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Gene Encoding Protein Responsible For Flocculation Property Of Yeast And Use Thereof

Abstract: Abstract GENE ENCODING PROTEIN RESPONSIBLE FOR FLOCCULATION PROPERTY OF YEAST AND USE THEREOF The present invention relates to a gene encoding a protein responsible for flocculation property of yeast and use thereof, in particular, a brewery yeast with appropriate flocculation property for producing desired alcoholic beverages, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast to which suitable flocculation property for brewing desired alcoholic beverages was imparted by controlling expression level of KRE9 gene encoding a protein responsible for flocculation property of brewery yeast, especially non-ScKRE9 gene specific to a lager brewing yeast, to alcoholic beverages produced with said yeast and to a method for producing said beverages.

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Patent Information

Application #
Filing Date
26 September 2008
Publication Number
12/2009
Publication Type
INA
Invention Field
BIOTECHNOLOGY
Status
Email
Parent Application

Applicants

SUNTORY LIMITED
1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8203

Inventors

1. NAKAO, YOSHIHIRO
C/O SUNTORY LIMITED, 1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8203
2. KODAMA, YUKIKO
C/O SUNTORY LIMITED, 1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8203
3. SHIMONAGA, TOMOKO
C/O SUNTORY LIMITED, 1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8203

Specification

DESCRIPTION GENE ENCODING PROTEIN RESPONSBLE FOR FLOCCULAHON PROPERTY OF YEAST AND USE THEREOF TECHNICAL HELD The present invention relates to a gene encoding a protein responsible for flocculation propaly of yeast and use thereof, in particular, a brewery yeast for producing alcoholic beverages with appropriate flocculation property, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, which shows appropriate flocculation property by controlling expression level of KRE9 gene encoding a protein responsible for flocculation property of brewery yeast, especially non-ScKRE9 gene specific to a lager brewing yeast, to a method for selecting said yeast, to a method for breeding said yeast and to a method for producing alcoholic beverages with said yeast. BACKGROUND ART Flocculation propaty of yeast used for fermentation in alcoholic beverages is very important. Flocculation property of yeast means property of forming aggregation as a result of interaction among each individual yeast cells, thus sedimenting to the bottom of the liquid in a liquid culture medium. For example, yeasts used for fermentation of lager-type beer, which is popularly drunk at the present day, are also called bottom fermenting yeast, because the yeast have a character of flocculating and sedimentmg to the bottom of fermentation broth near the end of fermentation. In beer brewing, yeast is recovered ster fermentation and the recovered yeast is used at the subsequent fermentation, which is called "Renjo" which means successive fermentation. Thus, flocculation property of yeast is very important property from the standpoint of working efficiency of brewing process. That is, yeast having poor flocculation property does not sediment at the end of fermentation, and there is a problem that extraneous steps such as centriiiigation are required for recova: it. On the other hand, yeast having undesirably high flocculation property may sediment during fermentation, which can result in immature termination of fermentation. In that case, flavor and taste of resultant product are also seriously influenced. Accordingly, it is very important to use yeast having suitable flocculation property for production of desired alcoholic beverages. Further, flocculation of yeast is actively studied, because efficiency is required especially in the field of industrial alcohol production (Novel agglutinative alcohol fermenting yeast, Japanese Examined Patent Publication (Kokoku) No. H6-36734) and wastewater treatment (Japanese Patent No. 3044284), as well as in the field of alcoholic beverage production. FLO gene family (FLOl, FLOS, FLOS, EL09, FLOIO, ELOll) and SELl, etc. have ever been recognized as genes responsible for flocculation property of yeast as a result of enormous quantity of investigations on flocculation property, which is of importance for industry, of yeast as stated above. In the investigation, for example, propaiy of FLOl gene was analyzed at molecular level (Bidard et al., YEAST, 11(9), 809, 1995). Method for controlling flocculation property of beer yeast was investigated as well using Flolp (Japanese Patent No. 3643404). Method of controlling flocculation property of yeast by regulating expression of FLOS gene, and method of judging flocculating property using nucleotide sequence of FLOS gene (Intemational Publication No. WOOl/040514), etc. are also reported. On the other hand, some of yeast cell surface proteins specific to flocculating yeast are known as well. However, knowledge of each proteins is not suflScient for research to control flocculation property of beer yeast. DISCLOSURE OF INVENTION Under the above situations, there has been a need for breeding yeasts having flocculation property suitable for production of desired alcoholic beverages to make high-efficiency production of alcoholic beverages possible by using a gene encoding a protein responsible for flocculation property of brewery yeast and said protein. The present inventors made extensive studies to solve the above problems and as a result, succeeded in identifying and isolating a gene encoding a protein responsible for flocculation property of yeast fi-om beer yeast. Moreover, the present inventors produced transformed yeast in which expression of the obtained gene was controlled to verify that flocculation property can be actually controlled, thereby completing the present invention. Thus, the present invention relates to a gene encoding a protein responsible for flocculation property of brewery yeast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a method for controlling flocculation property using a yeast in which the expression of said gene is controlled, or the like. More specifically, the present invention provides the following polynucleotides, a vector or a DNA fi-agment comprising said polynucleotide, a transformed yeast introduced with said vector or DNA fi-agment, a method for producing alcoholic beverages by using said transformed yeast, and the like. (1) A polynucleotide selected from the group consisting of (a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ]DN0:1; (b) a polynucleotide comprising a polynucleotide aicoding a protean consisting of the amino add sequence of SEQ ID N0.2; (c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2 in which one or more amino acids thereof are deleted, substituted, inserted and/or added, and having an activity of imparting flocculation properly to yeast; (d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID N0:2, and said protein having an activity of imparting flocculation property to yeast; (e) a polynucleotide comprismg a polynucleotide.which hybridizes to a polynucleotide consisting of a.nucleotide sequence complementary to the nucleotide sequence of SEQ ID "MQ-.l under stringent conditions, and which encodes a protdn havmg an activity of inparting flocculation property to yeast; and (f) a polynucleotide comprising a polynucleotide which hybridizes t6 a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the protein having the amino acid sequence of SEQ ID N0:2 under stringent conditions, and which encodes a protein having an activity of imparting flocculation property to yeast. (2) The polynucleotide according to (1) above selected fiom the group consisting of (g) a polynucleotide comprising a polynucleotide encoding a protem consisting of the amino acid sequence of SEQ ID NO: 2, or encoding the amino acid sequence of SEQ ID NO: 2 in which 1 to 10 amino acids tha-eof are deleted, substituted, inserted, and/or added, and wherein said protein has an activity of imparting flocculation property to yeast; (h) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino add sequaice of SEQ ID NO: 2, and having an activity of imparting flocculation property to yeast; and (i) a polynucleotide comprising a polynucleotide vAnch hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nudeotide sequence of SEQ ID NO: 1, imder high stringent conditions, which encodes a protein having an activity of imparting flocculation property to yeast. (3) The polynucleotide according to (1) above comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. (4) The polynucleotide according to (1) above comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. (5) The polynucleotide according to any one of (1) to (4) above, wherein the polynucleotide is DNA. (6) A polynucleotide selected from the group consisting of: (j) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to (5) above; (k) a polynucleotide encoding KNA that represses the expression of the polynucleotide (DNA) according to (5) above through anRNAi eiSect; (1) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript ofthe polynucleotide (DNA) according to (5) above; and (m) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to (5) above through a co-suppression efifect. (7) A protein encoded by the polynucleotide according to any one of (1) to (5) above. (8) A vector containing the polynucleotide according to any one of (1) to (5) above. (8a) The vector of (8) above, which comprises the expression cassette comprising the followuig components: (x) a promoter that can be transcribed in a yeast cell; (y) any ofthe polynucleotides described in (1) to (5) above linked to the promoter in a sense or antisense direction; and (z) a signal that can function in a yeast with respect to transcription termination and pplyadenylation of a RNA molecule. (8b) The vector of (8) above, which comprises the expression cassette comprising the following components: (x) a promoter that can be transcribed in a yeast cell; (y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense direction; and (z) a signal that can function in a yeast with respect to transcription termination and polyadenylation of a RNA molecule. (8c) The vector of (8) above, which comprises the expression cassette comprising the following components; (x) a promoter that can be transcribed in a yeast cell; (y) any ofthe polynucleotides described in (1) to (5) above linked to the promoter in a antisense direction; and (z) a signal that can function in a yeast with respect to transcription termination and polyadenylation of a RNA molecule, (9) A vector containing the polynucleotide according to (6) above. (10) A yeast into which the vector according to any one of (8) to (9) above has been introduced. (11) The yeast according to (10) above, wherein the flocculation property is increased. (12) The yeast according to (11) above, wherein the flocculation property is increased by increasing an expression level of the protein of (7) above. (13) A yeast, wherein the expression of the polynucleotide (DNA) according to (5) above is repressed by: (A) introducing the vector according to any one of (8) to (9) above; (B) disrupting the gene according to the polynucleotide (DNA) of (5) above; or (C) introducing a mutation into a promoter or genetically altering a promoter. (14) The yeast according to (13) above, wherein the flocculation property is decreased. (15) A method for producing an alcoholic beverage by using the yeast according to any one of (10) to (14) above. (16) The method according to (15) above, wha-ein the brewed alcoholic beverage is a malt beverage. (17) The method according to (15) above, wherein the brewed alcoholic beverage is vnne. (18) An alcoholic beverage produced by the method according to any one of (15) to (17) above. (19) A method for assessing a test yeast for its flocculation property, comprising usmg a primer or probe designed based on the nucleotide sequence of a gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast. (19a) A method for selecting a yeast having a high or low flocculation property by using the method in (19) above. (19b) A method for producing an alcoholic beverage (for example, beer) by using the yeast selected with the method in (19a) above. (20) A method for assessing a test yeast for its flocculation propaty, comprising: culturing the test yeast; and measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of unparting flocculation property to yeast. (20a) A method for selecting a yeast, which comprises assessing a test yeast by the method described in (20) above and selecting a yeast having a high or low expression level of gene encoding a protein having an activity of unpartmg flocculation property to yeast. (20b) A method for producmg an alcohohc beverage (for example, beer) by using the yeast selected with the method in (20a) above. (21) A method for selecting a yeast, comprising: culturimg test yeasts; quantifying the protein of (7) above or measuring the expression level of the gene having the nucleotide sequence of SEQ BD NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast; and selectmg a test yeast having an amount of the protein or the gene expression level according to the flocculation property of interest. (22) The method for selecting a yeast according to (21) above, comprising: culturing a reference yeast and test yeasts; measuring for each yeast the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast; and selectmg a test yeast having the gene expression liigher or lower than that in the reference yeast. (23) The method for selecting a yeast according to (21) above, comprising: culturing a reference yeast and test yeasts; quantifying the protein according to (7) above in each yeast; and selecting a test yeast having a larger or smaller amount of the protein than that in the reference yeast, That is, the method for selecting a yeast of (21) above, comprising: culturing plural yeasts; quantifying the protem of (7) aboye in each yeast; and selecting a yeast having a largo- or smaller amount of the protein among them. (24) A method for producing an alcoholic beverage comprising: conducting fermentation for producing an alcoholic bever^e using the yeast according to any one of (10) to (14) above or a yeast selected by the methods according to any one of (21) to (23) above, and adjusting flocculation property of yeast. According to the method for producmg alcoholic beverages using transformed yeast of the present invention, alcohohc beverages can be produced highly-efBciently by using yeasts having flocculation property suitable for production of desired alcoholic beverages. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows the cell growth with time upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660). Figure 2 shows the extract (sugar) consumption with time upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%). Figure 3 shows the expression profile of non-ScKRE9 gene in yeasts upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal. Figure 4 shows the resuh of flocculation property test of non-ScKRE9 disrupted strain. The vertical axis represents Segmentation Index indicating flocculation property. BEST MODES FOR CARRYING OUT THE INVENTION The present inventors isolated and identified non-ScEGRE9 gene encoding a protein responsible for flocculation property of brewery yeast based on the lager brewing yeast genome information mapped according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-283169. The nucleotide sequence of the gene is represented by SEQ ID NO: 1. Further, an amino acid sequence of a protdn encoded by the gene is represented by SEQ ID NO: 2. 1. Polvnucieotide of tfie invention First of all, the present invention provides (a) a polynucleotide comprising a polynucleotide of the nucleotide sequence of SEQ ID N0:1; and (b) a polynucleotide comprising a polynucleotide encoding a protein of the amino acid sequence of SEQ ID N0:2. The polynucleotide can be DNA orRNA The target polynucleotide of the present invention is not limited to the polynucleotide encoding a protein responsible for flocculation property daived from lager brewing yeast described above and may include other polynucleotides encoding proteins having equivalent functions to said protein. Proteins with equivalent flinctions include, for example, (c) a protein of an amino acid sequence of SEQ ID N0:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added and imparting flocculation property to yeast. Such proteins include a protein consisting of an amino acid sequence of SEQ ID N0:2 with, for example, 1 to 100,1 to 90,1 to 80,1 to 70,1 to 60,1 to 50,1 to 40, 1 to 39,1 to 38,1 to 37,1 to 36,1 to35,1 to34; 1 to33, Ito32,1 to31,1 to30,1 to29,1 to28,1 to27,1 to26,1 to25,1 to24, 1 to 23,1 to 22,1 to 21,1 to 20,1 to 19,1 to 18,1 to 17,1 to 16,1 to 15,1 to 14,1 to 13,1 to 12,1 to 11,1 to 10,1 to 9,1 to 8, 1 to 7,1 to 6 (1 to several amino acids), 1 to 5,1 to 4,1 to 3,1 to 2, or 1 amino acid residues tho-eof being deleted, substituted, inserted and/or added and imparting flocculation propoty to yeast. In general, the number of deletions, substitutions, msertions, and/or additions is preferably smaller. In addition, such proteins include (d) a protein having an amino acid sequence with about 60% or hi^er, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or hi^er, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99,6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or liigher identity with the amino acid sequence of SEQ ID N0:2, and imparting flocculation property to yeast. In general, the percentage identity is preferably higher. Flocculation property of yeast may be measured, for example, by a method described in Japanese Patent Application Laid-Open No. H8-205890. Furthermore, the present invention also contemplates (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID N0:1 under stringent conditions and which encodes a protein imparting flocculation property to yeast; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide complementary to a nucleotide sequence of encoding a protein of SEQ ID N0:2 under stringent conditions, and which encodes a protein imparting flpcculation property to yeast. Herein, "a polynucleotide that hybridizes under stringent conditions" refers to nucleotide sequence, such as a DNA, obtained by a colony hybridization technique, a plaque hybridization technique, a southern hybridization technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID N0:1 or polynucleotide encoding the amino acid sequence of SEQ ID N0:2 as a probe. The hybridization method may be a method desoibed, for example, in MOLECULAR CLONING 3rd Ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997, and so on. The term "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 32°C. "Moderate stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C. "High stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50°C. Under these conditions, a polynucleotide, such as a DNA, with higher homology is expected to be obtained efficiently at higher temperature, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, and one skilled in the art may appropriately select these factors to realize similar stringency. When a commercially available kit is used for hybridization, for example, Alkphos Dkect Labeling Reagents (Amersham Pharmacia) may be used. In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (w/v) SDS at 55°C, thereby detecting hybridized polynucleotide, such as DNA. Other polynucleotides that can be hybridized include polynucleotides having about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 15% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher. 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or Wgher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99,8% or higher or 99.9% or highor identity to polynucleotide encoding the amino add sequence of SEQ ID N0:2 as calculated by homology search software, such as FASTA and BLAST using default parametors. Identity between amino acid sequences or nucleotide sequences .may be determined using algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad Sci. USA, 87: 2264-2268, 1990; Proc. Natl. Acad Sci. USA, 90: 5873,1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et al, J. Mol. Biol. 215: 403, 1990). When a nucleotide sequence is sequenced usmg BLASTN, the parameters are, for example, score = 100 and word length = 12. When an amino acid sequence is sequenced using BLASTX, the parameters are, for example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, defeult parameters for each of the programs are employed. The polynucleotide of the present invention includes (j) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript oiHae polynucleotide (DNA) according to (5) above; (k) a polynucleotide Micodmg RNA that represses the ejq)ression of the polynucleotide (DNA) according to (5) above through RNAi effect; (1) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to (5) above; and (m) a polynucleotide encoding RNA that represses expression of the polynucleotide (DNA) according to (5) above through co-supression effect. These polynucleotides may be incorporated into a vector, which can be introduced into a cell for transfonnation to repress the expression of the polynucleotides (DNA) of (a) to (i) above. Thus, these polynucleotides may suitably be used when repression of the expression of the above DNA is preferable. The phrase "polynucleotide encoding RNA having a nucleotide sequence complementaiy to the transcript of DNA" as used ho-ein refers to so-called antisense DNA. Antisense technique is known as a method for repressing expression of a particular endogenous gene, and is described in various publications (see e.g., IBrajima and Inoue: New Biochemistry Experiment Course 2 Nucleic Acids rV Gene Replication and Expression (Japanese Biochemical Society Ed., Tokyo Kagaku Dozin Co., Ltd.) pp.319-347,1993). The sequence of antisense DNA is prefarably complementary to all or part of the endogenous gene, but may not be completely complementary as long as it can effectively repress the ejqiression of the gene. The transcribed RNA has preferably 90% or higher, and more preferably 95% or higher complementarity to the transcript of the target gene. The length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more. The phrase "polynucleotide encoding KNfA that represses DNA expression through RNAi effect" as used herein refers to a polynucleotide for repressmg expression of an endogenous gene through RNA interference (RNAi). The term "RNAi" refers to a phenomenon where when double-stranded RNA having a sequence identical or similar to the target gene sequence is introduced into a cell, the expressions of both the introduced foreign gene and the target endogenous gene are repressed. RNA as used herein includes, for example, double-stranded RNA that causes RNA interference of 21 to 25 base length, for example, dsRNA (double strand RNA), siRNA (small interfering RNA) or shRNA (short hairpm RNA). Such RNA may be locally delivered to a desired site with a delivery system such as liposome, or a vector that generates the double-stranded RNA described above may be used for local expression thereof Methods for producmg or using such double-stranded RNA (dsRNA, siRNA or shRNA) are known from many publications (see, e.g., Japanese National Phase PCT Laid-open Patent Publication No. 2002-516062; US 2002/0863S6A; Nature Genetics, 24(2), 180-183, 2000 Feb.; Genesis, 26(4), 240-244, 2000 April; Nature, 407:6802, 319-20, 2002 Sep. 21; Genes & Dev., Vol.16, (8), 948-958, 2002 Apr.15; Proc. Natl. Acad. Sci. USA, 99(8), 5515-5520,2002 Apr. 16; Science, 296(5567), 550-553,2002 Apr. 19; Proc Natl. Acad. Sci. USA, 99:9, 6047-6052, 2002 Apr. 30; Nature Biotechnology, Vol.20 (5), 497-500, 2002 May; Nature Biotechnology, Vol. 20(5), 500-505, 2002 May; Nucleic Acids Res., 30:10, e46,2002 May 15). The phrase "polynucleotide encoding RNA having an activity of specifically cleavmg transcript of DNA" as used herein generally refers to a ribo2yme. Ribo2yme is an RNA molecule with a catalytic activity that cleaves a transcript of a target DNA and inhibits the flinction of that gene. Design of ribozymes can be found in various known publications (see, e.g., KEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323: 349, 1986; Nucl. Acids. Res. 19: 6751,1991; Protein Eng 3:733,1990; Nucl. Acids Res. 19:3875,1991; Nucl. Acids Res. 19: 5125, 1991; Biochem Biophys Res Commun 186: 1271, 1992). In addition, the phrase "polynucleotide encoding RNA that represses DNA expression through co-supression effect" refers to a nucleotide that inhibits fimctions of target DNA by "co-supression". The term "co-supression" as used herein, refers to a phenomenon where when a gene havmg a sequence identical or similar to a target endogenous gene is transformed into a cell, the expressions of both the introduced foreign gene and the target endogenous gene are repressed. Design of polynucleotides having a co-supression effect can also be found in various publications (see, e.g., SmythDR: Curr. Biol. 7: R793,1997, MartienssenR: Gun". Biol. 6: 810,1996). 2. Protein of the present invention The present invention also provides proteins encoded by any of the polynucleotides (a) to (i) above. A preferred protein of the present invention comprises an amino acid sequence of SEQ ID N0:2 with one or several amino acids thereof being deleted, substituted, inserted and/or added, and imparts flocculation property to yeast. Such protein includes those having an amino acid sequence of SEQ ID N0:2 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and imparting flocculation property to yeast. In addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID N0:2 and imparting flocculation property to yeast. Such proteins may be obtained by employuag site-directed mutation described, for example, in MOLECULAR CLONING 3rd Ed., CURRENT PROTOCOLS MMOLECULARBIOLOGY, NUC. Acids. Res., 10: 6487 (1982), Proa Natl. Acad Sci. USA 79: 6409 (1982), Gene 34:315 (1985), Nuc. Adds. Res., 13:4431 (1985), Proc. Nail. Acad Sci. USA 82: 488 (1985). Deletion, substitution, insertion and/or addition of one or mare amino acid residues in an amino acid sequence of the protein of the invention means that one or more amino acid residues are deleted, substituted, inserted and/or added at any one or more positions in the same amino acid sequence. Two or more types of deletion, substitution, insertion and/or addition may occur concurrently. Hereinafter, examples of mutually substitutable amino acid residues are enumerated. Amino acid residues in the same group are mutually substitutable. The groups are provided below. Group A: leucine, isoleucine, noileucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butyiglydne, t-butylalanine, cyclohexylalanine; Group B: asparatic acid, glutamic acid, isoasparatic add, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagme, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: soine, threonine, homoserine; and Group G: phenylalanine, tyrosine. The protem of the present invention may also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). In addition, peptide synthesizers available from, for example. Advanced ChemTech, PerkinEhner, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimazu Corp. can also be used for chemical synthesis. 3. Vector of the invention and veast transformed with the vector The present invention then provides a vector comprising the polynucleotide described above. The vector of the present invention is directed to a vector including any of the polynucleotides described in (a) to (i) above or any of the polynucleotides described in 0 to (m) above. Generally, the vector of the present invention comprises an expression cassette including as components (x) a promoter that can transcribe in a yeast cell; (y) a polynucleotide described in any of (a) to (i) above that is linked to the promoter in sense or antisense du-ection; and (z) a signal that functions in the yeast with respect to transcription termination and polyadenylation of RNA molecule. According to the present invention, in order to highly express the protein of the invention described above upon brewing alcoholic beverages (e.g., beer) described below, these polynucleotides are introduced in the sense direction to the promoter to promote expression of the pol3mucleotide (DNA) described in any of (a) to (i) above. Further, in order to repress the above protein of the invention upon brewing alcoholic beverages (e.g., beer) described below, these polynucleotides are introduced in the antisense direction to the promoter to repress the expression of the polynucleotide (DNA) described in any of (a) to (i) above. In order to repress the above protein of the invention, the polynucleotide may be introduced into vectors such that the polynucleotide of any of the (j) to (m) is to be expressed. According to the present invention, the target gene (DNA) may be disrupted to repress the expression of the DNA described above or the expression of the protein described above. A gene may be disrupted by adding or deleting one or more bases to or from a region involved in expression of the gene product in the target gene, for example, a coding region or a promoter region, or by deleting these regions entirely. Such disruption of gene may be found in known publications (see, e.g., Proc Natl. Acad. Sci. USA, 76, 4951(1979), Methods in Enzymology, 101, 202(1983), Japanese Patent Application Laid-Open No.6-253826). Further, in the present invention, the expression level of a target gene can be controlled by introducing a mutation to a promoter or genetically altering a promoter by homologous recombination. Such mutation introducing method is described in Nucleic Acids Res. 29, 4238-4250 (2001), and such alteration of a promoter is described in, for example, Appl Environ Microbiol.,72, 5266-5273 (2006). A vector introduced in the yeast may be any of a multicopy type (YEp type), a single copy type (YCp type), or a chromosome integration type (Yip type). For example, YEp24 (J. R. Broach et al., ExPEimdENTALMANIPULAnONOFGENEEXPRESSlON, Academic Press, New York, 83, 1983) is known as a YEp type vector, YCp50 (M. D, Rose et al., Gene 60:237,1987) is known as a YCp type vector, and YIp5 (K. Strahl et al., Proc. Natl. Acad Sci. USA, 76: 1035, 1979) is known as a Yip type vector, all of which are readily available. Promoters/taminators for adjusting gene expression in yeast may be in any combination as long as thpy ftinction in the brewery yeast and they are not influenced by constituents in fermentation broth. For example, a promoter ofglyceraldehydesS-phosphate dehydrogenase gene (TDH3), or a promoter of 3-phosphoglycerate kinase gene (PGKl) may be used. These genes have previously been cloned, described in detail, for example, in M. F. Tuite et al., EsMO J., 1, 603 (1982), and are readily available by known methods. Since an auxotrophy marker cannot be used as a selective marker upon transformation for a brewery yeast, for example, a geneticin-resistant gene (G418r), a copper-resistant gene (CUPl) (Marin et al., Proc. Natl. Accd Set USA, 81, 337 1984) or a coulenin-resistant gene (fas2m, PDR4) (Junji Inokoshi et al.. Biochemistry, 64, 660, 1992; and Hussain et al.,. Gene, 101: 149, 1991, respectively) may be used. A vector constructed as desoibed above is introduced into a host yeast Examples of the host yeast include any yeast that can be used for brewing, for example, brewery yeasts for he&i, wine, and sake. Specifically, yeasts such as genus Saccharoniyces may be used. According to the present invention, a lager brewing yeast, for example, Saccharomyces pastoriams W34/70, etc., Saccharomyces carlsbergeitsis NCYC453 or NCYC456, etc., or Sacchuromyces cerevisiae NBRC1951, NBRC1952, NBRC1953 or NBRC1954, etc., may be used. In addition, whisky yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japan, and sake yeasts such as sake yeast #7 and 9 from the Brewing Society of Japan may also be used but not limited thereto. In the present invention, lager brewing yeasts such as SaccJtaromycespastorianns may be used preferably. A yeast transformation method may be a generally used known method. For example, methods that can be used include but not limited to an electroporation method (^eth. Enzym., 194: 182 (1990)), a spheroplast method (Proa Natl Acad Set USA, 75: 1929(1978)), a lithium acetate method (J. Bacteriology, 153:163 (1983)), and m^ods described in Proc. Natl. Acad Sci. USA, 75: 1929 (1978), METHODS IN YEAST GSNEncs, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual. More specifically, a host yeast is cultured in a standard yeast nutrition medium (e.g., YEPD medium (Genetic Engineering. Vol. 1, Plenum Press, New York, 117(1979)), etc.) such that OD600 nm will be 1 to 6. This culture yeast is collected by centrifugation, washed and pre-treated with alkali metal ion, preferably lithium ion at a concentration of about 1 to 2 M. After the cell is left to stand at about 30°C for about 60 minutes, it is left to stand with DNA to be introduced (about 1 to 20 )4g) at about 30°C for about another 60 minutes. Polyethyleneglycol, preferably about 4,000 Dalton of polyethyleneglycol, is added to a final, concentration of about 20% to 50%. After leaving at about 30°C for about 30 minutes, the cell is heated at about 42°C for about 5 minutes. Preferably, this cell suspension is washed with a standard yeast nutrition medium, added to a predetentiined amount offresh standard yeast nutrition medium and left to stand at about 30°C for about 60 minutes. Thereafter, it is seeded to a standard agar medium containing an antibiotic or the like as a selective marker to obtain a transformant. Other general cloning techniques may be found, for example, in MOLECULAR CLONING 3rd Ed., and METHODS IN YEAST GENETICS, ALABOFATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). 4. Method of producing alcoholic beverages according to the present invention and alcoholic beverages produced bv the method A yeast having flocculation property suitable for a target alcoholic beverages can be obtained by introducing the vector of the present invention or DNA fragments described above to a yeast suitable for brewing target alcoholic beverages to control expression level of the gene. Thus, alcoholic beverages can be produced highly-efficiently. That is to say, desired kind of alcoholic beverages can be produced highly-efficiently by controlling (elevating or reducing) flocculation property using yeasts into which the vector or DNA fragment of the present invention was introduced described above, yeasts in which expression of the polynucleotide (DNA) of the present invention described above was regulated (promoted or suppressed) or yeasts selected by the yeast assessment method of the invention described below for fermentation to produce alcoholic beverages. The target alcoholic beverages include, for example, but not limited to beer, beer-taste beverages such as sparkling liquor (hcppoushu), wine, whisky, sake and the like. Further, alcohol for practical use such as alcohol for fiiel is also included among them. In order to produce these alcoholic beverages, a known technique can be used except that a brewery yeast obtained according to the present invention is used in the place of a parent strain. Since materials, manufacturing equipment, manufacturing control and the like may be exactly the same as the conventional ones, there is no need of increasing the cost for producing alcoholic beverages. Thus, according to the present invention, alcoholic beverages can be produced highly-efficiently using the existing facility without increasmg the cost. 5. Yeast assessment method of the invention The present invention relates to a method for assessing a test yeast for its flocculation property by using a primer or a probe designed based on a nucleotide sequence of a gene having the nucleotide sequence of SEQ ID N0:1 and encoding a protein imparting flocculation property to yeast. General techniques for such assessment method is known and is described in, for example, WOOl/040514, Japanese Laid-Open Patent Application No. H8-205900 or the like. This assessment method is described in below. First, genome of a test yeast is prepared. For this preparation, any known method such as Hereford method or potassium acetate method may be used (e.g.. METHODS IN YEAST Gteea-ature controlled room at 15°C. Subsequent measurement procedures were performed in a temperature controlled room at 15°C. The yeast suspension mixed for 30 seconds by vortex mixer was poured into a buret, then immediately 2 mL of the suspension was collected as a 0 min sample. After leaving 10 min at rest, 2 mL of the suspension was collected as a 10 min sample. OD600 of the collected yeast suspensions were measured, respectively, and flocculation properties (Segmentation Index; SI) were calculated by formula below. The results are mdicated in Figure 4. The values were averages of measurement of n = 2. As indicated in Figure 4, SI = 117 in the disrupted strmn shows that flocculation property of 18 yeast was decreased by disruption of non-ScKRE9 by comparison with SI = 232 in the parent strain. Example 5; Construction of nonScKRE9-Highlv Expressed Strain The nonScKRE9/pCR2.1-TOPO described in Example 1 is digested with the restriction enzymes Sad and Not! to prepare a DNA fragment contairdng the entire length of the protein-encoding region. This fragment is ligated to pYCGPYNot treated with the restriction enzymes Sad and NotI, thereby constructing the nonScKRE9 high e?q)ression vector nonScKRE9/pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector. A gene inserted is highly expressed by the pyruvate kinase gene PYKl promoter. The geneticin-resistant gene G418 is mcluded as the selectable marker in the yeast, and the ampicillui-resistant gene Amp' as the selectable marker in Escherichia coli. Using the high expression vector prepared by the above method, the strain Saccharomyces pasteurianus Weihenstephaner 34/70 is transformed by the method desaibed in Japanese Patent Application Laid-open No. H07-3 03475. The transformants are sdected on a YPD plate medium (1% yeast extract, 2% polypeptone, 2% glucose and 2% agar) containing 300 mg/L of geneticin. Flocculation propaties of the highly expressed strain obtained by the method described above and the parent strain (W34/70 strain) are evaluated by the same method as Example 4. INDUSTRIAL APPLICABILITY The method of producing alcoholic beverages of the present invention may allow for highly-efficient production of alcoholic beverages by using a yeast having suitable for production of desired alcoholic beverages, because the flocculation property of yeast during fermentation can be controlled. CLAIMS 1. A polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDNO:!; (b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2; (c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ED N0:2 in which one or more amino acids thereof are deleted, substituted, inserted and/or added, and having an activity of irnparting flocculation property to yeast; (d) a polynucleotide comprising a polynucleotide encoding a protein havmg an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ DD N0:2, and said protein having an activity of imparting flocculation property to yeast; (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ E) N0:1 under stringent conditions, and which encodes a protein having an activity of impartmg flocculation property to yeast; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the protein having the amino acid sequence of SEQ ID NO .2 under stringent conditions, and which encodes a protein having an activity of imparting flocculation property to yeast. 2. The polynucleotide according to Claim 1 selected from the group consisting of (g) a polynucleotide conprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, or encoding the amino acid sequence of SEQ ID NO: 2 in which 1 to 10 amino acids thereof are deleted, substituted, inserted, and/or added, and wherein said protein has an activity of imparting flocculation property to yeast; (h) a polynucleotide comprising a polynucleotide encoding a protem having 90% or higher identity with the amino acid sequence of SEQ ID NO; 2, and having an activity of imparting flocculation property to yeast; and (i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, under high stringent conditions, which encodes a protein having an activity of imparting flocculation property to yeast. 3. The polynucleotide according to Claim 1 comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. 4. The polynucleotide according to Claim 1 comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. 5. The polynucleotide according to any one of Clauns 1 to 4, wherein the polynucleotide isDNA. 6. A polynucleotide selected from the group consisting of: (j) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to Claim 5; (k) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to Claim 5 through an RNAi effect; 0) a polynucleotides eh(X)ding RNA ha\dng an activity of spedfically cleaving a transcript of the polynucleotide (DNA) according to Claim 5; and (m) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to Claim 5 through a co-suppression effect. 7. A protein encoded by the polynucleotide accordmg to any one of Claims 1 to 5. 8. A vector containing the polynucleotide according to any one of Claims 1 to 5. 9. A vector containing the polynucleotide according to Claim 6. 10. A yeast into which the vector according to Claim 8 or 9 has been introduced. 11. The yeast according to Claim 10, wherein the flocculation property is increased. 12. The yeast according to Claim 11, wherein the flocculation proparty is increased by increasmg an expression level of the protein of Claun 7. 13. A yeast, wherein the expression of the polynucleotide pNA) according to Claim 5 is repressed by: (A) introducing the vector according to Claim 8 or 9; (B) disrupting the gene according to the polynucleotide (DNA) of Claim 5; or (C) introducing a mutation into a promoter or genetically altering a promoter. 14. The yeast according to Claim 13, wherein the flocculation property is decreased. 15. A method for producing an alcoholic beverage by using the yeast according to any one of Claims 10 to 14. 16. The method according to Claun 15, wherein the brewed alcoholic beverage is a malt beverage. 17. The method according to Claim 15, wherein the brewed alcoholic beverage is wine. 18. An alcoholic beverage produced by the method according to any one of Claims 15 to 17. 19. A method for assessing a test yeast for its flocculation property, comprising using a primer or probe designed based on the nucleotide sequence of a gene having the nucleotide sequence of SEQ E) NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast. 20. A method for assessing a test yeast for its flocculation property, comprising: culturing the test yeast; and measuring the expresaon level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast. 21. A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein of Claim 7 or measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast; and selecting a test yeast having an amount of the protein or the gene expression level according to the flocculation property of interest. 22. The method for selecting a yeast according to Claim 21, comprising: culturing a reference yeast and test yeasts; measuring for each yeast the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting 11 flociculation property to yeast; and selecting a test yeast having the gene expression higher or lower than that in the reference yeast. 23. The method for selecting a yeast according to Claim 21, comprising: culturing a reference yeast and test yeasts; quantifying the protein according to Claim 7 in each yeast; and selecting a test yeast having a larger or smaller amount of the protein than that in the reference yeast. 24. A method for producing an alcoholic beverage comprising; conducting fermentation for producing an alcoholic beverage using the yeast according to any one of Claims 10 to 14 or a yeast selected by the methods according to any one of Claims 21 to 23, and adjusting flocculation property of yeast.

Documents

Application Documents

# Name Date
1 5192-chenp-2008 abstract.pdf 2011-09-04
1 5192-CHENP-2008 FORM-6 08-09-2009.pdf 2009-09-08
2 5192-CHENP-2008 FORM-13 08-09-2009.pdf 2009-09-08
2 5192-chenp-2008 claims.pdf 2011-09-04
3 5192-chenp-2008 pct.pdf 2011-09-04
3 5192-chenp-2008 correspondence others.pdf 2011-09-04
4 5192-chenp-2008 correspondence-others.pdf 2011-09-04
4 5192-chenp-2008 form-3.pdf 2011-09-04
5 5192-chenp-2008 form 5.pdf 2011-09-04
5 5192-chenp-2008 description(complete).pdf 2011-09-04
6 5192-chenp-2008 form 3.pdf 2011-09-04
6 5192-chenp-2008 drawings.pdf 2011-09-04
7 5192-chenp-2008 form 1.pdf 2011-09-04
8 5192-chenp-2008 form 3.pdf 2011-09-04
8 5192-chenp-2008 drawings.pdf 2011-09-04
9 5192-chenp-2008 form 5.pdf 2011-09-04
9 5192-chenp-2008 description(complete).pdf 2011-09-04
10 5192-chenp-2008 correspondence-others.pdf 2011-09-04
10 5192-chenp-2008 form-3.pdf 2011-09-04
11 5192-chenp-2008 correspondence others.pdf 2011-09-04
11 5192-chenp-2008 pct.pdf 2011-09-04
12 5192-CHENP-2008 FORM-13 08-09-2009.pdf 2009-09-08
12 5192-chenp-2008 claims.pdf 2011-09-04
13 5192-CHENP-2008 FORM-6 08-09-2009.pdf 2009-09-08
13 5192-chenp-2008 abstract.pdf 2011-09-04