80% or more, preferably 85% or more, more preferably 90% or more, in homology with the gene (d) (base sequences described in SEQ ID NOs: 5 to 8). Preferably, it has 95%, 96%, 97%, 98% or 99% or more sequence identity.
  • the homology with the base sequences described in SEQ ID NOs: 5 to 8 can be determined by FASTA search or BLAST search.
  • the nucleotide sequence of the polynucleotide can be determined by the dideoxy method described in Science, 214: 1205 (1981).
  • the gene used in the present invention may be composed only of a polynucleotide encoding the above protein, but other base sequences may be added.
  • the base sequence to be added is not particularly limited, but includes a label (for example, histidine tag, Myc tag or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP), a promoter sequence, and Examples thereof include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum transition signal sequence, a secretory sequence, etc.).
  • the site to which these base sequences are added is not particularly limited, and may be, for example, the N-terminus or C-terminus of the translated protein.
  • the method of improving the fermentation ability of yeast ie, the method of accelerating
  • Reduced or lost function of the VTC complex means that the function of the VTC complex as a whole is decreased or lost, and that the function of any one component of the VTC complex is decreased or lost. Any of these may be used.
  • the decrease or loss of the function of the VTC complex includes those caused by the inhibition of the formation of the VTC complex formed from a plurality of constitutive factors and the factor that inhibits the function of the VTC complex. Reduction or loss of function of the yeast VTC complex results in, for example, accelerated fermentation.
  • Fermentation is a process in which microorganisms such as yeast decompose organic compounds to obtain energy to produce alcohols, organic acids, carbon dioxide, and the like.
  • the fermentation in the present invention is preferably alcoholic fermentation, more preferably ethanolic fermentation.
  • Ethanol fermentation is a reaction in which yeast acts on sugars such as butter sugar, fructose, and sucrose that can be used by yeast to produce ethanol and carbon dioxide.
  • the function of the VTC complex or the function of any one of the constituent elements of the VTC complex may be a conventionally known function or a new function to be found in the future. Examples include maintenance of stability of vacuolar proton ATPase, protein transport, microautophagy, and accumulation of polyphosphoric acid in the vacuole (Non-patent Documents 1, 2, 3, 4). Confirmation of these functions can be confirmed based on the methods disclosed in Non-Patent Documents 1, 2, 3, and 4. Furthermore, it includes the alcohol fermentation regulation function by the yeast VTC complex revealed in the present application and the reduction or loss of the function inherent to the VTC complex to be found in the future.
  • the reduction or loss of the function of the yeast VTC complex is preferably by means of suppressing the expression of a gene encoding any one of the constituent factors.
  • Suppression of the expression of the gene means suppression of the process of expressing a constituent factor encoded by the gene, and includes suppression of transcription of the gene, suppression of translation of the gene, and the like.
  • Suppression of the expression of the gene may be performed by bringing some drug or the like into contact with the yeast, or by introducing a gene mutation in the yeast.
  • the introduction of the mutation may be artificially performed using a gene modification technique, or may be naturally occurring.
  • the gene mutation may be introduced as long as it causes a decrease or loss of the function of the yeast VTC complex.
  • the gene mutation is introduced into a gene selected from genes encoding constituent factors forming the yeast VTC complex.
  • the expression level of the gene encoding the component that forms a VTC complex can be reduced.
  • Examples of the gene mutation that reduces the expression level of the gene include destroying the gene. Since any of the genes of the constituent factors forming the VTC complex is considered to decrease or lose the function of the VTC complex, the gene into which the gene mutation is introduced is not limited, but VTC1 It is preferable to disrupt at least one of the VTC3 and VTC4 genes, and it is more preferable to disrupt the VTC1 gene.
  • “Destroy a gene encoding a constituent element of a VTC complex” means to delete or mutate a coding region of a gene encoding a protein that is a constituent element of a VTC complex on the yeast genome. Deletion of a gene encoding a constituent factor of the VTC complex may delete the entire coding region or a part thereof. When the entire region is deleted, the region adjacent to the gene encoding the constituent element of the VTC complex may also be deleted widely. When a part is deleted, it is not particularly limited, but it is preferable to delete at least half of the coding region of the gene encoding the constituent element of the VTC complex.
  • a nonsense mutation or a frameshift mutation is preferably introduced at a site upstream of the coding region, and further downstream than that. It is preferable to delete the amino acid sequence encoded by this region or to have an irrelevant amino acid sequence.
  • a VTC complex constituent factor is encoded by inserting an irrelevant sequence (other gene sequence or marker gene unrelated to the VTC complex constituent factor) into the gene encoding the VTC complex constituent factor. It can also be mutated and destroyed. Disruption of a gene encoding a component of the VTC complex reduces or loses the function of the VTC complex encoded by the gene.
  • the VTC1 gene will be exemplified as a gene encoding a component of the VTC complex to be destroyed, and the gene destruction will be specifically described.
  • Examples of the method for destroying the VTC1 gene include a homologous recombination method and a genome editing technique.
  • An example of genetic modification using the CRISPR-Cas system, which is a genome editing technology, has also been reported in practical yeasts that are used in industry (J.JMicrobiol. Methods Vol.127 p.203-205 (2016)). . According to the genome editing technique, even a higher polyploid can be modified at once, which is advantageous.
  • the homologous recombination method is a method in which a homologous DNA fragment not containing a normal VTC1 gene is introduced into yeast cells, and homologous recombination is performed between the homologous DNA fragment and yeast genomic DNA.
  • the “homologous DNA fragment” refers to a DNA fragment that can be recombined with a target region in the genome by homologous recombination.
  • the homologous recombination method itself is well known, and those skilled in the art can prepare a desired homologous DNA fragment based on the base sequence of the genome or coding region.
  • the chain length of each homologous region is not particularly limited. Generally, the longer the chain length, the higher the efficiency of homologous recombination. However, since homologous recombination activity is strong in budding yeast, a homologous region of about 50 bp may be provided. .
  • a homologous DNA fragment is constructed by linking a region homologous to the upstream region of the VTC1 gene on the genome and a region homologous to the downstream region upstream and downstream of the mutant VTC1 gene sequence that does not encode normal VTC1,
  • the VTC1 gene sequence on the genome can be replaced with the mutant VTC1 gene sequence, so that the VTC1 gene on the yeast genome can be destroyed.
  • genomic base sequences of the upstream and downstream regions of the coding region can be obtained from the Saccharomyces genome database (http://www.yeastgenome.org/).
  • the self-cloning method described in Aritomi et al., Biosci. Biotechnol. Biochem., 68 (1), 206-214, 2004 can be used.
  • This cloning method is a method of replacing a normal gene in a genome with a mutant gene using a plasmid vector containing a drug resistance marker for gene introduction and a growth suppression marker for counter selection.
  • a foreign DNA sequence required only for gene introduction can be removed after introduction of the mutated gene, which is preferable for growing brewing yeast used in the food industry.
  • the method for disrupting the VTC1 gene can be performed as follows.
  • the two types of markers used are not particularly limited, and for example, a known marker such as YAP1 as a drug resistance marker and GIN11 as a growth suppression marker can be used.
  • a mutated VTC1 gene for example, a stop codon is introduced by mutating “TCT” of the fourth to sixth bases of the base sequence shown in SEQ ID NO: 1 to “TAA”
  • TAT mutating “TCT” of the fourth to sixth bases of the base sequence shown in SEQ ID NO: 1 to “TAA”
  • the yeast in which the plasmid sequence remains is selected.
  • yeasts that cannot grow due to the action of the growth-inhibiting marker but can lose their plasmid sequence due to homologous recombination can grow, so that the plasmid sequence is lost and only the normal VTC1 gene or mutant VTC1 gene is present in the genome.
  • the remaining yeast can be obtained. Whether or not the VTC1 gene remaining in the genome has a desired mutation can be examined by, for example, amplifying a region containing the mutation by PCR and performing a sequence analysis.
  • the VTC1 gene disruption strain can be obtained by selecting from among the yeasts that have been subjected to mutation treatment. That is, in the method of the present invention, the VTC1 gene may be disrupted by mutation treatment.
  • the method of the mutation treatment is not particularly limited, and any of physical mutation treatment such as ultraviolet irradiation and radiation irradiation and chemical mutation treatment in which treatment is performed with a mutation agent such as ethylmethanesulfonic acid may be used. From the mutant strains obtained by the mutation treatment, a VTC1 gene disruption strain can be selected by sequence analysis.
  • yeasts such as brewer's yeast, baker's yeast, and bioethanol yeast are usually diploids or higher polyploids. Therefore, when yeast for which the productivity of fermentation products by alcohol fermentation is improved by the method of the present invention is used for alcoholic beverage production, bread production, and bioethanol production, there is no particular limitation, but both diploid and VTC1 allyl are used together. It is preferable to use a disrupted VTC1 gene disruption strain.
  • a diploid gene-disrupted strain can be obtained by a well-known conventional method.
  • VTC1 gene disrupted strains can be prepared using haploids (a type and ⁇ type) having different mating types, and both can be joined to obtain a diploid VTC1 gene disrupted strain.
  • haploids a type and ⁇ type
  • a diploid VTC1 gene disruption strain can be obtained by preparing a yeast in which one of the VTC1 alleles is disrupted and then subjecting the yeast to VTC1 disruption treatment again.
  • VTC1 gene When the VTC1 gene is disrupted by the above method using a homologous DNA fragment, for example, different drug resistance markers may be used in the first gene disruption process and the second gene disruption process. If the above self-cloning method is used, a diploid yeast in which one of the VTC1 alleles has been disrupted does not have a marker gene in the genome. Can be used. When the above gene disruption method is adopted for diploid yeast, a strain in which the VTC1 allele is simultaneously disrupted may also be generated, so that the difference in the growth rate on the selective medium (the strain that has been disrupted simultaneously does not grow). Early) or based on the nucleotide sequence of the VTC1 gene region, a strain in which the VTC1 allele is simultaneously destroyed can also be selected.
  • a method for obtaining a haploid from a diploid yeast can be performed by a conventional method well known in the art.
  • the spore can be obtained by culturing in a known spore-forming medium to form a spore, and the spore can be germinated to obtain a haploid.
  • the yeast in which the VTC1 gene is disrupted has significantly higher fermentation ability and ethanol productivity than the wild yeast strain having the normal VTC1 gene.
  • the present invention includes producing a yeast having improved fermentability and productivity of fermentation products by reducing the expression level of a gene encoding a constituent factor of a VTC complex present on the yeast genome.
  • a method for reducing the expression of a gene encoding a constituent element of a VTC complex include, for example, a transcription promoter region in a coding region of a gene encoding a protein that is a constituent element of a VTC complex on a yeast genome. And a method of culturing the mutant yeast under transcriptional repression conditions.
  • the present invention includes producing yeast having improved fermentability and productivity of fermentation products by reducing the translation efficiency of a gene encoding a constituent factor of a VTC complex present on the yeast genome.
  • Examples of the method for suppressing the translation of the gene encoding the constituent factor of the VTC complex include a method using so-called antisense RNA. That is, a gene that encodes a constituent element of a VTC complex by integrating a gene that transcribes an antisense RNA against mRNA of a gene encoding a constituent element of the VTC complex into the yeast genome and overexpressing the antisense RNA. Translation of mRNA is suppressed.
  • the present invention relates to a method for producing a yeast for alcohol fermentation, which comprises reducing or losing the function of a VTC complex by carrying out a gene mutation in yeast, and a method for producing a VTC complex or a VTC complex by genetic mutation. Also included are yeasts for alcohol fermentation in which the function of the constituent factors is reduced or lost.
  • the yeast in the present invention may be any yeast that performs fermentation, and is not particularly limited. Examples include brewing yeast used for the production of alcoholic beverages, baker's yeast used for the production of bread, and bioethanol yeast used for the production of bioethanol. Examples of the brewing yeast include sake yeast, wine yeast, beer yeast, shochu yeast, and the like. Sake yeast is used to produce sake, wine yeast is used to produce wine, and shochu yeast is used to produce shochu. Laboratory yeasts can also be used in the present invention.
  • the yeast used in the present invention is not particularly limited, and examples thereof include yeasts belonging to the genus Saccharomyces, the genus Schizosaccharomyces or the genus Kluyveromyces, and the genus Zygosaccharomyces. Yeast belonging to the genus Saccharomyces is preferable.
  • the yeast belonging to the genus Saccharomyces is preferably Saccharomyces cerevisiae.
  • Saccharomyces acti Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti ), Saccharomyces bailii, Saccharomyces bisporus, Saccharomyces capensis, Saccharomyces dri ci om i s ), Saccharomyces coreanus, Saccharomyces renenensis, Saccharo Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces eupagycus, Saccharomyces exiguus, Saccharomyces exiguus, Saccharomyces oms Saccharomyces florentinus), Saccharomyces globosus, Saccharomyces heterogenicus, Saccharomyces hissepiensis, Saccharomyces hienipices,
  • Preferred brewer's yeast used in the present invention includes Saccharomyces cerevisiae (more specifically, No. 7 (K7)) for sake yeast, and Saccharomyces cerevisiae (more specifically, for yeast).
  • Ampoule yeast for yeast wine Saccharomyces baianus, etc., for beer yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces carlsbergensis, etc., for shochu yeast, Saccharomyces cerevisiae (more specifically, No. 2) for yeast yeast shochu.
  • Preferred bioethanol yeasts used in the present invention include Saccharomyces cerevisiae, Saccharomyces dairenensis, Saccharomyces transvalensis, Saccharomyces rosinii, Zygosaccharomyces bisporus and the like.
  • a sucrose non-assimilating yeast that selectively converts only reducing sugars to ethanol may be used.
  • yeast that is the subject of the present invention may be derived from any of a wild strain, a mutant strain, and a transformed strain.
  • yeast VTC1 gene-disrupted strains and VTC4 gene-disrupted strains laboratory yeast VTC1 gene-disrupted strains and VTC4 gene-disrupted strains created for the purpose of investigating the catalytic mechanism of the polyphosphate synthesis pathway in yeast are known. Patent Document 4).
  • the gene-disrupted yeast encoding the component of the VTC complex or the yeast in which the expression level of the gene is reduced is increased in carbon dioxide loss and the productivity of produced ethanol is improved
  • brewing yeast, baker's yeast, or bioethanol yeast in which a gene encoding a constituent factor of the VTC complex is disrupted or the expression level of the gene is reduced There are also no known brewing yeast, baker's yeast, or bioethanol yeast in which a gene encoding a constituent factor of the VTC complex is disrupted or the expression level of the gene is reduced.
  • the present invention provides novel brewer's yeast, baker's yeast, and bioethanol yeast whose fermentability and ethanol productivity are improved by disrupting a gene encoding a constituent factor of the VTC complex or reducing the expression level of the gene. It is to provide.
  • “fermentability is improved” means that the amount of carbon dioxide (carbon dioxide) generated during yeast fermentation is increasing. Since yeast decomposes glucose into ethanol and carbon dioxide by alcohol fermentation, the amount of carbon dioxide generated is generally used as a numerical value indicating the progress of alcohol fermentation of yeast, that is, ethanol production. The amount of carbon dioxide generated is a general value indicating the fermentation rate of yeast, and an increase in the amount of carbon dioxide generated suggests that the fermentation rate is accelerated. Further, as described above, since yeast decomposes glucose into ethanol and carbon dioxide by alcohol fermentation, the generation amount of ethanol increases simultaneously with the generation amount of carbon dioxide by the promotion of fermentation. In other words, the yeast having improved fermentability according to the method of the present invention has improved ethanol productivity.
  • a method for producing yeast having improved fermentability and ethanol productivity includes a step of destroying a gene encoding a constituent factor of the VTC complex or a step of reducing the expression level of the gene, but the produced yeast is untreated.
  • the method may further comprise a step of comparing the fermentability and ethanol productivity with the parent strain.
  • the present invention also includes a composition for alcohol fermentation containing yeast in which the function of the VTC complex is reduced or lost.
  • the composition for alcohol fermentation is a composition used for alcohol fermentation.
  • it is a composition containing yeast for alcohol fermentation in which the function of the VTC complex or the component of the VTC complex is reduced or lost due to gene mutation, more preferably a gene encoding the component of the VTC complex
  • a composition comprising yeast for alcohol fermentation in which at least one gene selected from is disrupted.
  • the yeast in the composition for alcohol fermentation may be cultured and collected by a method commonly used by those skilled in the art, and may be used as it is for the production of the composition, or may be post-treated by a known method.
  • rough purification may be performed by centrifugation or the like.
  • it may be dissolved or suspended in a solvent conventionally used in the art, such as physiological saline, phosphate buffered saline (PBS), or a medium.
  • a solvent conventionally used in the art, such as physiological saline, phosphate buffered saline (PBS), or a medium.
  • PBS phosphate buffered saline
  • a protective agent may be added to the composition for alcohol fermentation.
  • the present invention also extends to a method for producing a fermentation product using yeast, comprising performing alcoholic fermentation under conditions where the function of the VTC complex is reduced or lost in yeast. Reduction or loss of the function of the VTC complex in yeast can be performed by the means described above.
  • the method for producing a fermentation product of the present invention specifically includes the following steps. (1) A step of performing alcoholic fermentation by bringing the raw material and yeast into contact with each other under the condition that the function of the VTC complex is reduced or lost in the yeast. (2) A step of obtaining a fermentation product after performing alcoholic fermentation.
  • alcohol preferably ethanol
  • carbon dioxide are produced by allowing yeast to act on the saccharide derived from the raw material. Examples of raw materials include rice, wheat, grapes, rice bran, flour, molasses, cellulosic biomass, and the like, or those obtained by treating them.
  • the fermentation product may be any product as long as it is produced through fermentation with yeast.
  • fermented products include fermented beverages, fermented foods, and bioethanol.
  • the fermented beverage may be any beverage as long as it is a beverage produced by fermenting a raw material, and alcoholic beverages are exemplified. Examples of liquors include, but are not limited to, sake, wine, beer, shochu, whiskey, brandy and the like.
  • fermented foods food materials produced by fermenting ingredients as raw materials, or processed foods, for example, fermented bread dough or food processed by fermenting bread dough, alcoholic beverages, tea, vinegar, grains Examples of processed products (natto, soy sauce, miso, etc.), processed seafood products, processed vegetable fruits, dairy products, etc.
  • Liquors include brewed liquor and distilled liquor. Brewed liquor is produced by fermenting the main ingredients such as rice, wheat and grapes. Distilled liquor is produced by fermenting the main ingredients and then distilled. It is sake.
  • the fermentation methods of brewed liquor include simple fermentation, single double fermentation, and parallel double fermentation.
  • the brewed liquor by simple fermentation includes wine
  • the brewed liquor by simple double fermentation includes beer
  • the brewed liquor by parallel double fermentation includes sake (Japanese sake).
  • saccharification and fermentation proceed simultaneously in the same container.
  • the main raw materials including saccharides obtained by saccharifying saccharides derived from foods or polysaccharides derived from foods
  • separating a liquid from solid content after performing alcoholic fermentation are included.
  • wort is used as the main raw material.
  • Wort is a saccharified solution prepared by saccharifying starch by holding auxiliary materials such as malt and rice at an appropriate temperature (for example, 40 ° C. to 100 ° C.) for an appropriate period of time, heating and cooling.
  • auxiliary materials such as malt and rice
  • Steamed rice is used as the main raw material for the production of sake.
  • rice bran for example, Aspergillus oryzae
  • steamed rice and yeast is cultured in a fermentor so that rice starch is broken down into glucose by the koji enzyme.
  • Alcohol fermentation is performed using glucose.
  • the brewed liquor is obtained by separating the liquid from the solid content by extraction, pressing, filtration, or the like. If necessary, pressing, heating, aging, storage, preparation, lactic acid fermentation, filtration, and the like can be performed.
  • the conditions for alcoholic fermentation in the production of each brewed liquor vary depending on the brewed liquor to be produced, but can be carried out at a suitable temperature between 4 ° C. and 40 ° C. for a suitable period of 4 days to 70 days.
  • the fermented liquor after performing alcohol fermentation of the main raw material in the same manner as brewed liquor, the fermented liquor (fermented rice cake) after alcohol fermentation may be distilled and separated. Moreover, storage, aging, etc. can also be performed as needed.
  • distilled spirits, shochu, whiskey, brandy and the like are known. Barley, straw, rice, etc. can be used as the main raw material of shochu, whiskey uses wort, brandy uses grape or grape juice.
  • the alcoholic beverages are not limited to those described above, and any alcohol may be used as long as it is obtained through an alcohol fermentation process.
  • Bread dough is manufactured using flour as the main ingredient.
  • Wheat flour is mixed with yeast, water and other materials, and fermented.
  • fermentation is performed by fermentation at an appropriate temperature (for example, 27 ° C.) and a humidity of about 70 to 80% for 4 to 5 hours, and bread dough swells.
  • the fermentation after molding is exemplified by a temperature of 35 to 38 degrees and a humidity of about 80%.
  • the bread dough in the present invention is not limited as long as it has been subjected to at least one fermentation step in a state in which materials containing at least flour, yeast, and water are mixed, and does not necessarily have to undergo a plurality of fermentation steps.
  • Bread dough can be baked at a predetermined temperature to produce bread, or it can be used to produce processed foods such as pizza.
  • Soy sauce is produced using soybeans and wheat as main ingredients. In the production of soy sauce, soybeans are first steamed with pressurized steam in high pressure for a short time, roasted with wheat, and processed into the main ingredients. Spice is sprinkled on the combined soybean and wheat after processing, and the koji is propagated to produce koji.
  • the koji is mixed with saline and placed in a fermenter, cooled and sterilized, heated, and yeast is added for fermentation. After fermentation, the soy sauce is obtained by squeezing to remove excess oil and solids and performing sterilization, filtration and the like as necessary.
  • the main raw material for bioethanol may be any biological resource that contains carbohydrates.
  • plant-derived plant resources containing a large amount of saccharides, starch, or cellulose are preferred. More specifically, as plant resources, a cellulose system containing cellulose such as molasses, corn, switchgrass (perennials), pulp waste liquor, waste wood, rice straw, etc. obtained after refining sugar from sugarcane and sugar beet Biomass is mentioned.
  • Bioethanol production is performed by saccharification in which polysaccharides such as starch and cellulose contained in the main raw material are treated with a polysaccharide-degrading enzyme such as amylase to be decomposed into glucose, and alcohol fermentation by yeast using glucose.
  • the produced bioethanol may be recovered by a known method or a method developed in the future.
  • yeasts in which at least one gene among the genes encoding the components constituting the VTC complex of yeast is disrupted or whose expression level is reduced are higher than those conventionally used. It was found to have fermentability and ethanol productivity.
  • the present invention also includes a drug screening method for improving the alcohol-fermenting ability of yeast based on such findings.
  • medical agent for improving the alcohol fermentation ability screened in this invention can be used in the method of improving the alcohol fermentation ability of the yeast of this invention, and the manufacturing method of a fermentation product.
  • the method for screening a drug of the present invention includes a step of allowing a test compound to act on a VTC complex, and uses the test compound as an index to reduce or lose the function of the VTC complex. Specifically, those including the following steps are exemplified.
  • (Ii) A step of bringing the VTC complex into contact with the test compound and confirming the function of the VTC complex.
  • VTC complex Reduced or lost function of the VTC complex in yeast is a known function of the VTC complex, maintaining the stability of vacuolar proton ATPase, protein transport, microautophagy, or polyphosphate into the vacuole This can be confirmed by a decrease or loss of the accumulation function. Furthermore, it can also be confirmed by a decrease or loss of the function inherent to the VTC complex to be found in the future.
  • the drug obtained by the screening method of the present invention causes a decrease or loss in the function of the yeast VTC complex, and the alcohol fermentability of the yeast can be improved by contacting with the yeast.
  • Example 1 Preparation of VTC1 gene disruption strain, VTC2 gene disruption strain, VTC3 gene disruption strain, VTC4 gene disruption strain and measurement of fermentation ability thereof
  • Preparation of gene disruption strain Yaploid laboratory of yeast Saccharomyces cerevisiae
  • VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains BY4741 vtc1 ⁇ , BY4741 vtc2 ⁇ , BY4741 vtc3 ⁇ , BY4741 vtc4 ⁇ , respectively
  • the vertical axis represents the average value (g) of the amount of carbon dioxide generated. Error bars indicate standard deviation. A one-sided t-test with no assumed equal variance was performed on the average value of carbon dioxide generation. As shown in FIG. 1, all of the VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains showed a carbon dioxide generation amount higher than that of the wild strain.
  • each of the VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains shows a statistically significantly higher carbon dioxide generation amount than the wild strain (less than 5% of the risk rate), and the carbon dioxide generation amount in the VTC1 gene-disrupted strain is The value was 70.8% higher than that of the wild type. From the above results, it was clarified that the disruption of each gene encoding the constituent factor of the VTC complex leads to the promotion of alcohol fermentation.
  • Example 2 Production of VTC1 gene-disrupted baker's yeast strain and measurement of its fermentative ability
  • a haploid 3346 strain derived from 3346/3347 strain a diploid baker's yeast strain of Saccharomyces cerevisiae, was used as a wild strain.
  • a VTC1 gene disruption strain (3346 vtc1 ⁇ ) was prepared by a known method (Yeast vol.21 p.947-962 (2004)).
  • a specific manufacturing method is as follows.
  • PCR was performed using primers having base sequences homologous to the upstream region and downstream region of the ORF of the VTC1 gene. Then, a base sequence homologous to the upstream and downstream of the VTC1 gene was linked upstream and downstream of the drug resistance gene to prepare a VTC1 gene disruption fragment.
  • the primers used are as follows.
  • VTC1-S1 CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC (SEQ ID NO: 9)
  • VTC1-S2 CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG (SEQ ID NO: 10)
  • VTC1-Fw AACACTACGCCAGATTTCCA (SEQ ID NO: 17)
  • VTC1-Rv CCACGCTTACGATATTGGAA (SEQ ID NO: 18)
  • Yeast (3346 vtc1 ⁇ or wild type) was cultured in YPD medium for 24 hours at 30 ° C., then collected by centrifugation and washed twice with distilled water.
  • As bread dough containing low sucrose 20 g of bread-only flour (Nissin), 1 g of sucrose, 0.4 g of sodium chloride, 12.5 mL of water, and 0.4 g of yeast were kneaded and prepared.
  • 30 g of bread exclusive flour (Nissin) 9 g of sucrose, 0.15 g of sodium chloride, 16 mL of water, and 0.6 g of yeast were prepared as a high sucrose-containing bread dough.
  • the amount of carbon dioxide generated was measured using a Pharmagraph II-W (Atto). The fermentation test was repeated three times for each strain.
  • the horizontal axis represents fermentation time (h), and the vertical axis represents the average value (mL) of carbon dioxide generation.
  • a one-sided t-test was performed in the same manner as in Example 1.
  • the VTC1 gene-disrupted strain showed more carbon dioxide generation than the wild strain in both the low sucrose-containing bread dough and the high sucrose-containing bread dough.
  • the carbon dioxide generation amount of the VTC1 gene disrupted strain after 2 hours from the start of fermentation was 10.1 to 16.0% (low sucrose-containing bread dough) or 16.3 to 17.5% (high Sucrose-containing bread dough) and a high value. From the above results, it was revealed that the disruption of the VTC1 gene leads to the promotion of alcohol fermentation in the production of bread dough.
  • Example 3 Production of VTC1 Gene Disrupted Bioethanol Yeast Strain and Measurement of Fermentation Capacitance
  • a VTC1 gene disruption strain (NCYC3233-27c vtc1 ⁇ ) was prepared by a known method (Yeast vol.21 p.947-962 (2004)).
  • a VTC1 gene disruption strain was prepared in the same manner as in Example 2 except that pFA6a-natNT2 (including a clone nut resistance marker) was used as a plasmid containing a drug resistance gene.
  • Yeast (NCYC3233-27c vtc1 ⁇ or wild type) is cultured with shaking in YPD medium at 30 ° C overnight, and then 50 mL of sugarcane molasses medium (Brix 15, Inoculated with 0.025% ammonium sulfate). After stationary culture at 30 ° C., the amount of carbon dioxide generated was measured using a Pharmagraph II-W (Atto). The fermentation test used 2 to 3 independent clones for each strain.
  • FIG. 3 the horizontal axis represents fermentation time (h), and the vertical axis represents the average value (mL) of carbon dioxide generation.
  • a one-sided t-test was performed in the same manner as in Example 1.
  • the VTC1 gene-disrupted strain showed a carbon dioxide generation amount higher than that of the wild strain.
  • the amount of carbon dioxide generated by the VTC1 gene-disrupted strain after 42 hours from the start of fermentation was 4.8 to 6.0% higher than that of the wild strain, and a statistically significant difference was detected (risk rate 5). %Less than). From the above results, it was revealed that the destruction of the VTC1 gene leads to the promotion of alcohol fermentation in bioethanol production.
  • Example 4 Production of VTC1 gene disrupted strain, VTC2 gene disrupted strain, VTC3 gene disrupted strain, VTC4 gene disrupted strain Yeast Saccharomyces cerevisiae haploid laboratory strain BY4741 strain was used as a wild strain, VTC1, Disrupted strains of VTC2, VTC3, and VTC4 genes are prepared.
  • a plasmid containing a drug resistance gene those described in Yeast vol.21 p.947-962 (2004) can be used, and for example, pFA6a-hphNT1 or pFA6a-natNT2 can be used.
  • PCR was performed using primers having base sequences homologous to the upstream region and downstream region of each ORF of the VTC1, VTC2, VTC3, and VTC4 genes, and upstream of the drug resistance gene.
  • a base sequence homologous to the upstream and downstream of the VTC1, VTC2, VTC3, and VTC4 genes is ligated to the downstream to produce fragments for disrupting the VTC1, VTC2, VTC3, and VTC4 genes.
  • the primers designed to create the VTC1, VTC2, VTC3, and VTC4 gene disruption fragments are as follows.
  • VTC1-S1 CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC (SEQ ID NO: 9)
  • VTC1-S2 CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG (SEQ ID NO: 10)
  • VTC2-S1 AGTAGAAAGAACGACTACACCTCAACATAACGACACTTTTTTGACCGTACGCTGCAGGTCGAC (SEQ ID NO: 11)
  • VTC3-S1 TTAGAGCGAACAGCAGAATTTGTCCTTGGTTTTCAGAGTTTGAAACGTACGCTGCAGGTCGAC (SEQ ID NO: 13)
  • VTC3-S2 CTGGTACTTGTGTAATATATGTGTATATAAAAA
  • VTC1-Fw AACACTACGCCAGATTTCCA (SEQ ID NO: 17)
  • VTC1-Rv CCACGCTTACGATATTGGAA (SEQ ID NO: 18)
  • VTC2-Fw AAGAACGACTACACCTCAACA (SEQ ID NO: 19)
  • VTC2-Rv CATAAAAACACATGGTCTCAG (SEQ ID NO: 20)
  • VTC3-Fw AGCGAACAGCAGAATTTGTCC
  • VTC3-Rv GGTACTTGTGTAATATATGTGTG (SEQ ID NO: 22)
  • VTC4-Fw ATAAAAGAGCATAACAAG (SEQ ID NO: 23)
  • VTC4-Rv GATTATTACTTAATTATACAG (SEQ ID NO: 24)
  • VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains are considered to show carbon dioxide generation exceeding that of the wild strain.
  • yeast fermentability and ethanol productivity can be improved.
  • the present invention can be used in various industries such as fermented foods, fermented beverages, and bioethanol production.

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Abstract

The present invention pertains to a method for enhancing the fermentation capacity and ethanol yield of yeast. Specifically, the present invention pertains to: a method for enhancing the fermentation capacity of yeast that involves causing the decrease or loss of the vacuolar transporter chaperone complex (VTC complex) function in yeast; a fermentation product production method using yeast that involves carrying out alcoholic fermentation in conditions wherein the VTC complex function in the yeast is decreased or lost; a fermentation yeast preparation method that involves causing the decrease or loss of the VTC complex function in yeast by introducing a genetic mutation; yeast wherein the VTC complex function has been decreased or lost due to genetic mutation; and a screening method for drugs that enhance the fermentation capacity of yeast.

Description

酵母の液胞トランスポーターシャペロン複合体の機能欠損による発酵促進方法Fermentation promotion method by functional defect of yeast vacuolar transporter chaperone complex
　本発明は、酵母の液胞トランスポーターシャペロン複合体の機能欠損によって発酵能を向上させる方法に関する。 The present invention relates to a method for improving fermentability by functional defect of yeast vacuolar transporter chaperone complex.
　本出願は、参照によりここに援用されるところの日本出願、特願２０１６－０２５３２４号優先権を請求する。 This application claims the priority of Japanese Patent Application No. 2016-025324, which is incorporated herein by reference.
　近年、酵母サッカロミセス・セレビシエ（Saccharomyces cerevisiae）において、液胞トランスポーターシャペロン複合体（以下「ＶＴＣ複合体」）が見出された。ＶＴＣ複合体は、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐにより構成されるタンパク質複合体である。ＶＴＣ複合体は、液胞型プロトンＡＴＰａｓｅの安定性の維持や、タンパク質の輸送、ミクロオートファジー、液胞内へのポリリン酸の蓄積に関与することが報告されている（非特許文献１、２、３、４）。非特許文献１では、実験室株の酵母Ｗ３０３株を用いて、Ｖｔｃ１ｐをコードするＶＴＣ１遺伝子の破壊株が作製され、ＶＴＣ複合体についての解析が行われている。 Recently, a vacuolar transporter chaperone complex (hereinafter “VTC complex”) has been found in the yeast Saccharomyces cerevisiae. The VTC complex is a protein complex composed of Vtc1p, Vtc2p, Vtc3p, and Vtc4p. The VTC complex has been reported to be involved in maintaining the stability of vacuolar proton ATPase, protein transport, microautophagy, and polyphosphate accumulation in the vacuole (Non-Patent Documents 1 and 2). 3, 4). In Non-Patent Document 1, a VTC1 gene-disrupted strain that encodes Vtc1p is prepared using a laboratory yeast strain W303, and analysis of the VTC complex is performed.
　酵母は、パン生地等や清酒をはじめとする酒類等の発酵食品やバイオエタノール等の製造に用いられている。発酵食品やバイオエタノールの製造においては、一般的に、ブドウ糖などの糖類をサッカロミセス属の酵母によりエタノールと二酸化炭素に分解するアルコール発酵の工程が用いられる。 Yeast is used for the production of fermented foods such as bread dough and alcoholic beverages such as sake and bioethanol. In the production of fermented foods and bioethanol, an alcohol fermentation process is generally used in which sugars such as glucose are decomposed into ethanol and carbon dioxide by Saccharomyces yeasts.
　酵母によるアルコール発酵速度を高めることは、発酵食品やバイオエタノールの生産性の向上に直結する。酵母のアルコール発酵を促進する方法として、遺伝子組換え酵母を作製する方法が検討されている。本発明者らは、酵母のストレス応答性を改変することによりアルコール発酵を促進し得ることを見出し、ストレス応答転写因子をコードするＭＳＮ２遺伝子又はＭＳＮ４遺伝子を破壊した酵母（特許文献１）や、酵母の増殖が停止する定常期への移行に関する定常期移行促進因子をコードするＩＧＯ１遺伝子又はＩＧＯ２遺伝子を破壊した酵母（特許文献２）を作製した。しかしながら、ストレス応答性を改変する方法では、酵母のアルコール発酵の促進には限界があり、酵母のアルコール発酵を効果的に促進する方法の開発が望まれる。 Accelerating the rate of alcohol fermentation by yeast directly improves the productivity of fermented foods and bioethanol. As a method for promoting alcohol fermentation of yeast, a method for producing a genetically modified yeast has been studied. The present inventors have found that alcohol fermentation can be promoted by modifying the stress responsiveness of yeast, and yeast (Patent Document 1) in which MSN2 gene or MSN4 gene encoding a stress response transcription factor is disrupted, yeast The yeast (patent document 2) which destroyed the IGO1 gene or the IGO2 gene which encodes the stationary phase transition promotion factor regarding the transition to the stationary phase where the growth of the yeast stops is produced. However, in the method of modifying stress responsiveness, there is a limit to the promotion of yeast alcohol fermentation, and development of a method for effectively promoting yeast alcohol fermentation is desired.
　酵母においてどの遺伝子がアルコール発酵に関連しているかは未だ解明されておらず、アルコール発酵とＶＴＣ複合体との関連性についての報告もない。 It has not yet been clarified which genes are related to alcoholic fermentation in yeast, and there is no report on the relationship between alcoholic fermentation and the VTC complex.
特許第５５８５９５２号公報Japanese Patent No. 5585952 特許第５８２８４４７号公報Japanese Patent No. 5828447
　本発明は、酵母の発酵能を向上させる方法を提供することを課題とする。 An object of the present invention is to provide a method for improving the fermentability of yeast.
　本発明者らは、上記課題を解決するために鋭意研究した結果、酵母のＶＴＣ複合体の構成因子をコードする遺伝子を破壊することにより、顕著なアルコール発酵の促進をもたらすことを着目し、酵母のＶＴＣ複合体の機能を低下又は喪失させることにより、酵母のアルコール発酵能を向上させることができることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above problems, the present inventors have paid attention to the fact that disrupting a gene encoding a constituent factor of a yeast VTC complex brings about remarkable acceleration of alcohol fermentation. It was found that the alcohol fermentability of yeast can be improved by reducing or losing the function of the VTC complex, and the present invention has been completed.
　すなわち、本発明は、以下の構成からなる。
１.　酵母において、液胞トランスポーターシャペロン複合体（ＶＴＣ複合体）の機能を低下又は喪失させることを含む、酵母の発酵能を向上させる方法。
２.　酵母のＶＴＣ複合体の機能の低下又は喪失が、複数の構成因子から形成されるＶＴＣ複合体の形成阻害によるものである、前項１に記載の酵母の発酵能を向上させる方法。
３.　酵母のＶＴＣ複合体の機能の低下又は喪失が、酵母において遺伝子変異を導入することにより、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の発現量を低下させることによるものである、前項１または２に記載の酵母の発酵能を向上させる方法。
４.　酵母における遺伝子変異が、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の破壊である、前項３に記載の酵母の発酵能を向上させる方法。５.　ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子が、ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子、ＶＴＣ４遺伝子からなる群から選択される少なくとも１つの遺伝子である、前項３又は４に記載の酵母の発酵能を向上させる方法。
６.　ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子が、以下の（ａ）～（ｅ）からなる群から選択される少なくとも１つの遺伝子である、前項２～５のいずれかに記載の酵母の発酵能を向上させる方法：
（ａ）配列番号１～４のいずれかに記載のアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｂ）配列番号１～４のいずれかに記載のアミノ酸配列において１又は数個以上のアミノ酸残基が欠失、置換、挿入及び／又は付加されたアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｃ）配列番号１～４のいずれかに記載のアミノ酸配列と８０％以上の相同性を有するアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｄ）配列番号５～８のいずれかに記載の塩基配列からなるポリヌクレオチドを含む遺伝子；
（ｅ）上記（ａ）～（ｄ）からなる群から選択されるいずれかのポリヌクレオチドと相補的な塩基配列からなるポリヌクレオチドを含む遺伝子。
７.　前項１～６のいずれかに記載の酵母の発酵能を向上させる方法を含む、酵母を用いて発酵産物を製造する方法であって、酵母においてＶＴＣ複合体の機能が低下又は喪失している条件で、アルコール発酵を行うことを含む、発酵産物の製造方法。
８.　発酵産物がエタノールである、前項７に記載の発酵産物の製造方法。
９.　発酵産物が発酵飲料又は発酵食品である、前項７に記載の発酵産物の製造方法。
１０.　酵母において、遺伝子変異を導入することにより、ＶＴＣ複合体の機能を低下又は喪失させることを含む、アルコール発酵用酵母の作製方法。
１１.　遺伝子変異により、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の機能が低下又は喪失した、アルコール発酵用酵母。
１２.　遺伝子変異が、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の破壊である、前項１１に記載のアルコール発酵用酵母。
１３.　遺伝子変異により、ＶＴＣ複合体の機能が低下又は喪失した酵母であり、醸造酵母、パン酵母、又はバイオエタノール酵母のいずれかである酵母。
１４.　前項１１～１３のいずれかに記載の酵母を含有する、アルコール発酵用組成物。
１５.　酵母の発酵能を向上させるための薬剤のスクリーニング方法であって、被験化合物をＶＴＣ複合体に作用させる工程を含み、当該被験化合物がＶＴＣ複合体の機能を低下又は喪失させることを指標とする、スクリーニング方法。 That is, this invention consists of the following structures.
1. A method for improving the fermentability of yeast, comprising reducing or eliminating the function of a vacuolar transporter chaperone complex (VTC complex) in yeast.
2. The method for improving the yeast fermentability according to item 1 above, wherein the decrease or loss of the function of the yeast VTC complex is due to inhibition of formation of a VTC complex formed from a plurality of components.
3. Decreasing or loss of the function of the yeast VTC complex reduces the expression level of at least one gene selected from genes encoding the components of the VTC complex by introducing a genetic mutation in the yeast. 3. A method for improving the fermentability of yeast according to 1 or 2 above.
4. The method for improving the fermentability of yeast according to item 3 above, wherein the genetic mutation in yeast is disruption of at least one gene selected from genes encoding a component of the VTC complex. 5. At least one gene selected from genes encoding constituent elements of a VTC complex is at least one gene selected from the group consisting of a VTC1, VTC2, VTC3, and VTC4 genes, 4. A method for improving the fermentability of yeast according to 4.
6. At least one gene selected from genes encoding a component of a VTC complex is at least one gene selected from the group consisting of the following (a) to (e): A method for improving the fermentability of yeast according to any one of the following:
(A) a gene comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(B) a gene comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(C) a gene comprising a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(D) a gene comprising a polynucleotide comprising the base sequence set forth in any one of SEQ ID NOs: 5 to 8;
(E) A gene comprising a polynucleotide comprising a base sequence complementary to any polynucleotide selected from the group consisting of (a) to (d) above.
7. A method for producing a fermentation product using yeast, comprising the method for improving the fermentability of yeast according to any one of 1 to 6 above, wherein the function of the VTC complex is reduced or lost in the yeast. A method for producing a fermented product, comprising performing alcoholic fermentation under certain conditions.
8. The method for producing a fermentation product according to item 7, wherein the fermentation product is ethanol.
9. The method for producing a fermented product according to item 7, wherein the fermented product is a fermented beverage or a fermented food.
10. A method for producing a yeast for alcohol fermentation, comprising reducing or losing the function of a VTC complex by introducing a gene mutation in the yeast.
11. A yeast for alcoholic fermentation in which the function of a VTC complex or a component of a VTC complex is reduced or lost due to a gene mutation.
12. The yeast for alcoholic fermentation according to 11 above, wherein the genetic mutation is a disruption of at least one gene selected from genes encoding a component of the VTC complex.
13. A yeast whose function of the VTC complex is reduced or lost due to a gene mutation, and is a brewer's yeast, baker's yeast, or bioethanol yeast.
14. A composition for alcoholic fermentation comprising the yeast according to any one of 11 to 13 above.
15. A method for screening a drug for improving the fermentability of yeast, comprising a step of allowing a test compound to act on a VTC complex, wherein the test compound decreases or loses the function of the VTC complex. A screening method.
　本発明の方法により、酵母において、ＶＴＣ複合体の機能を低下又は喪失させた状態で発酵を行うことにより、効率的に発酵を促進させることができる。さらに、本発明により効率的に発酵を行うことにより、酵母の発酵を経て製造される発酵産物を良好に生産することができる。また本発明により、発酵能及びエタノール生産性の高い酵母の作製を体系的に行うことも可能となる。本発明に基づき作製されたＶＴＣ複合体の機能を低下又は喪失させた酵母は、様々な発酵食品、発酵飲料の製造、バイオエタノールの製造に有用であり、特に醸造において極めて有用である。 By the method of the present invention, fermentation can be efficiently promoted by performing fermentation in a state where the function of the VTC complex is reduced or lost in yeast. Furthermore, by performing fermentation efficiently according to the present invention, a fermentation product produced through yeast fermentation can be favorably produced. Moreover, according to the present invention, it is possible to systematically produce yeast having high fermentability and ethanol productivity. Yeast with reduced or lost function of the VTC complex produced according to the present invention is useful for producing various fermented foods, fermented beverages, and bioethanol, and is particularly useful in brewing.
ＶＴＣ１遺伝子破壊株、ＶＴＣ２遺伝子破壊株、ＶＴＣ３遺伝子破壊株、ＶＴＣ４遺伝子破壊株の発酵試験結果を示す図である。（実施例１）It is a figure which shows the fermentation test result of a VTC1 gene disruption strain, a VTC2 gene disruption strain, a VTC3 gene disruption strain, and a VTC4 gene disruption strain. (Example 1) ＶＴＣ１遺伝子破壊パン酵母株の発酵試験結果を示す図である。（実施例２）It is a figure which shows the fermentation test result of a VTC1 gene disruption baker's yeast strain. (Example 2) ＶＴＣ１遺伝子破壊バイオエタノール酵母株の発酵試験結果を示す図である。（実施例３）It is a figure which shows the fermentation test result of a VTC1 gene disruption bioethanol yeast strain. (Example 3)
　本発明において、酵母の各遺伝子の破壊株から成る酵母遺伝子破壊株ライブラリーを用いて実験室条件下で発酵試験を行い、各遺伝子破壊がアルコール発酵に与える影響を解析したところ、ＶＴＣ複合体の構成因子をコードする各遺伝子の破壊が顕著なアルコール発酵の促進をもたらすことを明らかにした。本発明はかかる知見に基づき達成されたものであり、酵母において、ＶＴＣ複合体の機能を低下又は喪失させることにより、酵母の発酵能を向上させる方法に関するものである。以下、本発明を詳細に説明する。 In the present invention, a fermentation test was performed under laboratory conditions using a yeast gene-disrupted strain library consisting of a yeast gene-disrupted strain, and the influence of each gene disruption on alcohol fermentation was analyzed. It was clarified that the disruption of each gene encoding the component factor significantly promoted alcohol fermentation. The present invention has been achieved based on such findings, and relates to a method for improving the fermentability of yeast by reducing or losing the function of the VTC complex in yeast. Hereinafter, the present invention will be described in detail.
［１．液胞トランスポーターシャペロン複合体（ＶＴＣ複合体）］
　ＶＴＣ複合体は、液胞膜に存在する液胞トランスポーターシャペロン複合体であり、酵母サッカロミセス・セレビシエにおいて見出された。ＶＴＣ複合体は、２以上の複数の構成因子から形成されるタンパク質複合体であり、各構成因子は分子量の比較的小さい膜透過タンパク質である。例えば、サッカロミセス・セレビシエのＶＴＣ複合体は、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐの４つの構成因子により形成される。ＶＴＣ複合体の形成には、各構成因子をコードする遺伝子の２以上が、それぞれ構成因子を発現し、発現した各構成因子が相互作用をすることが必要であると考えられている。例えば、サッカロミセス・セレビシエでは、ＶＴＣ複合体を形成するには、Ｖｔｃ２ｐ又はＶｔｃ３ｐのいずれか一方、Ｖｔｃ１ｐ、及びＶｔｃ４ｐが発現していること必要であると考えられている（非特許文献１）。 [1. Vacuolar transporter chaperone complex (VTC complex)]
The VTC complex is a vacuolar transporter chaperone complex present in the vacuolar membrane and was found in the yeast Saccharomyces cerevisiae. The VTC complex is a protein complex formed from a plurality of constituent factors of two or more, and each constituent factor is a membrane permeation protein having a relatively small molecular weight. For example, the Saccharomyces cerevisiae VTC complex is formed by four components, Vtc1p, Vtc2p, Vtc3p, and Vtc4p. For the formation of the VTC complex, it is considered that two or more genes encoding each component factor express the component factor, and the expressed component factors need to interact with each other. For example, in Saccharomyces cerevisiae, in order to form a VTC complex, it is considered that either Vtc2p or Vtc3p, Vtc1p, and Vtc4p must be expressed (Non-patent Document 1).
　ＶＴＣ複合体としては具体的には、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐの４つの構成因子により形成されるものが挙げられる。Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐタンパク質は、液胞トランスポーターシャペロン複合体の構成因子として機能する。本発明のＶＴＣ複合体における構成因子はそれぞれ、以下の（Ａ）～（Ｃ）に示されるタンパク質から選択されるタンパク質である：
（Ａ）配列番号１～４のいずれかに記載のアミノ酸配列からなるタンパク質；
（Ｂ）配列番号１～４のいずれかに記載のアミノ酸配列において、１又は数個以上のアミノ酸残基が欠失、置換、挿入及び／又は付加されたアミノ酸配列からなるタンパク質；又は、
（Ｃ）配列番号１～４のいずれかに記載のアミノ酸配列と８０％以上の相同性を有するアミノ酸配列からなるタンパク質。 Specific examples of the VTC complex include those formed by four components of Vtc1p, Vtc2p, Vtc3p, and Vtc4p. Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins function as components of the vacuolar transporter chaperone complex. Each of the components in the VTC complex of the present invention is a protein selected from the proteins shown in the following (A) to (C):
(A) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(B) a protein comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4; or
(C) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4.
　上記（Ａ）のタンパク質に関し、配列表に示される配列番号１～４は、サッカロミセス・セレビシエ由来のＶｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐタンパク質のそれぞれのアミノ酸配列を示す。Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐタンパク質は、ＶＴＣ複合体の構成因子として機能する。 Regarding the protein (A) above, SEQ ID NOs: 1 to 4 shown in the sequence listing represent the amino acid sequences of the Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins derived from Saccharomyces cerevisiae, respectively. Vtc1p, Vtc2p, Vtc3p, Vtc4p proteins function as components of the VTC complex.
　上記（Ｂ）又は（Ｃ）のタンパク質は、配列番号１～４のいずれかに示すアミノ酸配列を有するタンパク質に関して、機能的に同等の変異体、誘導体、バリアント、アレル、ホモログ、オルソログ、部分ペプチド、又は、他のタンパク質若しくはペプチドとの融合タンパク質であって、液胞トランスポーターシャペロン複合体の構成因子をコードする遺伝子を意図しており、その具体的なアミノ酸配列については限定されない。 The protein of the above (B) or (C) is a functionally equivalent mutant, derivative, variant, allele, homolog, ortholog, partial peptide, with respect to the protein having the amino acid sequence shown in any of SEQ ID NOs: 1 to 4. Or it is a fusion protein with other protein or peptide, Comprising: The gene which codes the component factor of a vacuolar transporter chaperone complex is intended, The concrete amino acid sequence is not limited.
　ここで、上記（Ｂ）における置換、欠失、挿入及び／又は付加されてもよいアミノ酸の数としては、上記機能を失わせない限り、その個数は制限されないが、部位特異的突然変異誘発法等の公知の変異導入法により置換、欠失、挿入及び／又は付加できる程度の数をいう。通常は、３０アミノ酸以内であり、好ましくは２０アミノ酸以内であり、さらに好ましくは１０アミノ酸以内であり、最も好ましくは５アミノ酸以内（例えば、５，４，３，２又は１アミノ酸以内）である。変異を導入したタンパク質が酵母に所望の形質を付与するかどうかは、そのタンパク質をコードする遺伝子を発現させ、ＶＴＣ複合体を構成し、ＶＴＣ複合体が機能するかどうかを調べることにより判断できる。また、ここでいう「変異」は、主には部位特異的突然変異誘発法等により人為的に導入された変異を意味するが、天然に存在する同様の変異であってもよい。そして、「変異」は、構成因子をコードする遺伝子の破壊を含む。 Here, the number of amino acids that may be substituted, deleted, inserted and / or added in (B) is not limited as long as the function is not lost, but site-directed mutagenesis The number which can be substituted, deleted, inserted and / or added by a known mutagenesis method. Usually, it is within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, and most preferably within 5 amino acids (for example, within 5, 4, 3, 2, or 1 amino acid). Whether or not a protein into which a mutation has been introduced imparts a desired character to yeast can be determined by expressing a gene encoding the protein, constructing a VTC complex, and examining whether the VTC complex functions. The “mutation” here means a mutation artificially introduced mainly by site-directed mutagenesis or the like, but may be a similar naturally occurring mutation. “Mutation” includes the destruction of a gene encoding a constituent factor.
　変異するアミノ酸残基においては、アミノ酸側鎖の性質が保存されている別のアミノ酸に変異されることが好ましい。例えばアミノ酸側鎖の性質としては、疎水性アミノ酸（Ａ、Ｉ、Ｌ、Ｍ、Ｆ、Ｐ、Ｗ、Ｙ及びＶ）、親水性アミノ酸（Ｒ、Ｄ、Ｎ、Ｃ、Ｅ、Ｑ、Ｇ、Ｈ、Ｋ、Ｓ及びＴ）、脂肪族側鎖を有するアミノ酸（Ｇ、Ａ、Ｖ、Ｌ、Ｉ及びＰ）、水酸基含有側鎖を有するアミノ酸（Ｓ、Ｔ及びＹ）、硫黄原子含有側鎖を有するアミノ酸（Ｃ及びＭ）、カルボン酸及びアミド含有側鎖を有するアミノ酸（Ｄ、Ｎ、Ｅ及びＱ）、塩基含有側鎖を有するアミノ酸（Ｒ、Ｋ及びＨ）、並びに芳香族含有側鎖を有するアミノ酸（Ｈ、Ｆ、Ｙ及びＷ）を挙げることができる。さらに、例えば、変異マトリクス（mutational matrix）によってアミノ酸を分類することも周知である（Taylor 1986, J, Theor. Biol. 119, 205-218; Sambrook, J. et al., Molecular Cloning 3rd ed. A7.6-A7.9, Cold Spring Harbor Lab. Press, 2001）。この分類を以下に要約すると、脂肪族アミノ酸（Ｌ、Ｉ及びＶ）、芳香族アミノ酸（Ｈ、Ｗ、Ｙ及びＦ）、荷電アミノ酸（Ｄ、Ｅ、Ｒ、Ｋ及びＨ）、正荷電アミノ酸（Ｒ、Ｋ及びＨ）、負荷電アミノ酸（Ｄ及びＥ）、疎水性アミノ酸（Ｈ、Ｗ、Ｙ、Ｆ、Ｍ、Ｌ、Ｉ、Ｖ、Ｃ、Ａ、Ｇ、Ｔ及びＫ）、極性アミノ酸（Ｔ、Ｓ、Ｎ、Ｄ、Ｅ、Ｑ、Ｒ、Ｋ、Ｈ、Ｗ及びＹ）、小型アミノ酸（Ｐ、Ｖ、Ｃ、Ａ、Ｇ、Ｔ、Ｓ、Ｎ及びＤ）、並びに微小アミノ酸（Ａ、Ｇ及びＳ）及び大型（非小型）アミノ酸（Ｑ、Ｅ、Ｒ、Ｋ、Ｈ、Ｗ、Ｙ、Ｆ、Ｍ、Ｌ及びＩ）が挙げられる。なお、上記括弧内はいずれもアミノ酸の一文字標記を表す。 The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids having aliphatic side chains (G, A, V, L, I and P), amino acids having hydroxyl group-containing side chains (S, T and Y), sulfur atom-containing side chains Amino acids (C and M) having carboxylic acids and amide-containing side chains (D, N, E and Q), amino acids having base-containing side chains (R, K and H), and aromatic-containing side chains And amino acids (H, F, Y and W) having Furthermore, it is also well known to classify amino acids by, for example, mutational matrix (Taylor 1986, J, Theor. Biol. 119, 205-218; Sambrook, J. et al., Molecular Cloning 3rd ed. A7 .6-A7.9, Cold Spring Harbor Lab. Press, 2001). This classification is summarized below: aliphatic amino acids (L, I and V), aromatic amino acids (H, W, Y and F), charged amino acids (D, E, R, K and H), positively charged amino acids ( R, K and H), negatively charged amino acids (D and E), hydrophobic amino acids (H, W, Y, F, M, L, I, V, C, A, G, T and K), polar amino acids ( T, S, N, D, E, Q, R, K, H, W and Y), small amino acids (P, V, C, A, G, T, S, N and D), and minute amino acids (A , G and S) and large (non-small) amino acids (Q, E, R, K, H, W, Y, F, M, L and I). In addition, all the inside of the said parenthesis represents the single letter mark of an amino acid.
　あるアミノ酸配列に対する１又は複数個のアミノ酸残基の欠失、挿入、付加及び／又は他のアミノ酸による置換により修飾されたアミノ酸配列を有するポリペプチドがその生物学的活性を維持することはすでに知られている。さらに、標的アミノ酸残基は、共通した性質をできるだけ多く有するアミノ酸残基に変異させることがより好ましい。 It is already known that a polypeptide having an amino acid sequence modified by deletion, insertion, addition and / or substitution by another amino acid of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. It has been. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.
　本明細書において「機能的に同等」とは、対象となるタンパク質が、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、又はＶｔｃ４ｐタンパク質と同等（同一及び／又は類似）の生物学的機能及び／又は生化学的機能を有することを意図する。本明細書において、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、又はＶｔｃ４ｐタンパク質の生物学的機能及び／又は生化学的機能としては、例えばＶＴＣ複合体を構成し、ＶＴＣ複合体を機能させることを挙げることができる。生物学的な性質には発現する部位の特異性及び／又は発現量等も含まれ得る。 As used herein, “functionally equivalent” means that the target protein has a biological function and / or biochemical function equivalent to (same and / or similar to) the Vtc1p, Vtc2p, Vtc3p, or Vtc4p protein. Intended to have. In the present specification, examples of the biological function and / or biochemical function of the Vtc1p, Vtc2p, Vtc3p, or Vtc4p protein include, for example, constituting a VTC complex and allowing the VTC complex to function. Biological properties may include the specificity of the site to be expressed and / or the expression level.
　上記（Ｃ）におけるアミノ酸配列の相同性は、アミノ酸配列全体（若しくは機能発現に必要な領域）で、少なくとも８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは、９５％、９６％、９７％、９８％、９９％又は９９．５％以上の配列の同一性を有する。配列の相同性は、ＢＬＡＳＴＮ（核酸レベル）又はＢＬＡＳＴＸ（アミノ酸レベル）のプログラム（Altschul et al. J. Mol. Biol., 215: 403-410, 1990）を利用して決定することができる。該プログラムは、Karlin及びAltschulによるアルゴリズムＢＬＡＳＴ（Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993）に基づいている。ＢＬＡＳＴＮによって塩基配列を解析する場合には、パラメーターは例えばscore = 100、wordlength =12とする。また、ＢＬＡＳＴＸによってアミノ酸配列を解析する場合には、パラメーターは例えばscore = 50、wordlength = 3とする。また、Ｇａｐｐｅｄ ＢＬＡＳＴプログラムを用いて、アミノ酸配列を解析する場合は、Altschulら（Nucleic Acids Res. 25: 3389-3402, 1997）に記載されているように行うことができる。ＢＬＡＳＴとＧａｐｐｅｄ ＢＬＡＳＴプログラムを用いる場合には、各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である。比較対象の塩基配列又はアミノ酸配列を最適な状態にアラインメントするために、付加又は欠失（例えば、ギャップ等）を許容してもよい。 The homology of the amino acid sequence in (C) above is at least 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% in the entire amino acid sequence (or a region necessary for functional expression). 96%, 97%, 98%, 99% or 99.5% or more of sequence identity. Sequence homology can be determined using the BLASTN (nucleic acid level) or BLASTX (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc.ANatl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993) by Karlin and Altschul. Yes. When the base sequence is analyzed by BLASTN, the parameters are set, for example, score = 、 100 and wordlength = 12. When the amino acid sequence is analyzed by BLASTX, the parameters are set, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. In order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed.
　また、本明細書において「相同性」とは、性質が類似のアミノ酸残基数の割合（homology、positive等）を意図しているが、より好ましくは、一致したアミノ酸残基数の割合（identity）である。なお、アミノ酸の性質については上述したとおりである。 In the present specification, the term “homology” intends the ratio of the number of amino acid residues having similar properties (homology, positive, etc.), but more preferably the ratio of the number of matched amino acid residues (identity) ). The properties of amino acids are as described above.
　ＶＴＣ複合体の構成因子をコードする遺伝子は、Ｖｔｃ１ｐ、Ｖｔｃ２ｐ、Ｖｔｃ３ｐ、Ｖｔｃ４ｐタンパク質のいずれかをコードする遺伝子であればいかなるものであってもよい。より具体的には、本発明のＶＴＣ複合体の構成因子をコードする遺伝子は、それぞれ、以下の（ａ）～（ｅ）に示されるポリヌクレオチドから選択される遺伝子である：
（ａ）配列番号１～４のいずれかに記載のアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｂ）配列番号１～４のいずれかに記載のアミノ酸配列において１又は数個以上のアミノ酸残基が欠失、置換、挿入及び／又は付加されたアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｃ）配列番号１～４のいずれかに記載のアミノ酸配列と８０％以上の相同性を有するアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
（ｄ）配列番号５～８のいずれかに記載の塩基配列からなるポリヌクレオチドを含む遺伝子；
（ｅ）上記（ａ）～（ｄ）からなる群から選択されるいずれかのポリヌクレオチドと相補的な塩基配列からなるポリヌクレオチドを含む遺伝子。 The gene encoding the constituent factor of the VTC complex may be any gene as long as it encodes any one of the Vtc1p, Vtc2p, Vtc3p, and Vtc4p proteins. More specifically, the genes encoding the constituent elements of the VTC complex of the present invention are genes selected from the polynucleotides shown in the following (a) to (e):
(A) a gene comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(B) a gene comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(C) a gene comprising a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
(D) a gene comprising a polynucleotide comprising the base sequence set forth in any one of SEQ ID NOs: 5 to 8;
(E) A gene comprising a polynucleotide comprising a base sequence complementary to any polynucleotide selected from the group consisting of (a) to (d) above.
　上記（ａ）～（ｃ）の遺伝子は、上記（Ａ）～（Ｃ）のタンパク質をコードする遺伝子である。 The genes (a) to (c) are genes encoding the proteins (A) to (C).
　上記（ｄ）の遺伝子に関し、配列表に示される配列番号５は、サッカロミセス・セレビシエ由来のＶｔｃ１ｐ（配列番号１に示すアミノ酸配列を有するタンパク質）をコードするＶＴＣ１遺伝子の塩基配列（ＯＲＦ）を、配列番号６は、サッカロミセス・セレビシエ由来のＶｔｃ２ｐ（配列番号２に示すアミノ酸配列を有するタンパク質）をコードするＶＴＣ２遺伝子の塩基配列（ＯＲＦ）を、配列番号７は、サッカロミセス・セレビシエ由来のＶｔｃ３ｐ（配列番号３に示すアミノ酸配列を有するタンパク質）をコードするＶＴＣ３遺伝子の塩基配列（ＯＲＦ）を、配列番号８は、サッカロミセス・セレビシエ由来のＶｔｃ４ｐ（配列番号４に示すアミノ酸配列を有するタンパク質）をコードするＶＴＣ４遺伝子の塩基配列（ＯＲＦ）をそれぞれ示す。なお、ＯＲＦとは、オープンリーディングフレームを意味する。後述する実施例に示すように、本発明においては、ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子及びＶＴＣ４遺伝子からなる群から選ばれる少なくとも１つの遺伝子を破壊、又は該遺伝子の発現量を低下させると、酵母の発酵能が向上すること及びエタノール生産性が向上することが見出された。 Regarding the gene of (d) above, SEQ ID NO: 5 shown in the sequence listing is the nucleotide sequence (ORF) of the VTC1 gene encoding Vtc1p (protein having the amino acid sequence shown in SEQ ID NO: 1) derived from Saccharomyces cerevisiae, No. 6 is the base sequence (ORF) of the VTC2 gene encoding Vtc2p derived from Saccharomyces cerevisiae (protein having the amino acid sequence shown in SEQ ID NO: 2), and SEQ ID NO: 7 is Vtc3p derived from Saccharomyces cerevisiae (SEQ ID NO: 3 The nucleotide sequence (ORF) of the VTC3 gene that encodes the protein having the amino acid sequence shown in SEQ ID NO: 8, and SEQ ID NO: 8 is the VTC4 gene encoding the Vtc4p (protein having the amino acid sequence shown in SEQ ID NO: 4) derived from Saccharomyces cerevisiae Base sequence (ORF) Show Re respectively. The ORF means an open reading frame. As shown in the examples described later, in the present invention, when at least one gene selected from the group consisting of VTC1 gene, VTC2 gene, VTC3 gene and VTC4 gene is destroyed or the expression level of the gene is reduced, It has been found that the fermentability of ethanol is improved and ethanol productivity is improved.
　ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子及びＶＴＣ４遺伝子は酵母全般にわたって広く存在すると考えられる。つまり、本発明に係る遺伝子には、種々の酵母に存在する、ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子及びＶＴＣ４遺伝子の相同遺伝子も含まれる。ここで、相同遺伝子を単離するための当業者によく知られた方法としては、ハイブリダイゼーション技術（Southern, E. M., Journal of Molecular Biology, Vol. 98, 503, 1975）及びポリメラーゼ連鎖反応（ＰＣＲ）技術（Saiki, R. K., et al. Science, vol. 230, 1350-1354, 1985, Saiki, R. K. et al. Science, vol.239, 487-491,1988）が挙げられる。すなわち、当業者にとっては、ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子及びＶＴＣ４遺伝子の塩基配列（例えば、配列番号５～８に記載の塩基配列）若しくはその一部をプローブとして、またＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子若しくはＶＴＣ４遺伝子と特異的にハイブリダイズするオリゴヌクレオチドをプライマーとして、種々の酵母からＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子及びＶＴＣ４遺伝子の相同遺伝子を単離することは通常行い得る。 VTC1 gene, VTC2 gene, VTC3 gene and VTC4 gene are considered to exist widely throughout yeast. That is, the genes according to the present invention include homologous genes of VTC1, VTC2, VTC3, and VTC4 genes that are present in various yeasts. Here, methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, E. M., Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction. (PCR) technology (Saiki, R. K., et al. Science, vol. 230, 1350-1354, 1985, Saiki, R. K. et al. Science, vol.239, 487-491, 1988) It is done. That is, for those skilled in the art, the VTC1 gene, VTC2 gene, VTC3 gene and VTC4 gene nucleotide sequences (for example, the nucleotide sequences described in SEQ ID NOs: 5 to 8) or a part thereof are used as probes, and the VTC1 gene, VTC2 gene, Isolation of VTC1 gene, VTC2 gene, VTC3 gene, and homologous gene of VTC4 gene from various yeasts using oligonucleotides that specifically hybridize with VTC3 gene or VTC4 gene as primers can be generally performed.
　上記（ｅ）の遺伝子は、上記（ａ）～（ｄ）のいずれかのポリヌクレオチドと相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするタンパク質をコードする遺伝子を意図している。 The gene of (e) is intended to encode a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide of any of (a) to (d) above. ing.
　ここで、ストリンジェントな条件とは、いわゆる塩基配列に特異的な２本鎖のポリヌクレオチドが形成され、非特異的な２本鎖のポリヌクレオチドが形成されない条件をいう。換言すれば、相同性が高い核酸同士、例えば完全にマッチしたハイブリッドの融解温度（Ｔｍ値）から１５℃、好ましくは１０℃、更に好ましくは５℃低い温度までの範囲の温度でハイブリダイズする条件ともいえる。例えば、一般的なハイブリダイゼーション用緩衝液中で、６８℃、２０時間の条件でハイブリダイズする条件を挙げることができる。一例を示すと、０．２５Ｍ Ｎａ２ＨＰＯ４，ｐＨ７．２，７％ＳＤＳ，１ｍＭ ＥＤＴＡ，１×デンハルト溶液からなる緩衝液中で温度が６０～６８℃、好ましくは６５℃、さらに好ましくは６８℃の条件下で１６～２４時間ハイブリダイズさせ、さらに２０ｍＭ Ｎａ２ＨＰＯ４，ｐＨ７．２，１％ＳＤＳ，１ｍＭ ＥＤＴＡからなる緩衝液中で温度が６０～６８℃、好ましくは６５℃、さらに好ましくは６８℃の条件下で１５分間の洗浄を２回行う条件を挙げることができる。他の例としては、２５％ホルムアミド、より厳しい条件では５０％ホルムアミド、４×ＳＳＣ（塩化ナトリウム／クエン酸ナトリウム）、５０ｍＭＨｅｐｅｓ ｐＨ７．０、１０×デンハルト溶液、２０μｇ／ｍｌ変性サケ***ＤＮＡを含むハイブリダイゼーション溶液中、４２℃で一晩プレハイブリダイゼーションを行った後、標識したプローブを添加し、４２℃で一晩保温することによりハイブリダイゼーションを行う。その後の洗浄における洗浄液及び温度条件は、「１×ＳＳＣ、０．１％ＳＤＳ、３７℃」程度で、より厳しい条件としては「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」程度で、さらに厳しい条件としては「０．２×ＳＳＣ、０．１％ＳＤＳ、６５℃」程度で実施することができる。このようにハイブリダイゼーションの洗浄の条件が厳しくなるほどプローブ配列と高い相同性を有するＤＮＡの単離を期待し得る。ただし、上記ＳＳＣ、ＳＤＳ及び温度の条件の組み合わせは例示であり、当業者であれば、ハイブリダイゼーションのストリンジェンシーを決定する上記若しくは他の要素（例えば、プローブ濃度、プローブの長さ及びハイブリダイゼーション反応時間など）を適宜組み合わせることにより、上記と同様のストリンジェンシーを実現することが可能である。例えば、当業者であれば、Molecular Cloning（Sambrook, J. et al., Molecular Cloning :a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)）等を参照することにより、こうした遺伝子を容易に取得することができる。 Here, stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions for hybridizing at a temperature ranging from the melting temperature (Tm value) of highly homologous nucleic acids, for example, perfectly matched hybrids, to 15 ° C, preferably 10 ° C, more preferably 5 ° C lower. It can be said. For example, the hybridization can be carried out in a general hybridization buffer at 68 ° C. for 20 hours. As an example, the temperature is 60-68 ° C., preferably 65 ° C., more preferably 68 in a buffer solution consisting of 0.25M Na 2 HPO 4 , pH 7.2, 7% SDS, 1 mM EDTA, 1 × Denhardt's solution. At 60 ° C. for 16 to 24 hours, and further in a buffer solution comprising 20 mM Na 2 HPO 4 , pH 7.2, 1% SDS, 1 mM EDTA, the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably Can include a condition in which washing is performed twice at 68 ° C. for 15 minutes. Other examples include 25% formamide, 50% formamide under more severe conditions, 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA. After prehybridization is performed overnight in a hybridization solution at 42 ° C., a labeled probe is added, and hybridization is performed by incubating overnight at 42 ° C. In the subsequent cleaning, the cleaning solution and temperature conditions are about “1 × SSC, 0.1% SDS, 37 ° C.”, and more severe conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”. Further, as stricter conditions, it can be carried out at about “0.2 × SSC, 0.1% SDS, 65 ° C.”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the conditions for hybridization washing become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, and hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. For example, those skilled in the art can refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, such a gene can be easily obtained.
　また、上記（ｅ）の遺伝子は、上記（ｄ）遺伝子（配列番号５～８に記載の塩基配列）と相同性において少なくとも８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは、９５％、９６％、９７％、９８％又は９９％以上の配列の同一性を有することが好ましい。なお、配列番号５～８に記載の塩基配列との相同性は、ＦＡＳＴＡ検索又はＢＬＡＳＴ検索により決定することができる。ポリヌクレオチドの塩基配列は、Science, 214: 1205 (1981)に記載されたジデオキシ法により決定され得る。 Further, the gene of (e) is at least 80% or more, preferably 85% or more, more preferably 90% or more, in homology with the gene (d) (base sequences described in SEQ ID NOs: 5 to 8). Preferably, it has 95%, 96%, 97%, 98% or 99% or more sequence identity. The homology with the base sequences described in SEQ ID NOs: 5 to 8 can be determined by FASTA search or BLAST search. The nucleotide sequence of the polynucleotide can be determined by the dideoxy method described in Science, 214: 1205 (1981).
　また、本発明において利用する遺伝子は、上記タンパク質をコードするポリヌクレオチドのみからなるものであってもよいが、その他の塩基配列が付加されていてもよい。付加される塩基配列としては、特に限定されないが、標識（例えば、ヒスチジンタグ、Ｍｙｃタグ又はＦＬＡＧタグなど）、融合タンパク質（例えば、ストレプトアビジン、シトクロム、ＧＳＴ、ＧＦＰ又はＭＢＰなど）、プロモーター配列、及びシグナル配列（例えば、小胞体移行シグナル配列、及び分泌配列など）をコードする塩基配列などが挙げられる。これらの塩基配列が付加される部位は特に限定されるものではなく、例えば、翻訳されるタンパク質のＮ末端であっても、Ｃ末端であってもよい。 In addition, the gene used in the present invention may be composed only of a polynucleotide encoding the above protein, but other base sequences may be added. The base sequence to be added is not particularly limited, but includes a label (for example, histidine tag, Myc tag or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP), a promoter sequence, and Examples thereof include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum transition signal sequence, a secretory sequence, etc.). The site to which these base sequences are added is not particularly limited, and may be, for example, the N-terminus or C-terminus of the translated protein.
［２．酵母の発酵能を向上させる方法］
　本発明において、ＶＴＣ複合体の機能の低下又は喪失させることにより、酵母の発酵能を向上させる方法、すなわち酵母による発酵を促進させる方法を包含する。ＶＴＣ複合体の機能の低下又は喪失とは、ＶＴＣ複合体全体としての機能が低下又は喪失していること、及び、ＶＴＣ複合体のいずれか１つの構成因子の機能が低下又は喪失していることのいずれであってもよい。また、ＶＴＣ複合体の機能の低下又は喪失は、複数の構成因子から形成されるＶＴＣ複合体の形成阻害やＶＴＣ複合体の機能を阻害する因子によって引き起こされるものも含まれる。酵母のＶＴＣ複合体の機能の低下又は喪失の結果、例えば、発酵の促進が引き起こされる。発酵とは、酵母などの微生物がエネルギーを得るために有機化合物を分解して、アルコール類・有機酸類・二酸化炭素などを生成する過程である。本発明における発酵は、好ましくはアルコール発酵であり、より好ましくはエタノール発酵である。エタノール発酵は、酵母が利用可能なブトウ糖、果糖、ショ糖などの糖類に酵母を作用させて、エタノールと二酸化炭素を生成させる反応である。 [2. Method for improving yeast fermentability]
In this invention, the method of improving the fermentation ability of yeast, ie, the method of accelerating | stimulating fermentation by yeast, is included by making the function of a VTC complex fall or lose | eliminate. Reduced or lost function of the VTC complex means that the function of the VTC complex as a whole is decreased or lost, and that the function of any one component of the VTC complex is decreased or lost. Any of these may be used. In addition, the decrease or loss of the function of the VTC complex includes those caused by the inhibition of the formation of the VTC complex formed from a plurality of constitutive factors and the factor that inhibits the function of the VTC complex. Reduction or loss of function of the yeast VTC complex results in, for example, accelerated fermentation. Fermentation is a process in which microorganisms such as yeast decompose organic compounds to obtain energy to produce alcohols, organic acids, carbon dioxide, and the like. The fermentation in the present invention is preferably alcoholic fermentation, more preferably ethanolic fermentation. Ethanol fermentation is a reaction in which yeast acts on sugars such as butter sugar, fructose, and sucrose that can be used by yeast to produce ethanol and carbon dioxide.
　ＶＴＣ複合体の機能、又はＶＴＣ複合体のいずれか１つの構成因子の機能としては、従来公知の機能であってもよいし、今後見出される新たな機能であってもよい。例えば、液胞型プロトンＡＴＰａｓｅの安定性の維持や、タンパク質の輸送、ミクロオートファジー、液胞内へのポリリン酸の蓄積等が例示される（非特許文献１、２、３、４）。これらの機能の確認は、非特許文献１、２、３、４に開示された方法に基づき、確認することができる。さらに、本願で明らかにされた酵母のＶＴＣ複合体によるアルコール発酵調節機能及び、今後見出されるＶＴＣ複合体が本来有している機能の低下又は喪失を含む。 The function of the VTC complex or the function of any one of the constituent elements of the VTC complex may be a conventionally known function or a new function to be found in the future. Examples include maintenance of stability of vacuolar proton ATPase, protein transport, microautophagy, and accumulation of polyphosphoric acid in the vacuole ( Non-patent Documents 1, 2, 3, 4). Confirmation of these functions can be confirmed based on the methods disclosed in Non-Patent Documents 1, 2, 3, and 4. Furthermore, it includes the alcohol fermentation regulation function by the yeast VTC complex revealed in the present application and the reduction or loss of the function inherent to the VTC complex to be found in the future.
　酵母のＶＴＣ複合体の機能の低下又は喪失は、構成因子のいずれか１つをコードする遺伝子の発現抑制する手段によることが好ましい。当該遺伝子の発現の抑制は、当該遺伝子がコードする構成因子を発現する過程を抑制することを意味し、当該遺伝子の転写の抑制、当該遺伝子の翻訳の抑制等を含む。当該遺伝子の発現抑制は、何らかの薬剤等を酵母に接触させて行っても良いし、酵母において遺伝子変異を導入することにより行ってもよい。変異の導入は、遺伝子改変技術を用いて人工的に行われたものであってもよいし、天然に生じたものであってもよい。遺伝子の変異の導入は、酵母のＶＴＣ複合体の機能の低下又は喪失をもたらすものであればいかなるものであってもよい。 The reduction or loss of the function of the yeast VTC complex is preferably by means of suppressing the expression of a gene encoding any one of the constituent factors. Suppression of the expression of the gene means suppression of the process of expressing a constituent factor encoded by the gene, and includes suppression of transcription of the gene, suppression of translation of the gene, and the like. Suppression of the expression of the gene may be performed by bringing some drug or the like into contact with the yeast, or by introducing a gene mutation in the yeast. The introduction of the mutation may be artificially performed using a gene modification technique, or may be naturally occurring. The gene mutation may be introduced as long as it causes a decrease or loss of the function of the yeast VTC complex.
　遺伝子変異は、酵母のＶＴＣ複合体を形成する構成因子をコードする遺伝子から選択される遺伝子に導入されることが好ましい。酵母のＶＴＣ複合体を形成する構成因子をコードする遺伝子に変異を導入することにより、ＶＴＣ複合体を形成する構成因子をコードする遺伝子の発現量の低下させることができる。当該遺伝子の発現量を低下させる遺伝子変異としては、当該遺伝子を破壊することが例示される。ＶＴＣ複合体を形成する構成因子のいずれの遺伝子を破壊しても、ＶＴＣ複合体の機能の低下又は喪失をもたらすと考えられるため、遺伝子変異を導入する遺伝子は限定されるものではないが、ＶＴＣ１、ＶＴＣ３、ＶＴＣ４遺伝子のいずれか少なくとも１つの遺伝子を破壊することが好ましく、ＶＴＣ１遺伝子を破壊することがより好ましい。 It is preferable that the gene mutation is introduced into a gene selected from genes encoding constituent factors forming the yeast VTC complex. By introducing a mutation into a gene encoding a component that forms a VTC complex in yeast, the expression level of the gene encoding the component that forms a VTC complex can be reduced. Examples of the gene mutation that reduces the expression level of the gene include destroying the gene. Since any of the genes of the constituent factors forming the VTC complex is considered to decrease or lose the function of the VTC complex, the gene into which the gene mutation is introduced is not limited, but VTC1 It is preferable to disrupt at least one of the VTC3 and VTC4 genes, and it is more preferable to disrupt the VTC1 gene.
「ＶＴＣ複合体の構成因子をコードする遺伝子を破壊する」とは、酵母ゲノム上のＶＴＣ複合体の構成因子であるタンパク質をコードする遺伝子のコード領域を欠失又は変異させることをいう。ＶＴＣ複合体の構成因子をコードする遺伝子の欠失は、コード領域の全体を欠失させてもよく、また一部を欠失させてもよい。全体を欠失させる場合には、ＶＴＣ複合体の構成因子をコードする遺伝子に隣接する領域も合わせて広く欠失させてもよい。一部を欠失させる場合には、特に限定されないが、ＶＴＣ複合体の構成因子をコードする遺伝子のコード領域の半分以上を欠失させることが好ましい。コード領域の変異によりＶＴＣ複合体の構成因子をコードする遺伝子を破壊する場合には、該コード領域の好ましくは中央よりも上流の部位にナンセンス変異又はフレームシフト変異を導入して、それよりも下流の領域によりコードされるアミノ酸配列を欠失させ又は全く無関係なアミノ酸配列とすることが好ましい。あるいは、ＶＴＣ複合体の構成因子をコードする遺伝子中に無関係な配列（ＶＴＣ複合体の構成因子とは無関係な他の遺伝子配列やマーカー遺伝子等）を挿入することによりＶＴＣ複合体の構成因子をコードする遺伝子を変異させ破壊することもできる。ＶＴＣ複合体の構成因子をコードする遺伝子の破壊により、該遺伝子によりコードされるＶＴＣ複合体の機能が低下又は喪失する。 “Destroy a gene encoding a constituent element of a VTC complex” means to delete or mutate a coding region of a gene encoding a protein that is a constituent element of a VTC complex on the yeast genome. Deletion of a gene encoding a constituent factor of the VTC complex may delete the entire coding region or a part thereof. When the entire region is deleted, the region adjacent to the gene encoding the constituent element of the VTC complex may also be deleted widely. When a part is deleted, it is not particularly limited, but it is preferable to delete at least half of the coding region of the gene encoding the constituent element of the VTC complex. When a gene encoding a component of the VTC complex is disrupted by a mutation in the coding region, a nonsense mutation or a frameshift mutation is preferably introduced at a site upstream of the coding region, and further downstream than that. It is preferable to delete the amino acid sequence encoded by this region or to have an irrelevant amino acid sequence. Alternatively, a VTC complex constituent factor is encoded by inserting an irrelevant sequence (other gene sequence or marker gene unrelated to the VTC complex constituent factor) into the gene encoding the VTC complex constituent factor. It can also be mutated and destroyed. Disruption of a gene encoding a component of the VTC complex reduces or loses the function of the VTC complex encoded by the gene.
　以下、破壊する対象のＶＴＣ複合体の構成因子をコードする遺伝子としてＶＴＣ１遺伝子を例示して、遺伝子の破壊を具体的に説明する。ＶＴＣ１遺伝子を破壊する方法としては、例えば、相同組換え法やゲノム編集技術等が挙げられる。産業上実用されている実用酵母においても、ゲノム編集技術であるCRISPR-Casシステムにより遺伝子改変を行った例が報告されている（J. Microbiol. Methods Vol.127 p.203-205 (2016)）。ゲノム編集技術によれば、高次倍数体であっても一度に遺伝子の改変を行うことができ、有利である。また相同組換え方法は、正常なＶＴＣ１遺伝子を含まない相同ＤＮＡ断片を酵母細胞中に導入し、この相同ＤＮＡ断片と、酵母ゲノムＤＮＡとの間で相同組換えを行わせる方法である。ここで、「相同ＤＮＡ断片」とは、相同組換えによりゲノム中の標的領域と組換えられ得るＤＮＡ断片のことを言う。相同組換え法自体は周知であり、当業者であれば上記ゲノム又はコード領域の塩基配列をもとにして、所望の相同ＤＮＡ断片を調製することができる。上記各相同領域の鎖長は特に限定されず、一般に鎖長が長い方が相同組換えの効率が高まるが、出芽酵母では相同組換え活性が強いため、５０ｂｐ程度の相同領域を設ければよい。 Hereinafter, the VTC1 gene will be exemplified as a gene encoding a component of the VTC complex to be destroyed, and the gene destruction will be specifically described. Examples of the method for destroying the VTC1 gene include a homologous recombination method and a genome editing technique. An example of genetic modification using the CRISPR-Cas system, which is a genome editing technology, has also been reported in practical yeasts that are used in industry (J.JMicrobiol. Methods Vol.127 p.203-205 (2016)). . According to the genome editing technique, even a higher polyploid can be modified at once, which is advantageous. The homologous recombination method is a method in which a homologous DNA fragment not containing a normal VTC1 gene is introduced into yeast cells, and homologous recombination is performed between the homologous DNA fragment and yeast genomic DNA. Here, the “homologous DNA fragment” refers to a DNA fragment that can be recombined with a target region in the genome by homologous recombination. The homologous recombination method itself is well known, and those skilled in the art can prepare a desired homologous DNA fragment based on the base sequence of the genome or coding region. The chain length of each homologous region is not particularly limited. Generally, the longer the chain length, the higher the efficiency of homologous recombination. However, since homologous recombination activity is strong in budding yeast, a homologous region of about 50 bp may be provided. .
　例えば、正常なＶＴＣ１をコードしない変異ＶＴＣ１遺伝子配列の上流及び下流に、ゲノム上のＶＴＣ１遺伝子の上流領域と相同な領域及び下流領域と相同な領域をそれぞれ連結して相同ＤＮＡ断片を構築し、これを用いて相同組換えを行えば、ゲノム上のＶＴＣ１遺伝子配列を変異ＶＴＣ１遺伝子配列と入れ替えることができるので、酵母ゲノム上のＶＴＣ１遺伝子を破壊することができる。 For example, a homologous DNA fragment is constructed by linking a region homologous to the upstream region of the VTC1 gene on the genome and a region homologous to the downstream region upstream and downstream of the mutant VTC1 gene sequence that does not encode normal VTC1, When the homologous recombination is performed using the VTC1, the VTC1 gene sequence on the genome can be replaced with the mutant VTC1 gene sequence, so that the VTC1 gene on the yeast genome can be destroyed.
　上記コード領域のさらに上流及び下流の領域のゲノム塩基配列は、Saccharomyces genome database（http://www.yeastgenome.org/）より取得することができる。 The genomic base sequences of the upstream and downstream regions of the coding region can be obtained from the Saccharomyces genome database (http://www.yeastgenome.org/).
　ＶＴＣ１遺伝子を破壊する他の方法としては、例えばAritomiらのBiosci. Biotechnol. Biochem., 68(1), 206-214, 2004に記載されるセルフクローニング法を利用することができる。このクローニング方法は、遺伝子導入用の薬剤耐性マーカーとカウンターセレクション用の生育抑制マーカーとを含むプラスミドベクターを利用して、ゲノム中の正常遺伝子を変異遺伝子に置き換える方法である。該方法によれば、遺伝子導入のためだけに必要な外来ＤＮＡ配列を変異遺伝子の導入後に除去できるため、食品産業で用いられる醸造酵母の育成に好ましい。具体的には、ＶＴＣ１遺伝子を破壊する方法は、以下のようにして行なうことができる。 As another method for disrupting the VTC1 gene, for example, the self-cloning method described in Aritomi et al., Biosci. Biotechnol. Biochem., 68 (1), 206-214, 2004 can be used. This cloning method is a method of replacing a normal gene in a genome with a mutant gene using a plasmid vector containing a drug resistance marker for gene introduction and a growth suppression marker for counter selection. According to this method, a foreign DNA sequence required only for gene introduction can be removed after introduction of the mutated gene, which is preferable for growing brewing yeast used in the food industry. Specifically, the method for disrupting the VTC1 gene can be performed as follows.
　用いる上記２種類のマーカーは特に限定されず、例えば、薬剤耐性マーカーとしてＹＡＰ１等、生育抑制マーカーとしてＧＩＮ１１等の公知のマーカーを用いることができる。これら２種類のマーカーを含むプラスミドベクター中に変異ＶＴＣ１遺伝子（例えば、配列番号１に示す塩基配列のうち第４～６番目の塩基の「TCT」を「TAA」に変異させることによりストップコドンを導入した変異遺伝子）を挿入し、ＶＴＣ１遺伝子中の適当な制限酵素部位（例えばＨｉｎｄＩＩＩサイト）で切断してリニア化したものを酵母細胞中に導入すれば、相同組換えにより当該リニア化プラスミドがゲノム中のＶＴＣ１遺伝子座に組み込まれる。その結果、ゲノム中にはプラスミド配列を介して正常ＶＴＣ１遺伝子と変異ＶＴＣ１遺伝子が縦列して存在するようになる。薬剤耐性マーカーにより、リニア化プラスミドがゲノム中に組み込まれた酵母を容易に選択することができる。 The two types of markers used are not particularly limited, and for example, a known marker such as YAP1 as a drug resistance marker and GIN11 as a growth suppression marker can be used. In the plasmid vector containing these two types of markers, a mutated VTC1 gene (for example, a stop codon is introduced by mutating “TCT” of the fourth to sixth bases of the base sequence shown in SEQ ID NO: 1 to “TAA”) Inserted into a yeast cell and then linearized by cutting at an appropriate restriction enzyme site (for example, HindIII site) in the VTC1 gene, the linearized plasmid is introduced into the genome by homologous recombination. At the VTC1 locus. As a result, the normal VTC1 gene and the mutant VTC1 gene are present in tandem in the genome via the plasmid sequence. A drug resistance marker allows easy selection of yeasts that have linearized plasmids integrated into their genome.
　その後、ゲノム中で縦列して存在する正常ＶＴＣ１遺伝子と変異ＶＴＣ１遺伝子との間で相同組換えが生じると、生育抑制マーカーを含むプラスミド配列が脱落する。そのため、生育抑制マーカーを発現させる条件下で（例えば、生育抑制マーカーがガラクトース誘導性過剰発現プロモーターの制御下にある場合には、ガラクトース培地上で）選択を行なえば、プラスミド配列が残存する酵母は生育抑制マーカーの作用により生育することができず、一方、相同組換えによりプラスミド配列が脱落した酵母は生育することができるので、プラスミド配列が脱落し正常ＶＴＣ１遺伝子又は変異ＶＴＣ１遺伝子のみがゲノム中に残存する酵母を得ることができる。ゲノム中に残存したＶＴＣ１遺伝子が所期の変異を有するか否かは、例えば変異を含む領域をＰＣＲで増幅してシークエンス解析を行なうことにより調べることができる。 Thereafter, when homologous recombination occurs between the normal VTC1 gene and the mutated VTC1 gene existing in tandem in the genome, the plasmid sequence containing the growth suppression marker is lost. Therefore, if selection is performed under conditions for expressing a growth suppression marker (for example, on a galactose medium when the growth suppression marker is under the control of a galactose-inducible overexpression promoter), the yeast in which the plasmid sequence remains is On the other hand, yeasts that cannot grow due to the action of the growth-inhibiting marker but can lose their plasmid sequence due to homologous recombination can grow, so that the plasmid sequence is lost and only the normal VTC1 gene or mutant VTC1 gene is present in the genome. The remaining yeast can be obtained. Whether or not the VTC1 gene remaining in the genome has a desired mutation can be examined by, for example, amplifying a region containing the mutation by PCR and performing a sequence analysis.
　また、ＶＴＣ１遺伝子破壊株は、変異処理を行なった酵母の中から選択して得ることもできる。すなわち、本発明の方法においては、変異処理によりＶＴＣ１遺伝子を破壊してもよい。変異処理の方法は特に限定されず、紫外線照射、放射線照射等の物理的変異処理、及びエチルメタンスルフォン酸等の変異剤で処理する化学的変異処理のいずれであってもよい。変異処理により得られた変異株の中から、シークエンス解析によりＶＴＣ１遺伝子破壊株を選択することができる。 Also, the VTC1 gene disruption strain can be obtained by selecting from among the yeasts that have been subjected to mutation treatment. That is, in the method of the present invention, the VTC1 gene may be disrupted by mutation treatment. The method of the mutation treatment is not particularly limited, and any of physical mutation treatment such as ultraviolet irradiation and radiation irradiation and chemical mutation treatment in which treatment is performed with a mutation agent such as ethylmethanesulfonic acid may be used. From the mutant strains obtained by the mutation treatment, a VTC1 gene disruption strain can be selected by sequence analysis.
　産業上実用されている酵母、例えば醸造酵母、パン酵母、バイオエタノール酵母等は通常二倍体かそれ以上の高次倍数体である。従って、本発明の方法によりアルコール発酵による発酵産物の生産性が向上された酵母を、酒類製造、製パン、バイオエタノール製造に用いる場合には、特に限定されないが、二倍体でＶＴＣ１アリルが共に破壊されたＶＴＣ１遺伝子破壊株を用いることが好ましい。二倍体の遺伝子破壊株は、周知の常法により得ることができる。具体例を挙げると、例えば、接合型の異なる一倍体（ａ型及びα型）でＶＴＣ１遺伝子破壊株を作製し、両者を接合させて二倍体のＶＴＣ１遺伝子破壊株を得ることができる。また、二倍体でＶＴＣ１アリルの一方が破壊された酵母を作製し、次いで、該酵母に対し再度ＶＴＣ１破壊処理を行なうことによって、二倍体のＶＴＣ１遺伝子破壊株を得ることもできる。 Industrially practical yeasts such as brewer's yeast, baker's yeast, and bioethanol yeast are usually diploids or higher polyploids. Therefore, when yeast for which the productivity of fermentation products by alcohol fermentation is improved by the method of the present invention is used for alcoholic beverage production, bread production, and bioethanol production, there is no particular limitation, but both diploid and VTC1 allyl are used together. It is preferable to use a disrupted VTC1 gene disruption strain. A diploid gene-disrupted strain can be obtained by a well-known conventional method. To give a specific example, for example, VTC1 gene disrupted strains can be prepared using haploids (a type and α type) having different mating types, and both can be joined to obtain a diploid VTC1 gene disrupted strain. Alternatively, a diploid VTC1 gene disruption strain can be obtained by preparing a yeast in which one of the VTC1 alleles is disrupted and then subjecting the yeast to VTC1 disruption treatment again.
　上記した相同ＤＮＡ断片を用いる方法によりＶＴＣ１遺伝子を破壊する場合には、例えば、１回目の遺伝子破壊処理と２回目の遺伝子破壊処理とで異なる薬剤耐性マーカーを用いればよい。上記セルフクローニング法を用いる場合であれば、ＶＴＣ１アリルの一方が破壊された二倍体酵母は、ゲノム中にマーカー遺伝子を有さないため、２回目でも１回目と同一の遺伝子破壊用プラスミドベクターを用いることができる。二倍体の酵母に対して上記遺伝子破壊方法を採用した場合には、ＶＴＣ１アリルが同時に破壊された株も生じ得るので、選択培地上での生育速度の違い（同時に破壊された株では生育が早い）や、ＶＴＣ１遺伝子領域の塩基配列に基づいて、ＶＴＣ１アリルが同時に破壊された株を選択することもできる。 When the VTC1 gene is disrupted by the above method using a homologous DNA fragment, for example, different drug resistance markers may be used in the first gene disruption process and the second gene disruption process. If the above self-cloning method is used, a diploid yeast in which one of the VTC1 alleles has been disrupted does not have a marker gene in the genome. Can be used. When the above gene disruption method is adopted for diploid yeast, a strain in which the VTC1 allele is simultaneously disrupted may also be generated, so that the difference in the growth rate on the selective medium (the strain that has been disrupted simultaneously does not grow). Early) or based on the nucleotide sequence of the VTC1 gene region, a strain in which the VTC1 allele is simultaneously destroyed can also be selected.
　二倍体の酵母から一倍体を得る方法は、当分野で周知の常法により行なうことができる。例えば、公知の胞子形成用培地中にて培養することにより胞子形成させて胞子を得て、該胞子を発芽させて一倍体を得ることができる。ＶＴＣ１遺伝子が破壊された酵母は、正常なＶＴＣ１遺伝子を有する酵母野生株と比較して、発酵能及びエタノール生産性が有意に高い。 A method for obtaining a haploid from a diploid yeast can be performed by a conventional method well known in the art. For example, the spore can be obtained by culturing in a known spore-forming medium to form a spore, and the spore can be germinated to obtain a haploid. The yeast in which the VTC1 gene is disrupted has significantly higher fermentation ability and ethanol productivity than the wild yeast strain having the normal VTC1 gene.
　本発明は、酵母のゲノム上に存在するＶＴＣ複合体の構成因子をコードする遺伝子の発現量の低下により発酵能及び発酵産物の生産性の向上した酵母を作製することを含む。ＶＴＣ複合体の構成因子をコードする遺伝子の発現を低下させる方法としては、例えば、酵母ゲノム上のＶＴＣ複合体の構成因子であるタンパク質をコードする遺伝子のコード領域の転写プロモーター領域を転写抑制型プロモーターで置換してなる変異型酵母を調製し、当該変異型酵母を転写抑制条件下で培養する方法が挙げられる。また、酵母におけるＶＴＣ複合体の構成因子をコードする遺伝子の転写に関わる領域に転写抑制活性のある塩基配列を挿入して作出したものを用いても良い。 The present invention includes producing a yeast having improved fermentability and productivity of fermentation products by reducing the expression level of a gene encoding a constituent factor of a VTC complex present on the yeast genome. Examples of a method for reducing the expression of a gene encoding a constituent element of a VTC complex include, for example, a transcription promoter region in a coding region of a gene encoding a protein that is a constituent element of a VTC complex on a yeast genome. And a method of culturing the mutant yeast under transcriptional repression conditions. Moreover, you may use what produced by inserting the base sequence which has transcription suppression activity in the area | region in connection with transcription | transfer of the gene which codes the component factor of the VTC complex in yeast.
　本発明は、酵母のゲノム上に存在するＶＴＣ複合体の構成因子をコードする遺伝子の翻訳効率を低下させることにより発酵能及び発酵産物の生産性の向上した酵母を作製することを含む。ＶＴＣ複合体の構成因子をコードする遺伝子の翻訳を抑制する方法としては、いわゆるアンチセンスＲＮＡを用いる方法が挙げられる。すなわち、ＶＴＣ複合体の構成因子をコードする遺伝子のｍＲＮＡに対するアンチセンスＲＮＡを転写する遺伝子を、酵母ゲノムに組み込み、当該アンチセンスＲＮＡを過剰発現させることで、ＶＴＣ複合体の構成因子をコードする遺伝子のｍＲＮＡの翻訳が抑制される。 The present invention includes producing yeast having improved fermentability and productivity of fermentation products by reducing the translation efficiency of a gene encoding a constituent factor of a VTC complex present on the yeast genome. Examples of the method for suppressing the translation of the gene encoding the constituent factor of the VTC complex include a method using so-called antisense RNA. That is, a gene that encodes a constituent element of a VTC complex by integrating a gene that transcribes an antisense RNA against mRNA of a gene encoding a constituent element of the VTC complex into the yeast genome and overexpressing the antisense RNA. Translation of mRNA is suppressed.
　本発明は、酵母において、遺伝子変異を行うことにより、ＶＴＣ複合体の機能を低下又は喪失させることを含む、アルコール発酵用酵母の作製方法、及び、遺伝子変異により、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の機能が低下又は喪失した、アルコール発酵用酵母も包含する。 The present invention relates to a method for producing a yeast for alcohol fermentation, which comprises reducing or losing the function of a VTC complex by carrying out a gene mutation in yeast, and a method for producing a VTC complex or a VTC complex by genetic mutation. Also included are yeasts for alcohol fermentation in which the function of the constituent factors is reduced or lost.
　本発明における酵母とは、発酵を行なういかなる酵母であってもよく、特に限定されない。例えば、酒類の製造に用いられる醸造酵母、パンの製造に用いられるパン酵母、バイオエタノールの製造に用いられるバイオエタノール酵母が挙げられる。醸造酵母としては、清酒酵母、ワイン酵母、ビール酵母、焼酎酵母等が例示される。清酒酵母とは日本酒の製造に、ワイン酵母とはワインの製造に、焼酎酵母とは焼酎の製造に用いられる酵母である。また、本発明においては実験室酵母も使用することができる。 The yeast in the present invention may be any yeast that performs fermentation, and is not particularly limited. Examples include brewing yeast used for the production of alcoholic beverages, baker's yeast used for the production of bread, and bioethanol yeast used for the production of bioethanol. Examples of the brewing yeast include sake yeast, wine yeast, beer yeast, shochu yeast, and the like. Sake yeast is used to produce sake, wine yeast is used to produce wine, and shochu yeast is used to produce shochu. Laboratory yeasts can also be used in the present invention.
　本発明において使用される酵母は、特に限定されるものではないが、例えばサッカロミセス（Saccharomyces）属、シゾサッカロミセス（Schizosaccharomyces）属又はクリベロミセス（Kluyveromyces）属、ザイゴサッカロミセス（Zygosaccharomyces）属に属する酵母が挙げられ、好ましくは、サッカロミセス属に属する酵母である。サッカロミセス属に属する酵母としては、好ましくはサッカロミセス・セレビシエ（Saccharomyces cerevisiae）である。より具体的には、これらに限定されるものではないが、サッカロミセス・セレビシエ、サッカロミセス・バイアヌス（Saccharomyces bayanus）、サッカロミセス・パラドクサス（Saccharomyces paradoxus）、サッカロミセス・アセチ（Saccharomyces aceti）、サッカロミセス・アムルケ（Saccharomyces amurcae）、サッカロミセス・ベイリー（Saccharomyces bailii）、サッカロミセス・ビスポーラス（Saccharomyces bisporus）、サッカロミセス・カペンシス（Saccharomyces capensis）、サッカロミセス・カールスベルゲンシス（Saccharomyces carlsbergensis）、サッカロミセス・チェバリエリー（Saccharomyces chevalieri）、サッカロミセス・シドリ（Saccharomyces cidri）、サッカロミセス・コレアヌス（Saccharomyces coreanus）、サッカロミセス・ダイレネンシス（Saccharomyces dairenensis）、サッカロミセス・デルブルッキー（Saccharomyces delbrueckii）、サッカロミセス・ディアスタチカス（Saccharomyces diastaticus）、サッカロミセス・ユーパジカス（Saccharomyces eupagycus）、サッカロミセス・エキシグス（Saccharomyces exiguus）、サッカロミセス・ファーメンタティ（Saccharomyces fermentati）、サッカロミセス・フロレンティヌス（Saccharomyces florentinus）、サッカロミセス・グロボサス（Saccharomyces globosus）、サッカロミセス・ヘテロジニカス（Saccharomyces heterogenicus）、サッカロミセス・ヒーニピエンシス（Saccharomyces hienipiensis）、サッカロミセス・インコンスピカス（Saccharomyces inconspicuous）、サッカロミセス・イヌシタトゥス（Saccharomyces inusitatus）、サッカロミセス・イタリカス（Saccharomyces italicus）、サッカロミセス・クロッケリアヌス（Saccharomyces kloeckerianus）、サッカロミセス・クルイベリ（Saccharomyces kluyveri）、サッカロミセス・ミクロエリプソデス（Saccharomyces microellipsodes）、サッカロミセス・モンタヌス（Saccharomyces montanus）、サッカロミセス・ムラキー（Saccharomyces mrakii）、サッカロミセス・ノルベンシス（Saccharomyces norbensis）、サッカロミセス・オレアセウス（Saccharomyces oleaceus）、サッカロミセス・オレアジノウス（Saccharomyces oleaginous）、サッカロミセス・パストリアヌス（Saccharomyces pastorianus）、サッカロミセス・プレトリエンシス（Saccharomyces pretoriensis）、サッカロミセス・プロストサードビー（Saccharomyces prostoserdovii）、サッカロミセス・ロゼイ（Saccharomyces rosei）、サッカロミセス・ロシニー（Saccharomyces rosinii）、サッカロミセス・ロウキシー（saccharomyces rouxii）、サッカロミセス・サイトアヌス（Saccharomyces saitoanus）、サッカロミセス・テルリス（Saccharomyces telluris）、サッカロミセス・トランスバーレンシス（Saccharomyces transvaalensis）、サッカロミセス・ユニスポラス（Saccharomyces unisporus）、サッカロミセス・ウバルム（Saccharomyces uvarum）、サッカロミセス・ベイファー（Saccharomyces vafer）、クリベロミセス・エスツアリー（Kluyveromyces aestuarii）、クリベロミセス・アフリカヌス（Kluyveromyces africanus）、クリベロミセス・ブルガリカス（Kluyveromyces bulgaricus）、クリベロミセス・デルフェンシス（Kluyveromyces delphensis）、クリベロミセス・ドブザンスキー（Kluyveromyces dobzhanskii）、クリベロミセス・ ドロソフィラム（Kluyveromyces drosophilarum）、クリベロミセス・ラクティス（Kluyveromyces lactis）、クリベロミセス・ロデリ（Kluyveromyces lodderi）、クリベロミセス・ファフィー（Kluyveromyces phaffii）、クリベロミセス・ファゼオロスポラス（Kluyveromyces phaseolosporus）、クリベロミセス・ポリスポラス（Kluyveromyces polysporus）、クリベロミセス・バヌデニー（Kluyveromyces vanudenii）、クリベロミセス・ベロネ（Kluyveromyces veronae）、クリベロミセス・ウィッカーハミー（Kluyveromyces wickerhamii）、クリベロミセス・ウィケニー（Kluyveromyces wikenii）、ザイゴサッカロミセス・ロウキシイ（Zygosaccharomyces rouxii）、ザイゴサッカロミセス・ビスポラス（Zygosaccharomyces bisporus）等を使用することができる。本願において使用される醸造酵母、パン酵母、バイオエタノール酵母の代表例としては、サッカロミセス・セレビシエが挙げられる。 The yeast used in the present invention is not particularly limited, and examples thereof include yeasts belonging to the genus Saccharomyces, the genus Schizosaccharomyces or the genus Kluyveromyces, and the genus Zygosaccharomyces. Yeast belonging to the genus Saccharomyces is preferable. The yeast belonging to the genus Saccharomyces is preferably Saccharomyces cerevisiae. More specifically, but not limited to, Saccharomyces acti, Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti, Saccharomyces aceti ), Saccharomyces bailii, Saccharomyces bisporus, Saccharomyces capensis, Saccharomyces dri ci om i s ), Saccharomyces coreanus, Saccharomyces renenensis, Saccharo Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces eupagycus, Saccharomyces exiguus, Saccharomyces exiguus, Saccharomyces oms Saccharomyces florentinus), Saccharomyces globosus, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces hienipices, Saccharomyces hienipis Saccharomyces italicus), Saccharomyces klo eckerianus), Saccharomyces kluyveri, Saccharomyces microellipsodes, Saccharomyces omacies, Saccharomyces omomacies, Saccharomyces oleaceus), Saccharomyces oleaginous, Saccharomyces pastorianus, Saccharomyces pretoriensis, Saccharomyces ac prossidom, Saccharomyces (Saccharomyces rosinii), Saccharomyces rouxii, Saccharomyces rouxii Romyces Cytoaneus (Saccharomyces saitoanus), Saccharomyces telluris, Saccharomyces transvaalensis, Saccharomyces chars vaum (Saccharomyces unisporus), Saccharomyces unisporus , Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces bulgaricus, Krumyveromyces delpheny (Kluyveromyces bulges) , Kluyveromyces lactis, Deli (Kluyveromyces lodderi), Kluyveromyces phaffii, Cliveromyces phaseo sporus, Kluyveromyces veroy 、 ver ii・ Wicker Hammy (Kluyveromyces wickerhamii), Kluyveromyces wikenii, Zygosaccharomyces rouxii, Zygosaccharomyces bisporus can be used. Representative examples of brewing yeast, baker's yeast, and bioethanol yeast used in the present application include Saccharomyces cerevisiae.
　本発明において使用される好ましい醸造酵母は、清酒酵母ではサッカロミセス・セレビシエ（より具体的にはきょうかい７号（Ｋ７））などが挙げられ、ワイン酵母ではサッカロミセス・セレビシエ（より具体的にはきょうかい酵母ブドウ酒用アンプル酵母）、サッカロミセス・バイアヌスなどが挙げられ、ビール酵母では、サッカロミセス・セレビシエ、サッカロミセス・パストリアヌス、サッカロミセス・カールスベルゲンシスなどが挙げられ、焼酎酵母ではサッカロミセス・セレビシエ（より具体的にはきょうかい酵母焼酎用２号）などが挙げられる。 Preferred brewer's yeast used in the present invention includes Saccharomyces cerevisiae (more specifically, No. 7 (K7)) for sake yeast, and Saccharomyces cerevisiae (more specifically, for yeast). Ampoule yeast for yeast wine), Saccharomyces baianus, etc., for beer yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces carlsbergensis, etc., for shochu yeast, Saccharomyces cerevisiae (more specifically, No. 2) for yeast yeast shochu.
　本発明において使用される好ましいバイオエタノール酵母は、サッカロミセス・セレビシエ、サッカロミセス・ダイレネンシス、サッカロミセス・トランスバーレンシス、サッカロミセス・ロシニー、ザイゴサッカロミセス・ビスポラスなどが挙げられる。なお、還元糖のみを選択的にエタノールに変換するショ糖非資化性酵母であってもよい。 Preferred bioethanol yeasts used in the present invention include Saccharomyces cerevisiae, Saccharomyces dairenensis, Saccharomyces transvalensis, Saccharomyces rosinii, Zygosaccharomyces bisporus and the like. A sucrose non-assimilating yeast that selectively converts only reducing sugars to ethanol may be used.
　また、本発明の対象となる酵母は、野生株、変異株及び形質転換株のいずれに由来するものであってもよい。酵母のＶＴＣ１遺伝子破壊株、ＶＴＣ４遺伝子破壊株としては、酵母におけるポリリン酸合成経路の触媒機構を調べる目的で作出された実験室酵母のＶＴＣ１遺伝子破壊株、ＶＴＣ４遺伝子破壊株が知られている（非特許文献４）。しかしながら、ＶＴＣ複合体の構成因子をコードする遺伝子破壊酵母又は遺伝子の発現量が低下した酵母において二酸化炭素減量の増加及び産生されるエタノールの生産性が向上することは知られておらず、また、ＶＴＣ複合体の構成因子をコードする遺伝子が破壊された又は該遺伝子の発現量の低下した醸造酵母、パン酵母、及びバイオエタノール酵母も知られていない。本発明は、ＶＴＣ複合体の構成因子をコードする遺伝子の破壊又は該遺伝子の発現量の低下により、発酵能及びエタノールの生産性が向上された醸造酵母、パン酵母、及びバイオエタノール酵母を新規に提供するものである。 Further, the yeast that is the subject of the present invention may be derived from any of a wild strain, a mutant strain, and a transformed strain. As yeast VTC1 gene-disrupted strains and VTC4 gene-disrupted strains, laboratory yeast VTC1 gene-disrupted strains and VTC4 gene-disrupted strains created for the purpose of investigating the catalytic mechanism of the polyphosphate synthesis pathway in yeast are known. Patent Document 4). However, it is not known that the gene-disrupted yeast encoding the component of the VTC complex or the yeast in which the expression level of the gene is reduced is increased in carbon dioxide loss and the productivity of produced ethanol is improved, There are also no known brewing yeast, baker's yeast, or bioethanol yeast in which a gene encoding a constituent factor of the VTC complex is disrupted or the expression level of the gene is reduced. The present invention provides novel brewer's yeast, baker's yeast, and bioethanol yeast whose fermentability and ethanol productivity are improved by disrupting a gene encoding a constituent factor of the VTC complex or reducing the expression level of the gene. It is to provide.
　本発明の酵母において「発酵能が向上している」とは、酵母の発酵時の二酸化炭素（炭酸ガス）発生量が増加していることを意味する。酵母はアルコール発酵によって、グルコースをエタノールと二酸化炭素に分解するので、二酸化炭素発生量は酵母のアルコール発酵すなわちエタノール生産の進行を示す数値として一般的に用いられている。二酸化炭素発生量は酵母の発酵速度を示す一般的な数値であり、二酸化炭素発生量が増加していることは、発酵速度が促進していることを示唆している。また、上述のとおり、酵母はアルコール発酵によって、グルコースをエタノールと二酸化炭素に分解するので、発酵の促進により二酸化炭素の発生量と同時にエタノールの発生量も増加する。すなわち、本発明の方法による発酵能が向上している酵母は、エタノール生産性が向上している。 In the yeast of the present invention, “fermentability is improved” means that the amount of carbon dioxide (carbon dioxide) generated during yeast fermentation is increasing. Since yeast decomposes glucose into ethanol and carbon dioxide by alcohol fermentation, the amount of carbon dioxide generated is generally used as a numerical value indicating the progress of alcohol fermentation of yeast, that is, ethanol production. The amount of carbon dioxide generated is a general value indicating the fermentation rate of yeast, and an increase in the amount of carbon dioxide generated suggests that the fermentation rate is accelerated. Further, as described above, since yeast decomposes glucose into ethanol and carbon dioxide by alcohol fermentation, the generation amount of ethanol increases simultaneously with the generation amount of carbon dioxide by the promotion of fermentation. In other words, the yeast having improved fermentability according to the method of the present invention has improved ethanol productivity.
　発酵能及びエタノール生産性を向上した酵母の作製方法は、ＶＴＣ複合体の構成因子をコードする遺伝子を破壊する工程又は当該遺伝子の発現量を低下させる工程を含むが、作製された酵母を未処理の親株と、発酵能及びエタノール生産性を比較する工程をさらに含んでもよい。 A method for producing yeast having improved fermentability and ethanol productivity includes a step of destroying a gene encoding a constituent factor of the VTC complex or a step of reducing the expression level of the gene, but the produced yeast is untreated. The method may further comprise a step of comparing the fermentability and ethanol productivity with the parent strain.
　さらに本発明は、ＶＴＣ複合体の機能を低下又は喪失させた酵母を含むアルコール発酵用組成物も包含する。アルコール発酵用組成物とは、アルコール発酵に用いられる組成物である。好ましくは、遺伝子変異により、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の機能が低下又は喪失した、アルコール発酵用酵母を含む組成物であり、より好ましくは、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子が破壊されたアルコール発酵用酵母を含む組成物である。アルコール発酵用組成物における酵母は、当業者が通常用いる方法により培養し、回収して、そのまま組成物の製造に用いてもよいし、公知の方法で後処理を行ってもよい。例えば、遠心分離などにより粗精製を行ってもよい。また、所望により粗精製を行った後、生理食塩水またはリン酸緩衝生理食塩水（PBS）、培地などの当該分野で従来から使用されている溶媒に溶解または懸濁させてもよい。また、所望により乾燥物としてもよい。乾燥は、凍結乾燥、噴霧乾燥等が例示される。アルコール発酵用組成物には、保護剤を添加してもよい。 Furthermore, the present invention also includes a composition for alcohol fermentation containing yeast in which the function of the VTC complex is reduced or lost. The composition for alcohol fermentation is a composition used for alcohol fermentation. Preferably, it is a composition containing yeast for alcohol fermentation in which the function of the VTC complex or the component of the VTC complex is reduced or lost due to gene mutation, more preferably a gene encoding the component of the VTC complex A composition comprising yeast for alcohol fermentation in which at least one gene selected from is disrupted. The yeast in the composition for alcohol fermentation may be cultured and collected by a method commonly used by those skilled in the art, and may be used as it is for the production of the composition, or may be post-treated by a known method. For example, rough purification may be performed by centrifugation or the like. In addition, if desired, after rough purification, it may be dissolved or suspended in a solvent conventionally used in the art, such as physiological saline, phosphate buffered saline (PBS), or a medium. Moreover, it is good also as a dried material if desired. Examples of the drying include freeze drying and spray drying. A protective agent may be added to the composition for alcohol fermentation.
［３．発酵産物を製造する方法］
　本発明は、酵母においてＶＴＣ複合体の機能が低下又は喪失している条件で、アルコール発酵を行うことを含む、酵母を用いて発酵産物を製造する方法にも及ぶ。酵母におけるＶＴＣ複合体の機能の低下又は喪失は、上述の手段によって行うことができる。本発明の発酵産物の製造方法は、具体的には以下の工程を含む。
（１）酵母においてＶＴＣ複合体の機能が低下又は喪失している条件で、原材料と酵母を接触させてアルコール発酵を行う工程。
（２）アルコール発酵を行った後に、発酵産物を得る工程。
　工程（１）のアルコール発酵においては、原材料由来の糖類に酵母を作用させることにより、アルコール（好ましくはエタノール）と二酸化炭素が生成する。原材料としては、米、麦、ブドウ、芋、小麦粉、糖蜜、セルロース系バイオマス等、又はこれらを処理したものが例示される。 [3. Method for producing fermentation product]
The present invention also extends to a method for producing a fermentation product using yeast, comprising performing alcoholic fermentation under conditions where the function of the VTC complex is reduced or lost in yeast. Reduction or loss of the function of the VTC complex in yeast can be performed by the means described above. The method for producing a fermentation product of the present invention specifically includes the following steps.
(1) A step of performing alcoholic fermentation by bringing the raw material and yeast into contact with each other under the condition that the function of the VTC complex is reduced or lost in the yeast.
(2) A step of obtaining a fermentation product after performing alcoholic fermentation.
In the alcohol fermentation of step (1), alcohol (preferably ethanol) and carbon dioxide are produced by allowing yeast to act on the saccharide derived from the raw material. Examples of raw materials include rice, wheat, grapes, rice bran, flour, molasses, cellulosic biomass, and the like, or those obtained by treating them.
　発酵産物は酵母による発酵を経て製造されるものであれば、いかなるものであってもよい。発酵産物としては例えば、発酵飲料、発酵食品、バイオエタノールが例示される。発酵飲料は、原材料となる食材を発酵させることにより作製される飲料であればいかなるものであってもよく、酒類が例示される。酒類としては、清酒、ワイン、ビール、焼酎、ウィスキー、ブランデー等が挙げられるが、これらに限定されるものではない。発酵食品としては、原材料となる食材を発酵させることにより作製される食品材料、又は加工食品を含み、例えば、発酵されたパン生地若しくはパン生地を発酵させて加工された食品、酒類、茶、酢、穀物加工品（納豆、醤油、味噌等）、魚介類加工品、野菜果実加工品、酪農製品等が例示される。 The fermentation product may be any product as long as it is produced through fermentation with yeast. Examples of fermented products include fermented beverages, fermented foods, and bioethanol. The fermented beverage may be any beverage as long as it is a beverage produced by fermenting a raw material, and alcoholic beverages are exemplified. Examples of liquors include, but are not limited to, sake, wine, beer, shochu, whiskey, brandy and the like. As fermented foods, food materials produced by fermenting ingredients as raw materials, or processed foods, for example, fermented bread dough or food processed by fermenting bread dough, alcoholic beverages, tea, vinegar, grains Examples of processed products (natto, soy sauce, miso, etc.), processed seafood products, processed vegetable fruits, dairy products, etc.
　発酵飲料として酒類を製造する場合を例示して説明する。酒類には、醸造酒、蒸留酒が含まれる醸造酒は、米・麦・ブドウ等の主原料を発酵させて製造した酒、蒸留酒は、主原料を発酵させた後、蒸留して製造した酒である。醸造酒の発酵方式には、単発酵、単行複発酵、並行複発酵がある。単発酵による醸造酒としてはワイン、単行複発酵による醸造酒としてはビール、並行複発酵による醸造酒としては、清酒（日本酒）が挙げられる。並行複発酵では糖化と発酵が同じ容器の中で同時に進行する。酒類の製造には、酵母においてＶＴＣ複合体の機能が低下又は喪失している条件で、主原料（食材由来の糖類若しくは食材由来の多糖類を糖化させた糖類を含む）を用いて、酵母によるアルコール発酵を行う工程、及びアルコール発酵を行った後に、液体を固形分から分離することにより、発酵産物を得る工程が含まれる。 An example of producing alcoholic beverages as a fermented beverage will be described. Liquors include brewed liquor and distilled liquor. Brewed liquor is produced by fermenting the main ingredients such as rice, wheat and grapes. Distilled liquor is produced by fermenting the main ingredients and then distilled. It is sake. The fermentation methods of brewed liquor include simple fermentation, single double fermentation, and parallel double fermentation. The brewed liquor by simple fermentation includes wine, the brewed liquor by simple double fermentation includes beer, and the brewed liquor by parallel double fermentation includes sake (Japanese sake). In parallel double fermentation, saccharification and fermentation proceed simultaneously in the same container. For the production of alcoholic beverages, the main raw materials (including saccharides obtained by saccharifying saccharides derived from foods or polysaccharides derived from foods) are used depending on the yeast under the condition that the function of the VTC complex is reduced or lost in yeast. The process of performing alcoholic fermentation and the process of obtaining a fermented product by isolate | separating a liquid from solid content after performing alcoholic fermentation are included.
　ワインの製造には、主原料として、適切に処理されたブドウ又はブドウ果汁を用いる。ブドウ又はブドウ果汁と酵母を発酵槽内で培養することにより、ブドウに含まれる糖類を用いてアルコール発酵が行われる。ビールの製造には、主原料として麦汁を用いる。麦汁は、麦芽と米などの副原料を適温（例えば４０℃～１００℃）で適当な時間保持してデンプンを糖化させ、加熱・冷却して作製した糖化液である。麦汁と酵母を発酵槽内で培養することにより、麦汁に含まれる糖類を用いてアルコール発酵が行われる。清酒の製造には、主原料として蒸米が用いられる。主原料の蒸米、酵母に加えて、麹菌（例えば、Aspergillus oryzae）を蒸米に散布して培養した麹を発酵槽に入れて培養することにより、米のデンプンが麹の酵素によりブドウ糖へと分解され、ブドウ糖を用いてアルコール発酵が行われる。アルコール発酵後、抽出、圧搾、濾過等により液体を固形分と分離することにより、醸造酒が得られる。必要に応じて、圧搾、加熱、熟成、貯蔵、調合、乳酸発酵、濾過等を行うことができる。各醸造酒の製造におけるアルコール発酵の条件は、製造する醸造酒によって異なるが、４℃～４０℃の間の適温において、４日～７０日の適当な期間で行うことができる。蒸留酒を製造するときは、醸造酒と同様に主原料のアルコール発酵を行った後、アルコール発酵後の発酵液（発酵醪）を蒸留して分離すればよい。また必要に応じて、貯蔵・熟成等も行うことができる。蒸留酒としては、焼酎、ウィスキー、ブランデー等が知られている。焼酎の主原料としては、大麦、芋、米等を用いることができ、ウィスキーは麦汁、ブランデーはブドウ又はブドウ果汁を用いる。酒類は、上記のものに限定されるのではなく、アルコール発酵工程を経て得られるものであれば、いかなるものであってもよい。 In wine production, properly processed grapes or grape juice is used as the main ingredient. By culturing grapes or grape juice and yeast in a fermenter, alcohol fermentation is performed using sugars contained in grapes. For the production of beer, wort is used as the main raw material. Wort is a saccharified solution prepared by saccharifying starch by holding auxiliary materials such as malt and rice at an appropriate temperature (for example, 40 ° C. to 100 ° C.) for an appropriate period of time, heating and cooling. By culturing wort and yeast in a fermenter, alcohol fermentation is performed using sugars contained in wort. Steamed rice is used as the main raw material for the production of sake. In addition to steamed rice and yeast as the main ingredients, rice bran (for example, Aspergillus oryzae) sprinkled on steamed rice is cultured in a fermentor so that rice starch is broken down into glucose by the koji enzyme. Alcohol fermentation is performed using glucose. After alcoholic fermentation, the brewed liquor is obtained by separating the liquid from the solid content by extraction, pressing, filtration, or the like. If necessary, pressing, heating, aging, storage, preparation, lactic acid fermentation, filtration, and the like can be performed. The conditions for alcoholic fermentation in the production of each brewed liquor vary depending on the brewed liquor to be produced, but can be carried out at a suitable temperature between 4 ° C. and 40 ° C. for a suitable period of 4 days to 70 days. When producing distilled liquor, after performing alcohol fermentation of the main raw material in the same manner as brewed liquor, the fermented liquor (fermented rice cake) after alcohol fermentation may be distilled and separated. Moreover, storage, aging, etc. can also be performed as needed. As distilled spirits, shochu, whiskey, brandy and the like are known. Barley, straw, rice, etc. can be used as the main raw material of shochu, whiskey uses wort, brandy uses grape or grape juice. The alcoholic beverages are not limited to those described above, and any alcohol may be used as long as it is obtained through an alcohol fermentation process.
　発酵食品として、パン生地、醤油を製造する場合を例示して説明する。パン生地は、主原料として小麦粉を用いて製造される。小麦粉を、酵母、水等の材料と混ぜ合わせ、発酵を行う。例えば、適温（例えば、２７℃）、湿度が７０～８０％程度で４～５時間発酵させることにより、発酵が行われ、パン生地がふくらむ。発酵後のパン生地に、更に小麦粉、食塩、砂糖、油脂等の材料を加えて捏ねる。この後、約２０分程度、発酵を行っても良い。パン生地の成型を行った後、再度、約４０分程度、発酵を行う。成型後の発酵は、温度は３５～３８度、湿度は８０％程度が例示される。本発明におけるパン生地は、少なくとも小麦粉、酵母、水を含む材料を混ぜ合わせた状態で、少なくとも１回の発酵工程を経たものであればよく、必ずしも複数回の発酵工程を経ていなくてもよい。パン生地は、所定の温度で焼き上げることによりパンを製造することもできるし、ピザ等の加工食品の製造に用いることもできる。醤油は主原料として、大豆と小麦を用いて製造される。醤油の製造においてはまず、大豆を加圧蒸気により高圧・短時間で蒸し、小麦を煎って、主原料の加工処理を行う。加工処理後の大豆と小麦を合わせたものに麹菌を散布し、麹菌を繁殖させて、麹を製造する。麹を食塩水と混合して発酵槽に入れ、冷却殺菌を行った後、加温し、酵母を添加して発酵を行う。発酵後、圧搾して、余分な油分や固形分を除き、必要に応じて、殺菌、濾過等を行うことにより、醤油が得られる。 An example of producing dough and soy sauce as fermented food will be described. Bread dough is manufactured using flour as the main ingredient. Wheat flour is mixed with yeast, water and other materials, and fermented. For example, fermentation is performed by fermentation at an appropriate temperature (for example, 27 ° C.) and a humidity of about 70 to 80% for 4 to 5 hours, and bread dough swells. Add flour, salt, sugar, fats and other ingredients to the dough after fermentation and knead. Thereafter, fermentation may be performed for about 20 minutes. After forming the dough, the fermentation is performed again for about 40 minutes. The fermentation after molding is exemplified by a temperature of 35 to 38 degrees and a humidity of about 80%. The bread dough in the present invention is not limited as long as it has been subjected to at least one fermentation step in a state in which materials containing at least flour, yeast, and water are mixed, and does not necessarily have to undergo a plurality of fermentation steps. Bread dough can be baked at a predetermined temperature to produce bread, or it can be used to produce processed foods such as pizza. Soy sauce is produced using soybeans and wheat as main ingredients. In the production of soy sauce, soybeans are first steamed with pressurized steam in high pressure for a short time, roasted with wheat, and processed into the main ingredients. Spice is sprinkled on the combined soybean and wheat after processing, and the koji is propagated to produce koji. The koji is mixed with saline and placed in a fermenter, cooled and sterilized, heated, and yeast is added for fermentation. After fermentation, the soy sauce is obtained by squeezing to remove excess oil and solids and performing sterilization, filtration and the like as necessary.
　バイオエタノールを製造する場合を例示して説明する。バイオエタノールの主原料としては、炭水化物を含む生物由来の資源であればいかなるものであってもよい。生産効率の面から糖類あるいはデンプン、セルロースを多く含む、植物由来の植物資源が好ましい。植物資源としてはより具体的には、サトウキビや甜菜から砂糖を精製した後に得られる糖蜜、トウモロコシ、スイッチグラス（イネ科の多年草）、パルプ廃液、廃材木、稲わら等のセルロースを含有するセルロース系バイオマスが挙げられる。バイオエタノール生産は、主原料に含まれるデンプン、セルロースなどの多糖類をアミラーゼなどの多糖分解酵素で処理してブドウ糖に分解する糖化と、ブドウ糖を用いた酵母によるアルコール発酵により行われる。産生したバイオエタノールは、公知の方法又は今後開発される方法により回収すればよい。 An example of producing bioethanol will be described. The main raw material for bioethanol may be any biological resource that contains carbohydrates. From the viewpoint of production efficiency, plant-derived plant resources containing a large amount of saccharides, starch, or cellulose are preferred. More specifically, as plant resources, a cellulose system containing cellulose such as molasses, corn, switchgrass (perennials), pulp waste liquor, waste wood, rice straw, etc. obtained after refining sugar from sugarcane and sugar beet Biomass is mentioned. Bioethanol production is performed by saccharification in which polysaccharides such as starch and cellulose contained in the main raw material are treated with a polysaccharide-degrading enzyme such as amylase to be decomposed into glucose, and alcohol fermentation by yeast using glucose. The produced bioethanol may be recovered by a known method or a method developed in the future.
［４．酵母のアルコール発酵能を向上させるための薬剤のスクリーニング方法］
　後述する実施例に記載のとおり、酵母のＶＴＣ複合体の構成因子をコードする遺伝子のうち少なくとも１つの遺伝子が破壊された、又は発現量が低下した酵母が、従来使用されてきた酵母よりも高い発酵能及びエタノール生産性を有することが見出された。本発明は、かかる知見に基づく、酵母のアルコール発酵能を向上させるための薬剤のスクリーニング方法も包含する。本発明においてスクリーニングされるアルコール発酵能を向上させるための薬剤は、本発明の酵母のアルコール発酵能を向上させる方法や発酵産物の製造方法において、用いることができる。 [4. Method for screening drug for improving alcohol-fermenting ability of yeast]
As described in Examples below, yeasts in which at least one gene among the genes encoding the components constituting the VTC complex of yeast is disrupted or whose expression level is reduced are higher than those conventionally used. It was found to have fermentability and ethanol productivity. The present invention also includes a drug screening method for improving the alcohol-fermenting ability of yeast based on such findings. The chemical | medical agent for improving the alcohol fermentation ability screened in this invention can be used in the method of improving the alcohol fermentation ability of the yeast of this invention, and the manufacturing method of a fermentation product.
　本発明の薬剤のスクリーニング方法は、被験化合物をＶＴＣ複合体に作用させる工程を含み、当該被験化合物がＶＴＣ複合体の機能を低下又は喪失させることを指標とするものである。具体的には、以下の工程を含むものが例示される。
（ｉ）ＶＴＣ複合体に作用させる被験化合物と、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の少なくとも１つを準備する工程。
（ｉｉ）ＶＴＣ複合体と被験化合物を接触させ、ＶＴＣ複合体の機能を確認する工程。
（ｉｉｉ）被験化合物の非存在下と比較して、ＶＴＣ複合体の機能を低下又は喪失させる被験化合物を、酵母のアルコール発酵能を向上させるための薬剤として選別する工程。 The method for screening a drug of the present invention includes a step of allowing a test compound to act on a VTC complex, and uses the test compound as an index to reduce or lose the function of the VTC complex. Specifically, those including the following steps are exemplified.
(I) A step of preparing a test compound that acts on the VTC complex and at least one of a VTC complex or a constituent factor of the VTC complex.
(Ii) A step of bringing the VTC complex into contact with the test compound and confirming the function of the VTC complex.
(Iii) A step of selecting a test compound that reduces or loses the function of the VTC complex as compared with the absence of the test compound as a drug for improving the alcohol-fermenting ability of yeast.
　さらに具体的には、以下の工程を含むものが例示される。
（ｉ）ＶＴＣ複合体に作用させる被験化合物と、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の少なくとも１つを発現する細胞（好ましくは酵母）を準備する工程。
（ｉｉ）被験化合物をＶＴＣ複合体と相互作用させるように、被験化合物の存在下で該細胞を培養し、その培養後の細胞におけるＶＴＣ複合体の機能の低下又は喪失を確認する工程。
（ｉｉｉ）細胞におけるＶＴＣ複合体の機能の低下又は喪失させる被験化合物を、酵母のアルコール発酵能を向上させるための薬剤として選別する工程。
　なお上記工程において用いられる細胞では、酵母のＶＴＣ複合体又はＶＴＣ複合体の構成因子の少なくとも１つが液胞膜に発現していることが好ましい。 More specifically, those including the following steps are exemplified.
(I) A step of preparing a test compound that acts on the VTC complex and a cell (preferably yeast) expressing at least one of the VTC complex or a component of the VTC complex.
(Ii) A step of culturing the cell in the presence of the test compound so that the test compound interacts with the VTC complex, and confirming a decrease or loss of the function of the VTC complex in the cultured cell.
(Iii) A step of selecting a test compound that decreases or loses the function of the VTC complex in cells as a drug for improving the alcohol-fermenting ability of yeast.
In the cells used in the above step, it is preferable that at least one of the yeast VTC complex or the constituent factor of the VTC complex is expressed in the vacuolar membrane.
　酵母におけるＶＴＣ複合体の機能の低下又は喪失は、ＶＴＣ複合体の公知の機能である、液胞型プロトンＡＴＰａｓｅの安定性の維持、タンパク質の輸送、ミクロオートファジー、又は液胞内へのポリリン酸の蓄積の機能の低下や喪失によって確認することができる。さらに、今後見出されるＶＴＣ複合体が本来有している機能の低下又は喪失によって確認することもできる。 Reduced or lost function of the VTC complex in yeast is a known function of the VTC complex, maintaining the stability of vacuolar proton ATPase, protein transport, microautophagy, or polyphosphate into the vacuole This can be confirmed by a decrease or loss of the accumulation function. Furthermore, it can also be confirmed by a decrease or loss of the function inherent to the VTC complex to be found in the future.
　本発明のスクリーニング方法で得られる薬剤は、酵母のＶＴＣ複合体の機能の低下又は喪失を引き起こすものであり、酵母に接触させることにより、酵母のアルコール発酵能を向上させることができる。 The drug obtained by the screening method of the present invention causes a decrease or loss in the function of the yeast VTC complex, and the alcohol fermentability of the yeast can be improved by contacting with the yeast.
　以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
（実施例１）ＶＴＣ１遺伝子破壊株、ＶＴＣ２遺伝子破壊株、ＶＴＣ３遺伝子破壊株、ＶＴＣ４遺伝子破壊株の作製及びその発酵能の測定
（１）遺伝子破壊株の作製
　酵母サッカロミセス・セレビシエの一倍体実験室株であるＢＹ４７４１株を野生株として使用し、公知の方法によってＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子の破壊株（それぞれ、ＢＹ４７４１ ｖｔｃ１Δ、ＢＹ４７４１ ｖｔｃ２Δ、ＢＹ４７４１ ｖｔｃ３Δ、ＢＹ４７４１ ｖｔｃ４Δ）を作製した（Science. 1999 Aug 6;285(5429):901-6. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.）。 (Example 1) Preparation of VTC1 gene disruption strain, VTC2 gene disruption strain, VTC3 gene disruption strain, VTC4 gene disruption strain and measurement of fermentation ability thereof (1) Preparation of gene disruption strain Yaploid laboratory of yeast Saccharomyces cerevisiae By using the BY4741 strain as a wild strain, VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains (BY4741 vtc1Δ, BY4741 vtc2Δ, BY4741 vtc3Δ, BY4741 vtc4Δ, respectively) were prepared by a known method (Science). 6; 285 (5429): 901-6. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.).
（２）遺伝子破壊株の発酵能の確認
　各酵母は、ＹＰＤ培地（酵母エキス１％、ペプトン２％、グルコース２％）において一晩３０℃で振とう培養した後、初期酵母密度がＯＤ６００＝０．１になるように、５ｍＬの高濃度グルコース含有ＹＰＤ培地（酵母エキス１％、ペプトン２％、グルコース２０％）を分注した試験管に植菌し、３０℃で振とう培養を行った。発酵試験は各株について独立した３クローンを用いた。植菌開始から３日後、電子天秤を用いて培養液の重量を測定し、植菌開始時の重量との差を二酸化炭素発生量とした。 (2) Confirmation of Fermentation Capacity of Gene Disrupted Strain Each yeast was cultured at 30 ° C. overnight in YPD medium (yeast extract 1%, peptone 2%, glucose 2%), and then the initial yeast density was OD600 = 0. .1 was inoculated into a test tube into which 5 mL of high-concentration glucose-containing YPD medium (yeast extract 1%, peptone 2%, glucose 20%) was dispensed, and cultured with shaking at 30 ° C. The fermentation test used 3 independent clones for each strain. Three days after the start of inoculation, the weight of the culture solution was measured using an electronic balance, and the difference from the weight at the start of inoculation was defined as the amount of carbon dioxide generated.
　結果を図１に示す。図１において、縦軸は二酸化炭素発生量の平均値（ｇ）を示す。エラーバーは標準偏差を示す。二酸化炭素発生量の平均値について、等分散を仮定しない片側ｔ検定を行った。
　図１に示されるとおり、ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子のいずれの破壊株も、野生株を上回る二酸化炭素発生量を示した。中でもＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子の各破壊株は、野生株と比べて統計的に有意に高い二酸化炭素発生量を示し（危険率５％未満）、ＶＴＣ１遺伝子破壊株における二酸化炭素発生量は、野生株と比べて７０．８％高い値を示した。
　以上の結果から、ＶＴＣ複合体の構成因子をコードする各遺伝子の破壊がアルコール発酵の促進につながることが明らかになった。 The results are shown in FIG. In FIG. 1, the vertical axis represents the average value (g) of the amount of carbon dioxide generated. Error bars indicate standard deviation. A one-sided t-test with no assumed equal variance was performed on the average value of carbon dioxide generation.
As shown in FIG. 1, all of the VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains showed a carbon dioxide generation amount higher than that of the wild strain. Among them, each of the VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains shows a statistically significantly higher carbon dioxide generation amount than the wild strain (less than 5% of the risk rate), and the carbon dioxide generation amount in the VTC1 gene-disrupted strain is The value was 70.8% higher than that of the wild type.
From the above results, it was clarified that the disruption of each gene encoding the constituent factor of the VTC complex leads to the promotion of alcohol fermentation.
（実施例２）ＶＴＣ１遺伝子破壊パン酵母株の作製、及びその発酵能の測定
　酵母サッカロミセス・セレビシエの二倍体パン酵母株である３３４６／３３４７株に由来する一倍体３３４６株を野生株に使用し、公知の方法（Yeast vol.21 p.947-962 (2004)）によってＶＴＣ１遺伝子破壊株（３３４６ ｖｔｃ１Δ）を作製した。具体的な作製方法は以下の通りである。 (Example 2) Production of VTC1 gene-disrupted baker's yeast strain and measurement of its fermentative ability A haploid 3346 strain derived from 3346/3347 strain, a diploid baker's yeast strain of Saccharomyces cerevisiae, was used as a wild strain. A VTC1 gene disruption strain (3346 vtc1Δ) was prepared by a known method (Yeast vol.21 p.947-962 (2004)). A specific manufacturing method is as follows.
　まず、薬剤耐性遺伝子を含むプラスミド（pFA6a-hphNT1：ハイグロマイシン耐性マーカーを含む）を鋳型として、ＶＴＣ１遺伝子のＯＲＦの上流領域及び下流領域のそれぞれと相同な塩基配列を持つプライマーを用いて、ＰＣＲを行い、薬剤耐性遺伝子の上流及び下流にＶＴＣ１遺伝子の上流及び下流と相同な塩基配列を連結させて、ＶＴＣ１遺伝子破壊用断片を作製した。用いたプライマーは以下の通りである。
　VTC1-S1：CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC（配列番号９）
　VTC1-S2：CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG（配列番号１０） First, using a plasmid containing a drug resistance gene (pFA6a-hphNT1: containing a hygromycin resistance marker) as a template, PCR was performed using primers having base sequences homologous to the upstream region and downstream region of the ORF of the VTC1 gene. Then, a base sequence homologous to the upstream and downstream of the VTC1 gene was linked upstream and downstream of the drug resistance gene to prepare a VTC1 gene disruption fragment. The primers used are as follows.
VTC1-S1: CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC (SEQ ID NO: 9)
VTC1-S2: CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG (SEQ ID NO: 10)
　ＶＴＣ１遺伝子破壊用断片を組み入れた組換えプラスミドを作製し、酵母３３４６株に導入し、薬剤耐性を指標にしてＶＴＣ１遺伝子破壊株を取得した。取得したＶＴＣ１遺伝子破壊株について、コロニーＰＣＲを行うことにより、ＶＴＣ１遺伝子が破壊されていることを確認した。確認用のプライマーは以下の通りである。
　VTC1-Fw：AACACTACGCCAGATTTCCA（配列番号１７）
　VTC1-Rv：CCACGCTTACGATATTGGAA（配列番号１８） A recombinant plasmid incorporating the VTC1 gene disruption fragment was prepared and introduced into the yeast 3346 strain, and a VTC1 gene disruption strain was obtained using drug resistance as an index. The obtained VTC1 gene disrupted strain was confirmed to have been disrupted by conducting colony PCR. Primers for confirmation are as follows.
VTC1-Fw: AACACTACGCCAGATTTCCA (SEQ ID NO: 17)
VTC1-Rv: CCACGCTTACGATATTGGAA (SEQ ID NO: 18)
　酵母（３３４６ ｖｔｃ１Δ又は野生株）を、ＹＰＤ培地において２４時間３０℃で培養し、その後遠心分離によって集菌し、蒸留水で２回洗浄した。低スクロース含有パン生地として、パン専用小麦粉（日清）２０ｇ、スクロース１ｇ、塩化ナトリウム０．４ｇ、水１２．５ｍＬ、酵母０．４ｇを捏ねて調製した。また、高スクロース含有パン生地として、パン専用小麦粉（日清）３０ｇ、スクロース９ｇ、塩化ナトリウム０．１５ｇ、水１６ｍＬ、酵母０．６ｇを捏ねて調製した。３０℃で静置培養し、二酸化炭素発生量をファーモグラフＩＩ－Ｗ（アトー）を用いて測定した。発酵試験は各株について３回の繰り返し実験を行った。 Yeast (3346 vtc1Δ or wild type) was cultured in YPD medium for 24 hours at 30 ° C., then collected by centrifugation and washed twice with distilled water. As bread dough containing low sucrose, 20 g of bread-only flour (Nissin), 1 g of sucrose, 0.4 g of sodium chloride, 12.5 mL of water, and 0.4 g of yeast were kneaded and prepared. Moreover, 30 g of bread exclusive flour (Nissin), 9 g of sucrose, 0.15 g of sodium chloride, 16 mL of water, and 0.6 g of yeast were prepared as a high sucrose-containing bread dough. After stationary culture at 30 ° C., the amount of carbon dioxide generated was measured using a Pharmagraph II-W (Atto). The fermentation test was repeated three times for each strain.
　結果を図２に示す。図２において、横軸は発酵時間（ｈ）、縦軸は二酸化炭素発生量の平均値（ｍＬ）を示す。実施例１と同様に片側ｔ検定を行った。
　図２に示されるとおり、低スクロース含有パン生地および高スクロース含有パン生地のいずれにおいても、ＶＴＣ１遺伝子破壊株は野生株を上回る二酸化炭素発生量を示した。また、発酵開始から２時間以降におけるＶＴＣ１遺伝子破壊株の二酸化炭素発生量は、野生株と比べて１０．１～１６．０％（低スクロース含有パン生地）または１６．３～１７．５％（高スクロース含有パン生地）と、高い値を示した。
　以上の結果から、ＶＴＣ１遺伝子の破壊がパン生地の製造におけるアルコール発酵の促進につながることが明らかになった。 The results are shown in FIG. In FIG. 2, the horizontal axis represents fermentation time (h), and the vertical axis represents the average value (mL) of carbon dioxide generation. A one-sided t-test was performed in the same manner as in Example 1.
As shown in FIG. 2, the VTC1 gene-disrupted strain showed more carbon dioxide generation than the wild strain in both the low sucrose-containing bread dough and the high sucrose-containing bread dough. Further, the carbon dioxide generation amount of the VTC1 gene disrupted strain after 2 hours from the start of fermentation was 10.1 to 16.0% (low sucrose-containing bread dough) or 16.3 to 17.5% (high Sucrose-containing bread dough) and a high value.
From the above results, it was revealed that the disruption of the VTC1 gene leads to the promotion of alcohol fermentation in the production of bread dough.
（実施例３）ＶＴＣ１遺伝子破壊バイオエタノール酵母株の作製、及びその発酵能の測定
　酵母サッカロミセス・セレビシエの二倍体バイオエタノール酵母株であるＰＥ－２株に由来する一倍体ＮＣＹＣ３２３３－２７ｃ株を野生株に使用し、公知の方法（Yeast vol.21 p.947-962 (2004)）によってＶＴＣ１遺伝子破壊株（ＮＣＹＣ３２３３－２７ｃ ｖｔｃ１Δ）を作製した。薬剤耐性遺伝子を含むプラスミドとして、pFA6a-natNT2（クロンナット耐性マーカーを含む）を用いた以外は、実施例２と同様の方法により、ＶＴＣ１遺伝子破壊株を作製した。 Example 3 Production of VTC1 Gene Disrupted Bioethanol Yeast Strain and Measurement of Fermentation Capacitance A haploid NCYC3233-27c strain derived from PE-2 strain which is a diploid bioethanol yeast strain of yeast Saccharomyces cerevisiae A VTC1 gene disruption strain (NCYC3233-27c vtc1Δ) was prepared by a known method (Yeast vol.21 p.947-962 (2004)). A VTC1 gene disruption strain was prepared in the same manner as in Example 2 except that pFA6a-natNT2 (including a clone nut resistance marker) was used as a plasmid containing a drug resistance gene.
　酵母（ＮＣＹＣ３２３３－２７ｃ ｖｔｃ１Δ又は野生株）は、ＹＰＤ培地において一晩３０℃で振とう培養した後、初期酵母密度がＯＤ６００ ＝ ０．１になるように、５０ｍＬのサトウキビ廃糖蜜培地（Ｂｒｉｘ １５、０．０２５％硫酸アンモニウム添加）に植菌した。３０℃で静置培養し、二酸化炭素発生量をファーモグラフＩＩ－Ｗ（アトー）を用いて測定した。発酵試験は各株について独立した２～３クローンを用いた。 Yeast (NCYC3233-27c vtc1Δ or wild type) is cultured with shaking in YPD medium at 30 ° C overnight, and then 50 mL of sugarcane molasses medium (Brix 15, Inoculated with 0.025% ammonium sulfate). After stationary culture at 30 ° C., the amount of carbon dioxide generated was measured using a Pharmagraph II-W (Atto). The fermentation test used 2 to 3 independent clones for each strain.
　結果を図３に示す。図３において、横軸は発酵時間（ｈ）、縦軸は二酸化炭素発生量の平均値（ｍＬ）を示す。実施例１と同様に片側ｔ検定を行った。
　図３に示されるとおり、ＶＴＣ１遺伝子破壊株は野生株を上回る二酸化炭素発生量を示した。発酵開始から４２時間以降におけるＶＴＣ１遺伝子破壊株の二酸化炭素発生量は、野生株と比べて４．８～６．０％高い値を示し、統計的に有意な差が検出された（危険率５％未満）。
　以上の結果から、ＶＴＣ１遺伝子の破壊がバイオエタノール製造におけるアルコール発酵の促進につながることが明らかになった。 The results are shown in FIG. In FIG. 3, the horizontal axis represents fermentation time (h), and the vertical axis represents the average value (mL) of carbon dioxide generation. A one-sided t-test was performed in the same manner as in Example 1.
As shown in FIG. 3, the VTC1 gene-disrupted strain showed a carbon dioxide generation amount higher than that of the wild strain. The amount of carbon dioxide generated by the VTC1 gene-disrupted strain after 42 hours from the start of fermentation was 4.8 to 6.0% higher than that of the wild strain, and a statistically significant difference was detected (risk rate 5). %Less than).
From the above results, it was revealed that the destruction of the VTC1 gene leads to the promotion of alcohol fermentation in bioethanol production.
（実施例４）ＶＴＣ１遺伝子破壊株、ＶＴＣ２遺伝子破壊株、ＶＴＣ３遺伝子破壊株、ＶＴＣ４遺伝子破壊株の作製
　酵母サッカロミセス・セレビシエの一倍体実験室株であるＢＹ４７４１株を野生株として使用し、ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子の破壊株を作製する。薬剤耐性遺伝子を含むプラスミドとして、Yeast vol.21 p.947-962 (2004)に記載のものを用いることができ、例えば、pFA6a-hphNT1又はpFA6a-natNT2等を用いることができる。薬剤耐性遺伝子を含むプラスミドを鋳型として、ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子の各ＯＲＦの上流領域及び下流領域のそれぞれと相同な塩基配列を持つプライマーを用いて、ＰＣＲを行い、薬剤耐性遺伝子の上流及び下流にＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子の上流及び下流と相同な塩基配列を連結させて、ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子破壊用断片を作製する。ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子破壊用断片を作成するために設計したプライマーは以下の通りである。
　VTC1-S1：CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC（配列番号９）
　VTC1-S2：CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG（配列番号１０）
　VTC2-S1:AGTAGAAAGAACGACTACACCTCAACATAACGACACTTTTTTGACCGTACGCTGCAGGTCGAC（配列番号１１）
　VTC2-S2:AATTCTGTCAAAATGAATTATCAGTTGACCCAGAAATCTGTCGCAATCGATGAATTCGAGCTCG（配列番号１２）
　VTC3-S1:TTAGAGCGAACAGCAGAATTTGTCCTTGGTTTTCAGAGTTTGAAACGTACGCTGCAGGTCGAC（配列番号１３）
　VTC3-S2:CTGGTACTTGTGTAATATATGTGTATATAAAAAATATACATGTTCATCGATGAATTCGAGCTCG（配列番号１４）
　VTC4-S1:CAATCAAATCGGCCAATAAAAGAGCATAACAAGGCAGGAACAGCTCGTACGCTGCAGGTCGAC（配列番号１５）
　VTC4-S2:TATGATTATTACTTAATTATACAGTAAAAAAAACACGCTGTGTATATCGATGAATTCGAGCTCG（配列番号１６） (Example 4) Production of VTC1 gene disrupted strain, VTC2 gene disrupted strain, VTC3 gene disrupted strain, VTC4 gene disrupted strain Yeast Saccharomyces cerevisiae haploid laboratory strain BY4741 strain was used as a wild strain, VTC1, Disrupted strains of VTC2, VTC3, and VTC4 genes are prepared. As a plasmid containing a drug resistance gene, those described in Yeast vol.21 p.947-962 (2004) can be used, and for example, pFA6a-hphNT1 or pFA6a-natNT2 can be used. Using a plasmid containing the drug resistance gene as a template, PCR was performed using primers having base sequences homologous to the upstream region and downstream region of each ORF of the VTC1, VTC2, VTC3, and VTC4 genes, and upstream of the drug resistance gene. In addition, a base sequence homologous to the upstream and downstream of the VTC1, VTC2, VTC3, and VTC4 genes is ligated to the downstream to produce fragments for disrupting the VTC1, VTC2, VTC3, and VTC4 genes. The primers designed to create the VTC1, VTC2, VTC3, and VTC4 gene disruption fragments are as follows.
VTC1-S1: CATTATCGAATACGATTAAACACTACGCCAGATTTCCACAATATGCGTACGCTGCAGGTCGAC (SEQ ID NO: 9)
VTC1-S2: CAGTTTGTGCGTAACCCACGCTTACGATATTGGAATTACAATTTCAATCGATGAATTCGAGCTCG (SEQ ID NO: 10)
VTC2-S1: AGTAGAAAGAACGACTACACCTCAACATAACGACACTTTTTTGACCGTACGCTGCAGGTCGAC (SEQ ID NO: 11)
VTC2-S2: AATTCTGTCAAAATGAATTATCAGTTGACCCAGAAATCTGTCGCAATCGATGAATTCGAGCTCG (SEQ ID NO: 12)
VTC3-S1: TTAGAGCGAACAGCAGAATTTGTCCTTGGTTTTCAGAGTTTGAAACGTACGCTGCAGGTCGAC (SEQ ID NO: 13)
VTC3-S2: CTGGTACTTGTGTAATATATGTGTATATAAAAAATATACATGTTCATCGATGAATTCGAGCTCG (SEQ ID NO: 14)
VTC4-S1: CAATCAAATCGGCCAATAAAAGAGCATAACAAGGCAGGAACAGCTCGTACGCTGCAGGTCGAC (SEQ ID NO: 15)
VTC4-S2: TATGATTATTACTTAATTATACAGTAAAAAAAACACGCTGTGTATATCGATGAATTCGAGCTCG (SEQ ID NO: 16)
　ＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子破壊を確認するために設計したプライマーは以下の通りである。
　VTC1-Fw：AACACTACGCCAGATTTCCA（配列番号１７）
　VTC1-Rv：CCACGCTTACGATATTGGAA（配列番号１８）
　VTC2-Fw:AAGAACGACTACACCTCAACA（配列番号１９）
　VTC2-Rv:CATAAAAACACATGGTCTCAG（配列番号２０）
　VTC3-Fw:AGCGAACAGCAGAATTTGTCC（配列番号２１）
　VTC3-Rv:GGTACTTGTGTAATATATGTG（配列番号２２）
　VTC4-Fw:ATAAAAGAGCATAACAAG（配列番号２３）
　VTC4-Rv:GATTATTACTTAATTATACAG（配列番号２４） Primers designed for confirming VTC1, VTC2, VTC3, and VTC4 gene disruption are as follows.
VTC1-Fw: AACACTACGCCAGATTTCCA (SEQ ID NO: 17)
VTC1-Rv: CCACGCTTACGATATTGGAA (SEQ ID NO: 18)
VTC2-Fw: AAGAACGACTACACCTCAACA (SEQ ID NO: 19)
VTC2-Rv: CATAAAAACACATGGTCTCAG (SEQ ID NO: 20)
VTC3-Fw: AGCGAACAGCAGAATTTGTCC (SEQ ID NO: 21)
VTC3-Rv: GGTACTTGTGTAATATATGTG (SEQ ID NO: 22)
VTC4-Fw: ATAAAAGAGCATAACAAG (SEQ ID NO: 23)
VTC4-Rv: GATTATTACTTAATTATACAG (SEQ ID NO: 24)
　作製したＶＴＣ１、ＶＴＣ２、ＶＴＣ３、ＶＴＣ４遺伝子破壊株はいずれも、野生株を上回る二酸化炭素発生量を示すと考えられる。 The produced VTC1, VTC2, VTC3, and VTC4 gene-disrupted strains are considered to show carbon dioxide generation exceeding that of the wild strain.
　本発明により、酵母の発酵能及びエタノール生産性を向上することができる。本発明は、発酵食品、発酵飲料の製造、バイオエタノール生産等といった種々の産業において利用可能である。 According to the present invention, yeast fermentability and ethanol productivity can be improved. The present invention can be used in various industries such as fermented foods, fermented beverages, and bioethanol production.

Claims (15)

  1. 酵母において、液胞トランスポーターシャペロン複合体（ＶＴＣ複合体）の機能を低下又は喪失させることを含む、酵母の発酵能を向上させる方法。 In yeast, the method to improve the fermentability of yeast including reducing or losing the function of a vacuolar transporter chaperone complex (VTC complex).
  2. 酵母のＶＴＣ複合体の機能の低下又は喪失が、複数の構成因子から形成されるＶＴＣ複合体の形成阻害によるものである、請求項１に記載の酵母の発酵能を向上させる方法。 The method for improving the fermentability of yeast according to claim 1, wherein the decrease or loss of the function of the yeast VTC complex is due to inhibition of formation of a VTC complex formed from a plurality of components.
  3. 酵母のＶＴＣ複合体の機能の低下又は喪失が、酵母において遺伝子変異を導入することにより、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の発現量を低下させることによるものである、請求項１または２に記載の酵母の発酵能を向上させる方法。 The decrease or loss of the function of the yeast VTC complex is due to a decrease in the expression level of at least one gene selected from genes encoding the components of the VTC complex by introducing a gene mutation in the yeast. The method for improving the fermentability of the yeast according to claim 1 or 2.
  4. 酵母における遺伝子変異が、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の破壊である、請求項３に記載の酵母の発酵能を向上させる方法。 The method for improving the fermentability of yeast according to claim 3, wherein the genetic mutation in the yeast is a disruption of at least one gene selected from genes encoding components of the VTC complex.
  5. ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子が、ＶＴＣ１遺伝子、ＶＴＣ２遺伝子、ＶＴＣ３遺伝子、ＶＴＣ４遺伝子からなる群から選択される少なくとも１つの遺伝子である、請求項３又は４に記載の酵母の発酵能を向上させる方法。 The at least one gene selected from the genes encoding the constituent elements of the VTC complex is at least one gene selected from the group consisting of the VTC1, VTC2, VTC3, and VTC4 genes. The method to improve the fermentative ability of yeast as described in 2.
  6. ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子が、以下の（ａ）～（ｅ）からなる群から選択される少なくとも１つの遺伝子である、請求項２～５のいずれかに記載の酵母の発酵能を向上させる方法：
    （ａ）配列番号１～４のいずれかに記載のアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
    （ｂ）配列番号１～４のいずれかに記載のアミノ酸配列において１又は数個以上のアミノ酸残基が欠失、置換、挿入及び／又は付加されたアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
    （ｃ）配列番号１～４のいずれかに記載のアミノ酸配列と８０％以上の相同性を有するアミノ酸配列をコードするポリヌクレオチドを含む遺伝子；
    （ｄ）配列番号５～８のいずれかに記載の塩基配列からなるポリヌクレオチドを含む遺伝子；
    （ｅ）上記（ａ）～（ｄ）からなる群から選択されるいずれかのポリヌクレオチドと相補的な塩基配列からなるポリヌクレオチドを含む遺伝子。     The at least one gene selected from the genes encoding the constituent elements of the VTC complex is at least one gene selected from the group consisting of the following (a) to (e): A method for improving the fermentability of yeast according to crab:
    (A) a gene comprising a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
    (B) a gene comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
    (C) a gene comprising a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 4;
    (D) a gene comprising a polynucleotide comprising the base sequence set forth in any one of SEQ ID NOs: 5 to 8;
    (E) A gene comprising a polynucleotide comprising a base sequence complementary to any polynucleotide selected from the group consisting of (a) to (d) above.
  7. 請求項１～６のいずれかに記載の酵母の発酵能を向上させる方法を含む、酵母を用いて発酵産物を製造する方法であって、酵母においてＶＴＣ複合体の機能が低下又は喪失している条件で、アルコール発酵を行うことを含む、発酵産物の製造方法。 A method for producing a fermentation product using yeast, comprising the method for improving the fermentability of yeast according to any one of claims 1 to 6, wherein the function of the VTC complex is reduced or lost in yeast. A method for producing a fermentation product, comprising performing alcoholic fermentation under conditions.
  8. 発酵産物がエタノールである、請求項７に記載の発酵産物の製造方法。 The method for producing a fermentation product according to claim 7, wherein the fermentation product is ethanol.
  9. 発酵産物が発酵飲料又は発酵食品である、請求項７に記載の発酵産物の製造方法。 The method for producing a fermented product according to claim 7, wherein the fermented product is a fermented beverage or a fermented food.
  10. 酵母において、遺伝子変異を導入することにより、ＶＴＣ複合体の機能を低下又は喪失させることを含む、アルコール発酵用酵母の作製方法。 A method for producing a yeast for alcohol fermentation, comprising reducing or losing the function of a VTC complex by introducing a gene mutation in the yeast.
  11. 遺伝子変異により、ＶＴＣ複合体又はＶＴＣ複合体の構成因子の機能が低下又は喪失した、アルコール発酵用酵母。 A yeast for alcoholic fermentation in which the function of a VTC complex or a component of a VTC complex is reduced or lost due to a gene mutation.
  12. 遺伝子変異が、ＶＴＣ複合体の構成因子をコードする遺伝子から選択される少なくとも１つの遺伝子の破壊である、請求項１１に記載のアルコール発酵用酵母。 The yeast for alcoholic fermentation according to claim 11, wherein the gene mutation is a disruption of at least one gene selected from genes encoding a component of the VTC complex.
  13. 遺伝子変異により、ＶＴＣ複合体の機能が低下又は喪失した酵母であり、醸造酵母、パン酵母、又はバイオエタノール酵母のいずれかである酵母。 A yeast in which the function of the VTC complex is reduced or lost due to a gene mutation, and is a brewery yeast, baker's yeast, or bioethanol yeast.
  14. 請求項１１～１３のいずれかに記載の酵母を含有する、アルコール発酵用組成物。 A composition for alcoholic fermentation comprising the yeast according to any one of claims 11 to 13.
  15. 酵母の発酵能を向上させるための薬剤のスクリーニング方法であって、被験化合物をＶＴＣ複合体に作用させる工程を含み、当該被験化合物がＶＴＣ複合体の機能を低下又は喪失させることを指標とする、スクリーニング方法。 A method for screening a drug for improving the fermentability of yeast, comprising a step of causing a test compound to act on a VTC complex, wherein the test compound decreases or loses the function of the VTC complex, Screening method.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006280268A (en) * 2005-03-31 2006-10-19 Chiba Univ Method for improving alcohol production efficiency by promoting cell proliferation
JP2014076046A (en) * 2012-09-19 2014-05-01 Saga Univ Method for enhancing ethanol production and fermentative performance of yeast by disruption of its mitophagy function

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006280268A (en) * 2005-03-31 2006-10-19 Chiba Univ Method for improving alcohol production efficiency by promoting cell proliferation
JP2014076046A (en) * 2012-09-19 2014-05-01 Saga Univ Method for enhancing ethanol production and fermentative performance of yeast by disruption of its mitophagy function

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
COHEN, ADIEL ET AL.: "A Novel Family of Yeast Chaperons Involved in the Distribution of V- ATPase and Other Membrane Proteins", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 38, 1999, pages 26885 - 26893, XP055408179 *
MULLER, OLIVER ET AL.: "Role of the Vtc proteins in V-ATPase stability and membrane trafficking", JOURNAL OF CELL SCIENCE, vol. 116, 2003, pages 1107 - 1115, XP055408169 *
MULLER, OLIVER ET AL.: "The Vtc proteins in vacuole fusion: coupling NSF activity to VO trans-complex formation", THE EMBO JOURNAL, vol. 21, no. 3, 2002, pages 259 - 269, XP055408167 *
NELSON, NATHAN ET AL.: "The Cellular Biology of Protein-Motive Force Generation by V-ATPase", THE JOURNAL OF EXPERIMENTAL BIOLOGY, vol. 203, 2000, pages 89 - 95, XP055408175 *
UTTENWEILER, ANDREAS ET AL.: "The Vacuolar Transporter Chaperone (VTC) Complex is Required for Microautophagy", MOLECULAR BIOLOFY OF THE CELL, vol. 18, 2007, pages 166 - 175, XP055408180 *

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