JP4537094B2 - Screening method for yeast genes for brewing - Google Patents

Description

  The present invention relates to a method for screening genes for industrial yeasts used in the production of alcoholic beverages such as beer and sake, alcohol for fuels, and particularly brewing yeasts used in the production of alcoholic beverages. Specifically, in the production of alcoholic beverages, a database of brewing yeast base sequence information, a method for selecting genes involved in improving productivity and / or flavor stabilization such as flavor stabilization and enhancement, and the gene disruption The present invention relates to a method for breeding a yeast suitable for brewing by controlling the expression of the gene by producing a yeast or a yeast in which the gene is highly expressed, and a method for producing an alcoholic beverage using the bred yeast.

Development of production technologies for alcohol for fuel, alcoholic beverages such as beer and sake using industrial yeasts has been carried out. In particular, in the production of alcoholic beverages using brewer's yeast, the development of technologies for improving flavors, such as improving the productivity of alcoholic beverages and stabilizing or improving flavors, has been actively promoted. .
The world's most drunk alcoholic beverage is beer, and the world production in 2001 is about 140 million kL. There are three types of beer depending on the type of yeast and the fermentation method. A naturally fermented beer fermented using yeast and microorganisms that have sown in the brewery, top fermenting yeast belonging to S. cerevisiae, fermented at a temperature of 20-25 ° C, and ale type that shortens the subsequent aging period There are three types of beer: Lager type beer that is fermented at a temperature of 6 to 15 ° C. using a bottom fermentation yeast belonging to Saccharomyces pastorianus (hereinafter abbreviated as S. pastorianus) and then aged at low temperature. Currently, more than 90% of the world's beer production is Lager type beer. Therefore, the bottom fermenting yeast used for brewing Lager type beer is most widely used in brewing beer.

In so-called fermentation production, which uses microorganisms, including the above brewing yeast, optimizing the fermentation process and selecting and breeding useful strains can improve productivity and product quality. It is important for improvement.
For example, in the case of beer brewing, the optimization of the beer brewing process involves monitoring yeast metabolites such as ethanol and other alcohols, esters, and organic acids, and controlling the temperature, aeration rate, or raw material concentration. Has been done. These controls treat the transfer of substances into and out of brewer's yeast cells and the metabolism of intracellular substances as a black box, and perform only superficial control. In addition, for example, a process control method that suppresses the oxygen supply during beer brewing has been attempted for the purpose of, for example, imparting a high flavor to alcoholic beverages. However, it may cause adverse effects such as delayed fermentation and reduced quality. Therefore, there has been a limit to improving productivity and quality by optimizing the fermentation process.

On the other hand, as a method for breeding useful industrial yeast, for example, useful brewer's yeast, a technique of selecting an excellent strain rather than breeding has been widely used. Beer brewing itself has been carried out long before the discovery of microorganisms by Pasteur, and in beer brewing, the traditional method of selecting the finest brewer's yeast used in breweries has been carried out actively. There are few examples of breeding beer yeast with excellent traits.
For example, as an active breeding method, there is a method using artificial mutagenesis by drugs or radiation, but brewing yeast, especially bottom fermentation yeast widely used in beer brewing, is a higher order polyploid. In many cases of such higher-order polyploid yeast, the desired mutant strain cannot be obtained unless all the alleles to be mutated are mutated. Thus, causing the desired mutation is not possible with higher order polyploid brewer's yeast, even though it is possible with haploid laboratory yeast.
In recent years, spores are isolated from bottom fermenting yeast, and breeding by mutation introduction and hybridization using them has been attempted (see, for example, Non-Patent Document 1). However, bottom fermented yeast, which is a higher polyploid, is difficult to isolate spores having proliferative ability due to a complicated chromosomal structure, and it is almost impossible to obtain a strain having a superior trait from among them.
Recently, it has become possible to introduce and express a target gene in brewing yeast using genetic engineering techniques. It became possible to breed yeast with However, compared to baker's yeast (S. cerevisiae, for example, see Non-Patent Document 2) whose whole genome base sequence is already known, the whole genome base sequence is not known in bottom fermentation yeast, and brewing of bottom fermentation yeast is not possible. There is very little knowledge about genes related to properties and the function of the genes in beer brewing.

  In recent years, analysis of enormous numbers of genes has been carried out using DNA arrays, DNA chips, and the like in which genes on the genome or DNA fragments outside the genes are fixed to a fixed support. For example, Olesen et al. Perform comprehensive gene expression analysis of bottom fermentation yeast during brewing using GeneFilters (manufactured by Research Genetics). However, since the whole genome base sequence of bottom fermenting yeast has not been clarified, it is unclear exactly which gene expression was monitored. As a result, analysis of substance metabolism of bottom fermenting yeast and breeding of useful yeast It is extremely insufficient as information for application to beer brewing process control.

  In recent years, 100 or more microorganism whole genome base sequences have already been determined including S. cerevisiae, Escherichia coli (for example, see Non-patent Document 4), and tuberculosis (for example, see Non-Patent Document 5) (for example, Non-Patent Document 6). Based on the determined base sequence, identification of the gene of the microorganism, comparison of the base sequence between the gene and a known gene is performed, and genetic, biochemical and molecular biological experiments are performed. Therefore, the function of a huge number of genes has been estimated. However, among brewing yeasts that are widely used in industry, bottom fermenting yeasts having heterogeneous ploidy have a particularly complicated chromosome structure (for example, Non-Patent Document 7), and assembly (operation for linking base sequences) Since it is expected to be difficult, determination of the whole genome base sequence has not yet been reported.

  In the production of specific alcohols or alcoholic beverages, for example, as technology development aimed at improving the flavor of alcoholic beverages, there is a technology for increasing the concentration of sulfurous acid in products. Sulfurous acid is a compound having a high antioxidant effect, is widely used as an antioxidant in the fields of food, beverages, pharmaceuticals, and the like, and is also used as an antioxidant in alcoholic beverages. For example, in wines that require long-term aging, sulfurous acid plays an important role in maintaining its quality. Also in beer brewing, it is known that the quality retention period will be prolonged depending on the increase in the concentration of sulfite contained in the product, and if the sulfite content in the product is increased, the flavor stability is excellent, and long-term A product having a quality retention period can be manufactured.

  The simplest way to increase the sulfite content in the product is to add sulfite. In Japan, the Ministry of Health, Labor and Welfare permits the addition of sulfurous acid to a residual sulfurous acid concentration of 350 ppm or less only for wine. However, in that case, since sulfurous acid is treated as a food additive, it is not preferable to add sulfurous acid to beer in consideration of the consumer's negative image of the food additive.

  However, yeast used in brewing produces hydrogen sulfide by reducing sulfate ions in the medium in order to biosynthesize sulfur-containing compounds such as sulfur-containing amino acids necessary for its life activity. Sulfurous acid is an intermediate metabolite. If this ability of yeast is used to produce sulfite during brewing and the sulfite can be efficiently discharged out of the cell, the sulfite content in the fermentation broth and product It was considered possible to increase the amount.

  As a method for increasing the concentration of sulfite in the fermentation liquid in the brewing process, there are a method by controlling the fermentation process and a method by breeding yeast for brewing. In the method based on the control of the fermentation process, the amount of sulfite produced during beer fermentation is inversely proportional to the initial oxygen supply, so the oxygen supply in the fermentation process is reduced to increase the amount of sulfite produced and to oxidize it. Attempts have been made to suppress it. However, this method is not practical because it has an adverse effect such as a decrease in the growth rate of yeast due to lack of oxygen, causing a delay in fermentation and a decrease in quality.

On the other hand, as described above, development of a method using genetic engineering techniques is being promoted for the method of breeding yeast for brewing. For example, in yeast sulfur metabolism, sulfurous acid (SO ₂ ) is an intermediate product in the biosynthetic pathway of sulfur-containing amino acids and vitamins. From sulfate ions (SO ₄ ²⁻ ) taken from outside the cells, sulfate ions (SO ^{_{4 2-) → APS (adenylylsulfate)}} → PAPS (phosphoadenylylsulfate) → sulfite ion (SO ₃ ^2-) that focuses on the fact that generated through a reduction reaction, wherein the sulfate ion ^{_{(SO 4 2-) → APS (}} adenylylsulfate MET3 gene involved in the reaction of APS (adenylylsulfate) → PAPS (phosphoadenylylsulfate) by increasing the gene copy number of the MET14 gene involved in the reaction (example, for example, an attempt to improve the ability of sulfite generation in yeast (for example, non Patent Document 8), also inhibited the reduction of sulfite ion (SO ₃ ^2-) by disruption of the MET10 gene increased sulfite production of yeast Cormorants and Example tried (for example, see Non-Patent Document 9) is disclosed. According to these attempts, the amount of sulfite produced by the MET10 gene disrupted strain increased to more than 10 times that of the parent strain. There were problems in putting these bred yeasts into practical use.

Therefore, although the development of breeding methods for industrial yeasts using genetic engineering techniques, such as brewing yeasts, has been promoted, the genes related to the brewing characteristics of brewing yeasts are not available due to the lack of genome information of brewing yeasts. At present, it is not possible to sufficiently select, analyze the function of the protein encoded by the gene, and use the knowledge for breeding.
Therefore, a method for breeding yeast that can exhibit the desired characteristics without impairing the fermentation rate or the quality of the product has not yet been established, and the development of this method is very not only in brewing but also in the industry using yeast. It is desired.
C. C. Gjermansen, Construction of a hybrid brewing Strain of Saccharomyces carlsbergensis by mating of meiotic segregants, Carlsberg Research Communications (Carlsberg Res. Commun.) 46, p1-11, 1981 A. A. Goffeau et al., The Yeast Genome Directory, 387, p5-105, 1997 K. Olesen et al., The dynamics of the Saccharomyces carlsbergensis brewing yeast transcriptome during a brewing yeast Saccharomyces carlsbergensis during the production scale of lager beer fermentation production-scale lager beer fermentation), FEMS Yeast Research, Vol. 2, p563-573, 2000 F. R. FR Blattner et al., The Complete Genome Sequence of Escherichia coli K-12, Science, Vol. 277, p1453-62, 1997 S. T. T. ST Cole et al., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence, Nature, Vol. 393 , P537-544, 1998 The National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html Y. Tamai et al., Bottom fermenting yeast, Saccharomyces pastorianus, there are two types of chromosomes (Co-existence of two types of chromosomes in the fermenting yeast, Saccharomyces pastorianus.), Yeast (Yeast) , Volume 10, p923-33, 1998 C. C. Korch et al., Proceeding of the European Brewery Convention Congress; Lisbon (Proc. Eur. Brew. Conv. Congress., Lisbon), p201-208, 1991 J. et al. Hansen et al., Inactivation of MET10 in brewer's yeast specifically increases SO2 formation during beer production. Biotechnology. Volume 14, p1587-1591, 1996 T. T. T. Sijen et al., Transcriptional and posttranscriptional gene silencing are mechanistically related., Current Biology, No. 1 Volume 11, p436-440, 2001 N. Goto et al., The sulfite resistance gene SSU1-R in wine yeast is an allele of SSU1 and has an upstream base sequence different from SSU1 (SSU1-R, a sulphite resistance gene of wine yeast, is an allele of SSU1 with a different upstream sequence.), Journal of Fermentation and Bioengineering (Volume 86, p427-433, 1998) D. In D. Avram et al., Saccharomyces cerevisiae, SSU1 encodes a plasma membrane protein that plays an important role in the network of proteins involved in sulfite tolerance in a network of proteins conferring sulfite tolerance in Saccharomyces cerevisiae), Journal of Bacteriol., 179, p5971-5974, 1997 H. H. Park et al., In Saccharomyces cerevisiae, SSU1 mediates sulfite efflux (SSU1 mediates sulphite efflux in Saccharomyces cerevisiae), Yeast, Vol. 16, p881-888, 2000

  The present invention produces a whole genome base sequence database (hereinafter sometimes abbreviated as “genome DB”) of brewing yeast used for the production of industrial yeasts, in particular, alcoholic beverages such as beer, and is used for brewing based on the database. It is an object of the present invention to provide a method for selecting a gene related to a target brewing characteristic by selecting a yeast gene, analyzing the function of the gene by destroying and / or highly expressing the gene, and selecting the gene. It is another object of the present invention to provide a method for breeding yeast exhibiting brewing characteristics related to the gene and a method for producing alcohol or alcoholic beverages with improved productivity and quality using the yeast. It is another object of the present invention to provide a gene of the yeast and a peptide encoded by the gene.

Many of the brewing yeasts widely used as industrial yeasts are higher-order polyploids, and among them, bottom fermentation yeasts are known to be heteroploids composed of at least two types of genomes. . One of them is thought to be a genome derived from S. cerevisiae whose entire genome sequence has been decoded, but the origin of the remaining genome has not been elucidated.
Therefore, the present inventors have found that the whole genome of bottom fermentation yeast, one of the brewing yeasts, is used to find an unidentified gene having a function unique to brewing yeast, which is the basis of the excellent brewing characteristics of brewing yeast. The base sequence was determined. And compared with the amino acid sequence registered in the genome DB of S. cerevisiae whose whole genome sequence has already been clarified, the function of the protein encoded by the brewing yeast gene was estimated. It has been clarified that the genes are broadly classified into Sc-type genes showing nearly 100% identity with S. cerevisiae and non-Sc-type genes showing about 70-97% identity in amino acid sequence. Furthermore, the bottom fermenting yeast is a complex in which Sc / non-Sc chimeric chromosomes, which are chimeras of both chromosomes, are mixed in addition to the Sc-type chromosome consisting of only the Sc-type base sequence and non-Sc type chromosome consisting of only the non-Sc type base sequence. Clarified that the structure. The structure of all chromosomes of the bottom fermentation yeast is shown in FIG. Based on the genomic information clarified in the present invention, the present inventors have discovered such an unexpected complex chromosome structure, and more specifically, (b) industrial (B) comparing the genomic base sequence with the entire genomic base sequence of S. cerevisiae, and (c) comparing the genomic base sequence of S. cerevisiae. By selecting the gene of the bottom fermenting yeast encoding an amino acid sequence having 70-97% identity with the amino acid sequence encoded by the gene, and (d) analyzing the function of the selected gene, The inventors have come up with a screening method for genes related to brewing characteristics peculiar to brewing yeast, characterized by identifying brewing characteristics to be imparted to yeast. Based on these findings, the present inventors have made further extensive studies and have completed the present invention.

That is, the present invention
(1) (b) Analyzing the whole genome base sequence of industrial yeast, (b) comparing the genome base sequence with the whole genome base sequence of Saccharomyces cerevisiae (hereinafter abbreviated as S. cerevisiae), C) selecting the industrial yeast gene encoding an amino acid sequence having 70-97% identity with the amino acid sequence encoded by the S. cerevisiae gene, and (d) analyzing the function of the selected gene. A method for screening genes involved in improving the productivity and / or improving the flavor in the production of alcohol or alcoholic beverages, characterized by identifying the characteristics that the gene imparts to yeast by
(2) The screening method according to (1) above, wherein functional analysis is performed using a DNA array in (d) above,
(3) The screening according to (2) above, wherein a DNA array consisting of the following base sequence or a base sequence complementary to the base sequence and having one or more oligonucleotides immobilized on a substrate is used. Method,
(A) a nucleotide sequence of 10 to 30 nucleotides in an open reading frame (or may be abbreviated as ORF in addition to the open reading frame) in the entire genome sequence of industrial yeast; and Base sequences that do not exist in other parts of the genomic base sequence,
(4) The screening method according to (2), wherein a DNA array in which an oligonucleotide that hybridizes with the oligonucleotide according to (3) under stringent conditions is immobilized on a substrate is used,
(5) The screening according to (2) above, wherein a DNA array consisting of the following base sequence or a base sequence complementary to the base sequence and having one or more oligonucleotides immobilized on a substrate is used. Method,
(A) a base sequence of 10 to 30 nucleotides in an untranslated region other than the open reading frame in the whole genome sequence of industrial yeast, and (a) in the other part of the whole genome sequence. Is a non-existent base sequence,
(6) The screening method according to (2), wherein an oligonucleotide that hybridizes under stringent conditions with the oligonucleotide having the base sequence according to (5) above,
(7) One or more nucleotides described in (3) above,
One or more nucleotides described in (4) above,
One or more nucleotides described in (5) above and
One or more nucleotides described in (6) above,
A screening method according to the above (2), wherein a DNA array in which nucleotides selected from two or more groups out of four groups comprising:
(8) The screening method according to (1) to (7) above, wherein the industrial yeast is a brewery yeast,
(9) The screening method according to (1) to (8) above, wherein the brewing yeast is brewer's yeast,
(10) a gene obtained by the screening method according to (1),
(11) The gene according to (10), wherein when the gene according to (10) is expressed in yeast, the concentration of sulfite in a culture solution of the yeast increases,
(12) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2, or DNA that hybridizes with the DNA under stringent conditions,
(13) In the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or 4, or the amino acid sequence represented by SEQ ID NO: 3 or 4, one to several amino acid residues are deleted and / or substituted and DNA encoding a polypeptide having an added amino acid sequence,
(14) A recombinant vector having the gene or DNA according to any one of (10) to (13),
(15) The recombinant vector according to (9), wherein a promoter and / or terminator is provided adjacent to the gene or DNA according to any of (10) to (13). ,
(16) The recombinant vector according to (15), wherein the promoter is a promoter that constitutively expresses,
(17) The recombinant vector according to (15) or (16) above, wherein the promoter is a promoter of a glyceraldehyde-3-phosphate dehydrogenase gene,
(18) A transformant comprising the gene, DNA or recombinant vector according to any one of (10) to (17),
(19) The transformant according to (18) above, which is a yeast of the genus Saccharomyces,
(20) In the polypeptide encoded by the gene or DNA according to any one of (10) to (13) above or the amino acid sequence of the polypeptide, one to several amino acid residues are deleted and / or A polypeptide having a substituted and / or added amino acid sequence,
(21) In the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or 4, or the amino acid sequence represented by SEQ ID NO: 3 or 4, one to several amino acid residues are deleted and / or substituted and A polypeptide having an added amino acid sequence,
(22) A method for producing alcohol or liquor, characterized by using the transformant according to (18) or (19),
(23) Yeast suitable for production of alcohol or alcoholic beverages characterized by controlling expression of the gene according to (10) or (11) or the gene on DNA according to (12) or (13) Breeding method,
(24) The method for breeding according to (23), wherein the yeast is a genus Saccharomyces,
(25) Yeast obtained by the breeding method according to (23) or (24),
(26) A method for producing alcohol or liquor using the yeast according to (25),
(27) Alcohol or liquor produced using the method for producing alcohol or liquor according to (26),
(28) a DNA array comprising one or a plurality of oligonucleotides fixed to a substrate, comprising the following base sequence or a base sequence complementary to the base sequence:
(1) a nucleotide sequence of 10 to 30 nucleotides in an open reading frame in the entire genome sequence of industrial yeast, and
(2) a base sequence that does not exist in other parts of the whole genome base sequence,
(29) a DNA array in which one or more oligonucleotides that hybridize under stringent conditions with the oligonucleotide having the base sequence according to (28) are immobilized on a substrate;
(30) a DNA array comprising one or a plurality of oligonucleotides fixed to a substrate, comprising the following base sequence or a base sequence complementary to the base sequence:
(1) A base sequence of 10 to 30 nucleotides in an untranslated region other than the open reading frame in the entire genome sequence of industrial yeast, and
(2) a base sequence that does not exist in other parts of the whole genome base sequence,
(31) a DNA array in which one or more oligonucleotides that hybridize under stringent conditions with the oligonucleotide having the base sequence according to (30) are immobilized on a substrate;
(32) one or more nucleotides described in the above (3),
One or more nucleotides described in (4) above,
One or more nucleotides described in (5) above and
One or more nucleotides described in (6) above,
A DNA array in which nucleotides selected from two or more of four groups consisting of:
About.

Create a whole genome sequence database of industrial yeasts, especially brewing yeasts used in the production of alcoholic beverages such as beer, and select genes that are considered to be related to the brewing characteristics of brewing yeasts based on the database. By analyzing the function of a gene by destroying and / or highly expressing the gene, a gene related to the target brewing characteristic can be selected. Further, by using the gene to control the expression of the gene, a yeast that exhibits brewing characteristics related to the gene is bred, and alcohol or alcoholic beverages having improved productivity and quality using the yeast, for example, It is possible to produce an alcoholic beverage having a high sulfurous acid content having an antioxidative action, excellent flavor stability, and a longer quality retention period.
In addition, it is possible to provide a DNA array based on a whole genome base sequence database of industrial yeasts, particularly brewing yeasts used in the production of alcoholic beverages such as beer. Furthermore, gene function analysis, yeast classification, nucleotide polymorphism detection, and the like using the DNA array can be performed.

  Examples of the industrial yeast in the present invention include brewing yeasts such as beer, wine and sake, yeasts used for the production of fuel alcohol, and the like. Specific examples include yeasts of the genus Saccharomyces. In the present invention, beer yeasts such as Saccharomyces pastorianus Weihenstephan 34/70, BH84, NBRC1951, NBRC1952, NBRC1953, NBRC1954, etc. are used. it can. In addition, whiskey yeasts such as S. cerevisiae NCYC90, wine yeasts such as association wines No. 1, 3, and 4 and sake yeasts such as association yeast sake nos. 7 and 9 can be used. .

The gene screening method of the present invention comprises: Compared with the base sequence, (c) the bottom fermenting yeast gene encoding an amino acid sequence having 70 to 97% identity with the amino acid sequence encoded by the S. cerevisiae gene is selected, and (d) further selected The brewing characteristics that the gene imparts to yeast are identified by analyzing the function of the gene.
Further, by using a gene related to brewing characteristics peculiar to brewing yeast obtained by the screening method of the present invention, expression control such as high expression of the gene in yeast or gene disruption of the gene is performed. Thus, yeast having excellent brewing characteristics can be bred. Therefore, a gene obtained by the gene screening method of the present invention and a peptide encoded by the gene, a breeding method for industrial yeast using the gene, a yeast obtained by the breeding method, and the yeast were used. The method for producing alcohol or liquor is also within the scope of the present invention.

(A) Determination of the whole genome base sequence of industrial yeast In order to determine the whole genome base sequence of industrial yeast, first, (a) genomic DNA was prepared from yeast, (b) shotgun library and (c) Kosumi. (D) using these library clones to prepare DNA fragments to be used for sequencing, (e) determining the nucleotide sequence of the library DNA fragments by a sequencing reaction, and (f) The process includes assembling DNA fragments to reconstruct the whole genome sequence.
The method used in (a) to (f) is not particularly limited, and may be performed according to known means. Preferred methods are described below.

  (A) Preparation of extraction and purification of total DNA is performed by conventional methods such as “Yeast a practical approach (IRL PRESS, 6.2.1, p228)”, “Biochemical Experimental Method 39 Yeast Molecular Genetics Experimental Method (Oshima It is preferably carried out according to a known method described in Taiji, Ed., Academic Publishing Center, p84-85, 1996). Specific examples of preferred DNA preparation methods are shown below.

  The yeast for preparing genomic DNA is cultured by a conventional method. As the medium, any of a natural medium and a synthetic medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the yeast and can efficiently culture the microorganism. For example, YPD medium (2% (w / w) glucose, 1% (w / w) yeast extract, 2% (w / w) polypeptone), etc. can be used. As a culture method, it is preferable to culture overnight at about 25 to 35 ° C. with shaking.

  After cultivation, the cells are collected from the culture solution by centrifugation. The obtained microbial cells are washed with a washing solution. Examples of the washing liquid include buffer A (50 mM Na phosphate, 25 mM EDTA, 1% (v / v) β-mercaptoethanol, pH 7.5) and the like. Genomic DNA from the washed cells is obtained by lysing the cell wall of the cells using zymolyase and SDS, etc., then removing proteins and the like using a phenol solution and a phenol / chloroform solution, and using ethanol etc. Can be carried out in accordance with a general method for preparing genomic DNA for precipitating, specifically, the following method can be exemplified.

  The cells obtained by culturing were washed with buffer A (50 mM Na phosphate, 25 mM EDTA, 1% (v / v) β-mercaptoethanol, pH 7.5), and then suspended in buffer A again. Add 10 mg of zymolyase-100T (Seikagaku Corporation) and gently shake at about 25-40 ° C. for about 30 minutes to 2 hours. After shaking, lysis is performed by adding a buffer containing SDS, such as buffer B (0.2 M Tris-HCl, 80 mM EDTA, 1% SDS, pH 9.5), and allowing to stand at about 60 to 70 ° C. for about 30 minutes. Let After lysis, cool on ice, add 5M potassium acetate, mix, and leave on ice for about 60 minutes. The obtained solution is centrifuged (for example, 5,000 g, 10 minutes, 15 ° C.), and an equal volume of ethanol is added to the collected supernatant to precipitate DNA, and immediately centrifuged (for example, 5,000 g, 10 minutes) 15 ° C.) to recover the DNA. The obtained precipitate was washed with 70% (v / v) ethanol, air dried, and then dissolved in a DNA suspension solution such as TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). A crude genomic DNA solution is obtained. The obtained crude genomic DNA solution was dissolved by adding cesium chloride and bisbenzimide, and this solution was subjected to ultracentrifugation (for example, 100,000 g, 17 hours, 25 ° C.) and then irradiated with UV light to visualize the DNA band. Collect the lower band. From the recovered DNA solution, bisbenzimide was extracted and removed using isopropanol saturated with a cesium chloride solution, and 4 times the volume of 0.3 M sodium acetate was added to and mixed with the recovered aqueous layer, and then the DNA was ethanol precipitated. Collect by centrifugation. The recovered DNA is treated with RNase, extracted with phenol / chloroform, and purified from the recovered aqueous layer by ethanol precipitation again. The precipitate collected by centrifugation is washed with 70% (v / v) ethanol, air-dried, dissolved in TE buffer, and a genomic DNA solution is prepared.

(B) Production of Shotgun Library As a method for producing a genomic DNA library using the genomic DNA of yeast prepared in (a) above, “Molecular Cloning, A laboratory Manual, Third Edition (2001)” The method described in “Molecular Cloning 3rd Edition”) can be used, but a method for preparing a shotgun library particularly suitable for use in determination of the total nucleotide sequence by the shotgun method is described below. The method can be illustrated.

  TE buffer is added to the genomic DNA prepared in (a), and the genomic DNA is fragmented using Hydroshear (produced by Jin Machines). After smoothing the ends of the genomic fragments using a DNA branding kit (manufactured by Takara Shuzo), etc., the fragments are fractionated by agarose electrophoresis, and the genomic fragments of about 1.5 to 2.5 kb are separated from the gel. Cut out and add DNA elution buffer such as MG elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS) to the gel and shake at about 25-40 ° C. overnight to DNA Elute. The DNA eluate is treated with phenol / chloroform and then ethanol precipitated to obtain a genomic library insert. Using T4 ligase (Takara Shuzo), the entire amount of the above insert and an appropriate vector such as pUC18SmaI / BAP (Amersham Biosciences) are ligated at about 10 to 20 ° C. for about 20 to 50 hours. The ligation reaction product is ethanol precipitated, and the resulting recombinant vector DNA is dissolved in an appropriate amount of TE buffer. The recombinant vector DNA is transformed into E. coli, for example, ELECTRO CELL DH5α (Takara Shuzo) strain, by means of electroporation or the like. Electroporation should be performed under the conditions indicated in the attached experiment manual.

  A transformed strain into which a recombinant vector containing a genomic DNA fragment has been introduced is selected on an appropriate selection medium. For example, when pUC18SmaI / BAP is used as the vector, the transformed strain is about 0.01 to 0.1 mg / mL ampicillin, about 0.1 mg / mL X-gal, about 1 mM isopropyl-β-D-thio. On LB plate medium containing galactopyranoside (IPTG) [LB medium containing 1.6% agar (10 g / L bactotryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0)] Selection is easy because white colonies are formed by culturing at 30 to 37 ° C. overnight. After the transformant obtained from the colony formed on the plate medium was statically cultured overnight at about 30 to 37 ° C. in a 384-well titer plate to which an LB medium containing about 0.1 mg / mL ampicillin was added. Add 50% glycerol aqueous solution in the same volume as LB and stir to make a glycerol stock. Glycerol stock can usually be stored at about -80 ° C.

(C) Preparation of cosmid library The genomic DNA obtained in (a) is partially digested with an appropriate restriction enzyme such as Sau3AI (Takara Shuzo). When using the obtained restriction enzyme-digested DNA fragment, a cosmid vector having a restriction enzyme digestion site that produces a sticky end that can be ligated to the fragment, for example, Super Cos I vector (Stratagene), Sau3AI digested DNA fragment It can be inserted into the BamHI site of the vector. Restriction enzyme treatment and ligation may be performed according to the attached protocol. The ligation product obtained by this method is packaged using Gigapack III Gold (manufactured by Stratagene), etc., and E. coli, for example, XL1-Blue MR strain (manufactured by Stratagene), etc. according to the attached experiment procedure manual. Introduce. This is smeared on an LB plate medium containing ampicillin and cultured at about 30-37 ° C. overnight. The obtained colonies were cultured in each well of a 96-well titer plate containing ampicillin-added LB medium at about 30 to 37 ° C. overnight, and then added with 50% glycerol aqueous solution in an amount equal to the LB and stirred to glycerol. Let it be stock. Glycerol stock can usually be stored at about -80 ° C.

(D) Preparation of DNA Fragment for Determining Base Sequence The entire base sequence of the brewer's yeast genome is determined mainly using the whole genome shotgun method. A DNA fragment whose base sequence is determined by this method is prepared from the shotgun library prepared in the above (b) using the PCR method. Specifically, a whole genome shotgun library-derived clone was inoculated using a replicator (manufactured by Gin Solution) into a 384-well titer plate in which about 50 μL of LB medium containing ampicillin was dispensed per well, Incubate statically overnight at about 30-37 ° C. Using the replicator (manufactured by Jin Solution Co., Ltd.), etc., to the 384-well reaction plate (manufactured by AB Gene Co., Ltd.), in which about 10 μL each of the PCR reaction solution [TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)] was dispensed. Then, PCR is performed using GeneAmp PCR System 9700 (Applied Biosystems) or the like using the protocol “DNA Research, 5, p1-9 (1998)” of Makino et al. And the inserted fragment is amplified.
Excess primers and nucleotides are removed using a PCR product purification kit (Amersham Biosciences) or the like, and this is used as a template for the sequence reaction.

  A DNA fragment from the cosmid library of (c) is prepared by the following method. Appropriate medium containing ampicillin, for example, 2 × YT medium (1.6% bactotryptone, 1% yeast extract, 0.5% sodium chloride, pH 7.0) was dispensed into each well of a 96-well plate. All cosmid library-derived clones are inoculated and cultured with shaking at about 30-37 ° C. overnight. A cosmid DNA is prepared from the culture solution using a plasmid automatic preparation machine KURABO PI-1100 (manufactured by Kurashiki Boseki Co., Ltd.) according to the protocol of Kurashiki Boseki Co., and used as a template for the sequence reaction.

(E) Sequence reaction The sequence reaction can be performed using a commercially available sequence kit or the like. Hereinafter, preferred examples in the present invention will be shown.
For approximately 2 μl of DYEnamic ET Terminator Sequencing Kit (Amersham Biosciences), M13_for primer (SEQ ID NO: 5) and M13_RV primer (SEQ ID NO: 6) (manufactured by Takara Bio Inc.) are used for the sequence reaction of shotgun DNA fragments. In the cosmid DNA sequencing reaction, a forward primer such as SS-cosF.1 (SEQ ID NO: 7) and a reverse primer such as SS-cosR.1 (SEQ ID NO: 8) are used. Mix the PCR product or cosmid DNA prepared in (d) to make about 8 μL of the sequencing reaction solution. The amount of primer and DNA fragment is about 1-4 pmol and about 50-200 ng, respectively. Using the reaction solution, a dye terminator sequence reaction of about 50 to 70 cycles is performed with Gene AmpPCR System 9700 (Applied Biosystems). When using a commercially available kit such as DYEnamic ET Terminator Sequencing Kit, follow the attached manual. The sample is purified using a MultiScreen HV plate (Millipore) according to the Millipore manual. The purified reaction product is ethanol precipitated, and the resulting precipitate is dried and stored in the dark at about 4 ° C. Analysis is performed using a commercially available sequencer and analyzer such as MegaBACE1000 Sequencing System (Amersham Biosciences) and ABI PRISM 3700 DNA Analyzer (Applied Biosystems) according to the attached manual.

(F) Genomic DNA can be reconstructed from the sequence data of the DNA fragment obtained by reconstruction (e) of genomic DNA by assembly (operation for aligning, superposing and linking base sequence fragments). All the work of genome base sequence reconstruction is performed on the UNIX (registered trademark) platform. For example, base call is performed by phred (The University of Washington), vector sequence mask is performed by Cross Match (The University of Washington), and assembly is performed by Phrap (The University of Washington). The contig resulting from the assembly is analyzed using a graphical editor such as consed (The University of Washington). A series of operations from base call to assembly can be performed at once by using the script phredPhrap attached to consed.

(B) Comparison with the entire genome sequence of S. cerevisiae Comparison of the genome sequence obtained in (a) with the entire genome sequence of S. cerevisiae And a process of creating a comparison database of each contig base sequence with the S. cerevisiae genomic base sequence and mapping to the S. cerevisiae genomic base sequence.

(G) Creation of a comparison database of cosmids, shotgun clone both-end base sequences and contig base sequences with S. cerevisiae genomic base sequences and mapping to S. cerevisiae genomic base sequences Widely used industrial yeasts such as bottom fermentation Yeasts such as S. pastorinus are considered to be natural hybrids between S. cerevisiae and its related species (such as S. bayanus) “Int. J. Syst Bacteriol. 35, p508-511 (1985). ) ”. Therefore, the base sequence of both ends of the cosmid clone obtained in (e) is subjected to a homology search using a homology search algorithm for the S. cerevisiae genomic base sequence. Select the homologous region above and determine its identity, and create a database. FIG. 2 shows an example of the% identity distribution map of the cosmid base sequence with the corresponding S. cerevisiae genomic base sequence. The base sequences of cosmids are roughly classified into a base sequence group having about 94% identity with the S. cerevisiae genomic base sequence and a base sequence group having about 84% identity. Therefore, a base sequence showing an identity higher than 94% is an Sc-type base sequence derived from S. cerevisiae, and a base sequence group showing about 84% identity is a non-Sc-type base derived from a S. cerevisiae related species genome. A gene having a Sc-type base sequence or a non-Sc-type base sequence is referred to as a Sc-type gene or a non-Sc-type gene, respectively.

  Similarly, a comparison database between the base sequences of both ends of the shotgun clone obtained in (e) and the S. cerevisiae genomic base sequence is prepared. Mapping of cosmid clones and shotgun clones onto the S. cerevisiae genome base sequence is performed based on the information obtained from the prepared comparison database (see, for example, FIG. 3). In addition, a comparison database is created (for example, see Table 1) between the contig base sequence obtained in (f) and the S. cerevisiae genomic base sequence, and mapping is performed. The mapping method is almost the same as the method described above. However, when the forward strand and the reverse strand of a cosmid or shotgun clone are present in different contigs, these contigs are linked (for example, see FIG. 4).

(C) Selection of bottom fermenting yeast gene encoding an amino acid sequence having 70-97% identity with the amino acid sequence encoded by S. cerevisiae gene
The step of selecting a bottom fermentation yeast gene encoding an amino acid sequence having 70-97% identity with the amino acid sequence encoded by the S. cerevisiae gene includes (h) identification of ORF and functional estimation. It is.

(H) Identification of ORF and function estimation The ORF (Open Reading Frame) in the base sequence assembled in (f) is identified. A preferred embodiment is specifically shown below. A program for identifying an ORF for six types of reading frames including a complementary strand for a sequence having a certain length from the start codon to the end codon, for example, a length of 150 base sequences or more, such as ORF finder (http: / /www.ncbi.nih.gov/gorf/gorf.html) to identify the ORF present in the base sequence assembled in (f).
The function estimation of the protein encoded by the extracted ORF is registered and published in the Saccharomyces Genome Database (SGD: http://genome-www.stanford.edu/Saccharomyces/). This is performed by homology search with the amino acid sequence of cerevisiae ORF.

On the other hand, the structure analysis of the brewing yeast chromosome can be performed using a commercially available DNA array and PCR.
Genomic DNA from brewing yeast or S. cerevisiae is prepared using QIAGEN Genomic Tip 100 / G (# 10243) and QIAGEN Genomic DNA buffer set (# 19060) according to the manual attached to the kit. 10 μg of this DNA was digested with DNaseI (Invitrogen) according to the method of Winzeler et al. “Science 281 p1194-1197, (1998)”, biotinylated with terminal transferase (Roche) and DNA array (Affymetrix Gene Chip Yeast Genome) S98 Array). Hybridization and detection of the brightness of the array may be performed using a Gene Chip analysis basic system manufactured by Affymetrix.

The brightness of the probes obtained from S. cerevisiae and yeast for brewing was compared using expression analysis software (Microarray Suite 5.0 (Affymetrix)), and the difference in luminance was compared with the signal ratio based on S. cerevisiae ( Signal log ratio) Next, the signal ratio of each probe is aligned for each chromosome using spreadsheet software (Microsoft Excel 2000), and the signal ratio is represented by a bar graph (see, for example, FIG. 5). For example, under the above-mentioned conditions, only a gene (Sc type gene) having nearly 100% identity with the base sequence of a gene derived from S. cerevisiae in the brewing yeast genome gene hybridizes. The signal ratio is considered to change due to the change in the number of Sc-type genes. In places where the value of the signal ratio changes greatly, the Sc-type chromosome consisting of the Sc-type base sequence and the non-Sc-type base sequence The existence of a chimeric chromosomal structure linked to the Sc-type chromosome (or vice versa) is presumed.
This chimera chromosome structure is designed based on the genome sequence of a brewing yeast determined by the shotgun method, and a primer having a base sequence of Sc type on one side and a non-Sc type on the other side is designed. Can be confirmed by performing PCR using as a template.
For PCR, TaKaRaLA Taq ^™ and the attached buffer are used, and the reaction is performed using TaKaRa PCR Thermal Cycler SP according to the attached manual.

  As a result of PCR, it is confirmed by 0.8% agarose electrophoresis that a DNA fragment of a certain length has been amplified from the brewing yeast. Here, when the genomic DNA of the laboratory strain S. cerevisiae is used as a PCR template, amplification of the DNA fragment is not observed. Furthermore, by confirming the base sequences at both ends of the DNA fragment amplified from the brewing yeast, it is clear that it matches the genomic base sequence determined by the shotgun method. It can be confirmed that a non-Sc type chromosome is linked (transferred) and a chimeric chromosome is formed.

(D) Gene function analysis The gene function analysis stage includes (i) gene selection process, (i ′) gene cloning process, (j) gene function analysis process by gene disruption, (k) gene height Includes gene analysis by expression.

(I) Selection of genes Examples of methods for selecting genes for functional analysis include, but are not limited to, a method based on ORF function estimation obtained in (h) above and a method using a DNA array described below. It is not something.
As a method using a DNA array, a gene expression analysis is used to identify a gene exhibiting characteristic expression under a certain condition, and a DNA-DNA hybridization is used to select a region having higher or lower brightness than other regions. A method of identifying genes having different numbers of genes or different base sequences by detection is included.

(I ′) Gene Cloning Based on the ORF information obtained in (i) above, the gene of the bottom fermenting yeast can be prepared and obtained by a conventional method described in Molecular Cloning 3rd edition and the like. That is, an oligonucleotide having a base sequence adjacent to the gene is synthesized, and the gene can be obtained by a normal PCR cloning method using the genomic DNA obtained from bottom fermentation yeast as a template. Examples of the base sequence of the DNA thus obtained include DNA having the base sequence represented by SEQ ID NO: 1 or 2.
For example, if the gene is gene (1), the DNA encoding gene (1) or the primer for amplifying gene (1) by PCR is synthesized using a polynucleotide synthesizer based on the above sequence information. You can also The DNA encoding the gene (1) includes not only DNA containing the base sequence of the gene (1) obtained above but also DNA that hybridizes with the DNA under stringent conditions. The DNA that hybridizes under stringent conditions is a colony hybridization method, plaque hybridization method, Southern blot hybridization method, etc. using the DNA having the base sequence of the gene (1) identified above as a probe. DNA obtained by using Specifically, DNA having at least 60% homology with the base sequence of gene (1), preferably DNA having 80% or more homology, more preferably DNA having 95% or more homology. be able to. Hybridization is described in [Molecular Cloning 3rd Edition], “Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)” (hereinafter referred to as “Current Protocols in Molecular Biology”), “DNA Cloning”. 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) ”.

More specifically, a clone containing the gene (1) is searched using the comparison database obtained in (g), and a DNA fragment containing a full-length structural gene is determined based on homology and positional information. Inserted shotgun clones can be obtained. If the clone selected from the shotgun library does not have the full length of the gene, a DNA fragment encoding the full length of the gene is obtained by PCR. For example, by using a synthetic DNA primer pair represented by SEQ ID NO: 13 and SEQ ID NO: 14, etc., a DNA fragment containing the gene can be obtained. Similarly, S.M. Using the genomic DNA of cerevisiae or bottom fermenting yeast as a template, PCR was performed using a primer pair designed based on SGD information that has already been published, and the Sc type gene corresponding to the non-Sc type gene of the bottom fermenting yeast was Get the full length. As the primer, for example, a synthetic DNA primer pair of SEQ ID NOs: 15 and 16 can be used, and a DNA fragment containing the Sc-type gene is obtained by this combination.
The non-Sc type DNA fragment or Sc type DNA fragment obtained as described above is inserted into an appropriate vector using the TA cloning kit or the like, for example, the pCR2.1-TOPO vector attached to the TA cloning kit, and the respective DNA Recombinant vector TOPO / non-Sc type gene containing fragment (recombinant vector having non-Sc type DNA fragment shall be TOPO / non-Sc type gene), or recombinant vector TOPO / Sc type gene (having Sc type DNA fragment) The vector can be a TOPO / Sc type gene). The nucleotide sequences of the obtained non-Sc type and Sc type DNA fragments can be examined by the Sanger method “F. Sanger, Science, 214, p1215, 1981”.

(J) Functional analysis of genes by gene disruption According to the method of literature “Goldstein et al., Yeast.15, p1541 (1999)”, plasmids containing drug resistance markers (eg pFA6a (G418 ^r ), pAG25 (nat1)) A DNA fragment for gene disruption can be prepared by PCR using a template. As a primer for PCR, for example, non-Sc type SSU1_for (SEQ ID NO: 17) / non-Sc type SSU1_rv (SEQ ID NO: 18) is used for disruption of non-Sc type gene, and ScSSU1_for (sequence) is used for Sc type gene disruption. Number: 19) / ScSSU1_rv (SEQ ID NO: 20) or the like is used. For disruption of the non-Sc type gene, a plasmid such as pPGAPAUR (AUR1-C) and a primer such as non-Sc type SSU1_for + pGAPAUR (SEQ ID NO: 21) / non-Sc type SSU1_rv + AUR1-C (SEQ ID NO: 22) are further used. You can also
The yeast for brewing is transformed with the DNA fragment for gene disruption produced by the method described above. Transformation may be performed according to the method described in JP-A-07-303475. The concentration of the selected drug may be determined appropriately by measuring the sensitivity of yeast as a host to the drug.
About the transformant obtained here, it can be confirmed by Southern analysis that the respective drug resistance markers used are introduced and each gene is destroyed. Specifically, first, genomic DNA extracted from a parent strain and a transformant is digested (for example, 37 ° C., 18 hrs) using a restriction enzyme suitable for fractionating Sc-type or non-Sc-type genes. Developed with 5% agarose gel electrophoresis and transferred to membrane. In the following, according to the protocol of Alkphos Direct Labeling Reagents (manufactured by Amersham), hybridization is carried out with a probe specific to the Sc type or non-Sc type gene (for example, 55 ° C., 18 hours), and the signal is detected by CDP-Star.

By performing a fermentation test using the parent strain and the gene-disrupted strain obtained in (j) above and comparing their fermentation characteristics, the function of the gene cloned in (i ′) can be confirmed. . The parent strain and the gene-disrupted strain obtained in (j) above can be cultured, for example, in a medium containing wort under the following conditions.
Wort extract concentration about 10-15%
Wort capacity 1-3L
Wort dissolved oxygen concentration about 8-10ppm
Fermentation temperature about 15 ℃
Yeast input about 8-12g Wet yeast cells / 2L wort Sampling the fermentation solution over time, and related to the function of the gene cloned by yeast growth (OD600), appearance extract concentration, (i ') Investigate changes in the concentration of possible substances over time. For example, when the function of the gene cloned in (i ′) is related to the discharge of sulfite, the time-dependent change of the sulfite concentration in the fermentation broth is measured. Sulfurous acid is quantified by collecting sulfurous acid in aqueous hydrogen peroxide by distillation under acidic conditions and then titrating with alkali (Japan Brewing Association revised BCOJ beer analysis method).

(K) Functional analysis of gene by high gene expression From the plasmid TOPO / non-Sc type gene obtained in (i ′), a DNA fragment containing the full length of the non-Sc type gene is cut out by appropriate restriction enzyme treatment. This is similarly inserted into a restriction vector-treated gene expression vector, such as pNI-NUT, to construct a vector (pYI-non-Sc type gene) for high expression of non-Sc type genes. Vector pNI-NUT URA3 as yeast chromosomal homologous recombination site includes nourseothricin-resistance gene (nat1) and ampicillin resistance gene as a selectable marker (Amp ^r). On the other hand, the vector (pNI-Sc type gene) for highly expressing the Sc type gene has a structure in which the non-Sc type gene portion of the pYI-non-Sc type gene is replaced with the corresponding Sc type gene. For the high expression of the non-Sc type gene or Sc type gene introduced here, for example, the promoter and terminator of glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) can be used.
The bottom fermentation yeast is transformed using the high expression vector produced by the method described above. Transformation is performed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and is preferably selected using a YPD plate medium containing an antibiotic or the like as a selection marker.

  Confirmation of high expression may be performed by the RT-PCR method or the like. The total RNA may be extracted using an RNeasy Mini Kit (manufactured by Qiagen) or the like according to the manual for total RNA isolation from yeast. Specific primers for the Sc-type gene include, for example, ScSSU1_for331 (SEQ ID NO: 23) / ScSSU1_982rv (SEQ ID NO: 24), and specific primers for the non-Sc type gene include, for example, nonSc-SSU1_for329 (SEQ ID NO: 25) / nonSc As an internal standard such as -SSU1_981rv (SEQ ID NO: 26), a gene constitutively expressed in yeast, such as PDA1, can be used. For example, PDA1_for1 (SEQ ID NO: 27) / PDA1_730rv (SEQ ID NO: 28) can be used as a specific primer at this time. The PCR product is developed by 1.2% agarose electrophoresis, then stained with ethidium bromide solution, and the signal value of each gene is normalized with the signal value of a gene used as an internal standard, such as PDA1, and compared with that of the parent strain. The high expression strain confirmed in this way can be used as a Sc type gene high expression strain and a non-Sc type gene high expression strain.

It is possible to confirm the function of the gene cloned in (i ′) by conducting a fermentation test using the parent strain and the gene high-expression strain obtained in (k) above, and comparing their fermentation characteristics. it can. Culture of the parent strain and the high gene expression strain obtained in (k) above can be performed under the conditions described in (j).
As in (j), the fermentation broth is sampled over time, and the amount of yeast growth (OD600), appearance extract concentration, change in the concentration of the substance related to the function of the gene cloned in (i ′), etc. are examined.

  Examples of the DNA obtained by the screening method of the present invention include DNA containing the base sequence of the non-Sc type gene obtained above and DNA that hybridizes with the DNA under stringent conditions.

  The DNA obtained by the screening method of the present invention includes, but is not limited to, single-stranded and double-stranded DNA. The DNA hybridized under stringent conditions with the DNA containing the base sequence of the non-Sc type gene obtained above includes a degenerate variant of the codon of the protein encoded by the gene. A degenerate variant refers to a polynucleotide fragment that differs in nucleotide sequence from the nucleotide sequence of a non-Sc gene selected in the present invention, but encodes the same amino acid sequence due to codon degeneracy.

  Specific examples include DNA having the base sequence shown in SEQ ID NO: 1 or 2, DNA that hybridizes with the DNA under stringent conditions, and the like. The DNA that hybridizes under stringent conditions is a colony hybridization method, plaque hybridization method, or Southern blot hybridization method using the DNA fragment having the base sequence of the non-Sc type gene identified above as a probe. It means DNA obtained by using etc.

  Hybridization is described in “Molecular Cloning 3rd Edition”, “Current Protocols in Molecular Biology”, “DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)”, etc. It can be performed according to the method that has been described. Specifically, DNA that can be hybridized is calculated using homology search software such as FASTA, BLAST, Smith-Waterman “Meth. Enzym., 164, p765 (1988)” using default (initial setting) parameters. DNA having at least 60% identity, preferably DNA having 80% identity or more, more preferably 95% identity or more, with the nucleotide sequence shown in SEQ ID NO: 1 or 2 DNA can be raised.

  Examples of DNA obtained by the screening method of the present invention include DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4 or DNA that hybridizes with the DNA under stringent conditions. it can.

  Examples of the polypeptide encoded by the DNA obtained by the screening method of the present invention include a DNA containing the ORF base sequence obtained above and a polypeptide encoded by the DNA that hybridizes with the DNA under stringent conditions. Or the polypeptide which has an amino acid sequence represented by sequence number: 3 or 4 is mentioned.

  Furthermore, a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted and / or substituted and / or added in the amino acid sequence of the polypeptide, and having substantially the same activity as that of the polypeptide. Peptides are also included in the present invention. The activity substantially the same as the activity of the polypeptide means the same activity as the activity represented by the intrinsic function or enzyme activity of the polypeptide before deletion and / or substitution and / or addition. is doing. The polypeptide is described in "Molecular Cloning 3rd Edition", "Current Protocols in Molecular Biology", "Nuc. Acids. Res., 10, 6487 (1982)", "Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) "," Gene, 34, 315 (1985) "," Nuc. Acids. Res., 13,4431 (1985) "," Proc. Natl. Acad. Sci. USA, 82, 488 ( 1985) "etc., and can be obtained using the site-directed mutagenesis method. For example, it can be obtained by introducing a site-specific mutation into DNA encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 or 4. The number of amino acid residues to be deleted and / or substituted and / or added is not particularly limited, but is a number that can be deleted and / or substituted and / or added by a known method such as the above-mentioned site-specific mutation method. 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

  In the amino acid sequence of the polypeptide of the present invention, one or more amino acid residues are deleted and / or substituted and / or added in any one or a plurality of amino acid sequences in the same sequence. It means that there are deletions and / or substitutions and / or additions of a plurality of amino acid residues, and deletions and / or substitutions and / or additions may occur simultaneously. Does not matter whether natural or non-natural. Natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine. , L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

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, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine B group: 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-diaminopropionic acid group E: proline, 3 -Hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine In addition, because the obtained mutant polypeptide has substantially the same activity as that of the polypeptide before mutation. Before the mutation There is at least 60% or more, usually 80% or more, especially 95% or more homology when calculated using the default (initial setting) parameters with the analysis software such as BLAST and FASTA with the amino acid sequence of the peptide. It is preferable.

  The polypeptide 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 using peptide synthesizers such as Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive, Shimadzu, etc. You can also.

  By using the present invention, the entire base sequence of the genome of industrial yeast can be determined, useful genes of industrial yeast can be identified, and the function of the gene can be estimated. In many cases, industrial yeast genes are industrially useful genes, and by classifying the genes based on their estimated functions, the characteristics of the yeast become clear and valuable for breeding industrial yeasts. Information can be obtained. For example, if the industrial yeast is a brewing yeast, the genes involved in improving the productivity and flavor in the production of alcoholic beverages are identified, and the genes are negative for improving productivity and improving flavor. If there is, the gene is destroyed and the expression is controlled, or the gene is not expressed by the antisense method or RNAi method (for example, see Non-Patent Document 10) or the expression is suppressed, or the expression is excellent. Yeast can be bred. In addition, if the gene is positive for improving productivity, improving flavor, etc., the gene can be highly expressed in yeast to breed industrially useful brewing yeast with excellent brewing characteristics. .

An example of breeding useful yeast using the gene obtained by the screening method of the present invention is shown below.
As described above, in beer brewing, if the sulfurous acid content in a product is increased, a product excellent in flavor stability and the like can be produced. Therefore, if the gene obtained by the screening method of the present invention is a gene related to sulfite production or excretion function, when transformant yeast introduced with the gene is cultured and expressed in the transformant yeast The sulfite concentration in the moromi is increased, and a product excellent in flavor stability and the like can be produced.
It is known that the bottom fermentation yeast reduces sulfate ions (SO ₄ ²⁻ ) taken from outside the cells to generate sulfite ions (SO ₃ ²⁻ ). However, since sulfite inhibits glyceraldehyde 3-phosphate dehydrogenase in the energy metabolic pathway and lowers the intracellular ATP concentration of the yeast itself, the yeast is released to the outside of the cell so that an excessive amount of sulfite does not accumulate in the cell. Has the function of discharging sulfurous acid. Here, for example, the SSU1 gene isolated as a gene complementary to a sulfite sensitive mutation (see, for example, Non-Patent Document 11). This SSU1 gene product is composed of 458 amino acid residues, and it is estimated from the results of structural analysis that it is a transporter having 9 to 10 transmembrane regions (see, for example, Non-Patent Document 12). Furthermore, it has already been proved that the SSU1 gene product is involved in the sulfite excretion by experiments using a strain highly expressing the SSU1 gene (see, for example, Non-Patent Document 13).

In general, bottom fermenting yeast has a high ability to produce sulfite, whereas top fermenting yeast hardly produces sulfite. By using the screening method of the present invention, for example, for the SSU1 gene, in addition to the Sc-type SSU1 gene possessed by both the bottom fermentation yeast and the top fermentation yeast, a non-Sc SSU1 gene derived from the bottom fermentation yeast can be selected. Similarly, non-Sc MET14 derived from bottom fermentation yeast can be selected for the MET14 gene encoding a protein involved in the production of sulfite. For example, the functions of the non-Sc type SSU1 and non-Sc type MET14 are greatly involved in the high sulfite-producing ability peculiar to the bottom fermentation yeast, and in order to aim for breeding of yeast having a higher sulfite-producing ability, It is effective to strengthen the type SSU1 gene, the non-Sc type MET14 gene, and the like.
The method for breeding yeasts with enhanced non-Sc type SSU1 gene and non-Sc type MET14 is specifically described in Examples.

  When the brewing yeast gene selected by the screening method of the present invention is introduced into the host yeast, the yeast used as the host is not particularly limited as long as it is a yeast that can be used for brewing. Any widely used yeast, for example, beer yeast such as BH84, NBRC1951, NBRC1952, NBRC1953, and NBRC1954 can be used. Furthermore, whiskey yeasts (for example, S. cerevisiae NCYC90), wine yeasts (for example, Association Wines No. 1, 3, 4, etc.) and sake yeasts (for example, Association yeast sake Nos. 7, 9, etc.) can be used as well. .

  The vector used when the gene is introduced into the host yeast is not particularly limited as long as it is a vector capable of expressing the gene introduced into the yeast. For example, a multicopy plasmid (YEp type), a single copy plasmid (YCp type), and the like. Any of the chromosomal DNA integration plasmids (YIp type) can be used. For example, YEp type vectors include YEp51 (JRBroach et al., Experimental Manipulation of Gene Expression, Academic Press, New York, 83, 1983), and YCp type vectors include YCp50 (MDRose et al., Gene, 60,237, 1987) and the like, YIp5 vectors (K. Struhl et al., Proc. Natl. Acad. Sci. USP, 76, 1035, 1979) and the like can be mentioned. These plasmids are commercially available and can be easily obtained.

The vector may have other sequences for preparing the expression of a gene introduced into yeast, specifically, a promoter, an operator, an enhancer, a silencer, a ribosome binding sequence, a terminator, and the like. In the above vector, the promoter and terminator for constitutively expressing the brewing yeast gene from the early stage of fermentation function in the brewing yeast and are not particularly limited as long as they are independent of the sulfite concentration in the fermentation broth. They may be used in any combination. For example, as the promoter, a promoter of glyceraldehyde 3-phosphate dehydrogenase (TDH3) gene, a promoter of phosphoglycerate kinase (PGK1) gene, or the like can be used. These promoters are known. For example, the PGK1 gene is described in detail in the known literature “MFTuite et al., EMBO J., 1, p603 (1982)” and can be easily obtained.
As long as the DNA obtained by the screening method of the present invention contains these other sequences necessary for the expression of the gene introduced into yeast, it may not be particularly provided on the vector side. When such other sequences are not included in the DNA, it is preferable to prepare other sequences separately and link them to the DNA in an operable state. Alternatively, when a higher expression level or specific expression regulation is expected, it is preferable to link other sequences commensurate with the DNA in an operable state.

The method for transforming the vector into host yeast may be performed according to known means. For example, the following methods can be used. For example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75, p1929 (1978)”, lithium acetate method “J. Bacteriology , 153, p163 (1983) ”,“ Proc. Natl. Acad. Sci. USA, 75, p1929 (1978) ”, and the like.
More specifically, the host yeast is a standard yeast nutrient medium (for example, YEPD medium “Genetic Engineering, vol. 1, Plenum Press, New York, 117 (1979)”), and the value of OD 600 nm is 1-6. Incubate to. The cultured yeast is collected by centrifugation, washed, and pretreated with alkali metal ions, preferably lithium ions, at a concentration of about 1-2 M. The cells are allowed to stand at about 30 ° C. for about 60 minutes, and then left at about 30 ° C. for about 60 minutes together with the DNA to be introduced (about 1 to 20 μg). Polyethylene glycol, preferably about 4,000 daltons, is added to a final concentration of about 20% to 50%. After standing at about 30 ° C. for about 30 minutes, the cells are heated at about 42 ° C. for about 5 minutes. Preferably, the cell suspension is washed with a standard yeast nutrient medium, placed in a predetermined amount of fresh standard yeast nutrient medium, and allowed to stand at about 30 ° C. for about 60 minutes. Thereafter, the transformant is obtained by planting on a standard agar medium containing an antibiotic or the like used as a selection marker.
In addition, as for general cloning techniques, “Molecular Cloning 3rd Edition”, “Methods in Yeast Genitics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)” and the like were referred to.

As a selection marker used for transformation, since an auxotrophic marker cannot be used in the case of brewing yeast, the G418 resistance gene (G418 ^r ) and the copper resistance gene (CUP1) “M.Marin et al., Proc. Natl. Acad. Sci. USA, 81, p 337, 1984 ”, cerulenin resistance gene (fas2m, PDR4) (“ Ogoshi Kaoru et al., Biochemistry, 64, p660, 1992 ”,“ M. Hussain et al., Gene, 101, p149, 1991 ”) and the like are available.

  The yeast for breeding bred in the present invention is not different from the case of using the parent strain in the growth and fermentability of the yeast. Therefore, the raw materials, production equipment, production management, etc. may be exactly the same as the conventional method. This is an important feature of the present invention. However, it goes without saying that conditions such as fermentation time may be varied as desired. For example, when breeding yeast for brewing with enhanced sulfite excretion capacity and producing alcoholic beverages using this yeast, only the content of sulfite excreted as described above changes, and in yeast growth and fermentation capacity, There is no change from the case of using the parent strain. Accordingly, the raw materials, production equipment, production management, etc. may be exactly the same as in the conventional method, and there is no increase in cost for producing alcoholic beverages with an increased sulfite content and improved flavor.

(E) Production of DNA Array A DNA array can be produced using the polynucleotide or oligonucleotide of the present invention obtained in (f) and (h) above. Specifically, a polynucleotide comprising the base sequence obtained in (f) above, a polynucleotide that hybridizes with the polynucleotide under stringent conditions, and / or a contiguous sequence in the base sequence possessed by these polynucleotides. A polynucleotide having a sequence consisting of at least 10 to 200 bases, a polynucleotide encoding a polypeptide consisting of an amino acid sequence encoding the ORF obtained in (h) above, and hybridizing with the polynucleotide under stringent conditions And a polynucleotide comprising an intergenic base sequence that is a DNA base sequence between ORFs obtained in (h) above and / or comprising at least 10 to 200 consecutive bases in the base sequence of these polynucleotides. Arrangement A polynucleotide having one or more preferably can be mentioned DNA array which is fixed to two or more to the solid support.
In the present invention, the DNA array refers to a polynucleotide array, a DNA chip, a DNA macroarray, a DNA array, or the like, in which one or a plurality of polynucleotides or fragments thereof are fixed on the surface of a solid support. . As the polynucleotide or oligonucleotide to be fixed to the solid support, the polynucleotide and oligonucleotide of the present invention obtained in the above (f) and (h) can be used. By fixing polynucleotides or oligonucleotides to a solid support at high density, the analysis described later can be carried out efficiently, but it is not always necessary to have high density. Devices such as an array robot for fixing at high density are commercially available from Takara Shuzo Co., Ltd. (GMS417 Arrayer) and the like, and the commercially available products can be used. Further, the oligonucleotide of the present invention may be directly synthesized on a solid support by a photolithographic method or the like [Lipshutz et al., Nat. Genet., 21, 20-24.
(1999)]. Usually, the length of the synthesized oligonucleotide is 10 to 30 bases, but is not limited thereto. The method for producing the DNA array is not particularly limited, and may be performed according to known means. Preferred methods are described below.

(L) DNA Array Manufacturing Process (l) -1 Substrate As a substrate material used for the DNA array of the present invention, any material that can stably immobilize oligonucleotides may be used. For example, synthetic resins such as polycarbonate and plastic, glass, and the like can be mentioned, but are not limited thereto. Although the form of the substrate is not particularly limited, for example, a plate-like or film-like substrate can be suitably used.

(L) -2 Selection of Oligonucleotides Oligonucleotides immobilized on the substrate surface of the DNA array according to the present invention include, for example, the DNA base sequence of the ORF estimated in (h) above or the intergenic gene estimated from (h) above. Selection can be made based on the DNA base sequence information of the region. For the production of a specific probe, for example, using GeneChip technology (Affymetrix), a unique (PM) probe can be selected for the entire genome base sequence. As a specific probe, for example, an oligonucleotide consisting of 10 to 30 bases in the ORF in the genome base sequence of industrial yeast, and the base sequence is not present in other parts of the whole genome base sequence, Oligonucleotides having a base sequence complementary to the base sequence of the oligonucleotide, and oligonucleotides having 10 to 30 bases that hybridize with these oligonucleotides under stringent conditions can be used. In addition, it is a folded nucleotide consisting of 10 to 30 bases in the untranslated region other than the ORF in the genome base sequence of industrial yeast, and the base sequence does not exist in other parts of the whole genome base sequence. Oligonucleotides, oligonucleotides having a base sequence complementary to the base sequence of the oligonucleotide, and oligonucleotides that hybridize with these oligonucleotides under stringent conditions can be used. The number of bases per oligonucleotide is not particularly limited, but is preferably 10 to 30 bases. 11-50 probes can be selected per region so that extensive quantification and reproducibility can be obtained in the analysis. In addition, for example, in the base sequence of bottom fermenting yeast having two types of closely related species genome, the base sequences are similar, so that one base of the PM probe is used to quantify the degree of non-specific hybridization in the analysis, For example, a mismatch (MM) probe that differs only in the central base can be designed.

(L) -3 Immobilization of oligonucleotides The method for immobilizing oligonucleotides on the substrate surface is not particularly limited, and known methods can be used. For example, a DNA array can be produced by integrating and synthesizing all PM and MM probes selected from the above on a substrate using GeneChip technology.

  Next, as an example of using a DNA array, gene expression analysis is used to identify genes that exhibit characteristic expression under certain conditions, industrial yeast classification, nucleotide polymorphism detection, and functional analysis using these. Although selection of genes will be described, gene analysis using a DNA array is not limited to these methods, and each method is not limited to the examples shown here.

(M) Gene expression analysis By using the DNA array of the present invention prepared in (l), it is possible to analyze, for example, changes in the expression genes of industrial yeast. For example, it is possible to identify a gene (group) whose expression level increases or decreases according to changes in the components of the medium or environmental changes. On the other hand, it is possible to identify a gene (group) exhibiting an expression behavior characteristic of industrial yeast, but is not limited thereto.
Gene expression analysis includes the steps of culturing industrial yeast, preparing mRNA from the cultured yeast, preparing labeled cRNA (or cDNA), hybridization, and data analysis. Each step will be described below with an example, but the present invention is not limited to this example.

(M) -1 Culture under various conditions Industrial yeast to be subjected to expression analysis is cultured in various environments according to the purpose. For example, in the case of identifying a genetic marker that responds to changes in components during culture, it can be cultured by the following method.
Industrial yeast is inoculated into an appropriate medium, for example, LZMM + 40 μM ZnSO4 medium, and cultured with shaking at 30 ° C. overnight. LZMM medium is yeast nitrogen base (Difco) without 0.17% amino acid, 0.5% ammonium sulfate, 20 mM sodium citrate (pH 4.2), 125 μM manganese chloride, 10 μM iron chloride, 2% maltose, It consists of 10 mM EDTA (pH 8.0). The yeast cells are collected and washed three times with an appropriate amount of sterilized water. The appropriate amount of yeast cells, for example, 500 ml of the yeast cells so that the absorbance at OD 600 nm is 0.25, 1) a zinc-depleted medium, For example, LZMM medium, 2) LZMM + 40 μM ZnSO4 medium, 3) oxidative stress medium, such as LZMM + 40 μM ZnSO4 + hydrogen peroxide medium (medium in which hydrogen peroxide is added to the LZMM + 40 μM ZnSO4 medium to 2 mM), 4 ) Inoculate in a carbon source starvation medium, for example, LZMM + 40 μM ZnSO4-Mal medium (medium obtained by removing maltose from the above LZMM + 40 μM ZnSO4 medium) and incubate at 30 ° C. with shaking. After culturing for an appropriate time, each yeast culture solution is recovered in an appropriate amount, for example, 50 mL, for RNA preparation.
It is also possible to collect yeast cells during beer test fermentation and use them in the following experiments.

(M) -2 Preparation of mRNA Total RNA can be prepared using a commercially available kit such as RNeasy (registered trademark) mini kit (manufactured by Qiagen) according to the attached manual.
Poly A messenger RNA (of Poly (A) + mRNA) can be prepared from each total RNA using a commercially available kit, for example, Oligotex Direct mRNA kit (Qiagen) according to the attached manual.

(M) -3 Preparation of labeled cRNA Labeled cRNA can be synthesized using a commercially available kit, for example, a bioarray high yield RNA transcript labeling kit (manufactured by Affymetrix) according to the attached manual. For example, biotin can be used as the labeling dye.

(M) -4 Hybridization To prepare a hybridization cocktail, for example, 5 μg of biotin-labeled cRNA, 1.7 μl of 3 nM control oligonucleotide B2 (manufactured by Affymetrix), 5 μl of 20-fold concentration Eucaliotic High Hybridization control (Affymetrix), 1 μl of 10 mg / mL salmon sperm DNA (Affymetrix), 1 μl of 50 mg / mL acetylated bovine serum albumin (Affymetrix), 50 μl of double concentration hybridization solution (Affymetrix) ), Add sterile water (Affymetrix) to a total volume of 100 μl, mix, and attach using a commercially available device such as a hybridization oven (Affymetrix). Hybridize the DNA array according to the manual. After hybridization for an appropriate time, for example, 16 hours, the hybridization cocktail is removed from the DNA array, and the DNA array is used according to the attached manual using a commercially available device, for example, GeneChip Fluidics Station (Affymetrix). After washing, the cells are stained with a suitable staining solution, such as a streptavidin-phycoerythrin solution. Streptavidin-phycoerythrin solution consists of 300 μl of 2 × concentrated female staining solution (Affymetrix), 24 μl of 50 mg / mL acetylated bovine serum albumin, 6 μl of 1 mg / mL streptavidin-phycoerythrin (Affymetrix), and 270 μl of Consists of sterilized water.

(M) -5 Data analysis For analysis and quantification of DNA arrays on solid supports, data analysis is performed using commercially available analysis software such as GeneChip Operating Software (GCOS; GeneChip Operating Software 1.0 manufactured by Affymetrix). Can be done. For analysis of the gene expression level, commercially available analysis software (for example, GeneSpring; manufactured by Silicon Genetics, ImaGene; manufactured by Fuji Film, Array Gauge; manufactured by Amersham Pharmacia Biotech, ImageQuant, etc.) can be used. As a result of the analysis of the gene expression level, it is possible to identify gene groups exhibiting characteristic expression under certain conditions, and to select these gene groups as targets for functional analysis. Furthermore, brewing conditions can be optimized based on the results of functional analysis.

(N) Classification of industrial yeasts Classification of industrial yeasts using the DNA array of the present invention can be performed, for example, by the following method. Preparation of genomic DNA from yeast and hybridization to the array may be performed using the methods described above. The detection of the brightness of the array can be performed using the above-described GCOS. By calculating the ratio of hybridization to the Sc-type and non-Sc-type probes of the DNA array of the present invention, the test strain and the industrial yeast whose total base sequence was determined, for example, Saccharomyces pastorianus weihenstephan 34/70 Homology can be calculated, and industrial yeasts can be classified according to the value.

(O) Nucleotide polymorphism detection Nucleotide polymorphisms can also be detected by analyzing the results of DNA-DNA hybridization in detail. The probe of the DNA array of the present invention used for the detection of nucleotide polymorphism detection is composed of an oligonucleotide (PM probe) having the same sequence as that of the industrial yeast for which the entire nucleotide sequence has been determined, for example, a base at the center position. Different oligonucleotides (MM probes) are used. By using this DNA array of the present invention, it can be judged that there is a high possibility that the base sequence of a gene that is hybridized more strongly with the MM probe than with the PM probe is different from that of the 34/70 strain. From the result of hybridizing the DNA of the test strain to the present DNA array, the nucleotide polymorphism can be detected if the brightness of the MM probe is, for example, 5 times higher than the brightness of the PM probe.

(P) Selection of gene for functional analysis Using the DNA array of the present invention, a gene for functional analysis can be selected. Preparation of genomic DNA from yeast, hybridization to a DNA array, and detection of the brightness of the DNA array may be performed using the methods described above. Among the probes of the DNA array of the present invention, the gene that did not hybridize with the test strain is the 34/70 strain in which the gene is deleted in the test strain or the base sequence is determined as the entire base sequence. It can be judged that there is a high possibility of difference. Moreover, it can be judged that the gene which showed the high brightness | luminance compared with the other probe has high possibility that the copy number is high in a test strain. There is a possibility that such a difference affects the brewing characteristics, and these genes can be selected as targets for functional analysis.
In addition, by using the DNA array of the present invention used for nucleotide polymorphism detection, a gene that is more strongly hybridized by the MM probe than the above-described PM probe is extracted, and the nucleotide polymorphism is detected, thereby analyzing the function. It is also possible to select a gene to perform.

〔Example〕
EXAMPLES Hereinafter, although the detail of this invention is described according to an Example, this invention is not limited to a following example.

Preparation of genomic DNA of Saccharomyces pastorianus Weihenstephan 34/70 strain (Saccharomyces pastorianus Weihenstephan 34/70, hereinafter abbreviated as 34/70 strain) The method described in “)” was partially modified. The strain 34/70 was inoculated into 200 mL of YPD medium (2% glucose, 1% yeast extract, 2% polypeptone), cultured at 30 ° C. until the absorbance at 660 nm of the culture solution reached 4, and then centrifuged. The cells were collected. The obtained microbial cells were washed with 25 mL of buffer A (50 mM sodium phosphate, 25 mM EDTA, 1% (v / v) β-mercaptoethanol, pH 7.5) and then suspended again in 25 mL of buffer A, 7 mg Zymolyase-100T (Seikagaku Corporation) was added and gently shaken at 37 ° C. for 60 minutes. Add 25 mL of Buffer B (0.2 M Tris-HCl, 80 mM EDTA, 1% SDS, (pH 9.5)) and let stand at 65 ° C. for 30 minutes, then cool on ice, add 12 mL of 5 M potassium acetate. Mix and leave on ice for an additional 60 minutes. The resulting solution was centrifuged at 5,000 g for 10 minutes at 15 ° C., and an equal volume of ethanol was added to the collected supernatant to precipitate the DNA, which was immediately centrifuged at 5,000 g for 10 minutes at 15 ° C. And recovered. The obtained precipitate was washed with 70% (v / v) ethanol, air dried, and then dissolved in 5 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to obtain a crude genomic DNA solution. It was. 4.06 g of cesium chloride and 840 μg of bisbenzimide (Hoechst33258) are added to 3.5 mL of crude genomic DNA solution and dissolved. After centrifugation at 100,000 g for 17 hours at 25 ° C., the DNA band is irradiated with UV light. Was visualized and the lower band was collected. The recovered DNA solution was extracted with isopropanol saturated with a cesium chloride solution to remove bisbenzimide (Hoechst33258), and 4 times volume of 0.3 M sodium acetate was added to the recovered aqueous layer and mixed, and then 3 times volume of ethanol was added. The DNA was precipitated by addition and recovered by centrifugation. The recovered DNA was dissolved in TE buffer containing 75 μg / mL RNase and kept at 37 ° C. for 5 minutes, and then phenol / chloroform extraction was repeated three times, and the recovered aqueous layer was purified again by ethanol precipitation. The precipitate collected by centrifugation was washed with 70% (v / v) ethanol, air-dried, and dissolved in TE buffer to prepare a genomic DNA solution.

Preparation of shotgun library The 34/70 strain genomic solution prepared in Example 1 was prepared to a concentration of 1 mg / mL using TE buffer, and 0.1 mL of the solution was prepared by Hydroshear (Jin Machines, speed6, cycle20). ), The genomic DNA was fragmented. After blunting the ends of the genomic fragments using a DNA Blunting kit (Takara Shuzo), fractionation was performed by 0.8% agarose electrophoresis, and a 1.5-2.5 kb genomic fragment was obtained. The DNA was cut out from the gel and eluted. The DNA eluate was treated with phenol / chloroform and then ethanol precipitated to obtain a genomic library insert. Using T4 ligase (Takara Shuzo), the total amount of the above insert and pUC18 SmaI / BAP (Amersham Biosciences) 0.5 μg were ligated at 15 ° C. for 15 hours.

  The ligation reaction product was ethanol precipitated and dissolved in 10 μL of TE buffer. 1 μL of the ligation solution was introduced by electroporation under the conditions shown in the attached experiment manual with respect to 40 μL of E. coli ELECTRO CELL DH5α (Takara Shuzo). The LB plate medium containing 0.1 mg / mL ampicillin, 0.1 mg / mL X-gal, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) [LB medium containing 1.6% agar (1 % Bactotryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0)] and cultured at 37 ° C. overnight.

  A transformant obtained from a colony formed on the plate medium was statically cultured overnight at 37 ° C. in a 384-well titer plate supplemented with 50 μL of LB medium containing 0.1 mg / mL ampicillin, and then 50% 50 μL of glycerol aqueous solution was added and stirred to use as a glycerol stock.

Preparation of cosmid library About 0.1 mg of 34/70 strain genomic DNA obtained in Example 1 was partially digested with Sau3AI (Takara Shuzo). Insertion of this fragment into the BamHI site of Super Cos I vector (Stratagene) was performed according to the manual. The ligation product obtained by this method was packaged using Gigapack III Gold (Stratagene) and introduced into E. coli XL1-BlueMR strain (Stratagene) according to the manual. This was applied to an LB plate medium containing 0.1 mg / mL of ampicillin and cultured at 37 ° C. overnight. The obtained colonies were cultured in a 96-well titer plate in LB medium (50 μL each well) containing 0.1 mg / mL ampicillin at 37 ° C. overnight, 50 μL of a 50% glycerol aqueous solution was added, and the mixture was stirred to prepare a glycerol stock.

Determination of Base Sequence (4-1) Preparation of DNA Fragment The total base sequence of the 34/70 strain genome was determined mainly using the whole genome shotgun method. A DNA fragment whose nucleotide sequence was determined by this method was prepared from the shotgun library prepared in Example 2 above using the PCR method. Specifically, a clone derived from the whole genome shotgun library was inoculated with a replicator (manufactured by Ginsolution) into a 384-well titer plate in which 50 μL of LB medium containing 0.1 mg / mL ampicillin was dispensed per well, The stationary culture was performed overnight at 37 ° C. The culture solution was transferred to a 384-well reaction plate (manufactured by AB Gene Co.) containing 10 μL of a PCR reaction solution [TaKaRa Ex Taq (manufactured by Takara Shuzo Co., Ltd.) using a replicator (manufactured by Jinsolution Co., Ltd.), and GeneAmp PCR PCR was performed using System9700 (Applied Biosystems) according to Makino et al. Protocol “DNA Research, 5, p1-9 (1998)” to amplify the inserted fragment. M13_for primer (SEQ ID NO: 5) and M13_rv primer (SEQ ID NO: 6) were used as primers. Thereafter, excess primers and nucleotides were removed using a PCR product purification kit (Amersham Biosciences), and this was used as a template for a sequence reaction.

  A DNA fragment from the cosmid library of Example 3 was prepared by the following method. Into each well of a 96-well plate containing 1.0 mL of 2 × YT medium (1.6% bactotryptone, 1% yeast extract, 0.5% sodium chloride, pH 7.0) containing ampicillin 50 μg / mL, all cosmetic drives A rally-derived clone was inoculated and cultured with shaking at 30 ° C. overnight. A cosmid DNA was prepared from the culture broth using an automatic plasmid preparation machine KURABO PI-1100 (manufactured by Kurashiki Boseki Co., Ltd.) according to the manual of Kurashiki Boseki Co., and used as a template for the sequence reaction.

(4-2) Sequencing reaction For 2 μL of DYEnamic ET Terminator Sequencing Kit (Amersham Biosciences), M13_for (SEQ ID NO: 5) / M13_rv (SEQ ID NO: 6) is used as a primer pair for the sequence reaction of shotgun DNA fragments. "DNA Research, 5, p1-9 (1998)" was used as a sequence reaction primer pair of cosmid DNA SS-cosF.1 (SEQ ID NO: 7) /SS-cosR.1 (SEQ ID NO: 8), The PCR product or cosmid DNA prepared in (4-1) above was mixed to prepare an 8 μL sequencing reaction solution. The amount of primer and DNA fragment is 3.2 pmol and 50-200 ng, respectively. Using this reaction solution, a 60-cycle dye terminator sequencing reaction was performed using GeneAmp PCR System 9700. The cycle parameters were according to the manual attached to the DYEnamic ET Terminator Sequencing Kit. The sample was purified using a MultiScreen HV plate (Millipore) according to the Millipore manual. The purified reaction was stored in the dark at 4 ° C. Analysis was performed using MegaBACE1000 Sequencing System (Amersham Bioscience) and ABI PRISM 3700 DNA Analyzer (Applied Biosystems) according to the attached manual. Data of 332,592 sequence obtained by MegaBACE1000 Sequencing System and 13,461 sequence obtained by 3700 DNA Analyzer were transferred to server Enterprise6500 (manufactured by Sun Microsystems) and stored. The data for 346,053 sequence corresponded to about 7 times the genome size.
Table 3 shows a list of PCR primers used in the examples.

Assembly (work to align and connect base sequence fragments)
All the operations of reconstructing the genomic base sequence from the sequence data of the 346,053 sequence DNA fragment obtained in Example 4 were performed on the UNIX (registered trademark) platform. Base calls were made with phred (University of Washington), vector sequence masks with Cross_Match (University of Washington), and assembly with Phrap (University of Washington). The contig obtained as a result of assembly was analyzed using the graphical editor consed (University of Washington). A series of operations from base call to assembly was performed in a lump using the script phredPhrap attached to consed.

Comparison database creation with S. cerevisiae genome sequence
S. pastorianus is considered to be a natural hybrid between S. cerevisiae and its related species “Int. J. Syst Bacteriol., 35, p508-511 (1985)”. Therefore, the homologous search by the homology search algorithm with respect to the S. cerevisiae genomic base sequence is performed on the both end base sequences (10,044 base sequences) of the cosmid DNA clone obtained in (4-2). Regarding the nucleotide sequence, the identification and identity of the homologous region on the S. cerevisiae genomic nucleotide sequence was determined, and a database was created. FIG. 2 shows an identity distribution map of the cosmid base sequence with the corresponding S. cerevisiae genomic base sequence. The base sequences of cosmids were broadly divided into a base sequence group showing an identity higher than 94% with the S. cerevisiae genomic base sequence and a base sequence group showing about 84% identity. A base sequence group showing an identity higher than 94% was an Sc type base sequence, and a base sequence group showing about 84% identity was a non-Sc base sequence. Similarly, a comparison database was prepared of the base sequences of both ends of the shotgun clone and cosmid clone obtained in (4-2) and the S. cerevisiae genomic base sequence. Table 1 is a table showing an example of a comparative database between the base sequences of both ends of 3,648 cosmid clones and the S. cerevisiae genomic base sequence. The homologous region and its identity on the S. cerevisiae genomic nucleotide sequence of each of the forward and reverse strands of the cosmid for which the nucleotide sequence was determined are shown.

  Based on the information obtained from the prepared comparative database, cosmid clones and shotgun clones were mapped onto the S. cerevisiae genome base sequence (FIG. 3). In addition, a comparison database was created between the contig base sequence obtained in Example 5 and the S. cerevisiae genomic base sequence, and mapping was performed. The mapping method is almost the same as the method described above. However, when the forward strand and the reverse strand of the cosmid or shotgun clone are present in different contigs, these contigs were linked (FIG. 4).

Identification of ORF and estimation of function ORF (Open Reading Frame) in the base sequence assembled in Example 5 was identified. Examples are specifically shown below. ORF finder (http://www.ncbi.nih.gov), a program that identifies ORFs for six types of reading frames, including complementary strands, for sequences with a length of 150 bases or more from the start codon to the end codon /gorf/gorf.html) was used to identify the ORF present in the base sequence assembled in Example 5. The function estimation of the extracted ORF was performed by homology search with the amino acid sequence of the ORF of S. cerevisiae registered and published in SGD. Table 2 is a table showing an example of the ORF name of S. cerevisiae corresponding to the function prediction result of the ORF existing in the non-Sc type genome. From left to right, ORF name, ORF polynucleotide length, ORF polypeptide length, ORF name of S. cerevisiae determined by homology search, identity, match length and gene function Indicates.

Analysis of chromosome structure by DNA microarray and PCR Preparation of genomic DNA from yeast using QIAGEN Genomic Tip 100 / G (# 10243: Qiagen) and QIAGEN Genomic DNA buffer set (# 19060: Qiagen) It was carried out according to the attached manual. 10 μg of this DNA was digested with DNaseI (manufactured by Invitrogen) according to the method “Science 281 1194-1197, (1998)” by Winzeler et al. S98 Array: manufactured by Affymetrix Co.). Hybridization and array brightness detection were performed using the Gene Chip analysis basic system manufactured by Affymetrix.
The luminance of probes obtained from S. cerevisiae S288C and 34/70 strains was compared using expression analysis software (Microarray Suite 5.0: manufactured by Affymetrix), and the difference in luminance was a signal based on S. cerevisiae S288C strain. Calculated as the ratio (Signal log ratio). Next, when the signal ratio of each probe is aligned for each chromosome using spreadsheet software (Microsoft Excel2000) and the signal ratio is represented by a bar graph, the number of points where the value of the signal ratio changes greatly as shown in FIG. It was found on some chromosomes. Under the hybridization conditions used in this experiment, only the Sc-type gene showing nearly 100% identity with S. cerevisiae hybridizes in the 34/70 strain genome. It is considered that a change in the signal ratio was caused by the change in the number of Sc-type genes, and in a place where the value of the signal ratio greatly changed, a non-Sc base sequence consisting of a non-Sc base sequence was obtained from a Sc type chromosome consisting of a Sc type base sequence. The existence of a chimeric chromosome structure on the Sc-type chromosome (or vice versa) was suggested.
Based on the genomic base sequence of the 34/70 strain determined by the shotgun method, this chimeric chromosome structure was based on two sets of primers (XVI-1 (L) cer-95894 (SEQ ID NO: 9) / XVI-1 (R) nonSc-106302rv (SEQ ID NO: 10), XVI-2 (L) cer-859737 (SEQ ID NO: 11) / XVI-2 (R) nonSc- 864595rv (SEQ ID NO: 12) was designed, and genomic DNA derived from the 34/70 strain was confirmed by PCR as a DNA fragment.
PCR was performed using TaKaRa LA Taq ^™ and the attached buffer and TaKaRa PCR Thermal Cycler SP according to the attached manual.

As a result of PCR, it was confirmed by 0.8% agarose electrophoresis that a DNA fragment of the expected length was amplified from the 34/70 strain, but as a PCR template, a laboratory strain S. cerevisiae X2180-1A was used. When the genomic DNA was used, amplification of the DNA fragment was not observed. Furthermore, when the nucleotide sequences at both ends of the DNA fragment amplified from the 34/70 strain were confirmed, it was revealed that the DNA sequence was indeed identical with the genomic nucleotide sequence determined by the shotgun method. It was confirmed that non-Sc-type chromosomes were linked (transferred).
From the above results, it was considered that the XVI chromosome has at least two types of chromosomes as shown in FIG. Using a similar method, confirmation was made regarding a region where the connection between the Sc-type chromosome and the non-Sc-type chromosome (or vice versa), that is, the presence of a chimeric chromosome structure was suggested. Such a chimeric chromosome structure of Sc-type chromosome and non-Sc-type chromosome was confirmed in at least 13 places in all chromosomes of 34/70 strain (FIG. 1).
By using genomic information, the complex chromosomal structure of bottom fermenting yeast was clarified, and it was clarified that 34/70 strains had at least 37 types of chromosomes.

Cloning of SSU1 gene of 34/70 strain Using the comparative database obtained in Example 6, a clone containing a non-Sc type SSU1 gene was searched, and the forward base sequence, reverse base sequence and S. cerevisiae of the shotgun clone Were obtained, and a shotgun clone SSS103_G08 was obtained in which a fragment of about 2.4 kb containing a full-length non-Sc type SSU1 gene was inserted from the position information.
This was selected from a shotgun library, and the full length of the non-Sc type SSU1 gene was obtained by PCR. SacI-nonSc-SSU1_for1 (SEQ ID NO: 13) and BglII-nonSc-SSU1_rv1460 (SEQ ID NO: 14) synthetic DNA were used as primers. By this combination, base numbers 1-1460 of the non-Sc type SSU1 gene were amplified, and a SacI-BglII fragment of about 1.5 kb was obtained.

For the Sc-type SSU1 gene, the full length of the gene was obtained by PCR using the 34/70 strain genomic DNA as a template and a primer pair designed based on SGD information. SacI-ScSSU1_for1 (SEQ ID NO: 15) and BglII-ScSSU1_rv1406 (SEQ ID NO: 16) synthetic DNA were used as primers. By this combination, base numbers 1 to 1406 of the Sc-type SSU1 gene were amplified, and a SacI-BglII fragment of about 1.4 kb was obtained.
The Sc-type and non-Sc-type SSU1 gene fragments obtained as described above were inserted into the pCR 2.1-TOPO vector attached to the kit by TA cloning kit (manufactured by Invitrogen), and these were inserted into TOPO / ScSSU1 and TOPO / It was set as nonSc-SSU1. The base sequences of the obtained Sc-type and non-Sc-type SSU1 genes were examined by the Sanger method “F. Sanger, Science, 214, p1215, 1981” (FIG. 10).

Preparation of SSU1 gene disruption strain PCR using plasmids containing drug resistance markers (pFA6a (G418 ^r ), pAG25 (nat1)) as a template according to the method described in the literature “Goldstein et al., Yeast.15 1541 (1999)” A fragment for gene disruption was prepared. As primers for PCR, nonSc-SSU1_for (SEQ ID NO: 17) / nonSc-SSU1_rv (SEQ ID NO: 18) is used for non-Sc SSU1 gene disruption, and ScSSU1_for (SEQ ID NO: 19) / for Sc-type SSU1 gene disruption. ScSSU1_rv (SEQ ID NO: 20) was used. Plasmid pPGAPAUR (AUR1-C) and primer nonSc-SSU1_for + pGAPAUR (SEQ ID NO: 21) / nonSc-SSU1_rv + AUR1-C (SEQ ID NO: 22) were further used for disruption of the non-Sc type SSU1 gene. In this way, two types of Sc-type SSU1 gene disruption fragments and three types of non-Sc-type SSU1 gene disruption fragments were prepared.
The bottom fermenting yeast strain BH96 was transformed with the gene disruption fragment prepared by the method described above. Transformation was performed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and the concentrations of selected drugs were geneticin 300 mg / L, nourseothricin 50 mg / L, and aureobasidin A 1 mg / L, respectively.

About the transformant obtained here, it was confirmed by Southern analysis that the respective drug resistance markers used were introduced and that each SSU1 gene was destroyed. First, genomic DNA extracted from a parent strain and a transformant was treated with restriction enzyme (37 ° C., 18 hrs) with NcoI for confirming Sc-type SSU1 gene disruption and with HindIII for confirming non-Sc-type SSU1 gene disruption. .Developed by 5% agarose gel electrophoresis and transferred to a membrane. In the following, according to the protocol of Alkphos Direct Labeling Reagents (manufactured by Amersham Pharmacia), hybridization with a probe specific to Sc type 1 or non-Sc type SSU1 gene (55 ° C., 18 hrs) was performed, and the signal was detected with CDP-Star .
In this way, each strain where gene disruption was confirmed
Sc-1 (ScSSU1 / Scssu1 :: G418 ^r )
Sc-2 (Scssu1 :: G418 ^r / Scssu1 :: nat)
nonSc-1 (nonSc-SSU1 / nonSc-SSU1 / nonSc-ssu1 :: G418 ^r )
nonSc-2 (nonSc-SSU1 / nonSc-ssu1 :: G418 ^r / nonSc-ssu1 :: nat)
nonSc-3 (nonSc-ssu1 :: G418 ^r / nonSc-ssu1 :: nat / nonSc-ssu1 :: AUR1-C)
Named.

Analysis of the amount of sulfite produced in beer test brewing A fermentation test using the parent strain and the gene-disrupted strains Sc-1 to nonSc-3 obtained in Example 10 was performed under the following conditions.
Wort extract concentration 12.75%
Wort capacity 2L
Wort dissolved oxygen concentration about 9ppm
Fermentation temperature 15 ° C constant yeast input 10g Wet yeast cells / 2L wort The fermentation liquor was sampled over time, yeast growth (OD600) (Fig. 7-a), appearance extract concentration (Fig. 7-b), sulfurous acid Changes with time in the concentration (FIG. 7-c) were examined. Sulfurous acid was quantified by collecting sulfurous acid in aqueous hydrogen peroxide by distillation under acidic conditions and then titrating with alkali (Japan Brewing Association revised BCOJ beer analysis method).
As a result, it was found that the amount of sulfite produced in the Sc-type SSU1 gene-disrupted strain was about the same as that of the parent strain, whereas that in the non-Sc-type SSU1 gene-disrupted strain was significantly reduced. This suggested that the non-Sc type SSU1 gene peculiar to bottom fermentation yeast greatly contributed to sulfite production.
In addition, at this time, the growth rate and extract consumption rate of the non-Sc SSU1 gene-disrupted strain are remarkably reduced, and excess sulfite accumulates in the cells due to lack of sulfite excretion ability, thereby inhibiting growth. Supporting results were obtained.

Preparation of SSU1 gene high expression strain A fragment of about 1.5 kb including the full length of non-Sc type SSU1 gene was excised from plasmid TOPO / nonSc-SSU1 described in Example 9 by restriction enzyme (SacI-BglII) treatment. This was similarly inserted into pNI-NUT treated with a restriction enzyme (SacI-BglII) to construct a non-Sc SSU1 gene high expression vector pYI-nonSc-SSU1. The vector pNI-NUT contains URA3 as a yeast chromosomal homologous recombination site, and includes a nuoseosricin resistance gene (nat1) and an ampicillin resistance gene (Amp ^r ) as transformation markers. On the other hand, the Sc-type SSU1 gene high expression vector pNI-SSU1 is a non-Sc-type SSU1 gene portion of the above-mentioned pYI-nonSc-SSU1 and has an S. This is a structure substituted with SSU1-R “J. Ferment. Bioeng., 86, p427, (1998)” derived from cerevisiae. The glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) promoter and terminator were used for the high expression of each SSU1 gene introduced here.
The bottom fermenting yeast strain BH225 was transformed with the high expression vector prepared by the method described above. Transformation was performed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and selection was performed on a YPD plate medium containing nourseothricin 50 mg / L.

  High expression was confirmed by RT-PCR. Total RNA was extracted using RNeasy Mini Kit (manufactured by Qiagen) according to the manual for total RNA isolation from yeast. ScSSU1_for331 (SEQ ID NO: 23) / ScSSU1_982rv (SEQ ID NO: 24) for the Sc-type SSU1 gene-specific primer, and nonSc-SSU1_for329 (SEQ ID NO: 25) / nonSc-SSU1_981rv (sequence) for the non-Sc-type SSU1 gene-specific primer No. 26), PDA1 which is a constitutive expression gene in yeast was used as an internal standard, and PDA1_for1 (SEQ ID NO: 27) / PDA1_730rv (SEQ ID NO: 28) was used as a specific primer. The PCR product was developed by 1.2% agarose electrophoresis, stained with ethidium bromide solution, and the signal value of each SSU1 gene was normalized with the signal value of PDA1 gene and compared with that of the parent strain. The strains that were confirmed to be highly expressed in this way were named Sc type SSU1 gene high expression strain and non-Sc type SSU1 gene high expression strain, respectively.

Analysis of Sulfurous Acid Emission in Beer Test Brewing A fermentation test using the parent strain and the SSU1 gene high expression strain obtained in Example 12 was performed under the following conditions.
Wort extract concentration 12.83%
Wort capacity 2L
Wort dissolved oxygen concentration about 9ppm
Fermentation temperature 12 ° C constant yeast input 10g wet yeast cells / 2L wort As in Example 11, the fermentation broth was sampled over time, and the yeast growth (OD600) (Fig. 8-a), appearance extract concentration ( FIG. 8-b) and the change with time of the sulfurous acid concentration (FIG. 8-c) were examined. The amount of sulfite produced was slightly higher in the Sc-type SSU1 high-expressing strain (19 ppm at the end of fermentation) than the parent strain (12 ppm in the same), whereas in the non-Sc-type SSU1 highly expressing strain (45 ppm in the same) ) Was seen. At this time, there was no difference in growth rate and extract consumption rate between the parent strain and the high expression strain.
From the above results, in the yeast in which the sulfite discharge pump of the bottom fermentation yeast disclosed by the present invention is constitutively expressed at a high level and the discharge ability of sulfite into beer is enhanced, the fermentation process and fermentation period are reduced. Without change, it became possible to specifically increase the amount of sulfurous acid that acts as an antioxidant for beer. This makes it possible to produce alcoholic beverages with excellent flavor stability and a longer quality retention period.

Cloning of MET14 gene of 34/70 strain Using the comparative database obtained in Example 6, a clone containing a non-Sc MET14 gene was searched, and the forward base sequence, reverse base sequence and S. cerevisiae of the shotgun clone were Were obtained, and a shotgun clone SSS134_O21 into which a fragment of about 1.9 kb containing a full-length non-Sc MET14 structural gene was obtained from the positional information was obtained.
This was selected from a shotgun library, and the full length of the non-Sc MET14 gene was obtained by PCR. As primers, synthetic DNAs of SacI-nonSc-MET14_for-21 (SEQ ID NO: 29) and BamHI-nonSc-MET14_rv618 (SEQ ID NO: 30) shown in Table 3 were used. This combination yielded a non-Sc MET14 gene SacI-BamHI fragment of about 0.6 kb.

For the Sc-type MET14 gene, the full length of the gene was obtained by PCR using the 34/70 strain genomic DNA as a template and a primer pair designed based on SGD information. SacI-ScMET14_for (SEQ ID NO: 31) and BamHI-ScMET14_rv (SEQ ID NO: 32) synthetic DNA were used as primers. By this combination, base numbers 1 to 609 of the approximately 0.6 kb Sc-type MET14 gene were amplified, and an approximately 0.6 kb SacI-BamHI fragment was obtained.
The Sc-type and non-Sc-type MET14 gene fragments obtained as described above were inserted into the pCR 2.1-TOPO vector attached to the kit using a TA cloning kit (manufactured by Invitrogen), and these were inserted into TOPO / ScMET14 and TOPO / It was set as nonSc-MET14.
The base sequences of the obtained Sc-type and non-Sc-type MET14 genes were examined by the Sanger method “Science, 214, p1215 (1981)” (FIG. 11).

High expression of each MET14 gene in a high expression strain of Sc-type SSU1 gene Insert a fragment of about 0.6 kb containing the Sc-type and non-Sc-type MET14 genes described in Example 14 into the multicloning site of pUP3GLP (Japanese Patent Laid-Open No. 2000-316559). Then, high expression vectors pUP3ScMET14 and pUP3nonSc-MET14 were constructed so that each MET14 gene was expressed by the promoter and terminator of the glyceraldehyde-3-phosphate dehydrogenase gene. On the other hand, the upper brewer's yeast KN009F strain was transformed with the Sc-type SSU1 gene high expression vector pNI-SSU1 described in Example 12 to produce the Sc-type SSU1 gene high expression strain FOY227. This FOY227 strain was transformed with the above-mentioned pUP3ScMET14 and pUP3nonSc-MET14 to prepare a Sc-type MET14 gene high-expression strain FOY306 and a non-Sc-type MET14 gene high-expression FOY307 strain in the Sc-type SSU1 high-expression strain.

Analysis of sulfite discharge in beer test brewing Sc type SSU1 gene high expression strain FOY227 obtained in Example 15, Sc type SSU1 high expression strain Sc type MET14 gene high expression strain FOY306 strain, non-Sc type MET14 gene high expression A fermentation test using the strain FOY307 and the parent strain KN009F was performed under the following conditions.
Wort extract concentration 12.84%
Wort capacity 1.5L
Wort dissolved oxygen concentration about 9ppm
Fermentation temperature 25 ° C constant yeast input 7.5g wet yeast cells / 1.5L wort As in Example 11, the fermentation broth was sampled over time, yeast growth (OD600), appearance extract concentration, sulfite concentration The change with time was examined. Although there was no difference between the strains in the amount of yeast growth and the amount of extract consumed, as shown in FIG. 9, the parent strain (KN009F) hardly produced sulfite as shown in FIG. 9 (0.32 ppm at the end of fermentation). On the other hand, the production amount of the F-type SFU1 gene FOY227 strain slightly increased (3.4 ppm), and the FOY306 strain that highly expressed the Sc-type MET14 further increased slightly. A significant increase (16.6 ppm) was observed in the non-Sc MET14 gene high expression strain.

  From the above results, in yeast that constitutively expresses the adenylyl sulfate kinase disclosed by the present invention and has enhanced sulfite production ability, without changing the fermentation process or fermentation period, The amount of sulfurous acid that acts as an antioxidant can be specifically increased. This makes it possible to produce alcoholic beverages with excellent flavor stability and a longer quality retention period.

Preparation of DNA microarray of bottom fermenting yeast A DNA microarray of bottom fermenting yeast was prepared based on the DNA base sequence of the ORF estimated from the whole genome base sequence information of the 34/70 strain and the region between the ORFs.

Preparation of DNA microarray (1) 22483 region from the whole genome sequence information of 34/70 strain, (2) 403 SGD-derived S. cerevisiae ORF regions not identified as Sc type ORF in 34/70 strain, 3) 27 regions from 20 S. pastorinus gene sequence information registered in Genbank, and (4) brewer's yeast using GeneChip technology (Affymetrix) based on 64 gene sequence region information as an internal standard. A PM (Perfect Match) probe (25 base oligonucleotide sequence) unique to the whole genome base sequence was designed. In order to obtain a wide range of quantification and reproducibility in the analysis, 11 probes were selected from 1 region for the regions selected in the above (1), (2), and (3), and 20 probes were selected from 1 region for (4). In addition, in order to quantify the degree of non-specific hybridization in each probe, an MM (MisMatch) probe having a different 13th base of the PM probe was also designed.
The designed all PM and MM probes were synthesized on a substrate using GeneChip technology to prepare a bottom fermentation yeast DNA microarray.

1) includes the following.
6307 A) DNA base sequences derived from non-Sc ORF, B) 7640 Sc base ORF-derived DNA base sequences, C) 28 34/70 strain mitochondrial ORF-derived DNA base sequences, D) 553 DNA base sequence region that was not identified as one of the above ORFs but had homology in the NCBI-BlastX homology search, E) a region between ORFs of 7955 above A) and B).

2) includes the following.
YBL108C-A, YBR074W, YFL061W, YIL165C, YGR165C, YJR052W, YDR223W, YAL025C, YAR073W, YFL057C, YLL015W, YJR105W, YLR299C-A, YNR073C, YDL246C, YHL049C, YHL049C, YHL049C, YHL0MR B, YAL037W, YAL063C-A, YAL064C-A, YAL064W, YAL065C, YAL068W-A, YAL069W, YAR009C, YAR020C, YAR042W, YAR047C, YAR053W, YAR060C, YAR061W, YAR062W, YW062W, YW06 YBL109W, YBL112C, YBR092C, YBR191W-A, YBR219C, YCL019W, YCL029C, YCL065W, YCL066W, YCL068C, YCL069W, YCL073C, YCL074W, YCL075W, YCL076W, YCR035C, YCR0W YCR108C, YDL003W, YDL037C, YDL064W, YDL073W, YDL094C, YDL095W, YDL096C, YDL136W, YDL143W, YDL152W, YDL191W, YDL200C, YDL201W, YDL247W-A, YDL248C, YDR014W, YDR015C YDR160W, YDR210C-D, YDR210W-B, YDR215C, YDR225W, YDR261C-D, YDR261W-B, YDR292C, YDR302W, YDR304C, YDR3 05C, YDR342C, YDR344C, YDR364C, YDR365W-B, YDR427W, YDR433W, YDR471W, YDR510C-A, YDR543C, YDR544C, YEL012W, YEL075W-A, YER039C-A, YER046W-A, YER056C-A, YER060W-A, YER060W YER138C, YER187W, YER188C-A, YER188C-A, YER190C-A, YFL002W-A, YFL014W, YFL019C, YFL020C, YFL030W, YFL031W, YFL051C, YFL052W, YFL053W, YFL054C, YFL055W, YFL056C, FL65C, YFL056C YGL028C, YGL041C, YGL052W, YGL210W-A, YGL259W, YGL262W, YGL263W, YGR034W, YGR038C-B, YGR089W, YGR107W, YGR109W-A, YIL082W-A, YGR122C-A, YGR146C, GR161W, YGR148C YGR271C-A, YGR290W, YGR295C, YHL009W-A, YHL009W-B, YHL015W-A, YHL046W-A, YHL047C, YHL048C-A, YHL048W, YHR032C-A, YHR032W-A, YHR039C-A, YHR043C, Y YHR071C-A, YHR071W, YHR141C, YHR165W-A, YHR179W, YHR180C-B, YHR180W-A, YHR182W, YHR193C, YHR193C-A, YHR207C, YHR211W, YHR213W-A, YHR216W, YHR217C, YHR218W-C, YHR218W-C, YHR218W-C YIL069C, YIL148W, YIL171 W, YIL174W, YIL176C, YIR018C-A, YIR041W, YIR042C, YIR043C, YIR044C, YJL012C-A, YJL014W, YJL062W-A, YJL136C, YJL175W, YJL222W-B, YJR024C, YJR05 YJR107W, YJR110W, YJR111C, YJR140W-A, YJR151C, YJR152W, YJR153W, YJR154W, YJR155W, YJR162C, YKL018W, YKL020C, YKL044W, YKL224C, YKL225W, YKR012C, YKR01C YKR041W, YKR042W, YKR052C, YKR053C, YKR057W, YKR062W, YKR094C, YKR102W, YKR103W, YKR104W, YLL014W, YLL030C, YLL037W, YLL038C, YLL043W, YLR065W, YLR029W, YLR029C, YLR029C YLR142W, YLR144C, YLR145W, YLR154C-G, YLR154W-A, YLR154W-B, YLR154W-C, YLR154W-E, YLR154W-F, YLR155C, YLR156W, YLR157C-B, YLR157W-C, YLR162W, YLR205C, YLR207W, LR YLR227W-B, YLR236C, YLR237W, YLR238W, YLR245C, YLR251W, YLR271W, YLR278C, YLR287C-A, YLR305C, YLR306W, YLR311C, YL R317W, YLR338W, YLR344W, YLR345W, YLR354C, YLR354W, YLR364W, YLR380W, YLR401C, YLR402W, YLR410W-B, YLR411W, YLR412C-A, YLR412W, YLR413W, YLR448W, YLR460C, YLR461W, YLR463C, Y4653 YMR143W, YMR175W-A, YMR247W-A, YMR247W-A, YMR324C, YMR325W, YNL020C, YNL035C, YNL054W-B, YNL243W, YNR034W-A, YNR075C-A, YNR077C, YOL038C-A, YOL053B, YOL053C YOL162W, YOL163W, YOL164W, YOL164W-A, YOL165C, YOL166C, YOL166W-A, YOR050C, YOR096W, YOR101W, YOR192C-B, YOR192C-C, YOR225W, YOR235W, YOR343W-B, YOR366W, YOR3831W, YOR381W-Y YOR384W, YOR385W, YOR386W, YOR387C, YOR389W, YPL003W, YPL019C, YPL023C, YPL036W, YPL048W, YPL055C, YPL060C-A, YPL175W, YPL194W, YPL197C, YPL257W-B, YPR002C-W, PR08C-A, PR YPR087W, YPR094W, YPR108W, YPR137C-B, YPR161C, YPR162C, YPR163C, YPR164W, YPR165W, YPR166C, YPR167C, YPR168W, YPR169W, YPR169W-A, YPR170C, YPR170W-A, YPR171W, YPR172W, YPR173C, YPR174C, YPR175W, YPR176C, YPR177C, YPR178W, YPR179C, YPR180W, YPR181C, YPR182W, YPR183W, YPR184W, YPR185W, YPR186C, YPR187C, PR12W, YPR188C

3) includes the following.
GenBank Accession Number; AY130327, BAA96796.1, BAA96795.1, BAA14032.1, NP_012081.1, NP_009338.1, BAA19915.1, P39711, AY130305, AF399764, AX684850, AB044575, AF114923, AF114915, AF114903, M81158, AJ229060, X12576, X00731, X01963 genes

4) includes the following.
GenBank registration number: J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, X03453.1, X03453.1, L38424.1, L38424.1, L38424. 1, X17013.1, X17013.1, X17013.1, M24537.1, M24537.1, M24537.1, X04603.1, X04603.1, X04603.1, K01391.1, K01391.1, K01391.1, J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, J04423.1, X03453.1, X03453.1, L38424.1, L38424.1, L38424.1, X17013. 1, X17013.1, X17013.1, M24537.1, M24537.1, M24537.1, X04603.1, X04603.1, X04603.1, V01288.1, V01288.1, V01288.1, X16860.1, X16860.1, X16860.1, L12026.1, L12026.1, L12026.1, Z75578.1, Z75578.1, Z75578.1, Z75578.1, Z75578.1, J01355.1, J01355.1, J01355. 1, gene of J01355.1, J01355.1

Identification of genetic markers that are highly expressed under zinc depletion conditions Preparation of mRNA The strain 34/70 was inoculated into 1 L of LZMM + 40 mM ZnSO4 medium and cultured with shaking at 30 ° C. overnight. LZMM medium is yeast nitrogen base containing 0.17% amino acid (Difco), 0.5% ammonium sulfate, 20 mM sodium citrate (pH 4.2), 125 μM manganese chloride, 10 μM iron chloride, 2% maltose, It consists of 10 mM EDTA (pH 8.0). The yeast cells are collected, washed 3 times with 300 mL of sterilized water, and 500 ml of 1) zinc-depleted medium (LZMM medium above) so that the absorbance at OD 600 nm is 0.25, 2) LZMM + 40 mM ZnSO4 medium, 3 ) Oxidative stress medium (LZMM + 40 μM ZnSO4 + hydrogen peroxide medium; medium in which hydrogen peroxide was added to 2 mM to the above LZMM + 40 μM ZnSO4 medium), 4) Carbon source starvation medium (LZMM + 40 μM ZnSO4-Mal medium; The above LZMM + 40 μM ZnSO4 medium in which maltose was removed was inoculated and cultured at 30 ° C. with shaking. After culturing for 6 hours, 50 mL of each yeast culture solution was collected for RNA preparation.
Total RNA was prepared using an RNeasy (registered trademark) mini kit (Qiagen) according to the attached manual.
Poly A messenger RNA (Poly (A) + mRNA) was prepared from each total RNA using Oligotex Direct mRNA Kit (Qiagen) according to the attached manual.

2. Preparation of biotin-labeled cRNA Biotin-labeled cRNA was synthesized using a bioarray high yield RNA transcript labeling kit (Affymetrix) according to the attached manual.

3. Hybridization 5 μg of biotin-labeled cRNA, 1.7 μl of 3 nM control oligonucleotide B2 (Affymetrix), 5 μl of 20-fold eucalitic hybridization control (Affymetrix), 1 μl of 10 mg / mL salmon sperm DNA (Affymetrix), 1 μl of 50 mg / mL acetylated bovine serum albumin (Affymetrix), 50 μl of double-concentration hybridization solution (Affymetrix) are mixed, and sterilized water (Affymetrix) to make a total volume of 100 μl Was added to prepare a hybridization cocktail. This was hybridized to a DNA microarray using a hybridization oven (Affymetrix) according to the attached manual. After 16 hours of hybridization, the hybridization cocktail was removed from the DNA microarray, and the DNA microarray was washed using a GeneChip Fullix Station (Affymetrix) according to the attached manual. Then, it dye | stained with 600 microliters streptavidin-phycoerythrin solution. Streptavidin-phycoerythrin solution is 300 μl of 2-fold concentrated female staining solution (Affymetrix), 24 μl of 50 mg / mL acetylated bovine serum albumin, 6 μl of 1 mg / mL streptavidin-phycoerythrin (Affymetrix), 270 μl of sterilization Prepared with water (Affymetrix).

4). Data Analysis The signal intensity of the microarray was detected using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0 Affymetrix). Normalization for adjustment of signal intensity in the comparative analysis was performed using an all probe set on GCOS. (1) Zinc depletion vs. zinc presence, (2) Oxidative stress vs. zinc presence, (3) Carbon source starvation vs. zinc presence, comparative files of gene expression were prepared using GCOS. Table 4 shows genes whose expression does not increase in the oxidative stress and carbon source starvation state, and whose expression is increased only in the zinc-depleted medium, and the signal log ratio indicating the degree of increase is 0.3 or more.
Sc-1159-1_at, Sc-1161-1_at, Sc-5030-1_at and Sc-2123-1_at are Sc-type YGL258W, YGL256W, YOL154W and YKL175W, respectively, and induces expression at the transcription level under zinc-depleted conditions (Higgins, VJ., Et al., Appl. Environ. Microbiol., 69: 7535-7540 (2003)). On the other hand, Lg-4216-1_s_at is a non-Sc type YKL175W (zinc ion transporter), and it has been confirmed that a gene having this function is induced upon zinc depletion.
From the results of this experiment, it was possible to identify genetic markers that are highly expressed under zinc-depleted conditions by using a DNA microarray prepared based on the entire genome sequence information of 34/70 strains.

  The probe set name indicates the name of the probe set placed on the DNA microarray. Detection indicates whether the transcript is detected (P; Present) or not detected (A; Absent) based on the GCOS detection algorithm (parameter; initial setting). The change is based on the GCOS change algorithm (parameter; initial setting), and the transcript is increased (I; Increase) or unchanged (NC; No Change). Each decrease (D; Decrease) is shown. When the two log results are compared, the signal log ratio indicates a decrease if it is negative, an increase if it is positive, and a numerical value indicates the magnitude of each. The gene name indicates from which gene region the probe was created. The type indicates whether it is a Sc-type gene (Sc) or a non-Sc-type gene (non-Sc).

Gene expression analysis of bottom fermentation yeast during beer test brewing A fermentation test using 34/70 strains was performed under the following conditions.
Wort extract concentration 12.84%
Wort capacity 2L
Wort dissolved oxygen concentration about 9ppm
Fermentation temperature 15 ° C constant yeast input 10g Wet yeast cells / 2L wort The fermentation liquor was sampled over time, and the yeast growth (OD600) (Fig. 12-a) and appearance extract concentration (Fig. 12-b) over time We examined changes. The bacterial cells at 42 hours from the start of fermentation were sampled, mRNA was prepared by the same method as in Example 18, labeled with biotin, and hybridized to the DNA microarray of bottom fermentation yeast.
Detection of the signal intensity (luminance) of the DNA microarray was performed using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0 manufactured by Affymetrix).
When the brightness (expression level) of the probe set of the Sc-type gene and the corresponding non-Sc-type gene was compared, there was one in which a significant difference was observed between the two. Table 5 shows examples of the SSU1 gene and the MET14 gene that are considered to be involved in the production of sulfite during fermentation. In the 34/70 strain, it was revealed that the non-Sc type SSU1 gene was expressed about 3.4 times that of the Sc type SSU1 gene, and the non-Sc type MET14 gene was expressed about 7 times that of the Sc type MET14 gene.

In order to examine whether the difference in brightness is due to the difference in hybridization efficiency of each probe set or the influence of cross hybridization between Sc type and non-Sc type, DNA- using a DNA microarray of bottom fermentation yeast DNA hybridization was performed. The genomic DNAs of the bottom fermenting yeast strain 34/70, the laboratory yeast (S. cerevisiae) S288C strain, and the Saccharomyces carlsbergensis IFO11023 strain were hybridized to the DNA microarray of the bottom fermenting yeast in the same manner as in Example 8. Luminance was measured. As shown in Table 6, the ratio of non-Sc type brightness to Sc type of SSU1 gene and MET14 gene of 34/70 strains is 1.0 to 1.3 times, which is very small compared to the ratio of expression level. It was. In addition, the S288C strain having no non-Sc type SSU1 gene and non-Sc type MET14 gene has extremely low brightness in the non-Sc type SSU1 probe and non-Sc type MET14 probe, and has no Sc type SSU1 gene and Sc type MET14 gene. It was also revealed that the possibility of cross-hybridization between Sc-type and non-Sc-type was extremely low because the strain had extremely low brightness in the Sc-type SSU1 probe and Sc-type MET14 probe. From the above, it was revealed that the non-Sc type SSU1 gene and the non-Sc type MET14 gene of the bottom fermenting yeast strain 34/70 were significantly expressed higher than the corresponding Sc type gene. These genes are thought to be candidates for genes involved in the ability of bottom fermenting yeast to produce high sulfite.
From the above results, it has been clarified that gene expression analysis using a DNA microarray of bottom fermentation yeast is effective in specifying genes to be subjected to functional analysis.

Classification of yeast for brewing using DNA array of bottom fermenting yeast Preparation of genomic DNA from yeast and hybridization to the array were carried out in the same manner as in Example 8, and the luminance of the DNA microarray was detected by Gene of Affymetrix. The analysis was performed using a chip analysis basic system (GCOS; GeneChip operating Software 1.0). As shown in Table 7, it was confirmed that 34/70 strains whose total nucleotide sequence was determined hybridized with 99% or more of the Sc-type and non-Sc-type probes of the DNA array of the present invention. In addition, when the same DNA-DNA hybridization was performed on other bottom fermenting yeasts, 99% or more of Sc-type and non-Sc-type probes in many cases (for example, Table 7, BH225 strain, BH232 strain, BH235 strain). It was confirmed that these strains are very closely related to the 34/70 strain. This indicates that the DNA array of the present invention can also be used for gene expression analysis of these strains. On the other hand, strains that hybridize to the probe of the DNA array of the present invention (for example, BH212 strain in Table 7) are strains that are taxonomically separated from 34/70 strains than the strains listed above. It has been suggested.
From the above results, it was possible to calculate the homology between the bottom fermenting yeasts, and to classify the brewing yeasts based on this.
In addition, genes that did not hybridize with the test strain among the probes of the DNA array of the bottom fermenting yeast were deleted in the test strain or the base sequence determined the entire base sequence. It is likely that it is different from stock. Moreover, it is highly possible that a gene showing high brightness compared to other probes has a high copy number in the test strain. These genes can be selected for functional analysis.

Detection of nucleotide polymorphism using DNA array of bottom fermenting yeast It was possible to detect nucleotide polymorphism by analyzing the results of DNA-DNA hybridization in detail. The probe of the DNA array of the present invention consists of an oligonucleotide (PM; perfect match) having the same sequence as that of the 34/70 strain whose entire base sequence has been determined, and an oligonucleotide (MM; mismatch) having a different base at the central position. Although it is configured, it is highly possible that the base sequence of the gene hybridized more strongly with MM than with PM is different from that of the 34/70 strain. From the results of hybridizing the DNA of the laboratory strain S288C to the DNA array of the present invention, those having a brightness of MM of 5 times or more of the brightness of PM were extracted and compared in nucleotide sequence. It was confirmed that a single nucleotide polymorphism can be detected.

It is a figure which shows a brewer's yeast whole-chromosome structure. White indicates Sc type and black indicates non-Sc type chromosome. Ellipses indicate centromeres. Each Roman numeral indicates the chromosome number of the corresponding S. cerevisiae. It is a figure which shows a non-Sc type | mold chromosome, It has shown that the black filling part has connected in the area | region. For example, nonScII-nonScIV indicates that nonScII and nonScIV are connected with a black-filled portion (300 kb). It is a figure which shows the identity distribution of the both-ends base sequence of 3648 cosmids prepared from the genomic DNA of 34/70 strain, and S. cerevisiae genomic base sequence. The horizontal axis indicates homology with S. cerevisiae, for example, the horizontal axis 84 indicates greater than 82% and less than 84% homology. The vertical axis shows the number of cosmid base sequences showing the identity. It is a figure which shows the example of mapping to the S.cerevisiae genome base sequence of a cosmid and a shotgun clone. (1) and (2) respectively show a gene present on the Watson chain on the S. cerevisiae XVI chromosome base sequence, a base sequence, and a gene present on the click chain. (2), (4), (5) and (6) are respectively a cosmid clone having a Sc-type base sequence, a cosmid clone having a non-Sc type base sequence, a shotgun clone having a Sc-type base sequence, and a non-Sc type. A shotgun clone having a base sequence is shown. An example of the mapping result to the S. cerevisiae genome base sequence of a brewer's yeast contig is shown. A shows a schematic diagram of S. cerevisiae chromosome XVI. B is an enlarged view of the 857-886 kb portion of chromosome XVI. The ordinate and abscissa indicate the contig identity (%) and the aligned position with respect to the S. cerevisiae genomic base sequence, respectively. The solid line and the numerical value above it indicate the contig and the contig number, respectively. The dotted line indicates that the contigs are connected using the forward and reverse strand information derived from the same shotgun and cosmid. The results show that the 34/70 strain genomic DNA was hybridized to a DNA microarray, and the signal intensity (Signal log ratio) compared with the S. cerevisiae S288C strain was aligned for the probe on chromosome XVI. Significant changes in the signal ratio are observed at the three locations indicated by arrows, suggesting that Sc and non-Sc type transfer occurs at this portion. The structure of the XVI chromosome of 34/70 strain estimated from the analysis result by DNA microarray and PCR is shown. It is a figure which shows the time-dependent change of the fermentation process in the beer test brewing using a SSU1 gene disruption strain. The horizontal axis of each of the three graphs represents the fermentation time, and the vertical axis represents a) yeast growth (OD600), b) appearance extract concentration (w / w%), and c) sulfite concentration (ppm), respectively. ing. It is a figure which shows a time-dependent change of the fermentation process in the beer test brewing using a SSU1 gene high expression strain. The horizontal axis of each of the three graphs represents the fermentation time, and the vertical axis represents a) yeast growth (OD600), b) appearance extract concentration (w / w%), and c) sulfite concentration (ppm), respectively. ing. It is a figure which shows the time-dependent change of the fermentation process in the beer test brewing using the MET14 gene high expression strain in Sc type | mold SSU1 high expression strain. The horizontal axis of the graph represents the fermentation time, and the vertical axis represents the sulfurous acid concentration (ppm). It is a figure which shows the base sequence of each SSU1 gene analyzed. It is a figure which shows the base sequence of each analyzed MET14 gene. It is a figure which shows the time-dependent change of the fermentation process in the beer test brewing using 34/70 strain. In each of the two graphs, the horizontal axis represents the fermentation time, and the vertical axis represents a) yeast growth amount (OD600) and b) appearance extract concentration (w / w%), respectively.

Claims (11)

DNA encoding a polypeptide having any of the following amino acid sequences and having sulfite excretion ability:
(a) In the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 3, 1 to 10 amino acid residues are deleted and / or substituted and / or added. Amino acid sequence; or
(b) an amino acid sequence having 95 % or more identity to the amino acid sequence represented by SEQ ID NO: 3. SEQ ID NO: polypeptide or SEQ ID NO: having the amino acid sequence represented by: in the amino acid sequence represented by 3, several amino acid residues 1 to have been deleted and / or substituted and / or additional amino acids The DNA according to claim 1, which has a sequence and encodes a polypeptide having a sulfite excretion ability .   A recombinant vector comprising the DNA according to claim 1 or 2.   The recombinant vector according to claim 2, wherein a promoter and / or terminator is provided adjacent to the DNA according to claim 1 or 2.   The recombinant vector according to claim 4, wherein the promoter is a promoter that constitutively expresses.   The recombinant vector according to claim 4 or 5, wherein the promoter is a promoter of a glyceraldehyde-3-phosphate dehydrogenase gene.   A transformant comprising the DNA according to claim 1 or 2 or the recombinant vector according to any one of claims 3 to 6.   The transformant according to claim 7, which is a yeast of the genus Saccharomyces.   A method for producing alcohol or liquor, comprising using the transformant according to claim 7 or 8. A polypeptide having any one of the following amino acid sequences and having sulfite excretion ability:
(a) In the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 3, 1 to 10 amino acid residues are deleted and / or substituted and / or added. Amino acid sequence; or
(b) an amino acid sequence having 95 % or more identity to the amino acid sequence represented by SEQ ID NO: 3. SEQ ID NO: polypeptide or SEQ ID NO: having the amino acid sequence represented by: in the amino acid sequence represented by 3, several amino acid residues 1 to have been deleted and / or substituted and / or additional amino acids The polypeptide according to claim 10, which has a sequence and has sulfite excretion ability .