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Ep 2275542 A2 (19) & (11) EP 2 275 542 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 19.01.2011 Bulletin 2011/03 C12N 15/10 (2006.01) C12Q 1/68 (2006.01) G01N 33/68 (2006.01) (21) Application number: 10179544.1 (22) Date of filing: 26.02.2007 (84) Designated Contracting States: • Kodama, Yukiko AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Shimamoto-cho, HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI Mishima-gun, SK TR Osaka 6188503 (JP) • Shimonaga, Tomoko (30) Priority: 28.02.2006 JP 2006117198 Shimamoto-cho, Mishima-gun, (62) Document number(s) of the earlier application(s) in Osaka 6188503 (JP) accordance with Art. 76 EPC: • Kanamori, Takeshi 07705679.4 / 1 994 150 Shimamoto-cho, Mishima-gun, (71) Applicant: Suntory Holdings Limited Osaka 6188503 (JP) Kita-ku, Osaka-shi Osaka 530-8203 (JP) (74) Representative: Vossius & Partner Siebertstrasse 4 (72) Inventors: 81675 München (DE) • Nakao, Yoshihiro Shimamoto-cho, Remarks: Mishima-gun, This application was filed on 24-09-2010 as a Osaka 6188503 (JP) divisional application to the application mentioned under INID code 62. (54) Method for identifying useful proteins of brewery yeast (57) The invention relates to a method for identifying of the target protein; (e) comparing the amino acid se- a useful protein of brewery yeast. More specifically, the quence determined in step (d) with the amino acid se- invention relates to (a) cultivating yeast under a prede- quence determined in advance based on all or a part of termined cultivation condition; (b) extracting a protein genome sequence information of bottom fermenting sample from the cultivation product of the yeast; (c) sep- yeast; (f) identifying the target protein and the gene en- arating the protein sample by a protein separation coding the target protein based on the results of compar- means, selecting a target peak or spot, and recovering ison; and (g) analyzing functions of the identified gene the target protein or a fragment thereof contained in the to identify characters given to the yeast by the gene. peak or spot; (d) determining the amino acid sequence EP 2 275 542 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 275 542 A2 Description Technical Field 5 [0001] The invention relates to a method for identifying useful proteins of brewery yeast. More specifically, the invention relates to a method for identifying useful proteins of brewery yeast and genes encoding the proteins by proteome analysis using genome sequence information of brewery yeast. [0002] The invention further relates to a gene identified by the above-mentioned method and uses thereof, in particular to breeding of brewery yeast useful for improving productivity of alcoholic beverages (for example beer) and/or for 10 improving flavor such as stabilization or enhancement of flavor, the alcoholic beverages produced by using the yeast, a production method thereof and the like. Background Art 15 [0003] In recent years, it is possible to introduce a target gene into desired cells and permits the gene to be expressed in the cell using genetic engineering techniques. Cells having desirable characters are prepared by gene engineering techniques with genes whose function have been analyzed using genome information. [0004] In the production of fuel alcohol using industrial yeasts and alcoholic beverages using brewery yeasts, tech- nologies for improving productivity and for stabilization and/or improvement of flavor have been successfully developed 20 by using genetic engineering methods. [0005] Proteome analysis is known as comprehensive protein analysis, and a method for identifying proteins and genes encoding the proteins as targets for genetic engineering. For example, it is used to identify the genes encoding said proteins separated by biochemical property and elucidate the function of the proteins by database searching (against known protein sequences). 25 [0006] An example of the most frequently used method in the analysis today is a peptide mass fingerprinting (PMF) method. A protein and a gene encoding the protein are identified by the PMF method, which comprises separating the protein by two-dimensional electrophoresis, digesting the protein with a protease such as trypsin, obtaining a mass spectrum of the resulting peptide mixture (peptide mass fingerprint) using a mass spectrometer, and comparing the mass spectrum obtained above with theoretical mass spectra calculated from amino acid sequences corresponding to 30 nucleotide sequences from a genome data base. [0007] A bottom fermenting yeast as one of the brewery yeast has been analyzed using a proteome analysis method (Joubert et al., Electrophoresis, 22, 2969 (2001)). However, it was not comprehensive analysis because almost half of the genome of bottom fermenting yeast has been unknown. Namely, the bottom fermenting yeast is an alloploid composed of at least two types of genome (Y. Tamai et al., Yeast, 10, 923 (1998)). One of the genomes is considered to be derived 35 from S. cerevisiae (Saccharomyces cerevisiae type; may be abbreviated as "Sc" hereinafter) whose genome has been sequenced (for example, see A. Goffeau et al., Nature, 387, 5 (1997)). However, remaining genome (Non- S. cerevisiae type; may be abbreviated as "Non-Sc" hereinafter) has not been sequenced Disclosure of Invention 40 [0008] Under the above-mentioned situations, it is desired to analyze whole genome of the bottom fermenting yeast for the comprehensive proteome analyses to identify proteins and the genes encoding the proteins useful for improving productivity and/or flavor of the alcoholic beverages. [0009] In view of the above-mentioned problems, the present inventors have sequenced genome of the bottom fer- 45 menting yeast. The bottom fermenting yeast was proved to have a genome structure having a Sc type nucleotide sequence group that shows over 94% identity and a Non-Sc type nucleotide sequence group that shows about 84% identity. The function of proteins encoded by the gene of the bottom fermenting yeast was inferred by comparing with the amino acid sequences that are registered in the genome database (DB) of S. cerevisiae whose genome sequences have been already elucidated. The results proved that the proteins of the bottom fermenting yeast are roughly divided 50 into two types: those with nearly 100% amino acid identity to S. cerevisiae (Sc type) and those with 70 to 97% (Non- Sc type). The present inventors have made intensive studies based on these discoveries, and have completed the invention. [0010] The invention provides a method for identifying desired proteins of yeast or genes encoding the proteins based on information of the analyzed genome sequence of the bottom fermenting yeast. [0011] In particular, the invention provides a method for identifying useful proteins of brewery yeast and genes encoding 55 the proteins. [0012] Specifically, the invention provides the following: [1] A method for identifying a target protein of yeast or a gene encoding the target protein comprising the steps of: 2 EP 2 275 542 A2 (a) cultivating yeast under a predetermined cultivation condition; (b) extracting a protein sample from the cultivation product of the yeast; (c) separating the protein sample by a protein separation means, selecting a target peak or spot, and recovering a target protein or a fragment thereof contained in the peak or spot; 5 (d) determining the amino acid sequence of the target protein; (e) comparing the amino acid sequence determined in step (d) with the amino acid sequence determined in advance based on all or a part of genome sequence information of the bottom fermenting yeast; (f) identifying the target protein and the gene encoding the target protein based on the results of comparison; and (g) analyzing functions of the identified gene to identify characters given to the yeast by the gene. 10 In addition, as used in the present specification, the term "cultivation product" broadly means those obtained by cultivation of yeast (or a yeast strain), and includes broth (or culture), culture precipitates, yeast cells contained therein, culture supernatant and the like. [2] A method for identifying a target protein of yeast or a gene encoding the target protein comprising the steps of: 15 (1) referring to a database comprising all or a part of genome sequence information of the bottom fermenting yeast based on the amino acid sequence of the target protein of the yeast; (2) identifying a gene encoding the target protein based on the result of reference; and (3) analyzing functions of the identified gene to identify characters given to the yeast by the gene. 20 [3] A method for identifying a target protein of yeast or a gene encoding the target protein comprising the steps of: (1) separating one or more proteins from a protein extract derived from yeast (including a protein directly extracted from yeast, a secreted protein in broth, and the like) and determining the amino acid sequences of 25 the one or more proteins; (2) referring to a database comprising all or a part of genome sequence information of the bottom fermenting yeast based on the amino acid sequence of the one or more proteins; (3) identifying the gene encoding the one or more proteins based on the results of reference; and (4) analyzing functions of the identified gene to identify characters given to the yeast by the gene. 30 [4] A method for identifying a target protein of yeast or a gene encoding the target protein comprising the steps of: (1) cultivating yeast under a predetermined cultivation condition; (2) extracting a protein sample from the cultured product of the yeast; 35 (3) determining the amino acid sequence of the one or more proteins contained in the protein sample; (4) comparing the amino acid sequence determined in step (3) with the amino acid sequence determined in advance based on all or a part of genome sequence information of the bottom fermenting yeast; (5) identifying the gene encoding the target protein based on the above- mentioned results of comparison; and (6) analyzing functions of the identified gene to identify characters given to the yeast by the gene.
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