DESCRIPTION
ALCOHOL ACETYL TRANSFERASE GENE AND USP THEREOF
TECHNICAL FIELD
The present
BACKGROUND ART


Laid-oprai Publication No. H06-253826), and increasing the amount of ester by destroying estca-ase geae EST2 in brewing yeast (%aDese Patrat Ldid-oipen Publication No. H09-234077).
DISCLOSl]RE OF INVENTION
As stated above, although a mutant strain is acquired for increasing the ester content of a product, Ifaere are cases in whk:b unexpected delays in fcnn ‘ xtation or increases in undesirable aromatic and flavor conponcots are obs’;ved as a result thereof tbus creating pioblems in the development of y ast for practical applicatioa Consequ’itly, there is need for a method for breeding yeast, capable of producing a desired amount of est’::s without in’airing the fermentation rate or product quality.
As a result of conducting wtensive studies to solve the above-mentioned problems, the inventors of the pres’ invention succeeded in identifying and isolating a gexie fiom Isi'ewer's yeast which encpdes an alcohol acetyl transfo:ase that demonstrates more advantageous efibcts than known proteins. In addition, the inventors of the present invention also confinned that the amount of ester fonned increases by producing a transformed yeast by iilserting and expressing the resulting gene in yeast, theroby leading to conviction of the pres’ invent!’
Namely, the present invention relates to a novel alcohol acetyl transfoase gene characteristioally present in brew’ yeast, aprotein encoded by said graie, atransformed yeast in which the e?q)ression' of said gene is r’julated, and a method for controllipg the amount of esta formed in a product by using yeast in which expression of said g’ has be’ regulated: More specifically, the present Invention provides the pofynucleotide indicated betow, a vector containing said polynucleotide,- d tran’rmed yeast in which said vector ha’ boen inserted, and a n’thod for producing an alcoholic beverage using said transfonned yeast. (1) A pplynucleotide selected from the group consisting o£
(a) a polynucleotide conpcisiiig a potynucleoiide consisti];)g of the nucleotide sequence ofSEQUiNO:!;
(b) a polynucleotide conpising a polynucleotide encoding a protein consisting of the amino acid sequeaice of SEQ ID N0:2;
(c) a polynucleotide conpising a polynucleotide encoding a protdn consisting of ‘ amino acid sequence of SEQ ID N0:2 with one or more amino adds thereof being deleted, substituted, ins’led and/or added, and having an alcohol acetyl transferase activity;
(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the anoino acid sequ’ice of SEQ ID N0:2, and having an alcohol acetyl transferase activity;
(e) a polynucleotide comprising a polynucleotide which hytn'Mizes to a polynucleotide

consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: I under stringent conditions, and whidi encodes a protein halving an alcohol acetyl transftiase activity, and
(f) a polynucleotide conpising a polynucleotide which hybrWizes to a polynucleotide
consisting of a nudeotide sequence con’lementaxy to the nucleotide sequence of the
polymicleotide encoding the protein of the amino acid sequence of SEQ ID N0;2 under stringent
conditions, and whidi ‘codes a protein having an alcohol acetyl transferase activity.
(2) The polynucleotide of (1) above selected fix)m the groiq) consisting of:
(g) a polynucleotide coinprising a polynucleotide encoding a pixytdn consisting of the
amino acid sequence of SEQ ID NO: 2, or encoding an amitio add sequetK’ of SEQ ID NO: 2
wherein 1 to 10 amino acids thereof is deleted, substituted, inserted, and/or added, and wbraein
said protein has an alcohol acetyl transferase activity;
(h) a polynucleotide comprising a polynucleotide encoding a pxctdn having 90% or hi’er identity with the amino acid sequence of SEQ 11’ NO: 2, and having an alcohol acetyl transferase activity; and
(9 a.polynucleotide con’rising a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybndkes to a nucleotide sequence conplementary to tibe nucleotide sequoice of SEQ ID NO; I under high stringent conditions, and which encodes a protein having an alcohol acetyl transferase activity.
(3) Tl2e polynucleotide of (1) above compming a polynucleotide consisting of the nucleotide sequwjce of SEQ ID NO: 1.
(4) The polynucleotide of (1) above conpasicg a polynucleotide ©needing a protein consisting of the amino acid sequeaice of SEQ ID NO: 2.
(5) The polynucleotide of any one of (1) to (4) above, wh«ein the polynucleotMe is DNA.
(6) A polynucleotide selected fiom the group oons’ting o£
Q) a polynucleotide oicoding RNA of a mcleotide sequence conQ>Iemi’ary to a transcrqpt of the polynucleotide (DNA) according to (5) above;
(k) a pofynucleot’e encoding RNA that represses the expression of the polynucleotide (DNA) according to (5) above through RNAi eflfect;
(1) a polynucleotide ‘icoding RNA having an activity of ‘ecifically cleaving a iranscr’ 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 tkougb co-sui’ression e£kct
(7) Aprotein encoded by the poj’deotide of any one of (1) to (5) abovei

(8) A vector con’nising the polynucleotide of any OM of (1) to (5) above.
(8a) The vector of (8) above, which conpises the esKpression cassette conpising the feUowing components:
(x) a' promoter ‘ can be transcribed in a yeast celt
(y) any of the polynucleotides desoribed in (I) to (5) above linked to the px’xnoter in a s’:ise direction OT an antise33se direction; and
(z) a signal that can fimction in a yeast wifli reject to tnmscription termination and polyadenylation of a UNA molecule.
(9) A vector con’jrising the polynucleotide of (6) above.
(10) A yeast, wherein the vector of (8) or (9) above iS introduced.
(11) The yeast of (10) above, wherein ester-producing ability is increased by introducing the vector of (8) above.
(12) A yeast, wha:ein an expression of the polynucleotide (DNA) of (5) above is repressed by introducing the vector of (9) above, or by disrupting a gene related to the polynucleotide OONA) of (5) above.
(13),'nie yeast of (11) above, whetem a esta-produdng ability is increased by inoeasit’ an ej’ftiession level of tiie protein of (7) above.
(14) A method fcr producing an alcoholic beverage by usti’ the yeast of any one of (10) to (13) above,
(15) The m’hod for producing an alcoholic beverage of (14) above, wherein the brewed alcoholic beverage is a malt beveiage.
(16) The method fbf producing an alcoholic beverage of (14) above, wherein the brewed alcobblic beverage is a wine.
(17) An alcoholicbeverage, which is produced ly’’wi’hod of any one of (14) to (16) above.
(18) A method jEbr assessing, a test yeast &r its ester-producing capability, comprising using aprimeiror apt?obe deseed based on a nucleotide sequence of an alcohol acetyl transferase gene having the nucleotide sequ’iceofSEQ ID NO: 1.
(18a) A method fi}r selecting a yeast having increased or dea:eased est’-pioducing capability by usii% the method in ‘18) above.
(18b) A method fcr iroducmg an alcoholic beverage (for exanqjfe, beer) by using the yeast selected v’tb the noetbod in (18a) above.
(19) A method fcr assessing a test yeast fcr its ester-producing capalalky, comprising:
culturing a test yi’st; and measurictg an expression level of an alcohol acetyl transferase gene
having the nucleotide sequence of SEQ ID NO: 1.

(20) A method for selecting a yeast, conprising: cuburing test yeasts; quantifying the
protein of (7) above or measuring an eTcpression level of an alcohol acdyl transferase gene having
the nucleotide sequence of SEQ ID NO; 1; and selecting a test yeast having said piotdn amount or
said gene expr’sion level accordintg to a target c’bility of producing ester.
(20a) A method for setecting a yeast, con’prising: culturing test yeasts; measuring a estcr-pioducing capability or an alcohol acetyl transferase activity of the protein of (7) above; and selecting a test yeast having a target capability of producixig ester or a taiget alcohol acetyl transferase activity:
(21) The method for sdbcting a yeast of (20) above, conpising: culturing a reference yeast and test yeasts; measuring an expression bvel of an alcohol acetyl transferase gatie hdving the nucleotide sequence of SEQ ID NO: 1 in each yeast; and selecting a test yeast havit’ the gene expressed higho* or lower than that in the reference yeast.
(22) The method for selecting a yeast of (20) above coirprising: culturing a reference yeast and test yeasts; quanti’nng the protein of (7) above in each yeast; and selecting a test yeast having said protein for a larger amount than that in the reference yeast That is, the method for selecting a yepst of (20) above comprising;.culturing plural yeasts; quantifying the protein of (7) above in each yeast; and sdecting a test yeast having a large amount of the protein from them
That is, the method for selecting a yeast of (20) above conpjsing: culturing plural yeasts;
*
quantij’dng the protein of (7) above in eadi yeast; and selecting a test yeast producing a large or ‘mall amount of the protein from them.
(23) A method for ptoduciog an alcoholic beverage ccnipising: conducting femientation
for producing an alcoholic; bever&ge using the yeast according to any one of (10) to (13) or a yeast
seilected by the method according to any one of (20) to (22); and adjusting the production amouiit
of ester.
According to the method for producing alcohols by using the transfbrmed yeast of the inventioi’' ester contents can be controlled so that alcohols v’ enhanced flavor can be produced
BRIEF DESCRIPnON OF DRAAVINGS
Figure 1 shows the cell growth with time upon beer fermentation test The horizontal axis represents fermentation time while the vertk’ axis r’resents optical density at 660 nm
(OD660).
Figure 2 shows the extract (sugar) consuiiq)tion widii time ip)n beer ‘mentation test The horizontal axis represents feaiD’tation tinie while Ihe ver’ concentration (w/w%).
Figure 3 shows the eT’ression profUe of nonScATF2 gene in yeasts upon beer

fiamentation test The horizontal axis represents feinentation time v’’e the vertical axis rq)reseQts the iutensi’ of detected signal
Figure 4 shows the cell growth with time upon fermentation test. The horizontal axis rep-esents foe’’itation time while the vertical axis rq’resraits optical density at 660 nm (OD660).
Figure S shows the retract (sugar) consumption with time upon beer lfem[ient3tion test. The horizontal axis represaits fennentation time while the vertical axis represents appda:aA extract concentration (wAv%).
BEST M0D£S FOR CARRYING OUT THE INVENTION
The present inventors conceived that it is possible to control est’ in products by increasing or decreasing an alcohol acetyl transferase activity of the yeast. Tte present inventors have studied based on this conception and as a result, isolated and identified a nonScATF2 geoQ encoding an alcohol acetyl transferase unique to lager brewing yeast based on the lager brewing yeast genome infermation mapped according to die method disclosed in Japanese Patent LaidOp’ Publication No. 2004-283169. The nucleotide sequaice Qf th6 gene isxepxsssedied by SEQ ID NO: L Furtfi’, an amino acid sequ’ice of a piotein exicoded by the gebe is repxes’ited bySEQIDNOiZ
First of all, the psiesent invention provides (a) a ppfynucleotide cotipising a polynucleotide consisting of the nucleotide Sjsqu’ice of SEQ ID N0:1; and (b) a poltynucleotide corrpjsing a polynucleotide encoding a protein of die amino acid sequence of SEQ ID N0:2. The pdlytmcleotide can be DNAor RNA.
The target polynucleotide of the present invention is not limited to the polynucleotide encoding an alcohol acd}i transferase gene derived tomlagerbxwiilig yeast described above and may include other polynucleotides encodihg proteins having equivalent functions to said protein. Protein? with equivalent fenctions include, fer exan’le, (c) a proteiii of an amino acid sequence of SEQ ID NO: 2 with one or more amino acids thereof being deleted, substituted, instiled and/or added and having an alcohol acetyl transferase activity.
Such proteins include aprotein consisting of an amino acid sequence of SEQ ID NO: 2 with,forexample,ltolOO, Ito90,lto80,lto70, Ito60,lto50, Ito40,lto39, It’ 37,lto36,lto35,lto34, lto33,lto32,lto31,lto30,lU)29,lto28,lto27,lto26,lto 25,1 to 24,1 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 sevKal amino acids), 1 to 5,1 to 4,1 to 3,1 to 2, or 1 amino acid residues thereof being deleted, substituted, inserted and/or added and

having an akohol acetyl transfo’e activity. In gaaeral, the numb’ of deletions, substitutions, insertions, and/or additions is pre’rably smaller: In additbn, such protdns include (d) a protdn having an amino add sequaice with about 60% or higher, about 70% or higher, 71% or higter, 72% or higt’; 73% or bi’ier, 74% or higher, 75% or higher, 76% or higjber, 77% or hi’i’, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higjicr, 85% or hig’, 86% or higb’, 87% or bigher, 88% or higho:, 89% or highCT, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or hjghea:, 98% or higher, 99% or higher, 99.1% or higher, 99’% or higher, 99.3% or hi’, 99.4% or higher, 99.5% or higher, 99.6% or highcs:, 99.7% or higher, 99,8% or higher, or 99.9% or higher identity with the amino acid sequence of SBQ ID NO: 2, and having an alcohol acetjd transferase activity. In general, tbepen’entageideiitity is prefbraU
Alcohol ace’l transferase activity may be measured, fi>r exanq}le’ by a method described in Japanese I’aid-open Pateait Publication No. 253826.
FurthmiK)re, the present invention also conten’lates (e) a po’nuckotide conpising a polynucleotide ‘hich hybridizes to a polynucleotide consisting pt a nucleotide sequence cpinplementary to the nucleotide sequence of SEQ ID NO; 1 under st’ aicodes a protein having an alcohol acetyl transferase activitjr, and (f) a polynucleotide coirprising a polynucleotide which hybridizes to a polynucleotide conptementary to a nucleotide sequence of encoding a pioteia of SEQ ID NO: 2 tmder string’ conditions, and which encodes a protein having an alcohol acdyl transferase activity.
Herein, "a poljmcleotide that l’/teidizes under stringent conditbns" refers to nucleotide sequmce’ sudi as a DNA, obtaiised t7y a.cobny faradization technique, a plaque hybnf’zation tedhnique, a southern h}4}ridization technique or the like using all or part of polynucleotide of a nucleotide sequence con’lementary to the nucleotide scquoice of SEQ ID NO: 1 or polynucleotide encoding the aznixio acid sequence of SEQ ID NO: 2 as a pcobe. The hybridization method may be a mediod described, &r example, in MOLECULAR CLDNINO 3rd Ed, CURRENTPROTOOOtS IN MOLECULAR BiDLOCJY, 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 conditio!’, "Low stringency conditions" are’ for example, 5 x SSC, 5 x Detihardf s solution, 0.5% SDS, 50% formamide at 32°C ‘Moderate stringency conditions" are, for example, 5 x SSC, S x D’ibardfs solution, 0.5% SDS, 50% formamide at 42°C. "High string’Ksy conditions" are, for exani’le, 5 x SSC, 5 x Denhardt’s solution, 0.5% SDS, 50% fonnamide at 50**C Vxidst these conditions, a polynucleotide, such as a DNA, with higjber homology is expected to be obtained efficiently at higher toi’erature, althongih multiple &ctors are involved in hybr’ization stringency including tenqperature’ probe

concentration, probe length, ionic strength, time, salt concentration and others, and one skilled in the art may appropriately select these Actors to realize similar stringency.
When a commercially available kit is used for hybridization, lor example, AUcphos Direct Labeling Reagents (Amersham Pharmacia) may be used. In this case, according to the attached protocol, after incubation with a labeled pxyhc 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 hs’ridized include polynucleotides having about 60% or higher, about 70% or higher, 71% or highca:. 72% or hi’ior, 73% or hig’, 74% or higher, 75% or highCT, 76% or hi’, 77% or higher, 78% or higher, 79% or higher, 80% or highw:, 81% or higher, 82% or hi’w, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or h’Jwa:, 89% or higha, 90% or M’hear, 91% or higher, 92% or higher, 93% or higjhfix, 94% or higher, 95% or higher, 96% or high’, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 993% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99,7% or higjwa:, 99.8% or higher or 99.9% or higher idratity to polymickotide encoding the amino acid sequwice of SEQ ID NO: 2 as calculated by homology search software, such as PASTA and BLAST using defeult parameters.
Identity between amino acid sequences or nucteotide sequ’ices may be determined using algprithm BLAST by Karlin and Altschul iPrvc Nail Acad ScL USA, 27: 2264-2268, 1990; Proa Natl Acad Set USA, 90: 5873,1993). Programs called BLASTN and JBLASTX based on BLAST algorithm have been developed (Altsdml SF et aL, J, Mol Biol 215:403,1990), When a nucleotide sequence is sfcquenced using BLASTN, the parameters are, for example, score - 100 and wod l’ogttx - 12. When an amino add sequence is sequenced using BLASTS the parameters are, for exaix’le, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, de&ult parameters for each of the programs are en’byed.
the polynucleotide of the pxsiN&. invention includes 0) a polynucleotide encoding jRNA having, a nucleotide sequence coniplementaiy to a transcnpt of the polynucleotide (DNA) according to (5) above; (k) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to (5) above fhroi’ RNAi effect; (1) a po’omcleotide encoding RNA having an activity of specifically cleaving a transoipt of the polynucleotide (DNA) according ‘ (5) above; ai’ (m) a polynucleotide encoding RNA that represses expression of the polynucleotide (DNA) according to (5) above thiou’ co-stp:ession effect Th’e polynucleotides may be incorporated into a vector, which can be introduced into a cell for tiansfonnation to iqxress the expression of the polynucleotides (DNA) of (a) to (i)' above. Thus, these polynucleotides may suitably be used when rqiression of the expres’n of the above

polynucleotide (DNA) is prs&rable.
The phrase "polymicleoticte encoding RNA having a nucleotide sequeaice conq)lementaiy to the transcript of DNA" as used herein refers to so-called antisense DNA. Antisense technique is known as a tnethod for repressing expression of a particular en(k»g€330U8 gejie, and is desaibed in various publications (see e.g., Hirajima and Inoue: New Biochemistry Experiment Course 2 Nucleic Acids IV Gene Rq)lication and Expression (Japanese Bioctonical Society Ed., Tokyo Kagaku Dozm Co., Ltd) pp319-347, 1993). The sequence of antisense DNA is preferably coi3[j)lementary to all or part of the endogenous gene, but may not be con?)letely co'n?)lem«ataiy as long as it can effectively repress the expressipn of the gene. The transcribed RNA has preferably 90% or higher, and more preferably 95% or higher conipleraentarity to the transcript of the target gene* The te)gth of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more prefcj:ably 500 bases or more.
The phrase "polynucleotide encoding RNA that rq)resses DNA expression through RNAi efifect'* as used her’ refers to a polynucleotide for rq)ressing e’essioh of an ‘idogenotis gaw througji RNA interfcr’ice (RNAi’. The term *'RNAi" refers to a phmomenon whae when double-strandied RNA having a sequence, identical or similar to the target gene sequoice is introduced into a cell, the eTq’ressions of both the introduced foreign gene and .the target endogenous gezxe are r’essed 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 intwiering RNA) or, shRNA (short hairpin RNA). Sudh RNA may be locally delivered to a desjred site with a delivery systean such as liposome, or a vector that g’erates the double-strauded RNA described above may be used for local expression tbsKot Methods for producirig or using such double-stranded RNA (dsRNA, siRNA oi; shRNA) are known fiom many publications (see, ag,, Jt’anese National Phase PCT LaM-opai Patent IhiblicdtionNo, 2002-51’062; US 2002/086356A; Nature Genetics, 24(2), 180-183, 2000 Feb.; Genesis, 26(4), 240-244, 2000 Apil; Nature, 407:6802, 319-20,2002 Sep. 21;- Geai’ & Dev,, V0LI6, (8), 948-958,2002 Apr.15; Proc. Natl. Acad. Sd.USA, 99(8), 5515-5520,2002 Apr. 16; Science, 296(5567), 550-553,2002 AF* 19; Proc NatL Acad ScL USA, 99:9, 6047-6052,2002 Apr, 30; Nature Biotedmology, VoL20 (5), 497-500, 2002 Msy; 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 cleaving transact of DNA" as used herein g’ierally refers to a ribozyme. Ribozyme is an RNA molecule with a catalytic activity that cleaves a transcr’t of a target DNA and inhibits the fiinctkm of that gene. Design of ribozymes can be found in various known publications (see» e.g., FEES Lett 228:228,1988; FEBS Lett 239: 285,1988; NucL Adds. Res. 17: 7059,1989; Nature 323:

349,1986; NucL Adds. Res. 19:6751.1991;ftoteinEng3:733,1990; NucL Acids Res, 19:3875, 1991; NucL Adds Res. 19: 5125,1991; Biochem Biopl’fls Res Comraun 186: 1271,1992). in addition, the phrase '"polynucleotide encodnig RNA that represses pNA expression thmugh co-supression'‘ effect" refers to a nucleotide that inhibits fiinctions of target DNA by "co-supression".
The tenn "co-sipession" as used herdn, refes to a phenomttron where when a gene having a sequence idcaitical or similar to a target endogenous gene is transfiwtned into a cell, the e?q>ressions of both the introduced fore’ gene and the target eindogenous gene are i’ressed Design of polynucleotides having a co-supression effect can also .be fotmd in various publications (se’ e.g., Smyth DR; Curr. Biol. 7; R793,1997, Marticnssen R: Cuir. BioL 6:810,1996).
I. PrptaJlP of Ifry pmmt lffY’9M
The present jnverition also provides proteins encoded by any of the polymcl’’ (i) above. A prefen’ protein of the preseait invention conpises an amino add sequence of SEQ ID N0:2 with one or several amino acids thereof being deleted, substituted, inserted mi/or aidded and hajs an alcohol acetyl trans&arase/activity.
Such protdn includes those having an anoino acid sequence of SEQ ID NO: 2 with amino add residues thereof of th’ number mentioned above being deleted, substituted, inserted and/or added and having.an alcphol acetyl transferase activity. In add£tk)n, such protein iocludes those having homobgy as described above with the amino acid sequence of SEQ ID NO: 2 and having an alcohol acetyl transferase activity.
Such |m)te]ns may be obtained tyy enq)loying site-directed mtitation described, fer
exan’le, in MoiBXnJUl CLONING 3id Ed., aJWlEOTPRDT(XX>I£JNNtoiJSa’ Ni4a
Acids. Res,, 10:6487 (1982), Prvc NatL Acad ScL USA 79:6409 (1982), Gene 34: 315 (1985), l’ud. Acids, Res,, 13; 4431 (1985), Proc. Natl Acad. Set USA 82:488 (1985).
Deletion, substitution, insertion ‘Kl’or addition of one or xnorearnino add residuesia an amino add sequence of the protdn of the invention means that one or more amino add residues are deleted, substituted, inserted and/or added at atiy one or more positions in the same amino add sequence. Two or more types of deletion, substitution, ins’tkm and/or addition may occur concurrently*
Herdnafter’ e’anples of mutually substitutable amino add residues are etuuna:ated Amino add residues in the same group are mutually substitutable. The groups are provided betow.
Group A: leucine, isoleucine, norleucine’ valine, norvalzne, alanine, 2-am2nobutanoic acid, methionine* o-methylserine, t-butylglycine, t-ljutylalanine, tive, Shima2U Coip* can also be used hx ch’Bical synthesis.
3> Vector of the jnventio’ and veast transformed Tyifli the vector
The present invention th’ provides a vector conpising the potynocleotide 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 the polynucleotides described in (j) to (m) above. Genprally, the vector of the present invention cotnprises an expre’siori cassette includmg as conp)nents(:iC) a promoter that can transcribe in a yeast cell; (y) apoVnucIeotide described in any of (a) to (i) above that is linked to the promoter in sense or.antisense direction; and (z) a signal that fimctions in the yeast with respect to transcription tennination and polyadenyiation of RNA molecule.
Ac’cM’rding to the preset mvention, jn order to highly ex|xres5 the protein of the invention described above x’n brewing akobolic beverages (e.g*, be’) desoibed betow, these polynucleotides are introduced in the sense directk)n to the promoter to promote expression pf the polynucleotide (DNA) described in any of (a) to (i) above. Furtho', in order to repiiess the above protean of the invention i’n brewing alcoholic beverages (e.g., beer) described befow, these polynucleotides are introduced in the antis3)se direction to the promoter to lepxess die expression of the polynucleotide PNA) described in' any of (a) to (i) above. In order to repress the above protein, of the invention, the polynucleotide n’y be introduced into vectors such that die polynucleotide of any of the (j) to (m) is to he expressed. According to the i«:esent invention, the target gene (DNA) may be dimipted to repress the expression of the polynucleotide (DNA) described above or the expression of the protein described above. A g’ie may be disrupted by adding or deleting one or more bases to or fiom a r’bn involved in e?q)re$sion of the g&a.e product in the target gene, for exan:%)le, a coding region or a |m)nK>ter region, or l’ i’ions entirety. Such disruptk)n of goie may be found in knovm publications (see, eg., Fxoc. Natl, Acad Sci USA, 76,4951(1979), Methods in Enzymobgy, 101,202(1983), Japanese Patent Laid-C)penPublicationNo.6-253826),

A vector introduced in the yeast may be any of a multicopy type (YEp type), a single copy type (YCp type), or a chrpmosome integration ‘/pe (Yip type). For example, YBpTA (J. R. Broach et aL, EXPERiMEr’ALMA>nPUiAllwOFGENEEXP’’ Acadmiic Press, New Yoric, 83, 1983) is known as a YEp type vector. YCp50 (M. a Rose et al, Ger’ 60: 237, 1987) is known as a YCp type vector, and Y’p5 (K Struhl et al, Proc. NatL Acad Sd. U&i,76: 1035, 1979) is known as a Yl’ type vector, all of \’iiich-are readily available.
Piomot»:s/tenmnators for adjusting gene e?q)ression in yeast may be in any combination as long as they fiinction in the brewery yeast and they are not influenced by consdtu’its in &rme3Qtadon brotk For exan’le, a promoter of glyc’alddiydes 3-phospbate dehydrogenase gene (TDH3), or a promoter of 3-phosphogIycerate kinase gene (PGKl) may be used TTiese genes have previously been ctoned, described in detail, for exan¥)le, in M. F. Tuite et al., EMBO X, 1,603 (1982), and are readily available by known methods,
Smce an auxotrophy marker cannot be iised as a selective maricer upon transfoimation for a brewery yeast, for exan’le, a geneticin-resistant |ene (Cf418r), a copper-resistant get’ (CUPl) (Marin et al, Pn>c NatL Acad ScL USA, 81, 337 1984) qr a ierulenin-resistant.gene (fes2m, PDR4) (Junji Inokoshi et al, Biochemistry, 64, ‘60,1992; and Hussain et al. Gene, 101; 149’ 1991, respectively) may be used,
A vector con’cted as described above is injtioduced into a host yeast. Exanples of ‘ host yeast include any yeast that can be used for brewing, for example, te ym’ and sake. Specifically, yeasts sudh as genus Saccharomyces may be used. According to the present invention, a lager brewing yeast, for exan?)le, SaccharonQ’ces pastorianus W34/70, eta, S’xccharofnyces carlsbergehsis NCYC453 or NCYC456, ete., or Saccharomyces cereyisiae NBRC1951, NBRC1952, NBRC1953 or NBRC1954, etc., may be used. In addition, whisky yeasts sudi as Saccharomyces cerevisiae NCYC90, wine yeasts such as wine yeasts #1,3 and 4 fix)m ifie Brewing Society of Jfpin, and sake yeasts sudi as sake yeast ‘ Society of Japan may also be used but not limited thereto. In the present invention, .lager brewing yeasts sucb as SaccharomycespOstoriam’s may be used prefiraUy.
A yeast transformation method may be a goierally used known method. For exanple, methods that can be used include but not limited to an dectrqporation method {Metk Enzym,, 194: 182 (1990)), a spheroplast method (Proc. NatL Acad Sd. USA, 75:1929(1978)), a lithium acetate method (J, Bacteriology, 153:163 (1983)), and methods described in Proa Natl. Acad. Sci USA, 75:1929 (1978), METHODS IN YEAST GENEHCS, 2000 Edition: A CoH Spring Harbor Laboratoiy Course Manual,
More specifically, a host yeast is cultured in a standard yeast nutritfon medium (e.g., YEPD medium (Gtaaetic Engineering. Vol. 1, Pkjium Press, New York, 117(1979)), etc.) such

that OD600 nm will be 1 to 6. This culture yeast is collected by c’rtri&gation, washed and pre-tKsated with alkali metal ion, preferably lithium ion at a ooncentmion of about 1 to 2 M After the ceH is left to stand at about 30°C fe about 60 minutes, it is left to stand ‘ introduced (about 1 to 20 ‘g) at about 30X for about another 60 minutes. Polyethyleneglycol, preferably abbut 4,000 Dalton of polyethylen’lycol, is added to a final conceiitration of about 20% to 50%. After teaVmg ‘ about 30°C for about 30 minutes, the cell is heated at about 42’C for about 5 minutes. Preferably, this ceil suspension is washed with a standard yeast nutrition medium, added to a pfed’ermined amount of fi’esh standard yeast nutrition medium and left to stand at about SO'‘C for about 60 minutes, Th’ieaftor, it is seeded to a standard agar medium containing an antibiotic or the like as a selective maiicer to obtain a transformant
Other general cloning techniques may be found, for exanq)le, in MOLECULAR CLCMSIO 3id Ed, and MEIHODS IN YfeAST GENETICS, A LABCMI’TORY MANUAL (Cold Spring Harbor Laboratory Piress, Cold Spring Haitor, NY)..
4, Method of nrodudng alcoholic bevemges according to the present inventton and alcoholic beverages produced lyvtfae method
The' vector of H:’ present invedion described above is introduced into a yeast suitable for
brewing a target alcoboHc product This yeast can be used to produce a desired alcoholk
beventgemth’ilutnced aroma and flavor with an edevatedcox’’ In addition, yeasts to
be sdected by the yeast assessm’t. nit’thodof the presoit invention described below l:;an also he used The taxget alcoholic bevere’es include, for exanple> but not linuted to beer, beear-laste bcm:agG8 such as sparkling liquor {hc’poushu), wine, whisky, sake and tibe like. Further, according to the present invention* desired alcoholic bever’es with reduced ester level can be produced using b:ew’ jneast m which the e’quression of the target gene was suppressed, if needed. That is to say, desired kind of alcoholic bevetages with conteoUed (elevated or reduced) level of ‘er can be pioduced by controlling (elevating or reducing) production amount olf ester using ye’iStB into which the vector of the present inventk>n was introduced desmbed above, yeasts in which e?5)ressk)n of the pofynucleotide (DNA) of the present irv«ition described above was suppressed or yea’ selected by tiie yeast assessment method oftbe invention desmbed below for form’t’on to produce alcoholic beverages.
In order to produce these alcoholic bevci:ages, a known technique can be used except that a brewery yeast obtained acoorcfing to the present invonition is used in t’ Since materials, manu&cturing equipment, manu&cturing control and the like may be exactly the same as the conventional ones, there is no need of inoreasii’ the cost for prodiicing alcoholic beverages widi an controlled content of esto. Tbus« accordiug to the present inventfon, alcoholic

beverages with excellent aroma and flavor can be produced using the existing &cility without increasing the cost.
S, Yeast asaegsmeat method of the invention
The present invention relates to a me«lK)d for assessing a test yeast for its ester-iproducmg capability by using a |»imer or a piobe designed based on a nucleotide sequence of an ateohol acetyl transferase gene having the nucleotkle sequence of SEQ ID NO:L Geiteral techniques for such assessment method is kiiown and is described in, for exan’Ie, WO01/040S14, J’anese Laid-open Patent PublicationNo. 8-205900 or the like. This as’essm’xt nwthod is described in below.
First, gaiome of a test yeast is prepared. For this prqyaration, any known mettod such as Hereford method or potassium acetate method may be? used (e.g., MIBIHODS IN YEAST GENEHCS, Cold Spring Harbor Laboratory Press, 130 (1990)), Using a primer or a probe designed based on a nticleotide sequence (preSst’ly, ORF sequence) of the alcohol acetyl tranpforase gem, the existence of the gene or a sequence ‘eciSc to the gene is determined in the test yeast gesjome obtained. The primer or tbeprobe may be designed acconjing to a known technique.
Detectkm of the gei]’ or the £pecific sequence ‘y be carried out by employing a k’ tednuque. For exito’Ie, a pojynucleotkle inchidtng part c’ all of the. specific sequence or a polynucleotide including a nucbotide sequence coniplementaty to said nucleotide Sequeaoce is used as one primer’ while a polynucleotide including part or all of the sequence upstream or downstream &om this , sequence or a polynucleotide inchiding a nucleotide sequence compl’Dentary to said nucleotide sequence, is used as another prim’ to aixq>Iify a nucleic add of the yeast t’ a PCR met’d, th£3:eby detemuning the existence of an:plified products and molixular weig’ of the amplified products. The number of b’es of polynucleotide used for a prinffiT iff generally 10 base pairs (bp) or more, and laeferably 15 to 25 Iq). bi g’ser’ the number of bases between the primers is suitably 300 to 2000 bp.
The reaction conditions for PCR are not particularly liniitedb’ forexanple, a
denaturation ten?)erature of 90 to 95’*0, an annealing temperature of 40 to 60°C, an elongation
ten’>erature of 60 to 75*"‘ and the number of cycle of 10 or more. The resulting reaction
product may be sqparated, for example, by electrophoresis using agarose gel to detamine the
molecular we’ of the an’lified product This method albws pnsdiction and assessment of the
capability of the yeast to produce esta as determined by whether the molecular weight of the
ait$Ii£ed product is a size that contains the DNAtnolecute of the Gpedfte 1’ addition, by
analyzing the nucleotide sequence ofihe an’lified product, the cq[)dbility may be predicted and/or

assessed more precisely.
Moreover, in the present invention, atest yeast is cuhiiTed to n’’ of the alcohol ace’l trans&grase gene haviqg tbe Bucleotide sequence of SEQ ID NO: I to assess thie test yeast for its ester-j’oducing c’ability. In measuring an e’qnession level of the alcohol acetyl transferase gmt, tbe test yeast is cultured and then mRNA or a pcotdn resulting fix)mtbe alcohol acetyl transferase gene is quantified. Tl’ quantification of niRKA or protean may be carried out by enploying a kmwn technique. For exanple, mRNA may be quantified, by Northern l’lmdization or quantitative RT-PCR, while protein may be quantified* for exaiDpIe, by Western hfotting (CuRR]’n* PROTOCOLS IN MOLBCULAR BIQLOGV, John Wiley & Sons 1994-2003),
Furthermore, test yeasts are cultured and repression levels of tfie alcohol acetyl transferase ge’ having the nucleotide sequence of SEQ H) NO; 1 are measured to select a test yeast with the gene expression level according to the target c’bility of producing ester, thereby selecting a yeast &voraBle hr brewing desired alcoholic beverages. In addition, a reference yeast and a test yeast maybe cultured so as to measure and conGpai’ the'expression level of tbe gene .in eadi, of tbe yeasts’ thereby selecting a &vorable test yeast. Nfore specifically, for ‘um’le, a reference yeast and one or more test yeasts are cultured and an expression level of tl’ alcohol acetyl transferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured in each yeasL By sdecting a tesi yeast with the gene e?q)ressed higher or \owet than that in the reference yeast, a yeast suitable for brewing alcoholic beverages can be sdected.
Alternatively, test .yeasts are cultured and a yeast with a higher or lower estor-producing c’ability or with a high’. or bwer alcohol acdjd transfi3!ase activity is sele’ a-yeast suitable for brewing desired alcoholic beverages.
In Ibese cases, the test yeasts or the reference yeast may be, for exan’le’ a yeast introduced with tbe vector of the invention, a yeast in'which an exi»:ession of a polynucleotide (DNA) of tbe invention has been controlled, an artificially mutated yeast or a naturally mutated yeast. The este’ a method described in Method of J. Am. Soc. Brew. Caiem. 49;152-157,1991. Alcohol acetyl transferase activity can be measured by, for exmiph, a method described in AppL Microbiol BiotechnoL S3:596-600, 2000. The mutation treatment may empby any methods including, for exan’le’ physical methods such as ultraviolet irradiation and radiation irradiation, and chemical methods associated vn&i treatme’ots with drugs such as EMS (ethylmethane sulphonate) and N-methyl-N-uitrosoguanidine (see, e.g., Yasuji Oshima Ed, BIOCHEMISTRY EXPERIMENTS voL 39, Yeast Molecular Gemtic Experiments, pp. 67-75, JSSP),
In addition, exan:’le$ofyeasts used as the refisrence yeast or the test yeasts inchide any

yeasts that can be used for brewing, for exan5)le, breweiy yeasts for beer, win’ More specifically, yeasts such as gams Saccharomyces may be used (eg., S pastorianus, S cerevisiae, and & carlsber'gmsis). According to the present invention, a lager brewing yeast, for exanq)le, Saccharomyces pastoriams W34/70; Saccharomyces caHsbergensis NCYC453 or NCJYC456J or Sacdmromyces ceremiae NBRC1951, NBRC1952, NBRC1953 or NBRC1954, etc., may be used Rather, wine yeasts such ‘ wine yeasts #1, 3 and 4 fiom the Brewing Society of Japan; and sake yeasts such as sake yeast #7 and 9 fiom the Brewing Society of J’)an may also be used but wX limited thcareto. In the present invention, lag’ brewing yeasts such as Saccharomyces pastorianus pmy preferably be used. The refermce yeast and the test yeasts may be selected fix)m tiic above yeasts in any combinatbn*
EXAMPLES
Hereinafter, the present invention will be described in more detail with reference to woddng ‘camples. The present inventkin, however, is not Hmited to the exaziples described betow.
Example 1; Cloning of Alcohol acetyl Iransferase Gene faoiiScATE2’
A specific novel alcohol acetyl transferase gene (nonScATT2) (SEQ ID NO: 1) fiom a lager brewing yeast w»:e fonnd’-as a result of a search utilizing the compmison databa£se desoibed in Japanese Patent Laid-Open Publication No. 2004-283169. Based on the acquired nucleotide sequ’uxj information, prinros nonScATF2_for (SEQ ID NO; 3) and nonScATF2_rv (SEQ ID NO: 4) were designed to anplify.the fbU-Iength goies, respectively PCR was carried out using chromo’mal DNA of a genome sequencing strain, Sacdiaromyces pastorianus Weihenst’han 34/70 strain, also abbreviated to **W34/70 strain", as a template to obtain DNA fiagniMits (about 6.7 kb) including the fall"length gene of nonScATFl
The thus-obtained nonScATF2 gene fi-agment was inserted into pCR2.1-TOPO vector (invitrogen) by TA cloning. The nucleotide sequences of nonScATF2 gene were analyzed according to Sai’gefs method (F. Sanger, Science, 214: 1215, 1981) to confirm the nucleotide sequence.
Example 2: Analysis of Exprnrfon of noiiScATF2 Gene dming Beer Feniientatlon Test
A be’ fermentation test was conducted using a lager brewing yeast> Saccharofryces pastorianus W34/70 strain and thfsi mRNA extracted fiom yeast cells during fermentation was analyzed by a DNA microarray’

Wort extract concentration 12.69%
Wort content 70 L
Wort dissolved oxyg’ concentration 8*6 ppm
Foin’xtationten];>cniture IS'‘C
Yeast pitching rate 12.8x 10’ cells/noL
Sanq>ling of fermentation liquor was performed with time, and variation with time of yeast growth amount (Fig. I) and apparent extract concentration (Fig. 2) was observed, SinialtaneoTisly> sampling of yeast cells was p’rfonned, and the prepared mRNA was subjected to be biotit’labeled and was hybridized to a beo- yeast DNA microarray* The signal was detected using GCOS; GcEneChip Operating Software 1.0 (manufectured by AfiEymetrix Co,X E’qjression pattern ofnonScATF2 gene is shown in Figure 3. AsaresuIt,itwascon&medthatnonScATF2 goie was expressed in the general beer fertneotatioa
ExampteS; ConstractionofttonScATFl Gene Hiehlv Expressed Strata
The nonScATF24)CR2.1-TOPO described in Example 1 was digested using the restoriction en2;yma8 Sad and NotI so as to prepare a DNA fiagment containing the entire l’jgth of the protein-encoding regioa This fragment was ligated to pYCGFYNot treated with the restriction ‘izymes Sad and NotI, thereby constructing the nonScATF2 high e?’xression vector nonScATF2’YCGPYNdt pYCGFYNot is &e YC’-type yeast ‘qwepsion vector. The inserted gene is highly expressed by the pyruvate kinase g’e PYKl promote* Hie geneticiurresistant g6ne G418' is included as the selectbn m’dcear in the y’ast, and the anq[>iciUinrresistant gene ksts’ is inchided as the selection marker in Escheridiia coU,
Using the high ejqpresssion vector prq)ared by the above method, the strain Saccharamyces pasteumnus Weibenstephaner 34/70 was transfom’ by the method described in Jg’anese Patent Laid-op’ Publication No, H7-303475. The transfoxmant was sdected in a YPD plate culture (1% yeast extract, 2% potypeptone, 2% glucose’ 2% agar) containing 300 mg/L of gcneticjn.
It should be noted ttiat strain W34/70-2Ae is a stjain W34/70-2, which is a ‘ore clone of Saccharomyces pastorianus Weihenstq)hflu W34/70 in which gene nonrScEHTl, which encodes hon*Sc£htlp, an alcohol acetyl transferase characteristic to brewei's yeast, has be’ destroyed Disruption of the BonScEHTl gene was carried out in accordance with a method described in the literature (Goldstein et al.. Yeast, 15,1541 (1999)), Fragmeaits for gene disruptwn were prq>ated by PCR using plamds containii’ a drug resistance marker (pFA6a(G418r), pA025(natl), pAG32(hph)) as templates. Prints consisting of nonScEHTl_deIta_for (SEQ ID NO. 7) and nonScETHljieltajrv (SEQ ID NO, 8) were used for the PCR pimers. A spore clone (W34/70-2) isobted from brewer's yeast Saccharomyces pastorianus strain W34/70 was

transfonned witii the ftagmcDts for gene disruption prepared as described above. Transformation was canied out according to the method described in Japanese Patsit Laid-open Publication No. H07-303475, and transformants were selected on YPD plate medium (1% yeast extract, 2% pol’peptone, 2% ‘cose» 2% agar) containing geneticin at 3(X) wg/U nourseotfaricin at 50 mg/L or hygromycin B at 200 mg/L.
Example 4: Analysis of Amounts of Ester Foimed to Beer TestBrewine
A fermentation test was conducted under the following conditions using the parent strain and the nonScATF2 h’y expressing stram obtained in Example 3.
Wort extract concentration: 12%
Wortvokime; IL
Wort dissolved oxygen concftitratioa' 9*5 ppm
FemKsntationten’erature: IS'‘C
yeast pitching rateS g/L
The j’n’tation teioth was san:p}ed ova: time to investigate the time-based changes in yeast, grovWh. (OD660) (Fig, 4) and e?dr’ consun’on (Fig. 5), Quantification of higher alcohol and extract concentrations at con’ktion of fem’itation was canied out using head space gas chromstogr£’ty(J, Am. Soc. Brew. Chem 49:152-157,1991).
Accordmg to Table 1, the amount of ethjd acetate formed at completion of fermentation was 37*0 ppm for the nonScATF2 tnghly e’ipressing strain in contrast to 33.0 ppm for the parent straia The anM>unt of isoamyl alcohol fomjed was 3.8 ppm for flie nonScATF2 highly e?qpressing strain in contrast to 2.9 ppm for the parent strain. On the basis of these results, the amounts of ethyl acetate and isoamyl alcohol formed by the nonScATF2 highly expressing strain were clearly demonstrated to incrfeased by 12 to 31%.

Unil;ppm
Values in par’xtheses indicate relative ‘‘es versus the parent straia
Emmnle fe DlsnmtJoii of nonScATFZ Gene
Fragmients for gene dismption are prepared by PCR using plasmids containing a dmg

resistance marker (pFA6a(G418r), pAG25(natlX pAO320?>h)).as tcn?)lates in accordance with a mediod described in the literature (Goldstein et al., Yeast, 15,1541 (1999)). Primers consisting of nonScATF2jieltajS)r (SEQ ED NO. 5) and nonScATF2_deltajrv (SEQ ID NO. 6) are used for thePCRprimeirs.
Brewer's yeast Saccharomyces pastoriamis strain W34/70 or a spore clone (W34/70-2) isolated from brevra's yeast Saccharomyces pastoriamis strain W34/70 is transfonned wifli the fragments for g«tie disruption prepared as described above. Transfonnatfon is carried out according to the method described in J’anese Patent Laid-open Publication No. H07.303475, and transfcmiants are selected on YPD plate medium (1% yeast extract, 2% polyp’jtone, 2% ghicose, 2% agar) containing geoeticin at 300 mg/)U nourseothricin at 50 mg/Lor hygron’in B at200mg/L.
Example 6; Analysis of Amounts of Ester Formed in Beer Test Brewing
A &nn’itation test is conducted under the fbUowing conditions using the parent strain and the noDScATF2 disrupted strain obtained in Example 5. Wort extract concentration: 12%
4
Wortvolmne: IL
Wort dissolved oxyg’ concentration: 10 ppm
Fermentation teiEperature: 15**C
yeast pitching rate: 5 g/L
The i’inentation bn’ih is san’led over time to investigate the tin’ growth (OD660) and extract conjsun’ytioa (Quantification of ester concentration at completion of fermentation is canied out according to the method described in J* Am. Soc. Brew. Chem. 49:152-157,1991 using head space gas chromatography.
IndttstriaaAppllcabilitv
According to the alcoholic beverage {Hoduction n::’thod of the ixresent inventbn, alcoholic beverages baviog sup’or aroma and flavor can be produced by increasing the content of esters which impart a fbrid aroma to products. In addition, in the case of malt beverages such as beer, £}r which an excessively high ester cont’ is not pre&tted, alcoholic bevetages baviog a mote desir’le axoma and flavor can be {Mtoduced by deoreasing the amount of ester contained therm This application claims benefit of Japanese Pat’’t Application Nos. 2005-266068 filed September 13, 200S and 2006-200892 filed July 24, 2006, which are herein incorporated by references in their entirety for all purposes. All other references cited above are also incorporated herein in their entirely &r all purposes.

CLAIMS
1. A polynucleotide selected the fop consisting o£
NO:

(i) a polynucleotide conjoining a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a nucleotide sequence to the nucleotide sequence of SEQ ID NO: 1 under high stringent conditions, and which encodes a proton having an alcohol acetyl transferees activity.

3. The polynucleotide of Claim 1 ionizing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
4. The polynucleotide of Claim 1 comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.
5. The polynucleotide of any one of Claims I to 4, wterein the polynucleotide is DNA.
6. A polynucleotide selected fiom the group consisting P£
(j) a polynucleotide encoding RNA of a nucleotide sequence con^IenMaitary to a transoipt of the polynucleotide (DNA) according to Claim 5;
(k) a polynucleotide tticodir^ RNA that represses the esqwession of the polynucteotide (DNA) according to Claim 5 through RNAi effect;
(1) a polynucteotide encoding RNA having an activity of specifically cleaving a transo^ of the polynucleotide (DNA) ^xoiding to Claim 5; and
(m) a polynucteotide eiKJOding RNA that represses e?qpressiqn of the polynucleotide (DNA) according to Qaim 5 through co-si^pression effect
7. A protein encoded by the polynucleotide ofany one of Claims 1 to5.
8. A vector cpngjrising the polynucleotide of any one of Claims I to 5,
9. A vector comprising the polynucleotide of Claim 6.
10. A yeast con5)risiDg ^ vector of Claim 8 or 9.
IL Tlie yeast of Claim 10, wherein a estcr-ptoducing ability is increased by introducing tiie vector of Cteim 8.
12- A yeast, wherein an expression of the polynucleotide (DNA) of Claim 5 is repressed by introdiwing the vector of Claim 9, or t^ diOTupting a g^e related to the polynucteotide (DNA) of Claim 5.
13. The yeast of Claim 11, ^^Aerein a ester-producing ability is incr^ised by increasing an expression level of the protdn of Claim 1

14. A method for producmg an alcoholic beverage by using the yeast of any one of Claims 10 to 13.
15. The method for producing an afcoholic beverage of Claim 14, wherein the brewed alcoholic beverage is a malt beverage.
16. The'method fi>r producing an alcoholic beverage of Claim 14, whearein the brewed alcoholic bevdrage is wine.
17. An alcoholic beverage produced by the mefliod of any one of Claims 14 to 16.
18. A method for assessing a test yeast fbr its ester-produdng capability, conprising using ajaim^ or a probe designed based on a nucleotide sequence of an alcohol acetyl transferafte gene having the nudeotiae sequence of SEQ ID NO: 1.
19. A n^od fbr assessing a test yeast for its ester-i»adudRg capability, oon5>rising: culturing a test yeast; and measuring an expression level of an alcohol acetyl transferase gene having tte nucleotide sequ^ice of SEQ ID NO: 1.
20. A method for selecting a yeast> con^rteing: culturing test yeasts; quantifying the ptot^ according to Claim 7 or measuring an expression level of an alcohol acetyl transferase gj^e having the nucleotide sequence of SEQ E) NO: 1; and selecting a test yeast having said protein amount or said g^)e eqyression level according to a l^get capability of ptodudhg ester.
i\. Hie tnisthod for selecting a yeast accordix]^ to Claim 20, conspising: cultutjng a xefbrenpe yeast and test yeasts; measuring an expression level of an alcohol acetyl trans&iase geifie having the nucleotide sequence of SEQ ID NO: 1 in each yeast; and sdecting a test yeast having the gene expressed higher or tower than that in the refeence yeast
22. The method for selecting a yeast according to Claim 20> comprising; culturing a refi»:ence ytost and test yeasts; quantifying the protdn according to Claim 7 in each yeast; and selecting a test yeast having said protein for a larger or smallo: amount than that in the refbence yeast

23. A method for producing an ateoholic beverage comprising: conducting fermentation for producing an alcoholic bevea:age using the yeast according to any one of Claims 10 to 13 or a yeast sdeded by the method according to any one of Claim the ptoductioii amount of ester.