DESCRIPTION
ESTERASE GENE AND USE THEREOF
TECHNICAL FIELD
The present invention relates to a esterase gene and to uses of the gene. The invention relates in particular to a brewer's yeast which produces alcoholic beverages with excellent aroma and flavor, alcoholic beverages produced using such a yeast, and a method of producing such alcoholic beverages. More specifically, the invention relates to lAHl gene which codes fer the esta*ase lahlp in brewer'^ yeast, particularly to a yeast whose capability of producing ester, which contribute to aroma and flavor of a product, is controlled by regulating the level of expression of the nonScIAHl gene characteristic to beer yeast and to amethod of producing alcoholic beverages using such a yeast.
BACKGROUND ART
Esters are an in5)prtant aromatic con?)onent of alcoholic beverages. Bi the case of rice wine, wine and, whiskey, an increase in the ester content is known to gjve flie beverage a florid aroma as well as cause it to be evaluated highly for its flavor. On the other hand, although esters are ah iirportant aromatic cofl5>onent of beer as well, an excess anM)unt of esters is disliked due to thq resulting ester smelL Thus, it is important to suitably control the amount of ester formed according to the type of alcoholic beverage.
Yeast producing high levels of esters have been developed in the past for the purpose of increasing the ester content of alcoholic beverages. Exanples of previously reported methods for effective isolatioii of yeast producing large aihounts of esters include a md&^ in which yeast, is subjected (or not subjected) to mutagenic treatment to obtain a strain which produces large amounts of caproic acid and is resist&nt to dmgs that inhibit fetty acid synthases such as cerulenin, as well as a strain which is resistant to leucine analogs such as 5,5,5-trifluoro-DL-leucine and produces large anaounts of isoamyl alcohol and isoamyl acetate (Japanese Patent AppUcation Laid-open No. 2002-253211), and a method in which a strain is acquired which is grown in medium containing a steroid having a pregnane backbone hydroxylated at position 3 (Japanese Patent Application Laid-openNo. 2002-191355),
On the other hand, exanples of previously reported methods involving the development of
yeast utilizing genetic engineering techniques include expressing high levels of the alcohol acetyl transferase gene ATFl of Saccharomyces corevisiae in brewing yeast (Japanese Patent ^implication

Laid-opra No. H06-062849), inhibiting the e?qpression of ATFl (Japanese Patrat Application Lakl-open No. H06-253826), ami increasing the amount of ester by destroying esterase gene EST2 in brewing yeast (Japanese Patent ^plication Laid-open No. H09-234077).
DISCLOSURE OF INVENTION
As stated above, although a mutant strain is acquired for increasing the ester content of a product, there are cases in which unexpected delays in fermentation or increases in undesirable aromatic and flavor con^nents are observed as a resultthereo:^ thus areaiing problems in the development of yeast for practical applicatioa Consequently, there is need for a method for breeding yeast capable of producing a desired amount of esters without in::pairing the fermratation rate or product quality.
As a result of conducting extensive studies to solve the above-mentioned problems, the inventors of the present invention succeeded, in identifying and isolating a gene from brewer's yeast which encodes an esterase that demonstrates more advantageous effects than known proteins. In addition, the inventors of the present invention also confirmed that the amount of ester formed decreases by producing a transformed yeast by inserting and expressing the resulting gene in yeast, and that the amount of ester formed increases by producing a transformed yeast in which expression of the resultmg gene is suppressed, thereby leading to con^Ietion of the present inventioa
Namely, the present invention relates to a novel esterase gene characteristically present in breweSr's yeast, a protein, mcoded by said gene, a transformed yeast in which the expression of said gene is regulated, and a method for controlling the amount of ester formed in a product by using yeast in which e5q)ression of said gene has been regulated More specifically, the pres^it invention provides the polynucleotide indicated below, a vector containing said polynucleotide, a transformed yeast in which said vector has been inserted, and a method for producing an alcoholic beverage using said transformed yeast.
(1) A polynucleotide selected from the group consisting of:
(a) a polynucleotide conprising a polynucleotide consisting of the nucleotide sequence of SEQIDNO:!;
(b) a polynucleotide conprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2;
(c) a polynucleotide con5)rising a polynucleotide encoding a protein consisting of the

amino acid sequence of SEQ ID N0:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a esterase activity,
(d) a polynucleotide conprising a polynucleotide encoding a protein having an amino acid sequ^ice haviag 60% or higher identity with the amino acid sequence of SEQ ID N0:2, and having a esterase activity;
(e) a polynucleotide con:5>rising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:l und^ stringent conditions, and which encodes a protein having a esterase activity, and
(f) a polynucleotide con:5)rising a polynucleotide which hybridizes to a polynucleotide consisting of aiiucleotide sequence compleirientary to the nucleotide sequence of the polynucleotide encoding the protein of the amino acid sequence of SEQ ID N0:2 under stringent conditions, and which encodes a protein having a esterase activity,
(2) Thepolyiiucleotide of (1) above selected from the group consisting of;
(g) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID
NO: 2, or encoding ^ amino acid sequence of SEQ ID NO; 2 wherein 1 to 10 amino acids thereof is
deleted, substituted, inserted, and/or added, and wherein said protein has a esterase activity;
(h) a polynucleotide encoding a protein having 90% or higher identity with the anano acid sequence of SEQ ID NO: 2, and having a esterase activity; and
(i) a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a nucleotide sequence con:]plementary to the nucleotide sequence of SEQ ID NO; 1 irndsr high stringent conditions, and which encodes a protein having a esterase activity.
(3) The polynucleotide of (1) above conprising a polynucleotide consisting of SEQ ID NO:L
(4) The, polynucleotide of (1) above con^rising a polynucleotide encoding a protein consisting of SEQ ID NO: 2.
(5) The polynucleotide of 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 of a nucleotide sequence coii5)lementary to a tramcript of the polynucleotide (DNA) according to (5) above;
(k) a polynucleotide encoding RNA that represses the e7q)ression of the polynucleotide (DNA) according to (5) above through RNAi eflFect;
(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 rqjresses expression of the polynucleotide (DNA) according to (5) above through co-suppression effect.

(7) A protein encoded by the polynucleotide of any one of (1) to (5) above.
(8) A vector conqjrising the polynucleotide of any one of (1) to (5) above.
(8a) The vector of (8) above, which corrprises the expression cassette comprising the following conponents:
(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 transcrq>tion termination and
polyadenjdation of a RNA molecule.
(9) A vector conprising 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 decreased by introducing the vector of (8) above.
(12) A yeast, wherein an expression of the polynucleotide (DNA) of (5) above is repressed by introducing the vector of (9) above, or by dismpting a gene related to the polynucleotide (DNA) of (5) above.
(13) The yeast of (11) above, whei"ein a ester-producing ability is decreased by increasing an expression level of the protein of (7) above.
(14) A method for producing an alcoholic beverage by using the yeast of any one of (10) to (13) above.
(15) The method for producing an alcoholic beverage of (14) above, wherein the brewed alcoholic beverage is a malt beverage.
(16) The method for producing an alcoholic beverage of (14) above, wherein the brewed alcoholic beverage is a wine. , ''
(17) An alcoholic beverage, which is produced by the method of any one of (14) to (16) above.
(18) A method for assessing a test yeast for its ester-producing capability, comprising using a primer or a probe designed based on a nucleotide sequence of a esterase gene having the nucleotide sequence ofSEQ ID NO: 1.
(18a) A metibod for selecting a yeast having a target ester-producing capability by using the method in (18) above.
(18b) A method for producing an alcoholic beverage (for exanfiple, beer) by using the yeast selected with the method m (18a) above.
(19) A method for assessing a test yeast for its ester-producing capability, con:q>rising:
culturing a test yeast; and measuring an ejqjression level of a esterase gene having the nucleotide

sequence of SEQ ID NO: 1.
(20) A method for selecting a yeast, conprising: culturing test yeasts; quantifying the
protein of (7) above or measuring an expression level of a esterase gene having the nucleotide
sequence of SEQ ID NO: 1; and selecting a test yeast having said protein amount or said gene
expression level according to a target capability of producing ester.
(20a) A method for selecting a yeast, conprising: culturing test yeasts; measuring a estQ'-producing capability or a esterase activity, and selecting a test yeast having a target capability of producing ester or a target esterase activity.
(21) The method for selecting a yeast of (20) above, conprising: culturing a reference yeast and test yeastsr measuring an e3q>ression fevel of a estei-ase gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast; and selecting a test yeast having the gene e?q5ressed higher or fower than that in the reference yeast.
(22) The method for selecting a yeast of (20) above conqjrising: cxilturing a reference yeast and test yeasts; quantifying the protein of (7) above in each yeast; and selectmg a test yeast having said protein for a larger or smaller amount than that in the reference yeast. That is, the method for selecting a yeast of (20) above con^rising: culturing plural yeasts; quantifying the protein of (7) above in each yeast; and selecting a test yeast having a large or small amount of the protein from them.
(23) A method for producing an alcohoUc beverage comprising: conducting fermentation for. producing an alcoholic beverage using the yeast according to any one of (10) to (13) or a yeast selected by the method according to any one of (20) to (2!2); and adjusting the production amount of ester.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the.ceU growth with time upon beer fennentation test. The horizontal axis rq)resents fennentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 2 shows the extract (sugar) consun5)tion with tinie upon beer feraientation test. The horizontal axis represents fennentation time while the vertical axis represents ^parent extract concentration (w/w%).
Figure 3 shows the expression profile of nonScIAHl 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 cell growth with time upon fennentation tesL The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660), The symbol "lAHl" denotes a nonScIAHl highly e?q)ressed strain.

Figure 5 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%). The symbol ''lAHl" denotes a nonSclAHl highly expressed strain.
Figure 6 shows the cell growth with time upon fermentation test. The horizontal axis represmts fermentation time while the vertical axis represents optical density at 660 nm (OD660). The symbol "iahV denotes a nonScIAHl diaiipted strain-Figure 7 shows the extract (sugar) consuniption with time upon bear fermentation test. The horizontal axis represaits fermentation time while the vertical axis represents appsrcai extract concentration (w/w%). The symbol "iahl" denotes a nonScIAHl disrupted strain.
BEST MODES FOR CARRYING OUT THE INVENTION
The present inventors conceived that it is possible to control ester in products by increasing or decreasing a esterase activity of the yeasL The present inventors have studied based on this conception and as a result, isolated and identified a nonScIAHl gene encoding a esterase xmique to lager brewing yeast based on the lago* brewing yeast genome information m^ped 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 protein encoded by the gene is represented by SEQ ID NO: 2.
L Polvnttcleotide of the invention
First of all, the present invention provides (a) a f)olynucleotide comprising a polynucleotide
of the nucleotide sequence of SEQ ID NO: 1; and (b) a polynucleotide conq^rising a polynucleotide
encoding a protein of the amino acid sequence of SEQ ID NO:2. The polynucleotide can be DNA
orRNA. '
The target polynucleotide of the present invention is not limited to the polynucleotide encoding a esterase gene derived from lager brewing yeast described above and may include other ' polynucleotides encoding protems having equivalent functions to said, proteia Proteins with equivalent fimctions include, for exanple, (c) a protein of an amino acid sequence of SEQ ID NO: 2 with one or more amino acids th^eof being deleted, substituted, inserted and/or added and having a esterase activity.
Such proteins include a protein consisting of an amino acid sequence of SEQ ID NO: 2 with, for exanple, 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 to 35,1 to 34,1 to 33,1 to 32,1 to 31,1 to 30,1 to 29,1 to 28,1 to 27,1 to 26,1 to 25,1 to 24,1 to 23,1 tx) 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 toll, 1 to 10,1 to 9,1 to 8,1 to 7,1 to 6 (1 to several amino acids), lto5, lto4,1 to 3,1 to 2, or

1 amino acid residues thereof being deleted, substituted, inserted and/or added and having a esterase activity. In general, the number of deletions, substitutions, insertions, and/or additions is preferably smaller. In addition, such proteins include (d) a protein having an amino acid sequence with about 60% or higher, 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 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 higher identity with the amino acid sequence of SEQ.ID NO: 2, and having a esterase activity. In general, the percentage identity is preferably higher.
EstCTase activity may be measured, for exan5)le, by a method described in Appl. Microbiol. BiotechnoL 53: 596-600,2000,
Furthermore, the present invention also contenq>lates (e) a polynucleotide comprising a polynucleotide which hj^bridizes to a polynucleotide consisting of a nucleotide sequence con:?)lementary to the nucleotide sequence of SEQ ID NO: 1 xmdo: stringent conditions and which encodes a protein having a esterase activity; and (f) a polynucleotide con5)rising a polynucleotide which hybridizes to a polynucleotide coirplementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 under stringent conditions, and which encodes a protein having a esterase activity.
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 con^lementary to the nucleotide sequence of SEQ ID NO: 1 or polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd Ed., CUREENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997, and so on.
The t^m "stringent conditions" as used herein may be any of low stringency conditions, moderate string^icy conditions or high stringency conditions. "Low stringency conditions" are, for exan^le, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 32°C. "Moderate stringency conditions" are, for exan:^)^, 5 x SSC, 5 x Denhardfs solution, 0.5% SDS, 50% formamide at 42°C. "High stringraicy conditions" are, for exan^le, 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 efiSciently at higher temperature, although mult^le fectors are involved in hybridization stringency including tenperature, probe concentration, probe length, ionic straigth, time, salt concratration and oth^, and one ddlled in the art may

appropriately select these fectors to realize similar stringency.
When a commercially available kit is used for hybridization, for example, Alkphos Direct 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 oDntaining 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, 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 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 high©:, 99.8% or higher or 99.9% or hi^er identity to polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as calculated by homology search software, such as FASTA and BLAST using defeult parameters.
Idaitity between amino acid sequences or nucleotide sequences may be determined using algorithm BLAST by Karlin and Altschul {Proc. Natl Acad, Scl 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 sequoiced using BLASTN, the parameters are, for exan^le, score = 100 and word length = 12. When an amino acid sequence is sequenced using BLASTX, the parameters are, for exan^le, score =^ 50 and word laigth = 3. When BLAST and Gapped BLAST programs are used, defeult parameters fi)r each of the programs are ernployed.
The polynucleotide of the present invention includes (j) a polynucleotide encoding RNA having a nucleotide sequence con^lementary to a transcrq)t 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 throu^ 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-suppression effect. These polynucleotides may be incorporated into a vector, which can be introduced into a cell for transformation to repress the expression of the polynucleotides (DNA) of (a) to (i) above. Thus, these polynucleotides may suitably be used when repression of the e7q)ression of the above DNA is prefeable.
The phrase "polynucleotide encoding RNA having a nucleotide sequence complementary

to the transcript of DNA" as used herein refers to so-called antisense DNA. Antisense technique is known as a method for rq)ressing expression of a particular endogenous gene, and is described in various publications (see e.g., Hirajima and Inoue: New Biochemistry Experiment Course 2 Nucleic Acids IV Gene Rq^lication and E?g>ression (Japanese Biochemical Society Ed, Tokyo Kagaku Dozin Co., Ltd) pp.319-347,1993). The sequence of antisense DNA is preferably conplementary to all or part of the endograous gene, but may not be con^letely conplementary as long as it can effectively repress the e^q^ression of the gene. The transcribed RNA has preferably 90% or higher, aiid more preferably 95% or higher con^lementarity 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 mdre.
The phrase "polynucleotide encoding RNA that represses DNA ©spression through RNAi effect" as used herein refers to a polynucleotide for repressing expression of an endogenous gene through RNA interference (RNAi). The temi "RNAi" refers to a phenomoion where when double-straiKied RNA having a sequence id^tical or sinailar to the target gene sequence is introduced into a cell, the expressions of both the introduced foreign gene and the target radogenous gene are rq)ressed RNA as used herein includes, for exan^le, double-stranded RNA that causes; RNA interference of 21 to 25 base length, for CKsxapk, dsRNA (double strand RNA), siRNA (smaU interfering RNA) or shRNA (short hairpin RNA). Such RNA.naay be locally delivered to a desired site with a deUvery system such as Iposome, or a vector that generates the double-stranded RNA described above may be tised &r local e^qxression thereof Methods for {Hodiicing or using such double-stranded RNA (dsRNA, siRNA or shRNA) are known fix)m many publications (see, e.g., Japanese National Phase PCT Laid-open Patent Publication No. 2002-516062; US 2002/086356A; Nature Genetics, 24(2), 180-183,2000 Feb.; Genesis, 26(4), 240-244,2000 ^ril; Nature, 407:6802, 319-20, 2002 Sep. 21; Genes .& Dev., Vol.1^^ (8), 948-958, 2002 Apr.15; Prop. NatL Acad. Sci. USA., 99(8), 5515-5520,2002 Apr. 16; Science, 296(5567), 550-553,2002 Apr. 19; ProcNatL 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 havii^ an activity of specifically cleaving transa:5)t of DNA" as used herein generally refers to a ribozyme. Ribo2yme is an RNA molecule with a catalytic activity that cleaves a transcript of a target DNA and inhibits the fimction of that gene. Design of ribozymes can be found in various known publications (see, e.g., FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; NucL Acids. Res. 17: 7059, 1989; Nature 323: 349, 1986; Nucl. Acids. Res. 19:6751,1991; Protein Eng 3:733,1990; Nucl. Acids Res. 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-suppression effect" refers to a nucleotide that inhfcits fiinctions of target DNA by "co-suppression".
The term "co-suppression" as used herein, refers to a phenomenon where when a gene having 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-suppression effect can also be found in various pubUcations (see, e.g., Smyth DR: Curr. Biol. 7: R793,1997, Martienssen R: Curr. BioL 6: 810,1996).
2. Protein of the present invention
The present invention also provides prpteins encoded by any of the. polynucleotides (a) to (i) above. A preferred protein of the present invention con^rises an amino acid sequence of SEQ ID NO:2 with one or sevetal anraio acids thereof being deleted, substituted, inserted and/or added, and has a esterase activity.
Such protein inchides those having an amino acid sequence of SEQ ID NO: 2 with amino acid residues thereof of the number mentioned above being deleted, substituted, insaled and/or added and having a esterase activity. In addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID NO: 2 and having a esterase activity.
Such proteins maybe obtained by en^loying site-directed mutation described, for exan^le,
in MOLECULAR CLOMNG 3rd Ed, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Nua Adds. Res,,
10: 6487 (1982), Pma Natl Acad Sci. USA 79: 6409 (1982), Gene 34: 315 (1985), Nua Acids, Res., 13:4431 (1985), Proc, Natl Acad, Sci USA 82:488 (1985).
Deletion, substitution, insertion and/or addition of one or more amino acid residues in an amino acid sequence of the protein of the invention means thiat one or more amino iacid residues are deleted, substitutpd, inserted 9nd/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, exan^les of mutaaUy 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, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: asparatic acid, glutamic acid, isoasparatic acid, isoglutamic acid, 2-aminoadipic acid, 2-aniinosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; and Group G: phenylalanine, tyrosine.
The protein 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 exanq^le, Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimazu Corp. can also be used for chemical synthesis.
3. Vector of fee invention and veart transformed with tiiie 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 the polynucleotides described in (j) to (m) above. ' Generally, the "vector of the present invention conqiris^ an expression cassette including, as con5)onents (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 direction; and (z) a signal that fiinctions in the yeast with respect to transOTption termination and polyadenyiation of RNA molecule.
According to the present invention, in order to highly e?^ess the protein of the invention described above upon brewing alcoholic beverages (e.g., beer) described below, these polynucleotides are introduced in the seme direction to the promoter to promote expression of the polynucleotide (DNA) desoibed in any of (a) to (i) above. Further, in orda: to repress the above protein of the invention upon brewing alcoholic beverages (e.g., beer) descdbed below, these potynucleotides are introduced in the antisense direction to the promoter to repress the ejq)ression 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 disrapted to repress the expression of the DNA described above or the expression of the protein described above. A gene may be dismpted by adding or deleting one or more bases to or from a region involved in expression of the gene product in the tstrgd gene, for exarqple, a coding region or a promoter region, or by deleting these regions entirely. Such disruption of gene may be found in known publications (see, eg., Proc. Natl Acad. Sci USA, 76, 4951(1979) , Methods in. Enzymology, 101,202(1983), Japanese Patent Application Laid-OpenNo.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 chromosome integration type (Yip type). For exanple, YEp24 (J. R. Broach et aL, EXPERMENTTALMAI^UIATIONOFGENEEXPI^ Academic Press, New Yoric, 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 Stnahl et aL, Proa Natl Acad. Sci USA, 76: 1035, 1979) is known as a Yip type vector, all of which are readily available.

PromotCTs/terminators for adjusting gene expression in yeast may be in any combination as long as they function in the brewery yeast and they are not influenced by constituents in fermentation broth. For example, a promoter of glyceraldehydes 3-phosphate dehydrogenase gene (TDHS), or a promoter of 3-phDsphoglycerate kinase gene (PGKl) may be used These genes have previously been cloned, described in detail, for example, in M. P. Tuite et aL, EMBO1, 1, 603 (1982), and are readify available by known methods.
Since an auxotrophy marker cannot be used as a selective marker upon transformation for a brew^ yeast, for exanple, a geneticin-resistant gene (G418r), a coppa"-resistant gene (CUPl) (Marin et aL, Proa Natl Acad, Set USA, 81,337 1984) or a cerulenin-resistant gene (fes2m, PDR4) (Junji Inokoshi et dL, Biochemistry, 64, 660, 1992; and Hussain et aL, Gene, 101: 149, 1991, respectively) maybe used.
A vector constructed as described above is introduced into a host yeasL Exanples of the host yeast include any yeast that can be used for brewing, for exanple, brewery yeasts for beer, wine and sake. Specifically, yeasts such as genus Saccharomyces may be used According to the present invention, a lager brewing yeast, for exan5)le, Saccharomyces pastorianus W34/70, etc., Saccharomyces carlsbergensis NCYC453 or NCYC456, etc., or Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRCi953 or NBRC1954, etc., may be used. In addition, whisky yeasts such as Saccltaromyces cerevisiae NCYC90, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japian, and sake yeasts such as sake yeast #7 and 9 from the Brewing Societyof Japan may also be used but not limited thereto. In the present invention, lager brewing yeasts such as Saccharomyces pastorianus maybe used preferably.'
A yeast transformation method may be a generally used known method For example, methods that can be used include but not liauted to an electroporation method (Metk Enzytn,, 194: 182 (1990)), a spheroplast method {Proc. Natl AcadSci USA, 75: 1929(1978)), a lithium acetate method {J. Bacteriology, 153:163 (1983)), and methods described mProc, Natl Acad, Set USA, 75: 1929 (1978), METHODS IN YEAST GENEncs, 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 ran will be 1 to 6. This culture yeast is collected by centri&gation, 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 |ig) 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 predetermined amount of fresh 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 exanple, in MOLECULAR CLONING 3rd Ed, and METHODS IN YEA.ST GENEncs, A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
4. Method of produdng alcoholic beverat^es flgcnrding to the present invention and alcoholic
beverages produced bv the method
The vector of the present invention described above is introduced into a yeast suitable for
brewing a target alcoholic product. This yeast can be used to produce a desired alcoholic beverage
with enhanced aronoa and flavor with an elevated content of ester. In addition, yeasts to be selected
by the yeast assessment melhod of the present invention described below can also be used. The
target alcoholic beverages include, for exan5)le, but not limited to beer, beer-taste beverages such as
sparkling liquor {happoiishu\ wine, whisky, sake and the like. Further, according to the preseiA
invention, desired alcoholic beverages with reduced ester level can be produced using brewery yeast
in which the expression of the target gene was suppressed, if needed That is to say, desired kind of
alcohoUc beverages with controlled (elevated or reduced) level of ester can be produced by
controlling (elevating or reducing) production amount of esta: using yeasts into which the vector of
the present invention was introduced described aboye, yeasts in which e?q)ression of the
polynucleotide (DNA) of the present invention described above was suppressed or yeasts selected by
the yeast assessment method of the invention described below for fermentation to produce alcoholic
beverages. , ,
In order to produce these alcoholic beverages, a known teclmique can be used except that a brewery yeast obtained according to the present invention is used in the place of a parent straia Since materials, manufecturing equipment, manufecturing control and the like may be exactly the same as the conventional ones, there is no need of increasing the cost for producing alcohoUc beverages with an controlled content of ester. Thus, according to the present invention, alcoholic beverages with excellent aroma and flavor can be produced using the existing feciUty without increasing the cost.
5. Yeast assessment method of the invention
The present invention relates to a method for assessing a test yeast for its estor-producing c^ability by using a primer or a probe designed based on a nucleotide sequence of a esterase gene

having the nucleotide sequence of SEQ ID NO: 1. Gena-al techniques for such assesanent method is known and is described in, for example, WOOl/040514, Japanese Laid-Open Patent Application No. 8-205900 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., MeraODS IN YEAST GENETICS, Cold Spring Harbor Laboratory Press, 130 (1990)). Using a prim^ or a probe designed based on a nucleotide sequence (preferably, ORF sequence) of the esterase gene, the existence of the gene or a sequence specific to the gene is detennined in the test yeast genome obtained. The primer or the probe may be designed according to a known technique.
Detection of the gene or the specific sequence may be carried out by enq)toying a known technique. For exaiiqjle, a polynucleotide including part or all of the specific sequence or a polynucleotide including a nucleotide sequence conplementary to said nucleotide sequence is used as one primer, while a polynucleotide including part or aU of the sequence upstream or dowtistream fi-om this sequence or a polynucleotide including a nucleotide sequence conplementary to said nucleotide sequence, is used as anotha: primer to an^lify a nucleic acid of the yeast by a PCR method, thereby detemiining the existence of amplified products and molecular weight of the amplified products. The number of bases of polynucleotide used for a {mmer is generally 10 base pairs (bp) or more, and preferably 15 to 25 bp. In general, the number of bases between the primers is suitably 300 to 2000 bp.
- The reaction conditions for PCR are not particularly limited but may be, for exanqjle, a denaturation tenq)erature of 90 to 95°C, an annealing tenperature of 40 to 60°C, an elongation tenq^erature of 60 to 75°C, and the number of cycle of 10 or more. The resulting reaction product may be separated, for exanple, by electrophoresis using agarose gel to detamine the molecular weigltf of the amplified produles. The present invention, however, is not hmited to the exan5)Ies described
below.
Example 1: rinninp of Esterase Gene fnonScIAHl)
A specijac novel esterase gene (nonScIAHl) (SEQ ID NO: 1) from a lager brewing yeast were foimd, as a result of a search utilizing the conq)arison database described in Japanese Patent Application Laid-Open No. 2004-283169. Based on the acquired nucleotide sequence information, primers nonScIAHlJor (SEQ ID NO: 3) and nonScIAHl^rv (SEQ ID NO: 4) were designed to an5)lify the fiill-length genes, re^ectiveiy. PCR was carried out using chromosomal DNA of a genome sequencing strain, Saccharomyces pastoricaius Weihenstephan 34/70 strain, also abbreviated to "W34/70 strain", as a teirplate to obtain DNA fragments (about 0.7 kb) including the M-length gene of nonScIAHl.
The thus-obtained nonScIAHl gene fragment was inserted into pCR2.1-T0P0 vector (Invittogen) by TA cloning. The nucleotide sequences of nonScIAHl gene w^e analyzed according to Sanger's method (F. Sanger, Science, 214: 1215, 1981) to confirm the nucleotide sequence.
Example 2; Analysis of Expression of nonScIAHl Gene during Beer Fermentation Test
- A beer fermentation test was conducted using a lager brewing yeast, Saccharomyces
pastonanus W34/70 strain aiid then mRNA extracted from yeast cells during fermentation was aioalyzed by a DNA inicroaxray.
Wort extract concentration -12.69%
Wort content 7p L
Wort dissolved oxygen concentration 8.6 ppm
Fermentation tenq>erature 15X
Yeast pitching rate 12.8x10^ cells/mL
Sampling of fermentation liquor was performed with time^ and variation with time of yeast growth amount (Fig. l) and ^parent extract concentration (Fig. 2) was obsoved Simultaneously, san^ling of yeast cells was performed, and the prepared mRNA was subjected to be biotin-labeled and was hybridized to a beer yeast DNA microarray. The signal was detected using GCOS; GeneChip Opemting Software 1.0 (manufectured by Afifymetrix Co.). Expression pattern of nonScIAHl gene is shown in Figure 3. As a result, it was confirmed that nonScIAHl gene was

expressed in the general beer fermentation
Example 3: Constniction of nonScIAHl Gene Highly Expressed Strain
The nonScIAHl/pCR2,l-TOPO described in Exanple 1 was digested using the restriction enzymes Sad eqd NotI so as to prepare a DNA fragment containing the entire length of tiie protein-encoding region. This fragment was ligated to pYCGPYNot treated with the restriction enzymes Sad and NotI, thereby constructing the nonScIAHl high ejqjression vector nonScIAHl/pYCGPYNot pYCGPYNot is the YCp-type yeast e?q)ression vector. The inserted gene is highly e3q)ressed by the pyruvate kinase gene PYKl promoter. The geneticin-resistant gene G418^ is included as the selection marker in the yeast, and the anpicillin-resistant gene Anp"^ is included as the selection marker in Escherichia colL
Using the high e^qjression vector prepared by the above method, the strain Saccharomyces pasteurianus Weihenstephaner 34/70 was transformed by the method described in Japanese Patent Application Laidropen No. H7-303475. The transformant was selected in a YPD plate culture (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) containing 300 mg/L of geneticiii.
Exambie 4: Analysis of Amounts of Ester Formed in Beer Fermeatation Test
A fernjentation test was Conducted under the following conditions using the parent strain (W34/70 strain) and the nonScIAHl highly expressed stram obtained in Exanq)le 3,
Wort extrapt concoitration: 12%
Wort volume: 2 L
Wort dissoh^ed oxygen concentration; 8 ppm
Fermentation tCT:5)erature: r5°C
Yeast pitching rate: 5 g/L
The fermentation broth was sanpled over time to investigate the time-based changes in yeast growth (OD660) (Fig. 4) and extract consumption (Fig. 5). Quantification of higher alcohol and extract concentrations at completion of feraientation was carried out using head space gas chromatography (J. Am. Soc. Brew. Chem. 49:152-157,1991).
The amount of ethyl acetate formed at completion of fermentation was 24,0 ppm for the

nonScIAHl highly expressed strain in contrast to 34.4 ppm for the parent strain as described in Table 1. The amount of isoamyl acetate formed was 0.2 ppm for the nonScIAHl highly expressed strain in contrast to 2.1 ppm for the parent straia On the basis of these results, the amoimts of ethyl acetate and isoamyl acetate formed were clearly demonstrated to decreased by 30 to 90% by high e?g)ression of no^ScIAHl.

Example 5: DisniPtJon of nonSrTATTI Gene
Fragments for gene disruption were prepared by PCR using plasmids cont^ning a drug resistance marker (pFA6a(G418r)5 pAG25(natl), pAG32(hph)) as tenplates in accordance with a method described in the literature (Goldstein et aL, Yeast, 15,1541 (1999)). Primers consisting of nonScIAHi_delta_for(SEQIDNC). 5)andnonScIAHl_delta_rv(SEQIDNO. 6) were used for the
PCR primers.
A spore clone (W34/70T2) isolated fronibrewer*s yeast Saccharbmyces pastorianus strain W34/70 was transformed with the fragmrats for gene disraption priepared as described above. Transformation was carried out according to the method described in Japanese Patent Application ^ Laid-open No. H07-303475, and transformants were selected on YPD plate medium (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) containing geneticin at 300 mg/L, nourseothricin at 50 mg/L or hygromycin B at 200 mg/L.
Example 6: Analysis nf Ammmts of Ester Formed in Beer Fermentation Test
A fomentation test was conducted under the following conditions using the parent strain (W34/70-2 strain) and the nonScIAHl disrupted strain obtained in Exanople 5.

I
Wort extract concentration: 13%
Wort volume: 1 L
Wort dissolved oxygen concentration: 8 ppm
Fermentation tenq>erature: 15°C
Yeast pitcljing rate: 5 g/L
The fermentation broth was sampled over time to investigate the time-based changes in yeast growth (OD660) (Fig. 6) and extract consumption (Fig. 7). Quantification of ester concentration at completion of fermentation was carried out using head space gas chromatography (J. Am. Soc. Brew. Chem. 49:152-157,1991).
The amount of ethyl acetate formed at completion of fermentation was 28.1 ppm for the nonScIAHl disrupted strain in contrast to 26.2 ppm for the parent strain as described in Table 2. The amount of isoam5d acetate formed was 2;9 ppm for the nonScIAHl dismpted strain in contrast to 2.3 ppm for the parent strain. On the basis of liiese results, the amounts of ethjd acetate and isoamyl acetate formyed were clearly demonstrated to increased by 7 to 26% by disruption of nonScIAHl.

Values in parentheses indicate relative values versus the parent straia
INDUSTRIAL APPLICABILITY
According to the alcoholic beverage production method of the present invention, alcoholic beverages having superior aroma and flavor can be produced because the method can control the content of esters.
More specifically, according to the alcoholic beverage production method of the present invention, alcoholic beverages having superior aroma and flavor can be produced because the

method can increase the content of esters which impart a florid aroma to products. In addition, in the case of nfialt beverages such as beer, for which an excessively high ester content is not preferred, alcoholic beverages having a more desirable aroma and flavor can be produced because the method can also decrease the amount of ester contained thereia

• CLAIMS
1. A polynucleotide selected from the group consisting of:
• a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDNO:!;
• a polynucleotide comprising a polynucleotide coding a protein consisting of the amino acid sequence of SEQ ID N0:2;
• a polynucleotide conprising a polynucleotide acceding a protein consisting of the amino acid sequence of SEQ ID N0:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a esterase activator,
• 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 having a esterase activator,
• a polynucleotide conprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide esquire complementary to the nucleotide sequence of SEQ ID N0:1 under string rat conditions, and which encodes a protein having a esterase activity; and
• a polynucleotide a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence to the nucleotide sequence of the polynucleotide encoding the protein of the amino acid sequence of SEQ ID NO:2 under stringmt conditions, and which encodes a protein having a esterase activity.
2. The pQlynucleotide of Claim 1 selected from the group consisting of:
(g) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID
NO: 2, or encoding an amino acid sequence of SEQ ID NO: 2 wherein 1 to 10 amino adds thereof is
deleted, substituted, inserted, and/or added, and wherein said protein has a esterase activity;
(h) a polynucleotide encoding a protein having* 90% or higher identity with the amino acid sequence of SEQ E) NO: 2, and having a esterase activator, and
(i) a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under high stringent conditions, and which encodes a protein having a esterase activity.
• The polynucleotide of Claim 1 comprising a polynucleotide consisting of SEQ ED NO: L
• The polynucleotide of Claim 1 Conan rising a polyrmcleotide encoding a protein

• consisting of SEQ ID NO: 2.
• The polynucleotide of any one of Claims 1 to 4, wherein the polynucleotide is DNA.
• A polynucleotide selected from the group consisting of:
(j) a polynucleotide encoding RNA of 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 Clairn 5 through Ran effect;
(1) a polynucleotide encoding RNA having ah activity of specifically cleaving a transcript of the polynucleotide (DNA) according to Claim 5; and
(l) a polynucleotide encoding RNA that represses egression of the polynucleotide (DNA) according to Claim 5 through co-suppression effect.
• A proton encoded by the polynucleotide of any one of Claims 1 to 5.
• Vector conprising the polynucleotide of any one of Claims 1 to 5.
• A vector conprising the polynucleotide of Claim 6.

• A yeast cortqjrising the vector of Claim 8 or 9.
• The yeast of Claim 10, wherein producing ability is decreased by introducing the vector of Claim 8.
• A yeast, whb"ein an expression of the polynucleotide (DNA) of Claim 5 is repressed by introducing the vector of Claim 9, or by disrupting a gene related to the polynucleotide (DNA) of Claims.
• The yeast of Claim 11, wherein a ester-producing ability is decreased by increasing an expression level of the protein of Claim 7.
• A method for producing an alcoholic beverage by using the yeast of any one of Claims 10 to 13.

• The method for producing an alcoholic beverage of Claim 14, wherein the brewed alcoholic beverage is a malt beverage,
• The method for producing an alcoholic beverage of Claim 14, wherein the brewed alcoholic beverage is wine.
• An alcoholic beverage produced by the method of any one of Claims 14 to 16.
• A method fer assessing a test yeast fer its estear-produciiig capability, comprising using a primer or a probe designed based on a nucleotide sequence of a esterase gene having the nucleotide sequence of SEQ ID NO: 1.
• A method for assessing a test yeast for its producing capability, surprising: culturing a test yeast; and measuring an expression level of a esterase gene having the nucleotide sequence of SEQ ED NO: 1.
• A method for selecting a , contriving: culturing test yeasts; quantifying the protein according to Claim 7 or measuring an expression level of a esterase gene having the nucleotide sequence of SEQ ID NO: l;.and selecting a test yeast having said protein amount or said gene expression level according to a target capability of producing ester.
 2L The uietfaod for selecting a yeast according to Claim 20, composing: culturing a reference yeast and test yeasts; measuring an expression level of a esterase gene having the nucleotide. sequence of SEQ ID NO: 1 in each yeast; and selecting a test yeast having the gene expressed higher or lower than that in the reference yeast.
 22. The method for selecting a yeast according to Claim 20, comprising: culturing a reference yeast and test yeasts; quantifying the proton according to Claim 7 in each yeast; and selecting a test yeast having said protein for a largo- or smaller amount than that in the reference yeast.

•
• 23. 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 13 or a yeast selected by the method according to any one of Claims 20 to 22; and adjusting the production amount of ester.
•