WO2007097073A1 - Gène codant pour une sous-unité régulatrice de l'activité d'une acétolactate synthase et utilisation dudit gène - Google Patents

Gène codant pour une sous-unité régulatrice de l'activité d'une acétolactate synthase et utilisation dudit gène Download PDF

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WO2007097073A1
WO2007097073A1 PCT/JP2006/322059 JP2006322059W WO2007097073A1 WO 2007097073 A1 WO2007097073 A1 WO 2007097073A1 JP 2006322059 W JP2006322059 W JP 2006322059W WO 2007097073 A1 WO2007097073 A1 WO 2007097073A1
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polynucleotide
yeast
seq
protein
amino acid
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Japanese (ja)
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Yoshihiro Nakao
Yukiko Kodama
Tomoko Shimonaga
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Suntory Limited
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    • 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
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • 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
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/004—Genetically modified microorganisms
    • 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
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/006—Yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a gene encoding a acetolactate synthase activity regulatory subunit and use thereof. More particularly, the present invention relates to a brewer's yeast producing a liquor with excellent flavor, a liquor produced using the yeast, a method for producing the same. More specifically, the present invention relates to a product by suppressing the expression level of a gene ILV6 encoding a lactic acid synthase activity-regulating subunit of brewing yeast, particularly the nonScILV6 gene characteristic of bi / yeast. The present invention relates to a vicinal diketone which is an off-flavor of the yeast, particularly a yeast with reduced diacetyl production, and a method for producing an alcoholic beverage using the yeast. Background Technology ''s yeast producing a liquor with excellent flavor, a liquor produced using the yeast, a method for producing the same. More specifically, the present invention relates to a product by suppressing the expression level of a gene ILV6 encoding a lactic acid synth
  • diacetyl (DA) odor is one of the typical off-flavors in brewed liquors such as beer, sake and wine.
  • DA odor (expressed as stuffy odor or butter odor in beer, or scented odor in sake) is caused by the presence of vicinal diketones (hereinafter referred to as VDK) mainly consisting of DA in the product,
  • VDK vicinal diketones
  • VDKs in liquors are broadly divided into DA and 2,3-pentanedione (PD).
  • DA and PD are produced by non-enzymatic reactions that do not involve yeast, using ⁇ -case lactic acid and hyase 'hydroxybutyric acid as precursors, valine and isoleucine biosynthesis intermediates, respectively.
  • VM DA and PD
  • -acetohydroxy acids hyacetolactic acid and -acetohydroxybutyric acid
  • Yeast breeding that stably reduces these can not only facilitate the production management of alcoholic beverages, but also expand the possibilities of new product development.
  • acetolactic acid synthase is an enzyme that converts pyruvate or ⁇ -oxobutyric acid to ⁇ -acetolactic acid or ⁇ -acetohydroxybutyric acid, respectively.
  • a gene encoding yeast acetolactic acid synthase It is known that ILV2 and ILV6 exist, ILV2 encodes the active subunit and ILV6 encodes the regulatory subunit.
  • Japanese Patent Application Laid-Open No. 2002-291465 discloses a method of obtaining mutant strains that are sensitive to analogs of these branched amino acids and selecting a DA low accumulation strain from them.
  • genetically engineered yeasts that regulate the expression level of the ILV3 gene are also reported.
  • the enzymatic activity of the acetate hydroxy acid reductoisomerase encoded by the ILV5 gene increased 5-7 times, and the amount of VM produced decreased to about 40%.
  • An object of the present invention is to breed a VDK, particularly a gene encoding a protein capable of reducing the generation of DA odor in yeast, and to cultivate a VM low-productivity yeast using the protein, and to produce an alcoholic beverage with excellent flavor. It is possible to manufacture.
  • the present invention provides a gene encoding a novel acetolactic acid synthase activity regulating subunit characteristically present in peel yeast, a protein encoded by the gene, a transformed yeast in which expression of the gene is regulated,
  • the present invention relates to a method for controlling the VDK concentration in a product, particularly the DA concentration, by using yeast in which gene expression is regulated.
  • the present invention uses the following polynucleotide, vector or DNA fragment containing the polynucleotide, transformed yeast introduced with the vector or DNA fragment, and the transformed yeast. Providing methods for producing alcoholic beverages.
  • a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO: 2 and having ability to regulate acetolactic acid synthase activity;
  • polynucleotides having a nucleotide sequence of SEQ ID NO: 1 or polynucleotides having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 are hyperprecipitated under highly stringent conditions;
  • polynucleotide according to (1) above which comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.
  • test yeast is cultured, and the protein described in (7) above is quantified, or the gene encoding the case-lactic acid synthase activity-regulating subunit having the base sequence of SEQ ID NO: 1.
  • a method for selecting yeast comprising measuring an expression level and selecting a test yeast having a production amount of the protein or an expression level of the gene according to a target total vicinal diketone production ability or total diacetyl production ability.
  • Reference yeast and test yeast are cultured and the expression level of the gene encoding the lactic acid synthase activity regulatory subunit having the nucleotide sequence of SEQ ID NO: 1 is measured in each yeast.
  • the method for selecting yeast according to (20) above, wherein a test yeast having a low expression is selected.
  • the reference yeast and the test yeast are cultured, the protein described in (7) above in each yeast is quantified, and the test yeast having a smaller amount of the protein than the reference yeast is selected.
  • 20. The method for selecting yeast according to 2). That is, culturing a plurality of yeasts, quantifying the protein described in (7) above in each yeast, and selecting a test mother having a small amount of the protein among them, wherein the yeast described in (20) above is selected Selection method.
  • FIG. 1 is a diagram showing the change over time in the amount of yeast grown in beer test brewing.
  • the horizontal axis shows the fermentation time, and the vertical axis shows the value of 0D660.
  • Fig. 2 is a diagram showing the change over time in the amount of extract consumed in beer test brewing.
  • the horizontal axis shows fermentation time, and the vertical axis shows appearance extract concentration (w / w%).
  • FIG. 3 shows the expression behavior of the nonScILV6 gene in yeast during brewing of beer.
  • the horizontal axis shows the fermentation time, and the vertical axis shows the detected signal brightness.
  • the present inventors have isolated and identified a nonScILV6 gene encoding a acetolactate synthase activity regulatory subunit specific to brewer's yeast, based on the brewer's yeast genome information decoded by the method disclosed in JP-A-2004-283169.
  • This base sequence is shown in SEQ ID NO: 1.
  • the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2.
  • VDK and its precursor ⁇ -acetohydroxy acid are collectively referred to as “all vicinal diketones”.
  • DA and its precursor hyase lactic acid are collectively referred to as “all diacetyl”. 1: Polynucleotide of the present invention
  • the present invention relates to (a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and 0)) a polynucleotide comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide may be DNA or RNA.
  • the polynucleotide targeted by the present invention is not limited to the polynucleotide that codes for the above-mentioned lactic acid synthase activity-regulating subunit derived from brewer's yeast, and is functionally equivalent to this protein.
  • functionally equivalent proteins include: (c) an amino acid sequence of SEQ ID NO: 2, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and Z or added; Examples include proteins having the ability to regulate lactic acid synthase activity.
  • 'As such a protein, in the amino acid sequence of SEQ ID NO: 2, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 -40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1 -30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1 -20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1 -10, 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, -3, 1-2, 1 Consisting of an amino acid sequence in which one amino acid residue is deleted, substituted, inserted and / or added, and A protein having an ability to regulate trilactic acid synthase activity.
  • such a protein includes (d) the amino acid sequence of SEQ ID NO: 2, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88 % Or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 % Or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, '99 .5% or higher, '99 .6% or higher, 99.7% or
  • the present invention also relates to (e) a protein that is hybridized under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and that has the ability to regulate acetate lactate synthase activity.
  • polynucleotide that hybridizes under stringent conditions is a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like with all or a part of the probe as a probe.
  • the hybridization method for example, the method described in 'Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997, etc. can be used.
  • stringent conditions refers to low stringency conditions, Either stringent conditions or highly stringent conditions may be used.
  • Low stringent conditions are, for example, conditions of 5 X SSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide.
  • the “medium stringent conditions” are, for example, conditions of 5 X SS 5 X Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C.
  • “High stringent conditions” are, for example, 5 X SS (:, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
  • Polynucleotides with high homology (eg, DNA) force S can be expected to be obtained efficiently, however, factors that affect the stringency of high-pridition include temperature, probe concentration, and probe length. Several factors such as ionic strength, time, and salt concentration are conceivable, and those skilled in the art can achieve the same stringency by selecting these factors as appropriate.
  • Alkphos Direct Labeling Reagents manufactured by Amersham Almacia
  • the protocol attached to the kit incubate the labeled probe overnight, then remove the membrane in the primary wash buffer containing 0.1% (w / v) SDS under 55 conditions. After washing, the hybridized polynucleotide (eg DNA) can be detected.
  • polynucleotides that can be hyper-predicted are those that encode the amino acid sequence of SEQ ID NO: 2 when calculated using the same parameters as FASTA, BLAST, etc. About 60% or more, About 70% or more, 71% or more, .72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, .78% or more, 79% or more, 80 % Or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 Polynucleotides having% or more, 99.5 or more, 99.6 or more, 99.7 or more
  • the polynucleotide of the present invention comprises (j) a polynucleotide that encodes a polynucleotide having a base sequence complementary to the transcript of the polynucleotide (MA) described in (5) above; ) A polynucleotide that encodes an RNA that suppresses the expression of the polynucleotide (DNA) described in (S) above by the RNAi effect; (1) a specific transcript of the polynucleotide (MA) described in (5) above; And (m) a polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) described in (5) above by a co-suppression effect.
  • polynucleotides can be suppressed in the expression of the polynucleotides (DNA) (a) to (i) in transformed cells into which the vector has been introduced and further incorporated into the vector. Therefore, it can be suitably used for suppressing the expression of the polynucleotide (DNA).
  • polynucleotide encoding RNA having a base sequence complementary to a transcription product of DNA refers to so-called antisense DNA.
  • Antisense technology is known as a method for suppressing the expression of specific endogenous genes, and is described in various literatures (for example, Hirashima and Inoue: Shinsei Chemistry Laboratory 1 Nucleic Acid IV Remnants. Expression (refer to pp. 319-347, 1993, etc.).
  • the sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof, but may not be completely complementary as long as the gene expression can be effectively suppressed.
  • the transcribed RNA preferably has a complementarity of 90% or more, more preferably 95% or more, to the transcript of the target gene.
  • the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more.
  • RNAiJ is a phenomenon in which when a double-stranded RNA having the same or similar sequence as the target gene is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both suppressed.
  • RNA used here include double-stranded RNA that causes RNA interference of 21 to 25 bases, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin)
  • dsRNA double strand RNA
  • siRNA small interfering RNA
  • shRNA short hairpin
  • RNA can be locally delivered to a desired site by a delivery system such as a ribosome, and this can be achieved by using a vector that produces the above double mRNA.
  • Methods for preparing and using such double-stranded RNA are known from many literatures (Japanese translations of PCT publication No.
  • the “polynucleotide encoding RNA having an activity of specifically cleaving DNi transcript” generally refers to a ribozyme.
  • Lipozyme is an RNA molecule that has catalytic activity, and inhibits the function of the gene by cleaving the target MA transcript.
  • Various known literatures can also be referred to for the design of lipozymes (for example, FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323). : 349, 1986; Nucl. Acids. Res. 19: 6751, 1991; Protein Eng 3: 733, 1990; Nucl. Acids Res.
  • RNA that suppresses MA expression by co-suppression effect refers to a nucleotide that inhibits the function of the target DNA by “co-suppression”.
  • co-suppression refers to the introduction of a gene having a sequence identical or similar to a target endogenous gene into a cell by transformation: the introduced foreign gene and the frictional endogenous gene. It refers to each phenomenon in which expression is suppressed.
  • Protein of the Present Invention-The present invention also provides a protein encoded by any of the polynucleotides (a) to (i).
  • a preferred protein of the present invention comprises: an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2; It is a protein having the ability.
  • Such a protein comprises an amino acid sequence in which the number of amino acid residues as described above is deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, and And a protein having the ability to regulate the activity of the enzyme.
  • Examples of such a protein include a protein having the amino acid sequence having the homology as described above with the amino acid sequence of SEQ ID NO: 2 and having the ability to regulate acetolactic acid synthase activity.
  • deletion, substitution, insertion and Z or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues in one or more amino acid sequences in the same sequence It means that there are amino acid residue deletion, substitution, insertion and / or addition, and two or more of deletion, substitution, insertion and addition may occur simultaneously. Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
  • Group A Leucine, Isoleucine, Norleucine, Norin, Norpaline, Alanine, 2-Aminobutanoic acid, Methionine, 0-Methylserine, t-Butylglycine, N-Butylalanin, Cyclohexylalanin;
  • Group B Aspartic acid, Glutamic acid, Isoaspartic acid , Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; group C: asparagine, glutamine;
  • group D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diamino Propionic acid;
  • Group E proline, 3-hydroxyproline, 4-hydroxyproline;
  • Group F serine, threonine, moserin;
  • Group G phenylalanin, tyrosine.
  • the protein of the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • a vector according to the present invention contains the polynucleotide (for example, DNA) described in any one of (a) to (i) above.
  • the vector of the present invention usually has (X) a promoter that can be transcribed in yeast cells so as to suppress the expression of the polynucleotide (DNA) described in any of (a) to (i) above.
  • polynucleotides are introduced into a vector containing the polynucleotide according to any one of (j) to (! II) so that the polynucleotide can be expressed.
  • the target gene is introduced into a vector containing the polynucleotide according to any one of (j) to (! II) so that the polynucleotide can be expressed.
  • the target gene is introduced into a vector containing the polynucleotide according to any one of (j) to (! II) so that the polynucleotide can be expressed.
  • the target gene is introduced into a vector containing the polynucleotide according to any one of (j) to (! II) so that the polynucleotide can be expressed.
  • Gene destruction is the gene in the target gene This can be done by adding or deleting single or multiple bases within the region involved in the expression of the product, for example, the coding region ⁇ 3 promoter region, or by deleting these entire regions.
  • known literature can be referred to (for example, Proc. Natl. Acad. Sci. USA, 76, 4951 (1979), Methods in Enzymology, 101, 202 (1983), (See Kaihei 6-253826).
  • any of a multicopy type (YEp type), a single copy type (YCp type), and a chromosomal integration type (Yip type) can be used.
  • YEp type J.. Broach et al., Experimental Manipulat ion of Gene Express ion, Academic Press, New York, 83, 1983
  • YCp50 MD Rose et al., Gene, 60, 237, 1987
  • YIp5 K. Struhl et al., Proc. Natl. Acad. Sci. USA, 76, 1035, 1979
  • Yip type vectors can be easily obtained. .
  • any combination may be used as long as it functions in brewing yeast and is not affected by components in mash.
  • a promoter of the dalyceraldehyde 3 phosphate dehydrogenase gene (TDH3) and a promoter of the 3-phosphodarylate kinase gene (PGK1) can be used.
  • TDH3 dalyceraldehyde 3 phosphate dehydrogenase gene
  • PGK1 3-phosphodarylate kinase gene
  • dieneticin resistance gene G418r
  • copper resistance gene are not available in the case of brewer's yeast.
  • CUP1 Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 1984
  • cerulenin resistance gene fas2ni, PDR4
  • fas2ni cerulenin resistance gene
  • PDR4 Ashigaki, et al., Biochemistry, 64, 660, 1992, respectively
  • Hussain et al., Gene, 101, 149, 1991 can be used.
  • the vector constructed as described above is introduced into the host yeast.
  • the host yeast include any yeast that can be used for brewing, for example, brewery yeast for beer, wine, sake and the like. Specific examples include yeasts of the genus Saccharomyces, and in the present invention, beer yeasts such as Saccharomyces pas torianus W34 / 70, Saccharomyces carlsberg, etc. Saccharomyces carl sbergens is NCYC453, NCYC456 etc., Saccharomyces cerevis iae NBRC195 NB C1952, NBRC1953, NBRC1954 'etc. can be used.
  • whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as association wine, No. 1, 3 and 4, etc., sake yeasts such as association yeast sake 7 and 9 etc. Yes, but not limited to this.
  • beer yeast such as Saccharomyces pastorianus is preferably used.
  • yeast transformation method a publicly known method can be used.
  • electroboration method “Meth. Enzymol., 194, pl82 (1990)”
  • Spheroplast method Proc. Natl. Acad. Sci. USA, 75 p 1.929 (1978)
  • the lithium acetate method “L Bacteriology , 153, pl63 (1983) ", Proc. Natl. Acad. Sci. USA, 75 pl929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual It can be implemented, but is not limited to this.
  • the host yeast is transformed into a standard yeast nutrient medium (for example, YEPD medium "Genetic Engineering. Vol. 1, Plenum Press, New York, 117 (1979)") with an OD600nm value of s 1-6.
  • a standard yeast nutrient medium for example, YEPD medium "Genetic Engineering. Vol. 1, Plenum Press, New York, 117 (1979)"
  • alkali metal ions preferably lithium ions
  • the cells are allowed to stand for about 3 (about 60 minutes at TC and then with the DNA to be introduced (about 1 to 20 g) at about 30 ° C. for about 60 minutes.
  • Polyethylene glycol preferably about 4, Add 000 daltons of polyethylene glycol to a final concentration of about 20% to 50% ..
  • this cell suspension is washed with standard yeast nutrient medium, placed in a predetermined amount of fresh standard yeast nutrient medium, and allowed to stand for about 3 (about 60 minutes at TC. Then select Transformants are obtained by planting on a standard agar medium containing antibiotics used as markers.
  • the desired liquor is reduced in the amount of VDK, particularly A, and excellent in flavor.
  • a yeast introduced with the above-described vector of the present invention a yeast in which expression of the above-described polynucleotide (DNA) of the present invention is suppressed, or a yeast selected by the yeast evaluation method of the present invention described below is selected. It is possible to produce a desired liquor and a liquor with a reduced VDK content, particularly DA content, by performing fermentation for liquor production and reducing the VDK production amount, particularly DA production amount.
  • alcoholic beverages to be covered include, but are not limited to, beer-taste drinks such as beer and happoshu, wine, whiskey, and sake.
  • alcoholic beverages with excellent flavor can be produced using existing facilities without increasing costs.
  • the present invention uses a primer or a probe designed on the basis of a nucleotide sequence of a gene that codes for a lactate synthase activity regulatory unit having the nucleotide sequence of SEQ ID NO: 1. Or, it relates to a method for evaluating DA production ability.
  • a general method of such an evaluation method is known, and is described in, for example, W001Z040514, JP-A-8-205900, and the like. This evaluation method is briefly explained below.
  • test yeast genome prepares the test yeast genome.
  • any known method such as Hereford method or potassium acetate method can be used (for example, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, pl30 (1990)).
  • a gene encoding a acetolactate synthase activity regulatory subunit in the obtained genome Using a primer or probe designed based on the nucleotide sequence (preferably 0RF sequence), it is examined whether the gene or the gene-specific sequence exists in the genome of the test yeast.
  • the primer or probe can be designed using a known method.
  • Detection of a gene or a specific sequence can be carried out using a known method.
  • a polynucleotide containing a part or all of a specific sequence or a polynucleotide containing a base sequence complementary to the base sequence is used as one primer, and the other primer is more than this sequence.
  • Amplification of yeast nucleic acid by PCR using a polynucleotide containing a part or all of the upstream or downstream sequence or a polynucleotide containing a base sequence complementary to the base sequence, and the presence or absence of the amplified product Measure the molecular weight of an object.
  • the number of bases of the polynucleotide used for the primer is usually 10 bp or more, preferably 15 to 25 bp.
  • the base number of the sandwiched portion is usually 300 to 2000 bp.
  • the reaction conditions of the PCR method are not particularly limited. For example, denaturation temperature: 90 to 95 ° C, annealing temperature: 40 to 60 ° C, extension temperature: 60 to 75, cycle number: 10 times or more, etc. Conditions can be used.
  • the obtained reaction product is separated by electrophoretic method using agarose gel or the like, and the molecular weight of the amplified product can be measured. This method predicts and evaluates the ability of the yeast to produce vicinal diketone (VDK) or total diacetyl (DA) depending on whether the molecular weight of the amplified product is large enough to include the DNA molecules of the specific part. . Further, by analyzing the base sequence of the amplified product, the above performance can be predicted and evaluated more accurately.
  • the test yeast is cultured, and the expression level of the gene coding for the acetate lactate synthase activity regulatory unit having the base sequence of SEQ ID NO: 1 is measured, whereby all of the test yeast is obtained.
  • the ability to produce vicinal diketone (VDK) or total diacetyl (DA) can also be evaluated.
  • the expression level of the gene encoding the lactic acid synthase activity-regulating subunit can be measured by culturing the test yeast, and then measuring the transcript of the gene encoding the acetolactic acid synthase activity-regulating unit. It is possible by quantifying the quality. Quantification of ⁇ or protein can be performed using a known method. Quantification of mRNA is, for example, Northern Hybridization By quantitative RT-PCR, protein quantification can be performed, for example, by Western blotting (Current Protocols in Molecular Biology, John Wiley & Sons 1994-2003).
  • the test yeast is cultured, and the expression level of the gene encoding the acetolactic acid synthase activity-regulating subunit having the nucleotide sequence of SEQ ID NO: 1 is measured, and the desired total vicinal diketone (VDK) production ability
  • a yeast suitable for brewing a desired liquor can be selected by selecting a yeast having the gene expression level according to the total diacetyl (DA) production ability ⁇ ).
  • the reference yeast eg, genome decoding strain Saccharomyces pastorianus' byhenstefan 34/70
  • the test yeast are cultured, the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast is determined.
  • a yeast suitable for brewing a desired liquor can be selected.
  • test yeast by culturing the test yeast and selecting a yeast having a low ability to produce total vicinal diketone (VDK) or total diacetyl (DA), or a low acetolactate synthase activity, the desired alcoholic beverage can be brewed.
  • a suitable test yeast can be selected.
  • examples of the test yeast or the reference yeast include a yeast introduced with the vector of the present invention described above, and the above-described polynucleotide (DNA) of the present invention. Fermentation in which expression is suppressed, yeast in which the expression of protein poor according to the present invention described above is suppressed, yeast that has been subjected to mutation treatment, naturally-mutated yeast, and the like can be used.
  • VDK or DA production ability can be measured by known methods.
  • the amount of total vicinal diketone can be determined by the method described in Drews et al., ⁇ . Fur Brau., 34, 1966. Quantification of the total amount of diacetyl can be carried out, for example, by the method described in J Agric Food Chem. 50 (13): 3647-53, 2002.
  • the acetate lactate synthase activity can be measured, for example, by the method of Pang et al. (Biochemistry, 38, 5222-5231 (1999)).
  • Mutation treatment can be performed by any method, for example, physical methods such as ultraviolet irradiation and radiation irradiation, chemical methods using chemical treatment such as EMS (ethyl methanesulfonate), N-methyl-N-nitrosoguanidine, etc.
  • physical methods such as ultraviolet irradiation and radiation irradiation
  • chemical methods using chemical treatment such as EMS (ethyl methanesulfonate), N-methyl-N-nitrosoguanidine, etc.
  • Good for example, edited by Taiji Oshima, Biochemical Experimental Method 39 Yeast Molecular Genetics Experimental Method, p 67-75, Society Press
  • yeasts that can be used as the reference yeast or the test yeast include any yeast that can be used for brewing, for example, brewery yeast for beer, wine, sake and the like. Specific examples include yeasts of the genus Saccharomyces (for example, Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces cerevisiae / cis). In the present invention, beer yeasts such as Saccharomyces pastorianus ( Saccharomyces pas tor ianus) W34 / 70, Saccharomyces carlsbergens is NCYC453, NCYC456, Saccharomyces cerevis iae NBRC195 L 195RC4, NB Can be used.
  • Saccharomyces pastorianus Saccharomyces pas tor ianus
  • Saccharomyces carlsbergens is NCYC453, NCYC456, Saccharomyces cerevis iae NBRC195 L 195RC4, NB Can be used.
  • grape yeast for example, association wine sake No. 1, 3, 4 etc.
  • sake yeast for example, association yeast sake no. 7, 9 etc.
  • beer yeasts such as Saccharomyces pastorianus are preferably used.
  • the reference yeast and the test yeast may be selected from any combination of the above yeasts.
  • the beer was brewed using the brewer's yeast Saccharomyces pastorianus bihenstefan W34 / 70. Wort extract concentration 12. 69%
  • the fermentation broth was sampled over time, and changes in the yeast growth (Fig. 1) and extract concentration (Fig. 2) over time were observed.
  • yeast cells were sampled, and the prepared mRNA was labeled with piotin and hybridized to a beer yeast MA microarray.
  • Signal detection was performed using a GeneChip operating system (GCOS; GeneChip, Operating Software 1.0, manufactured by Affymetrix).
  • GCOS GeneChip operating system
  • the expression pattern of the nonScILV6 gene is shown in FIG. From this result, it was confirmed that the non'ScILV6 gene was expressed in normal beer fermentation.
  • Example 2 NonScILV6 gene disruption
  • NonScILV6_delta_for SEQ ID NO: 3
  • nonScILV6_delta_rv SEQ ID NO: 4
  • the spore clone strain (W34 / 70-2) isolated from the brewer's yeast Saccharomyces pastorianus strain W34 / 70 is transformed with the gene disruption fragment prepared by the method described above. Transformation is carried out by the method described in Japanese Patent Application Laid-Open No. 07-303475, and includes dieneticin (300 mg / L), nourseoihricin (50 mg / L) or hygromycin B (Hygroiycin B) (200 mg / L).
  • YPD plate medium 1% yeast extract,
  • VDK Yeast input 5g Wet yeast cells ZL wort Sampling fermented koji over time, and examine changes in yeast growth (OD660) and extract consumption over time.
  • the total amount of VDK in the cocoon is determined by reacting VDK (DA and PD) with hydroxylamine and measuring the absorbance of the complex formed by the reaction of the resulting darioxime derivative with divalent iron ions (Drews et al. al., Mon. fur Brau., 34, 1966).
  • the precursors ⁇ -acetolactic acid and ⁇ -acetohydroxybutyric acid are converted into DA and PD by gas scrubbing (oxidative decarboxylation) in advance, respectively.
  • VDK amount Industrial applicability
  • the production amount of VDK, which is off-flavor in the product, particularly DA production is reduced, and it becomes possible to easily produce alcoholic beverages with excellent flavor.

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Abstract

La présente invention concerne un gène codant pour une sous-unité régulatrice de l'activité d'une acétolactate synthase et l'utilisation dudit gène. L'invention concerne en particulier une souche de levure de bière servant à produire une boisson alcoolisée ayant une bonne flaveur, une boisson alcoolisée produite à l'aide de cette souche de levure et un procédé de production de ladite boisson. Cette invention concerne plus particulièrement une souche de levure dont la capacité de production de dicétones vicinales, en particulier de diacétyle, responsables d'une flaveur atypique d'un produit, a été réduite par réduction du niveau d'expression du gène ILV6 codant pour IIv6p, une acétolactate synthase dans une souche de levure de bière, en particulier du gène ILV6 non Sc propre à la levure de bière, ainsi qu'un procédé de production d'une boisson alcoolisée à l'aide de ladite souche de levure.
PCT/JP2006/322059 2006-02-23 2006-10-30 Gène codant pour une sous-unité régulatrice de l'activité d'une acétolactate synthase et utilisation dudit gène WO2007097073A1 (fr)

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JP2006047566A JP2009165351A (ja) 2006-02-23 2006-02-23 アセト乳酸シンターゼをコードする遺伝子及びその用途
JP2006-047566 2006-02-23

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CULLIN C. ET AL.: "Functional Analysis of YCL09C: Evidence for a Role as the Regulatory Subunit of Acetolactate Synthase", YEAST, vol. 12, 1996, pages 1511 - 1518, XP003017234 *
SIEW SIEW PANG ET AL.: "Expression, Purification, Characterization, and Reconstitution of the Large and Small Subunits of Yeast Acetohydroxyacid Synthase", BIOCHEMISTRY, vol. 38, 1999, pages 5222 - 5231, XP003017235 *

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