DESCRIPTION
GENE ENCODING PROTEIN HAVING PROMOTING
AND USE THEREOF
TECHNICAL HELD
The present invention relates to a gene encoding a protein having a trdialose synthesis-promoting and use thereof in particular, a yeast for practical use with superior resistance propaty to drynras and/or resistance prcpaty to low-temperature storage, alcoholic produced with said yeast, and a method for producing said beverages. More particularity, the present invention relates to a yeast, whose resistance property to dryness and/or resistance propraty to bw- enhanced by amplifying expresaon level of TSLl gene a protein Tsllp having a trehalose synthesis-promoting activity in brewor's yeast, especially non-ScTSLl gsne spedfic to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc. Furtha:, the yeast of the presait invention is useM as a baker's yeast or an industrial as well.
BACKGROTUND ART
Beer characterized by a process recovering yeasts after fermentation and using the recovered yeasts at fermentation, which is called "Renjo". The yeasts are stored in the presence of ethanol in a tank whose temperature is kept at approximately 0 to 3°C. When the yeasts die during the stora^ not only the next fermentation process is mterfbed, but also constituents of the yeast cells released by cell lysis may impart unfevorable taste to product. Tiered^, it is very important for allowing some variance to deagn production process and fw stable production of quality products to use yeasts with superior resistant property to low-temperature storage.
"Raijo" may be terminated at a certain times of fermentation is carried out The number of times of "Renjo" may -vary accoxling to fermentation conditiom or propaties of yeasts used in the process- A process to develop yeasts for fermeaitation freshly is called prop^ation. Yeasts are
subcu!(,tured several times enlarging scales of culture aiccessively during the propagation process. Because propagation process requires from several days to several weeks, it brin^ great advantages
m production efficiency if term of the process is shortened or yeast ceils which are large-scale
pre-cultured are able to be stored stably for extended period of tune at low temperature or under dry
condition.
CoEcemii^ a method for prcducii^ dry yeast maintaining high viable cell ratio,

improvemKit of drying equipment, or improvement of manufacturing conditions such as temperature or addition of emulsifiere, etc. have been made. For example, Lndrying method is not practical to be used at industrial production scale because, thou^ it can maintain extremely lyg^ viable cell ratio, but at the same time it takes a lot of time and cost.
Regarding low-temperature reastance of yeast, some experiments designed to impiuve refiigeration-reastant property mainly of baker's yeast were reported. This is because Saccharomyces cerevisiae, which is a baker's yeast, has poor low-temperature storage property in con^arison' with brewer's yeast for beer or sake, vrfiich can ferment at low temperature. For example, baka"'s yeasts having refrigeration-resistant property and drying-resistant propKty were found out mainly by screening methods in Japanese Patent Application Laid-open No. Hn-155559 Md J^anese Patent Aj^lication Laid-open No. 2003-304864. Further, regarding exan^les' utilizing genetic engineering techniques, trehalose highly accumulating strains by disruption of NTHl, which is a trehalase gene, is reported in J^anese Patent Application Laid-open No. HlO-117771 and a strain highly accumulating specific amino adds such as alanine by disruption of CARl, whidi is an arginase gene, is rqjorted in Jqsanese Patent Application Laid-opai No. 2001-238665.
DISCLOSURE OF INVENTION
Under the above situations, there has been a need to make high-efficiency production of alcoholic beverages or usefiii materials possible by using a gene encoding a protein responsible for drying and/or low-temperature storage-reastant jHopaty of brewery yeast and said protein.
The present invKitors made extensive studies to solve the above problems and as a result, succeeded iii identifyh^ and isolating a gene encoding a protan having a trehalose synthesis-promoting actisily from beer yeast Moreova-, the present raventors produced transfonned yeast m which the obtained gene was expressed to veriiy that drying-resistant prq)erty and/or low-temperature storage-resistant property can be actually improved, tha-eby completing the present invention.
Thus, tiie present invention relates to a gene encoding a a prolan having a trehalose syndiesis-promoting activity of*)rewery yeast, to a protan encoded by'said gene, to a transformed , "yeast in which the e:q«ession of said gene is controlled, to a method for enhancir^ drying-resistant propert^^ andlor low-tai^eraliire aorage-resistant property of yeast using a yeast in which the ejgjre^on of said gene is controlled, or the like.. More specifically, the present mvention provides the following polynucleotides, a vector comprisii^ said polynucleotide, a transformed yeast introduced" with said vector, a iiwthod for producing alcoholic beva:ages by using said transformed yeast, and the like.

(1) A polynucleotide selected from the group consisting of:
(a) a polymKileotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDN0:1;
. (b) a polynucleotide con^rising a polynucleotide aicoding a protein consisting of the amino.add sequence of SEQ ID N02;
(c) a polynucleotide comprising a polynucleotide aicoding a protein consisting of tiie amino add sequence of SEQ ID N0:2 in vAdch one or more amino acids theareof are deleted, substituted, inserted and/or added, and having a trdialose synthesis-promoting activity;
(
(y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense or antisense direction; and
(z) a signal that can function in a yeast with respect to transcription termination and polyadenylation of a KNA molecule.
(7b) The vector of (7). above, which comprises the e^^resaon cassette comprising the following components:
(x) a promoter that can be transcribed in a yeast cell;
(y) any of the polynucleotides described m (1) to (5) above linked to the promoter in a sense direction; and
(z) a signal that can fimction in a yeast with respea to ti'anscription termination and polyadenylation of a KNA molecule.
(8) A yeast into which the vector acconling to any one of (7) to (7b) above has been
introduced-
(9) ■ The yeast (yeast for practical use) according to (8) above, wherein drying-resistant
propoly is increased. The "yeast for practical use" means that a yeast which possesses practical
value such as brewer's (brewery) yeast, baker's yeast or industrial yeast, &c.
(10) The yeast according to (8) above, wherein low-temperature stor^e-resistant
property is increased.
(11) The yeast acccffding to (9) abo\«, wheran the drying-reastant property is increased
; by increasing an expre^on levd of the protdn of (6) diove.
(12) ITie yeast.according to (10) above, wherdn the low-temperature storage-resistant
property is increaseid by increasing an es^ression level of th^rotein of (6) above,
"^ (12a) The yeast according to any one.of (9) to (12) above, wherdn the yeast is a brewey
" yeast.
(13) A method^jr producii^ an alcoholic beverage by using the yeast according to any
one of (8) to (12a) above.

(14) The method according to (13) above, wha-ein the brewed alcoholic Devra-age is a mak beverage.
(15) The method according to (13) above, who^ the brewed alcoholic beverage is wina
(16) An alcoholic beverage produced by the method according to any one of (13) to (15) above.
(17) A method for asse^ing a test yeast fiar its diying-resistant prapsrty and/ra-low-tempaature storage-r^stant property, cmnprising using a primer or probe designed based on the nucleotide SHiuence of a gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protdn having a trehalose ^mthesis-promoting activity.
(17a) A method fta- selecting a yeast having an ino-e^ed drying-resistant property and/or . low-tenq)eralure storage-resistant properly by using the method described in (17) above.
(17b) A method for producing an alcohoUc beverage (for example, beer or alcohol for industrial use, etc.) by using the yeast selected with the method described m (17a) above.
(17c) A method for producing an usefiil materials (for exan^le, protdn) t^ using the yeast selected with the method desaibed in (17a) above.
(18) A method for assessing a test yeast for its drying-resistant propaty and/or
low-tanperature storage-resistant property, comprising: culturing the test yeast; and measuring the
ejqjression level of the gene having the nucleotide sequKice of SBQ ID NO: 1 and encotfing a
prolan having a trehalose ^thesis-promoting activify,
(18a) A method for selecting a yeast having a high drying-resistant propaty and/or low-temperature storage-resistant prc^aty, vAach conprises assessing a test yeast by the method described in (18) above and sdecting a yszst having a high expresaon level of gene enco&ig a protein having a trehalose synthesis-promoting activity.
(18b) A method for producing an alcoholic beverage (for exan^le, beer) by using the yeast selected with the method desaibed in (18a) above.
(18c) A method for produdng an useful material (for example, jiotein) by using the yeast selected witii the meShod described in (18a) above,
(19) A method for sdectmg a yeast, con^irisu^: cuhuring test yeasts; quaiitifying the pro^n of (6) above or measuring the ejqsression level of the gene having ibs nucleotide sequence of SEQ E) NO: 1 and erKodmg a protan having a trehalose ^fntiiesis-promoting activity; and selecting a test yeast having an amount of Ifae protein or the gene expression level according to fevorable diying-rsistant propaty and/or low-ten^)a'atLire storage-resistant property.
(20) The method for selecting a yeast according to (19) above, con^rising: culturing a reference yeast and test yeasts; measuriag for eadi yeast the esqiresaon level of the gene having the

nucleotide sequence of SEQ ID NO: 1 and encoding a protean having a trehalose synthesis-promoting activity; and selecting a test yeast having the gene expression higher than that in the reference yeast.
(21) The ra^hod for selecting a yeast according to (19) above, conqjrising: culturing a reference yeast and test yeasts; quantifymg the protdn according to (6) above in each yeast; and selecting a test yeast having a larger amount of the protein than that in the referaice yeast.
(22) A method for producing an alcoholic beverage comprising: conducting fermentation using the yeast accorduig to any one of (8) to (12a) above or a yeast selected by the methods according to any one of (19) to (21) above.
The transformed yeast of the present invention is able to keep high viable cell ratio during dry storage or low-tempea'ature storage. Therefore, when it is used for brewTi^ and so on, panfijhiess of conservmg yeast can be eliminated. Furthra-, it is ejected to contribute to quality stabOizadoa MDreover, dry yeast is suitable for long-storage, and it is very advantageous to distribution or transportation due to its reduced virei^t. It is also useful as microorganisms for industrial application sucii as industrial alcohol production or production of useful proteins. The yeast-of the present invention also useful as an industrial yeast as well.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the cell growth with time upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 2 shows the extract (sugar) consumption with iiaie upon beer fermentation test. The horizontal axis represents fermentation lime vMe the vertical axis represents apparent extract concentration (w/w%).
Figure 3 shows the expression profile of non-ScTSLl gene in yeasts upon beer fermentation test. The horizontal axis represents fomentation time while the vertical axis rqjresents the intensity of detected agnal.
Figure 4 shows the result of dr^dng-resistant property test of parent Srain and non-ScTSLI highly expressed strain.
BEST MODES FOR CARRYING OUT THE INVENTION
The present inventors isolated and identified non-ScTSLI gene encodmg a protran havu^ a trehalose synthesis-promoting activity of brewery yeast based on the lager brewing yeast genome information mapped according to the method disclosed in J^anese Patent j^pUcation Laid-Open No. 2004-283169. The nucleotide sequence of the gene is represented by SEQ ID NO: 1. Further,

an amino add sequence of a protein encoded by the gene is represental by SEQ ID NO: 2.
1. Pdvnudeotide of flie invaitioii
Firet of all, the present invention provides (a) a polynudeotide con^rising a polynucleotide of the nucleotide sequaice of SEQ ID N0:1; and (b) a polynucleotide comprising a polynucleotide enccdmg a protdn of the amino add sequence of SEQ ID N0:2. The polynucleotide can be DNA orRNA
The taiget pofynucleotide of the present invention is act limited to the polyaudeoitide eiMX)dmg a protan having a trehalose synthesis-promoting activity described above and may include other pofynucleotides encoding protans having equivalent fimctions to said protdin. Proteins with equivalent fimctions indude, for exanyjle, (c) a protein of an amino add sequence of SEQ ID N0;2 with one or more amino adds thereof bemg delete4 substituted, inserted and/or added and having a trehalose synthesis-promoting activity.
Sudi protdns include a protan consisting of an amino add sei^ence of SEQ ID N0:2 with, for example, 1 to 100, 11o90, lto80, 1 to 70,1 to 60,1 to 50,1 to 40, lto39, ito3S, lto37, Ito 36,1 to35,1 to34, 1 to33, 1 to32,1 to31,1 to30,1 to29, 1 to28,1 to27,1 to26, Ito25,1 to24, Ito 23, Ito 22, Ito 21, Ito 20, Ito 19, Ito 18, Ito 17,1 to 16, Ito 15, Ito 14, Ito 13, Ito 12, Ito 11,1 to 10,1 to 9,1 to 8,1 to 7,1 to 6 (1 to several ammo adds). 1 to 5,1 to 4,1 to 3,1 to 2, or 1 amino add residues thaeof being deleted, substituted, inserted and/or added and having a trehalose synthesis-promoting activity. In general, the number of ddetions, substitutions, insertions, and/or additions is preferably smaller. In addition, such protdns include (d) a protean having an amino add sequaice witii about 60% ex higho-, about 70% or higher, 71% or higher, 72% or Hgher, 73% or highCT 74% a: higher, 75% or higher, 76% or higter, 77% or higher, 78% or higher, 79% or higher, 80% or hi^iH', 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 hi^ier, 91% or hi^er, 92% or higher, 93% or hi^er, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or bigha-, 99.2% or higher, 99.3% or higher, 99.4% or hi^er, 99.5% «-higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, ra 99.9% or higher identity witii tiie amino add sequence of SEQ ID N0:2, and havii^ a trehalose syntheas-promoting activity. In general, die percentage identity is preferably higho:.
Trehabse symhesis-promoting activity may be evaluated by n^asuring trehalose synthase activity and/or trehalose level, for example, by a method described in Walto- et al., I Biol. Chem., 273,33311-33319(1993),
Furthermore, the preseifi; invenrion also conten^lates (e) a polynucleotide con^rising a polynucleotide wMch i^rii^zes to a polymjcleotide consistii^ of a nudeotide sequence

complementary to the nucleotide sequence of SEQ ID N0:1 under stringent conditions ana wmcn encodes a protein having a trehaJose synthesis-pranwMii^ acrivity; and (fi a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide complementary to a nucleotide sequaice of encoding a protein of SEQ ID N0;2 under shingent cojiditions, and which encodes a pratein having a trehalose ^Tithesis-promoting activity.
Herein, "a polynucleotide that hybridizes under stringent conditions" refers to nucleotide sequence, such as a DNA, obtaned by a coloiQf hybridization technique, a plaque l^bridization technique, a southern h56ridi2a.tion technique or the like uang all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID N0:1 or polynucleotide encoding the amino acid sequence of SEQ ID N0:2 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd Ed., CUKEENT PROTOCOLS IN ■ MOLECULAR BIOLOGY, John Wil^ & Sons 1987-1997, and so oa
The term "strir^ent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low strii^ency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formanside at 32°C. "Moderate stringency conditions" ar^ for ©candle, 5 x SSC, 5 x Denhardt's solution, 0.5%) SDS, 50% formamide at 42°C. "Kgh stringaicy conditions" ^e, for exampie, 5 x SSC, 5 x Daihardt's solution, 0.5% SDS, 30% formamide at 50°C. Underthese conditions, a polynucleotide, such as a DNA, witii higher homolo^ is expected to be obtained efScicntly at higher temperature although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic arength, time, salt concentration and others, and one ^Ued in the art may appropriately select these factors to realize similar stringency.
When a commercially available kit is used for hybridization, for example, Alkphos Direct LabeUng 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 pimaiy wash buffer containing 0.1% (wA-) SDS at 55°C, thereby detecting hybridized polynucleotide, such as DNA.
Other polynucleotides that can be hybridized include polynucleotides having about €0% 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 hi^a-, 85% or higher, 86% or higher, 87% or higher, 88% or higher, S9% or hi^er, 90% or higher, 91 % or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higha:, 96% or higher, 97% or higher, 98% or hi^er, 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 higho" or 99.9% or higher identity to polynucleotide encodii^ the amino acid

Jequence of SEQ ID N0:2 as calculated by homology search software, such as FASTA and BLAST amsg d^uk parameters.
Identity between amino acid sequences or nucleotide sequences may be detomined using algorithm BLAST by Karlin and Altschul (B-oa Natl Acad Set USA, S7: 2264-2268, 1990; Proc. NailAcadSci. C/&4,90: 5873,1993). Programs called BLASTN and BLASTX based on BLAST a^orithm have been developed (Altschul SF et al., ^ Mol. Biol 215: 403, 1990). When a nucleotide sequence is sequenced usmg BLASTN, the parametM:s are, for ©cample, score = 100 and woni length = 12, When an amino acid sequence is sequenced using BLASTX, the parametas are, for example, score = 50 and word laigth = 3. When BLAST and Griped BLAST programs are used, defeult parameters for eadi of the programs are employed.
2. ft-Qtan of the prsent inveotion
The i^esent invention also provides proteins encoded by any of the polynucleotides (a) to (i) above. A preferred proton of the presat invention comprises an amino add sequence of SEQ ID N0:2 with one or several ammo adds tho-eof being ddetal, substitiited, inserted and/or added, and having ati^ehalose synthesis-promoting activity.
Such protein includes those having an amino add sequence of SBQ ID N0:2 with amino add residues thereof of the number mentioned above beuig deleted, substituted, inserted and/or added and having a trehalose synthesis-promoting activity. In addition, such |M-otKn indudes those having homology as dsaibed above with the amino add sequence of SEQ ID N0:2 and having a trehalose synthesis-promoting activity.
Such protdns may be obtained by enqiloying site-directed mutation desaibed, for example, in MOLECULAR CLCMNG 3rd Ed, CXiRRENTfeOTOCOi^ INMOI^CULARBIOLXXIY, NUC. Acids. Hes., 10:6487(19S2\Proc. Natl. Acad Sci. USA 79: 6409 (1982), Gene 34:315 (1985),Nita Acids. Res., 13:4431 (1985), Proc. Nad Acad Sci. USA 82:488 (1985).
Deletion, substitution, insertion and/or addition of one or more amino add readues in an amino add sequence of the protein of the invention means that one M" more amino acid residues are ddeted, sub^tuted, inserted and/or added at any one or more poshions in the same amino acid sequence. Two ca* more types of ddetion, substitution, insertion and/or addition may occur amcurrently.
Hereinafter, examples of mutually substitutable amino acid residues are enumerated. Amino add residues in the same ©x>up are imrtuaUy substitutable. The groups are provided below.
Group A: leudne, isoleudne, norleudne, valine, norvaline, alanine, 2-aminobutanoic add, methionine, o-methylserine, t-bu^lglycine, t-butylalanine, cyclohexylalanine; Group B: asparatic add, glutamic- add, isoasparatic add, isoghitamic add, 2-aminoadipic acid, 2-aminosuberic add;

G^"OUBX!: Jspara^e, ghitamine; Group D: lysine, arginine, ornithine, 2,4.diaminQbutanoic acid, 2,3-diarainopropionic acid; Group E: proline, 3-hydroxyproIine, 4-hydroxyproHne: Group F: serine, threonine, homoserine; and Group G: phenylalanine, tyrosine.
The protein of t!K present invention may also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyioxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). In addition, p^de synthesizers available fiom, for example. Advanced ChemTech, PerkinEImer, Pharmacia, Protdn Technology Instrument, Synthecell-Vega, Pa'Septive, SHmazu Coip. can also be used for chemical synthesis.
3. Vector of the invaitioD and veast transformed wifli liie 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 polynucleotideB descrftied in (a) to (i) above or ar^ of the polynucleotides described in (j) to (m) above. Generally, the vector of the presait invention comprises an expression cassette including as con^jonents (x) a j^omoter 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 agnal that ftinctions in the yeast with respect to transcriptioii tMmination and polyadenylatiQn of RNA. molecule. Further, in order to hi^y e?^ress the protein of the invention, these polynucleotides are preferably introduced in the sense direction to the promota: to promote egression of the polynucleotide (DNA) desoibed in any of (a) to (i) above.
A vector introduced in the yeast may be any of a multicopy type (YEp type), a single copy type (YCp type), or a chromosome integration type (Yip type). For example, YEp24 (J. R. Broach et al., EXPERIMEOTALMANIPULA-TIONOFCSNEEXPRSSION, Academic Press, New York, 83,1983) is known as a YEp type vector, YCp50 (M. D. Rose et al.. Gene 60: 237, 1987) is known as a YCp ^e vector, and YIp5 (K. Struhl et al., Proc. Noll Acad ScL USA, 76: 1035, 1979) is known as a Yip type vector, all of which are readily available.
Promoters/tKiDinatOTs for adjusting gene e)^ression in yeast may be in any combination as long as they function in the yeast for practical use and they are not mflueaced by constituents in fermentation broth. For example, a promoto- of glyceralddiydes 3-pho^hate dehydrogenase gene CrDH3), or a promoter of 3-phosphoglycerate kinase gene (PGKl) may be used. These gaies have previously been cloned, described in detail, for example, in M. F. Tuite et aL, M^O J., 1, 603 (1982), and are readily available by known methods.
Since an auxotrophy marker cannot be used as a selective marker upon iransformation for a yeast for practical use, for example, a geneticin-resistant gene (G418r), a copper-resistant gene (OJPl) (Marin et al., Proc. Natl. Acad Sci. USA, 81, 337 1984) or a cerulenin-resistant gene {fas2m.

PDR4) (Junji Inokoshi et al., Biochemistry, 64,660,1992; and Hussain et al., Gene, 101:149,19^1, respectively) may be used.
A vector constructed as described above is introduced into a host yeast Examples of the host yeast include ai^ yeast 6'east for practical use) tiiat can be used for brewing, for example, brewery yeasts for beer, wine and sak^ baker's yeast, yeast for producing industrial alcohol or yeast for produdng useful proteins and so on. Spedfically, yeasts such as genus Saccharcxnyces may be used. According to the present invention, a lager brewing yeast, for example, Sacchcff-onryces pa^ricmis W34/70, rtc., Sacchanmyces carlsbergensis NCYC453 or NCYC456, etc., or Sacdtaromyces cerevisiae NBRC1951, NBS.C1952, NBRC1953 or 1SIBRC1954, etc., may be used. In addition, whisbf yeaas aich as Saccks-on^Kxs cerevisiae NCYC90, wine yeasts aich as wine yeasts #1, 3 and 4 fiom the Brewing Soddy of J^an, and sake yeasts such as sake j«ast #7 and 9 fi-om the Bowing Society of Japan, baker's yeast such as NBRC0555, NBRC1346 or NBRC2043, etc., may also be used but not limited therdo. In the present invention, lager brewing yeasts such as Saccho'cmycespastoriioms may be used preferably.
A yeast transformation method may be a generally used known method. For exanqjle, methods that can be used mchide but not limited to an dectroporation method (Merfi. Enzym., 194: 182 (1990)), a spheroplast method {Proc. Natl Acad Set. USA, 75: 1929(1978)), a lithium acetate m^od {J. Bacteriology, 153:163 (1983)), and methods desaibed mProc. Natl Acad Set USA, 75: 1929 (197S), METHODS IN YEAST CSMETICS, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual.
More spedfically, a k)st yeast is caihured in a standard yeast nutrition medium (e.g., YEPD medium (Genetic Engineering, Vol. 1, Plenum Press, New Yoric, 117(1979)), ^.)sucb that OD600 nm will be 1 to 6. This culfeire j^ast is collected by ceniiifugation, wi^ed 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 aboirt 30°C for about 60 minutes, i is left to stand with DNA to be introduced (about 1 to 20 \ig) at about 30°C fw about another 60 minutes. Polyethylen^ycol, preferabty about 4,000 Datton of poiyethylen^ycol, is added to a final concentration of about 20% to 50%. After leaving at about 30°C fijr about 30 minutes, the cell is heated at about 42°C for about 5 minutes. Preferably, tius cell suspension is wa^ied with a standard yeast nutrition mecBum, added to a pretktermmed amonnt of fi^esh standard yeast nutrition medium and left to stand at about 30°C fcir about 60 minutes. Thereafter, it js seeded to a standard agar nwdium containing an antibiotic OT the like as a selective marker to obtain a transformant
Other genaal doning techniques may be found, for eiample, in MOIECULAR ClXMNG 3rd Ed, and METHCfDS IN YEAST Cfeffincs, A LABORATCEY MANUAL (Cold Spring Harbor Laboratoiy Press, Cold Spring Harbor, NY).

4. Method of producing alcoholic beverages according to the present invention and alcoholic
beverages produced by the mefliod
A ye^ hssm% a supaior drying-resistant property and/or low-tempKature storage-resistant property can be obtained by iatroducing the vector of the present invention described above to a yeast. Further, a yeast having a superior drying-resistant property and/or low-temperature stor^e-resistaitt prqiaty can be obtained by selecting a y^st by the yeast assessment method of the present invention described below. The target use of yeasts obtained in the present invention include, for example, but not limited to, brewing alcoholic beverages such as beer, wine, "wMsky, sake and the like, baking bread, manufectming use&I materials such as industtial alcohol production and prediction of useful proteins.
In order to produce these products, a bwwn technique can be used except that a yeast for practical use obtmned according to the present invention is used in the place of a parent stnon. Since starling mataials, manu&:turing equipment, manufecturing control and the like may be the same as the convaitional ones; it can be performed without increasir^ cost.
5. Yeast assKsment method of the invention
The presait invention relates to a metiiod for assessing a test yeast for its dryii^-resistant property and/'or iow-temperature sloiage-ie^stani property by usujg a prima: or a piobs desgned based on a nucleotide sequence of a gene havii^ the nucleotide sequence of SEQ ID N0:1 and encoding a protein having a trehalose synthesis-promoting activity. Genaal technique for such assessment method is known and is descaibed in, for example, WOOl/040514, JapaneseLaid-Open Patent Application No. H8-205900 or the hke. This assessment method is described in below.
First, genome of a test yeast is prepared. For this pr^aration, any known method such as Hereford mefeod or potasaum achate metiiod may be used (e.g., MElHCffis w YEAST GENETICS, Cold Spring Harbor Laboratory Press, 130 (1990)). Using a primer or a probe designed based on a nucleotide sequCTce ^sreferably, ORF sequence) of the gene encoding a protein havii^ a trehalose synthesis-promotii^ activity, the efflstence of die gene or a sequence spedfic to the graie is daermined in the test yeast gaiome obtained. The primer or the probe m^ be designed according
to aknown technique.
IMection of the gene or tiie specific sequence may be carried out by aaploying a known technique. For exan^le, a polynucleotide including part or all of the specific sequence or a polynucleotide including a nucleotide sequence complementary to said nucleotide sequence is used as one primer, while a polynucleotide indudit^ part or all of the sequence upstream or downstream from this sequence or a polynucleotide including a nucleotide sequence con:5)lementary to said

micleotide Sequence, is used as anotha primer to amplify a nucleic acid of the yeast by TPCR method, tha^eby determining the existence of ainplified products and molecular wdght of the amplified produtas. The number of bases of polynucleotide used fir a primer is generally 10 base pars (bp) or more, and preferably 15 to 25 bp. In general, the number of bases between the prime's is suitably 300 to 2000 bp.
The reaction conditions for PCR are not particularly limited but may be, for example, a denaturalion temperature of 90 to 95''C, an annealing taiqjerature of 40 to 60°C, an elongation temperature of 60 to 75°C, and tiie number of cycle of 10 or more. The resulting reacdon product may be separated, for exanqjle, by electrt^horesis using agarose gel to detamine the molecular weight of the anqjhfied product. This method allows prediction and assessment of the drying-resistant property and/or low^empaature storage-reastant property of ye^t as determined by wheths- the molecular weight of the amplified product is a size that contains the DNA molecule of the specific part, in addition, by analj'zing tte nucleotide secpience of the amplified product, the propaly may be predicted and/or assessed more precisely.
MoreovOT, in the present invention, a test yeast is cultured to measure an expression level of the gene encoding a protean ha^^l^ a trehalose synthesis-promoting activiiy havii^ fte nucleotide sequence of SEQ ID N0:1 to assess the test yeast for its drying-resdSant property and/or low-temperature storage-resistant property. Measurement of expression level of the gene encodir^ a protein having a trehalose synthesis-promoting activity can be performed by culturing test yeast and then quantifyii^ mRNA or a protan resulting fi^jm the gene. The quantification of mRNA or pro^n may be carried out by anployir^ a known technique. For example, mRKA may be quantified, by Northern hybridization or quantitative RT-PCR, while protem may be quantified, for example, by WKtem blotting (CuRREOTl^TOCca^INMOLEcnjiJiRBiOU)GY, John Wiley & Sons 1994-2003),
Furthamore, test yeasts are cultured and expression levels of tlK gene encoding a protein having a trehalose syntheas-promoting activity having the nucleotide sequence of SEQ ID NO: 1 are meaaired to select a test yeast with the gene e)q)resaon level according to the target trehalose-pToducing ability, therdiy a yeast fevorable for toewing desired alcoholic bevesr^es can be selected. In addition, a referraice yeast aiid a test yast may be cultured so as to measure and compare the expression level of the gene in eadi of the yeasts, thffei>y a fevorable test yeast can be selected. IVfore spedfically, for eiample, a reference yeast and one or more test yeasts are cultured and an expresaon level of the gene encoding a protdn having a trehalose sjmtiwsis-promoting activity having tlws nucleotide sequence of SEQ ID N0:1 is measured in eadi yeast. By selecting a tea yeast witii the gene expressed higher than that in the reference j«ast, a yeast suitable for brewing desired alcoholic beverages or production of usefid materials can be selected

Altematively, test yeasts are cuJtured and a yeast with a hi^ trdialose-producing abihty is selected, thereby a yeast suitable for brewing desired alcoholic beverages or production of useful materials can be selected
In these cases, the test yeasts or the reference yeast may be, for example, a yeast introduced with the vector of the invention, an artificially mutated yeast or a naturally mutated yeast. The trehalose ^thesis-promoting activity can be evaluated by measuring trehalose synthesis-promoting activity and/or trehalose level by, for example, a method described in Wahra" et al., X Biol. Chem., 273, 33311-33319 (1993). The mutation treatment may employ any methods including, for example, physical methods such as ultraviolet irradiation and radiation irradiation, and chemical methods assodated with treatments with dm^ sxki as EMS (ethyknethane sv^phooate) and N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima Ed., BIOCHEMSTRYEXPEEOMENTS vol. 39, Yeast Molecuhr Genetic E^iments, pp. 67-75,3SSP).
In addition, examples of yeasts used as the reference yeast or the test yeasts mclude any yeasts (yeasts for practical use), for example, brewery yeasts for beer, wine, sake and the like or baker's yeast, yeast for produdng industrial alcohol or yeast for producing usefiil proteins, etc. More spedfically, yeasts siKh as genus Saccharomyces may be used {e.g., S. pastcuiamis, S. cerevisiae. and S. carlsbergetms). According to the present invention,, a lager brewing yeast, for example, Sacdiaromyces pastoriamis W34/70; Saccharomyces carhhergensis NCYC453 or NCYC456; or Saccharomyces cerevisiae NBRC1951, MBRC1952, ]>ffiRC1953 or NBRC1954, etc., may be used. Further, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of J^an; aiid s^e yeasts such as sake yeast #7 and 9 frran the Brewng Society of Japan, baker's yeast such as NBRC0555, NBRC1346 and NBRC2043, etc., may also be used but not limited thereto. In the preseait invenlion, lager brewing yeasts such as Saccharomyces pastoiiarms may prefwably be used. The reference yeast and the test yeasts may be selected from the above yeasts in any combination.
EXAMPLES
Iferdnafter, the present invention will be desoibed in more detail with reference to working examples. The present havention, however, is not lunited to fte examples described betow.
Ikampk 1: Cioaing of Gmc Encoding Protaa Having Treludose Synthesis-pramoting Activitv fnoa-ScTSLU
A gene encodmg a protem havii^ a trehalose synthesis-promoting aclmty of lager brevrtng yeast (non-ScTSLl) (SEQ ID NO: 1) was found as a result of a search utilizmg the comparison

diabase described in Japanese Patent Application Laid-Open No. 2004-283169. Based on the acquired Qucleotide sequKKe infonnalion, primers nDn-ScTSLl_fOT (SEQ ID NO: 3) and non-ScTSLl_rv (SEQ ID NO: 4) were deseed to amplify the fiiU-length of the gene. PCR was carried out uang dhromosomal DNA of a gKiome s^uenrang strain, Sacdiaromyces pastoriamis Weihenstephan 34/70 (sometimes abbreviated as 'W34/70 strain"), as a ten^Iate to obtain DNA fiagtneots indudii^ the fiiU-laigth gene of non-ScTSLI,
The non-ScTSLI gene fr^ments thus obtained wae inserted into pC2t2.1-TOPO vector (Invitragen) by TA clonir^. The micleotkie sequence of the non-ScTSLI gaie wae analyzed by Sai^sinethod(F. Sanger, Science, 214:1215,1981) to confirm the nudeotidesequKice.
Example 2; Analysis of EsDression of nop-ScTSLl Gene during Beer Fennentation
A beer fermentation test was conducted using a lager brewing ^ast, SacchfflX)myces pastwianus W34/70, and mRNA extracted from the lager brewing yeast during fermentation was detected by a beo- yeast DNA microarray.
Woit extract concentration 12.69%
Wort content 70 L
Wort dissolved oxygen concentration S.6 ppm
Fermraitalion temperature 15°C
Yeast pitching rate 12.8x 10^ cells/mL
The fermentation litpior was sampled over time, and the time-course changes m amount of yeast cell growth (Fig. 1) and apparait exirart concentration (Pig. 2) were observed. Simultaneously, yeast cells vi^re san^led to pr^are mRNA, and the prepared mRNA was labeled with biotin and was hybridized to a beer yeast DNA microarray. The signal was detected using GeneChip Operatii^ system (GCOS; GeneChip Operaing Software 1.0, manufectured by Affymetrix Co). Expression pattan of the non-ScTSLI gene is shown in Figure 3. Tlus result confirmed the expression of the non-ScTSL 1 gene in flie general beer fermentation.
Example 3: Constmction of non-ScTSLl BSghlv Expressed Strain
The non-ScTSLl/pCR2.1-TOPO described in Exanq)le 1 was digested vrith the restriction enzymes Sad and NotI to prepare a DNA fragment containing ^ raifiiB length of the protdn-encotidg region. This fragment was ligated to pYCGPYNot treated vnfti the restriction enzymes SacI and NotI, thereby constructing the non-ScTSLl high expression vector non-ScTSLl/pYCGPYNot. pYCGPYNot is a YCp4ype yeast expression vector. A gene

mserted is highly e^essed by the pyruvate kinase gene PYKl promote. The geneticin-reastant gene G418'" is included as the selectable marker in ihs yeast, and the anqjicillin-resistant gene Amp' as the selectable marker in Escherichia coli.
Using the H^ esqjresaon vectco: prepared by the above method, an AIL4004 stram was transformed by the method described in Japanese Patent Application Laid-open No. H07-303475. The transformants were selected on a YPD pUite medium (1% yea^ ^tract, 2% polypeptone, 2% glucose and 2% agar) containing 300 mg/L of geneticin.
Example 4: Evaluation of Brying-r^istant Property of non-ScTSLl Highly Expressed Strain
Dryii^-resistant properties of the parent strain (ArL4004 Srain) and the non-ScTSLI highly ej^iressed srain obtained by the method described in Example 3 were evaluated by a method described below.
One platinum loopfUl of each yeast was inoculated into 10 mL of wort containing 100 mgl. of gen^cin, and stirred at 30°C overnight (precultivation). The precultivation liquid was inoculated into 10 mL wort containing 100 mg/L of gendicin to make its OD660 = 0.5, then main culture was initiated. The culture was continued for 2 days until tiie growth of the yeast reached stationary phase. Turbidity of the culture was measured at the completion of the culture, then the-cuiture liquid was au^ended in sterile water to make its CD = 2. One hundred microiiter (100 nL) of the suspension thus obtained was dispensed into a 1.5 oiL microtube, then the yezst cells were dried by evaporation for 1 hour using a reduced-pressure concentrator (DNAllO SpeedVac (registered trademark), manufectured by ThermoSavant).
Viable cdl ratio was measured by a method described below. The dried yeast cells obtained above were resuspended in 50 pL of sterile wata-, then 50 pL of 0.02% methylene blue solution (pH 4.5) was added to the su^ension. Blue-stained yeast ceUs which had lost reducing power were considered as dead yeast cells. Then the suspension was observed under a microscope, and viable cell ratio was measured using a Cell Vital Analyzer System (DA cell countK; manufactured by Yamato Scientific Co., Ltd.). The cells were counted urttil the population reached more than 2000 cells to minimize experimental esmr.
As mdicated in F^e 4, viable cell ratio of the highly-expressed strain was 36.7%, though viable cell ratio of the parent strain was 19.9%. It was demonste-ated by the results that dr3^ng-resistant property of yeast was increased by Mgh expression of non-ScTSLl.
Example 5: Evaluation of Low-temneratttre Resistant Property of non-ScTSLI Highly Expressed Strain

tow-temperature reastant property d the parent strain (AJL4004 strain) and the non-ScTSLI highly expressed strain obtained by the method described in Example 3 are evaluated by the method described bebw. Mne hundred microliter (900 pL) of the yeast si^eoaons cultured by the method descifeed in Example 4 and prq)ared as OD560=2 are dispensed into two microtubes, respecbvely. One hundred nuCToliter (100 pL) of sterile wato" is added to one of the microtubes, on the other hand, 100 |jL of 99.5 % ethanol is added to another one (final concentration is 10%). The suspensions are stored at 5°C for 4 weeks, then viable cell ratios are measured by the same method as Exan^le 4.
INDUSIHLa. APPLICABILITY
According to the present invention, yeast can be storal stably for extended period of time, because drjing-reastant prop&ty ani/or low-temperature storage^resistant property can be eaiiranrad by the present inventioa Accordingly, efficiency of brewing alcoholic beva^es (such as beer), production of bread, or manufecturing usefiil materials such as industrial alcohol production or production of use&il protdns, etc. can be improved by the present inventioa

CLAIMS
1. A polynucleotide selected from the group consisting of
(a) a polynucleotide comprising a polynudeotide consisting of the nucleotide sequence of SEQIDNO:!;
(b) a polynucleotide comprising a polynucleotide acceding a protdn consisting of the amino add sequence of SEQIDNO :2;
(c) a polynucleotide comprising a polynudeotide acceding a protein consisting of the amino acid sequence of SEQ ID N0:2 in vMch one or more amino adds thereof are ddaed, substituted, inserted and/or added, and having a trehalose synthesis-printings activity,
(d) a polynucleotide comprising a pol3'nucleotide encoding a protein an amino add sequraice having 60% or higha- identity with the amino add sequence of SEQ ID N0:2, and said protein havmg a trehalose synthesis-promotmg activity;
(e) a polynucleotide comprising a polynucleotide vAich hybridizes to a polynucleotide consisting of a nudeotide sequence complementary to the nucleotide sequence of SEQ ID NO:l under stringent conditions, and encodes a protean a trehalose synthesis-promoting activity; and
(f) a polynucleotide comprising a polynucleotide which hybridizes to a polynudeotide consisting of a nudeotide sequence complementary Lo the nucleotide sequence of the polynudeotide encoding the protein having the amino acid sequence of SEQ ID N0;2 under strmgent conditions, and which encodes a protein having a trehalose synthesis-promoting activity.
2. The polynudeotide according to Claim 1 selected from the group consisting of
(g) a polynucleotide comprising a polynudeotide encoding a protein consisting of the
amino add sequence of SEQ ID NO; 2, or encoding the amino add sequence of SEQ ID NO: 2 in
which 1 to 10 ammo adds tha-eof are deleted, substituted, inserted, and/or added, and w^ierein said
protein has a trehalose synthesis-promotmg activity;
(h) a polynucleotide con^rising a polynudeotide encoding a protein having 90% or fa^er identity with the ammo add sequence of SEQ ID NO: 2, and having a trehalose syntiiesis-promoting activity, and
(i) a polynucleotide comprising a polynucleotide wimh hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or w4iich hybridizes to a polynucleotide consistii^ of a nudeotide sequence con^lementaiy to the nudeotide sequence of SEQ ID NO: 1, under high stringent conditions, wiiich encodes a protein having a trehalose synthesis-promotmg activity.

3. The polynucleotide according to Claim 1 comprising a polynucleotide consisting of the nucleotide seqitenceof SEQIDNO: 1.
4. The polynucleotide accordh^ to Claim 1 conqmsing a polynucleotide encoding a protein consisting of the amino add sequmce of SEQ ID NO: 2.
5. The polynucleotide according to any one of Claims 1 to 4, wherein the polynucleotide isDNA.
6. A protan encoded by the polynucleotide according to any one of Qaims 1 to 5.
7. A vector containing the pofynucleotide according to any one of Claims 1 to 5.
8. A yeast into which the vectcraccordmg to Claim 7 has beai introduced
9. The yeast according to Claim 8, wdierein drying-resistant property is increased.

10. The yeast according to Claim 8, wiierem low-temperature storage-reastant property is increased.
11. The yeast according to Claim 9, vrfiadn the drying-reastant prc^erty is increased by incre^ng an ^pression level of the protean of Claim 6.
12. The yeast according to Claim 10, ^^te"an the low-teir^erature storagecesistant propea^ is increased by increasing an expression level of the protein of Claim 6.
13. A method for produdng an alcoholic beverage by usiog the yeast acctading to any one of Claims 8 to 12.
14. The method according to Cl^m 13, wheran the brewed alcoholic beverage is a malt bever^e.
15. The method acoordii^to Claim 13, wharon the brewedalcoholicbever^eB wine.

16. An alcoholic beverage produced by the method according to any one of Claims 13 to
15.
17. A method for assessing a test yeast for its diying-resistant property and/or
low-temperature storage-resistant property, comprising using a primer or probe designed based on
the nucleotide sequence of a gene having the nucleotide sequence of SEQ ID NO: 1 afld encoding a
trehalose synthesis-promoting activity.
IS. A method for assessing a test yeast for its drying-resistant property and/or low-temperature storage-resistant property, comprising: cultuiing the test yeast; and measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a trehalose synthesis-promotiDg activity.
19. A method for selecting a yeast, conqirising: cukuiing test yeasts; quantifying the
proton of Claim 6 or measuring the expression level of the gaie having the nucleotide sequence of
SEQ ED NO: 1 and encodii^ a protein having a trehalose synthesis-promoting activily; and selecting
a test yeast having an amount of the protein or the gene expression level accordmg to fevorable drying-resistant properly and/or low-temper^uie storage-re^stant property.
20. The method for selecting a yeast according to Claim 19, comprising: cultuiing a
reference yeast and test yeasts; measurii^ for each yeast the expresaon level of the gene having the
nucleotide secpience of SEQ ID NO: 1 and encoding a trehalose synthesis-promoting activity; and
selecting a test yeast having the gene expression higher than that in the reference yeast.
21, The method for selecting a yeast according to Claim 19, comprisii^: culturing a
refCTence yeast and test yeasts; quantifying the prolan according to Claim 6 in each yeast; and
sdectmg a test yeast having a largo- amount of the protein than that in the reference yeast.
22, A method for producing an alcoholic beverage comprismg: conducting fomentation
ming the yeast accordii^ to any one of Claims 8 to 12 or a yeast selected by the methods according
to any one of Claims 19 to 21.