DESCRIPTION
SULFATE ADENYLTRANSFERASE GENE AND USE THEREOF
TECHNICAL FIELD
The present invention relates to a sulfete adenyltransferase gene and use thereof.in particular, a brewery yeast for producing alcoholic beverages with enhanced flavor stability alcoholic beverages produced with said yeasty and a method for producing said beverages. More particularly the present invention relates to a yeast, whose sulfite-producing capability that contribute to a product's flavor, is adjusted by controlling expression level of MET3 gene encoding brewery yeast sulfete adenyltransferase Met3p, for example the non-ScMET3 gene specific to a lager brewing yeast, and to a method for producing alcoholic beverages with said yeast.
BACKGROUND ART
Sulfite has been known as a compound having high anti-oxidative activity, and thus has been widely used in the fields of food, beverages, pharmaceutical products or the like (for example, . Japanese Patent Application Laid-Open Nos. H06-040907 and 2000-093096). In alcoholic beverages, sulfite has been used as an anti-oxidant. For- example, in yiew of an important role in quality maintenance for wine that needs long time aging, addition of up to 350 ppm (parts per million) of residual concentration is permitted by the Ministry of Health, Welfere and Labor in Japan. Further, it is also known that shelf life (quality maintaihed period) varies depending upon sulfite concentration of a product in beer brewing. Thus, it is quite inportant to increase the content of this conpound from the viewpoint of flavor stability or the like.
The easiest way to increase the sulfite content in a product is to add sulfite. However, sulfite is treated as a food additive, resulting in some problems such as constraint of product development and the food additive related negative image of consumers.
In the meanwhile, yeast produces, by biosynthesis, sulfur containing compounds required for yeast life cycle. Sulfite is produced as an intermediate metabolite. Thus, with use of the capability of yeasts, sulfite content in a product can be increased without.addition of sulfite.
Methods of increasing sulfite content in a fermentation liquor during brewing process
include (1) a method based on process control, and (2) a method based on breeding of yeast. In the
method based on a process control, since the amount of sulfite produced is in inverse proportion to
the amount of initial oxygen supply, amount of oxygen to be supplied can be reduced to increase
amotmt of sulfite produced and to prevent oxidation.
On the other hand, gene manipulation techniques are used in the method based on breeding

of yeast. In sulfur metabolism of yeasts, sulfite is an intermediate produced in biosynthesis of sulfur-containing amino acids or sulfiar-containiEg vitamins. Sulfite is produced by reduction of three step reactions of sulfete ions taken up fi-om outside of cells.
The MET3 gene is a gene encoding an enzyme that catalyzes a first reaction; and the
MET14 gene is a gene encoding an enzyme that catalyzes a second ruction. Korch et al. attempted
to increase a sulfite-produdng capability of yeasts by increasing the expression level of the MET3
gene and the METl^gene, and found that MET14 is more effective (C. Kerch et al.. Proa Eur
Brew. Conv. Conger., Lisbon, 201-208, 1991). The MET 3 gene used was isolated from wine
yeasts. The obtained results indicated about 0.8 ppin which corresponds to about 10%. of sulfite
content in an actual beq: product. Also, Hansen et al. atterqjted to increase production amount of
sulfite by disrupting a METIO gate ^coding a reductasefcr sulfite to prevent reduction of sulfite
produced (X Hansen et aL, Nature Biotech.^ 1587-1591,1996).
Further, Fujimura et aL attenpted to increase sulfite content in beer by increasii^
e?q)ression level of a non-ScSSUl gene unique to a lager brewing yeast among SSUl genes
' encoding sulfite ion efflux punq) of yeast to promote excretion of sulfite to outside the fimgal body
(Fujimura et aL, Abstract of 2003 Annual Conference of the Japin Society for Bioscience,
Biotechnology and Agrochem, 159,2003).
DISCLOSURE OF INVENTION
Nevertheless, new methods and materials are needed for increasing the yeast-produced
• ■ •
amount of sulfite to improve the shelf-life and flavor stability of the alcohohc beverage produced by the yeast.. As mentioned above, the easiest way to increase sulfite content in a product is addition of extraneous- or non-yeast produced sulfite. However, it is desirable to minimize use of food additives in view of recent consumers' preference, Le., avoidance of food additives and use of natural materials. Thus, it is desirable to achieve sulfite content effective for flavor stability without adding sulfite from outside. However^ the method based on a process controLas described above may not be practical^ since shortage of oxygen inay cause decrease an growth rate, resulting in delay in fermentation and qiiality loss.
'^ Further, in. breeding of yeast using gene n^nipulatibn techniques, there is a report stating
« that ten times or more sulfite content was achieved (r|Iansen et al., Nature Biotech., 1587-1591,
1996). However, there ste problems such as delay in fermentation and increase of undesirable
flavor ing'cdients such as acetaldehyde and l-propanol. Thus, the yeast is not good for practical

use. Thus, there has been a need for a method for breeding yeast capable of producing abundant amount of sulfite without impairing the fermentation rates and quality of the products.
The materials and methods disclosed herein solve the above problems, and as a result succeeded in identifying and isolating a gene encoding sulfete adenyltransferase from lager brewing yeast which has advantageous effects than the existing proteins. Moreover, a yeast was transformed by introducing and expressing with the obtained gene to confirm that the amount of sulfite produced was incr^sed, thereby completing the present invention.
Thus, the present invention relates to a novel sulfete adenyltransferase gene existing specifically in a lager brewing yeast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a iriediod for controlling the amount of sulfite in a product by using a yeast in which the expression of said gene is cortfroUed. More specifically, the present invention provides the following polynucleotides, a vector con:q)rising said polynucleotide, a transformed yeast introduced with said vector, a method for producing alcoholic beverages by using said ti'ansfonned yeast, and the like.
(1) A polynucleotide selected from the group consisting of
(a) a polynucleotide con^rising a polynucleotide consisting of the nucleotide sequence of
. SEQE)N0:1;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the
amino acid sequence of SEQ ID N0:2;
(cy a polynucleotide coniprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2 with oiie or more amino acids thereof being deleted, substituted, inserted and/or added, and hkving a sulfete adenyltransferase activity;
.(d) a polynucleotide, corcprising 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 sulfete adenyltransferase activity;
(e) a polynucleotide. comprising a polynucleotide which hybridiz.es to a polynucleotide consisting of a nucleotide sequence complementary to'the nucleotide sequence of SEQ ID N0:1 under stringent conditions, and which encodes a protein having a sulfate adenyltransferase activity; and
' (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence corr5)lementary 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 sulfate adenyltransferase activity.
(2) The polynucleotide 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 aKX)ding an amino acid sequence of SEQ ID NO: 2 wherein 1 to 10 ammo acids thereof is deleted, substituted, inserted, and/or added, and wherein said protein has a sulfete adenyltransferase activity;
(h) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ED NO: 2, and having a sulfete adenyltransferase activity; and
V. (i) a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ED NO: 1 uiider stringent conditions, and which encodes a protein having a sulfete adenyltransferase activity.
(3) The polynucleotide of (1) above con5>rising a polynucleotide consisting of SEQ ID NO: 1.
(4) The polynucleotide of (1) above conprising a polynucleotide encoding a protein consisting of SEQ E) 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 conplementary to a transcript 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 through RNAi effect;
(I) a polynucleotide encoding RNA having an activity of specifically cleaving a transcrpt
of the polynucleotide (DNA) according to (5) above; and
(m) a polyimcleotide encoding RNA that represses e3q)ression of the polynucleotide (DNA) according to (5) above through co-supression effect.
(7) A protein encoded by the polynucleotide of any one of (1) to (5) above.
(8) A vector conoprising the polynucleotide of any one of (1) to (5) above.
r
(8a) The vector of (8) above, < which con:5)rises the expression cassette comprising the • following con5)onents:
(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 or antisense direction; and*
(z) a signal that can ftinction in a yeast with respect to transcription teraiination and polyadenylation of a RNA molecule.
(9) A vector conqjrising the polynucleotide of (6) above.
^ (10) A yeast, wherein the vector of (8) or (9) abdrve is introduced
(II) The yeast'of'(lO) above, whaein sulfite producing ability is enhanced by introducing
the vector .of (8) above.

(12) A yeastj wherein an e?q)ression of the polynucleotide (DNA) of (5) above is repressed by introducing the vector of (9) above, or by disrupting a gene related to the polynucleotide (DNA) of (5) above.
(13) The yeast of (10) above, wherein a sulfite-producing ability is elevated by increasing an expression level of the protein of (7) above.
(14) A method for producing an alcoholic liquor by using the yeast of any one of (10) through (13) above.
(15) The method for producing an alcoholic Uquor of (14) above, wherein the brew is a malt liquor.
(16) The method for producing an alcoholic liquor of (14) above, wherein the brew is a wine.
(17) An alcoholic Uquor, which is produced by the method of anyone of (14) through (16) above.
(18) A method for assessing a test yeast for its sulfite-producing ability, comprising using a primer or a probe designed based on a nucleotide sequence of a sul&te adenyltransferase gene having the nucleotide sequence of SEQ E) NO: 1.
(18a) A method for selecting a yeast having a high or low sulfite-producing ability by using the method in (18) above.
(18b) A method for producing an alcohohc Uquor (for exan5)le. beer) by using the yeast selected with the method in (18a) above.
(19) A method for assessing a tost yeast'for its sulfite-producing capabiUty, comprising:
culturing a test yeast; and measuring an' expression level of a sulfite adenyltransferase gene having
the nucleotide sequence of SEQ ID NO: 1.
(19a) A method for selecting a yeast having a high sulfite-producing abiUty, which comprises assessing a test yeast by the method described 'in (19) above and selecting a yeast having a high expression level of sulfite adenyWansferase gene.
•(19b) A method for producing an alcohoUc Uquor (for example, beer) by using the yeast selected with the method in (19a) above.
(20) A method for selecting a yeast, comprising: culturing .test yeasts; quantifying the
protein of (7) above of measuring an expression level of a sulfate adenyltransferase gene having the
nucleotide sequence of SEQ ID NO: 1; and selecting a test yeast having said protein amount or said
gene ejq^ression level according to a target capabiUty of producing sulfite.
, (21) The method for selecting a yeast of (20) ab^ve, conprising: culturing a reference yeast and test yeasts; measuring an expression level of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast; and selectiag a test yeast having the gene

expressed higher or lower than that in the reference yeast.
(22) The method for selecting a yeast of (20) above conprising: culturing a reference yeast and test yeasts; quantifying the protein of (7) above in each yeast; and selecting a test yeast having said protein for a larger or smalla* amoimt than that in the reference yeast.
(23) A method for producing an alcoholic beverage conpising: conducting fermentation for producing an alcoholic beverage using the yeast according to any one of (lO).to (13) or a yeast selected by the method according to any one of (20) to (22); and adjusting the production amount of sulfite.
According to the method for producing alcoholic beverages by using a yeast transformed with a sulfete adenyltransferase polynucleotide operably linked to a vector, the content of sulfite having an anti-oxidativ^ activity in a product can be increased so that alcoholic beverages can be produced with enhanced flavor and inproved shelf life.
BRIEF DESCRIFnON OF DRAWINGS
Figure 1 shows the cell growth with time upon beer brewing testing. The horizontal axis rqwresents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 2 shows the sugar consunption with time upon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 3 shows the expression behavior of non-ScMET3 gene in yeasts ipon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents the brightness of detected signal
Figure 4 shows the cell growth with time upon brewing testing using non-ScMET3-highly e>p-essed strains. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 5 shows the sugar consunqrtion with time upon beer brewing testing using non-ScMET3"hi^y e?5}ressed strains. The horizontal'axis represents fermentation time while the vertical axis represents ^jparent extract concentration (w/w%)-
Figure 6 shows the ^sulfite concentmtion in finished beer /using non-ScMET3-highly e?q)ressed strains.
BEST^ODES FOR CARRYING OUT THE INVENTION
, Iri the known method of increasing expression^level of a sulfite ion efQux pun^, suitable femientation rate can be maintained since superfluous sulfite is not accumulated in a fimgal body. However,.there is a possibility ttiat biosynthetic reaction of sulftirous add in the ftingal body can be a

limiting fector. Thus, disclosed herein are materials and methods that enhance sulfite production by enhancing reduction pathway from sulfete ion which is a staring material to sulfiirous acid.
The present inventors have studied based on this conception and as a result, isolated and identified non-ScMET3 gene encoding a sulfete adenyltransferase unique to lager brewing yeast based on the lager brewing yeast genome information mapped according to the method disclosed in Japanese Patent AppHcation Laid-OpenNo. 2004-283169. The nucleotide sequence of the gene is rq)resented 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 Polynucleotide of the invention
First of alls the.present invention provides (a) a polynucleotide comprising a polynucleotide of the nucleotide sequence of SEQ ID N0:1; and (b) a polynucleotide comprising a poljoaucleotide encoding a protein of the amino acid sequence of SEQ ID N0:2. The polynucleotide can be DNA orRNA.
The target polynucleotide of the present invention is not limited to the polynucleotide encoding a sulfete adenyltransferase gene derived from lager brewing yeast and may include othei' - polynucleotides encoding proteins having equivalent functions to said protein. Proteins with equivalent fimctions include, for example, (c) a protein of an amino acid ^sequence of SEQ ID NO: 2 with one or more amino acids thereof being deleted, substituted, inserted and/or added and having sulfate adenyltransferase activity.
Such proteins include a protein consisting of ah amino acid sequence of SEQ ED NO: 2 with, for exan^jle, 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, l.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 to 22, 1 to 21,1 to 20,1 to 19,1 to 18,1 to 17,1 to 16,1 to 15,1 to 14,1 to 13,1 to 12, Ito 11, Ito 10, lto9, lto8, lto7, lto6(ltoseveraUminoacids), 1 to 5,1 to 4,1 to 3,1 to 2, or 1 amino acid residues thereof being deleted, substituted, inserted and/or added and having a sul&te adenyltransferase 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% br 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% br higher, 84% or hi^er, 85% or higher, 86% orhigher, 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 liigher, '96^o or higher, 97% or higher, 98% or higher, 99% or higher, 99.1%. ot'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 sulfete adenyitransferase activity. In genial, the percentage identity is preferably higher.
Sulfete adenyitransferase activity may be measured, for exan:ple, by a method of Klonus et al. as described mPlantJ, 6(1): 105-12,1994 Jul.
Furthmnore, the present invention also contenplates (e) a polynucleotide corcprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence conplem©atary to the nucleotide sequence of SEQ ID NO: 1 undo- stringent conditions and which encodes a protein having sulfete adenyitransferase activity; and (f) a poljmucleotide con:q)rising a polynucleotide which hybridizes to a polynucleotide conplementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 under stringmt conditions, and which encodes a protein having sulfete adenyitransferase 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 con5}lementary to the nucleotide sequaice of SEQ ID NO: 1 or DNA encoding the amino acid sequence of SEQ ID NO: 2 as a probe. The hybridization method may be a method - described, for exanqjle, in MOLECULAR.CLDNING 3rd Ed, CURRENT PROTOCOLS m MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997.
The term "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for exan:5)le, 5 x SSQ 5 x Denhardt's solution, 0.5% SDS, 50% formainide at 32°C. "Moderate stringency conditions" are, for exan5)le, 5 x SSC, 5 x Denhardt*s solution, 0.5% SDS, 50% formamide at 42°C. "High.stringency conditions" are, for exan[q>le, 5 x SSQ 5 x Denhardfs solutipn,.0.5% SDS, 50% formamide at 50°C. Undo: these conditions, a polynucleotide, such as a DNA, with higher homology is expected to be obtained' efficiently at higjier temperature, although multiple fectors are involved in hybridisation stringency including ierrq)©rature, probe concentration, probe length, ionic strength, time, salt concentration and others, and one skilled in the art may appropriately select these fectors to realize similar stringency.
When a commercially*available kit is used for hybridization, for exan5)le, Alkphos Direct Labeling Reagents (Ainersham Pharmacia) may be used In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (w/v) SDS at 55^0, thareby.detecting hybridized 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 highesr, 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 to DNA encoding the amino acid sequence of SEQ HD NO: 2 as calculated by homology search software, such as FASTA and BLAST using defeult parameters.
Identity between amino acid sequences or nucleotide sequences may be determined using algorithm BLAST by Karlin and Altschul {Proa Natl Acad. Set USA, 87: 2264-2268, 1990; Proc. Natl. Acad. Sci WX4, 90: 5873, 1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et aL, J. Mol Biol 215: 403, 1990). When a nucleotide sequence is sequenced using BLASTN, the parameters are, for example, score = 100 and word length = 12. When an amino acid sequence is sequenced using BLASTX, the parameters are, for exQxrsph, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, defeult parameters for each of the programs are en^jloyed.
The polynucleotide of the present invention includes 0 a polynucleotide encoding RNA - having a nucleotide sequence coniq^lementary to a transcript 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 through RNAi effect; (I) 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-suprisssion 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 expression of the above DNA is preferable.
The phrase "polynncleotide 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 repressing e?q)ression of a particular endogenous gene, and is described in various pubHcations (see e.g., Hirajima and Inoue: New Biochemistry Experiment Course 2 Nucleic Acids IV Gene Replication and E^^ression (Japanese Biochemical Society Ed., Tokyo Kagaku Dozin Co., Ltd.) pp.319-347,1993). The sequence of antifense DNA is preferably complementary
I
to all or part of the -endogenous gene, but may not be completely cornplementary as long as it can effectively repress the expression of the gene. The transcribed RNA has preferably 90% or higher, and more preferably 95% or higher complementarity to the transcript of the target gene. The length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably

500 bases or more.
The phrase "polynucleotide eacoding RNA that represses DNA expre^ion through KNAi
effect'- as used herein refers to a polynucleotide for repressing e?q)ression of an endogenous gene
through RNA interference (RNAi). The term "RNAi*' refers to a phenomenon where when
double-stranded RNA having a sequence identical or similar to tiie target gene sequence is
introduced into a cell, the expressions of both the introduced foreign gene and the target endogenous
gene are rqoressed, RNA as used herein includes, for exarr^le, double-stranded RNA that causes
RNA interference of 21 to 25 base length, for exanple, dsRNA (double strand RNA), siRNA (small
interfering RNA) or shRNA (short hairpin RNA). Such RNA may be locally delivered to a desired
site with a delivery system such as Iposoine, or a ved:or that generates the double-stranded RNA
described above may be used for bcal ejqjtession thereof. Methods for producing or using such
double-stranded RNA (dsRNA, siRNA or shKNA) are known from many publications (see, e.g.,
J^anese 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 April; Nature, 407:6802,
319-20, 2002 Sep. 21; Genes & Dev., Vol.16, (8), 948-958, 2002 Apr.l5; Proc. Natl. Acad. Sci.
USA, 99(8), 5515-5520,2002 Apr. 16; Science, 296(5567), 550-553,2002 Apr. 19; Proc Natl. Acad.
. Sci USA, 99:9, 6047-6052, 2002 Apr. 30; Nature Biotechnology, Vol.20 (5), 497-500, 2002 May;
Nature Biotechnology, Vol. 20(5), 500-505, 2002 May; Nucleic Acids Res., 30:10, e46,2002 May
15).
The phrase "polynucleotide encoding RNA having an activity of specifically cleaving
transcript of DNA" as used herein gena-ally refers *to a ribozyme. Ribozynae is an RNA molecule
with a catalytic activity that cleaves a transcript of a target DNA and inhibits tiie fimction of that gene.
Design of ribozynaes can be found in various known pubUcations (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 Eng3:733,1990; NucL Acids Res. 19: 3875,1991; NucL Acids
Res. 19: 5125, 1991; Biochem Biophys Res Cbnmiun 186: 1271,'1992). In addition, the phrase
"polynucleotide encoding RNA that represses DNA expression through Co-siq)ression effect" refers
to a nucleotide that inhibits ftmctions of target DNA by "co-supression".
The term "co-sip'ession" 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 th^ target endogenous gene are repressed.
Design of polynucleotides having a co-supression effect can also be'found in various publications
- (see, e.g.,.Smydi DR: Cun\ BioL 7: R793,1997, Martied^sen R: Curr. BioL 6: 810,1996).
2. Protein of Ifae present inventjon

The present invention also provides proteins encoded by any of the polynucleotides (a) to (f) above. A preferred protein of the present invention comprises an anaino acid sequence of SEQ ID N0:2 with one or several amino acids thereof being deleted, substituted, inserted and/or adde4 and has sulfete adenyltransferase activity.
Such protein includes those having an amino acid sequence of SEQ E) NO: 2 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a sulfate adenylti-ansferase activity, hi addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID NO: 2 and having sul&te adenyltransferase activity.
Such proteins may be obtained by employing site-directed mutation described, for example, in MOLECULAR CLONING 3rd Ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Nua Acids. Res.,
10: 6487 (1982),Proa Natl. Acad Sci USA 79: 6409 (1982), Gene34: 315 (19^5%Nuc. 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 that one or more amino acid residues are
deleted, substituted, inserted and/or added at any one or more positions in the same amino acid
- sequence. Two or more types of deletion, substitution, insertion and/or addition may occur
concurrently.
Hereinafter, exan:^les of mutually substitutable amino acid residues are enumerated. Amino acid Residues in the same group are mutually substitutable. The groups are provided below.
Group A: leucine, isoleucine, norleucinej valine, norvahne, alanine, 2-aininobutanoic acid, methionine, o-mdhylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: asparatic acid, glutamic acid, isoaspar^tic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyprohne; 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, pq)tide synthesizers available from, for exan^le, Advanced ChemTech, PerkinElmer, Pharmacia, ■ Protein Technology Instrument, Synthecell-Vega, Pa*Septive, Shimazu Corp. can also be used for chemical synthesis.
■" 3. Vector of the inveption and yeast trapsformed withvthe 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 conprises an expression cassette including as conponmts (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 promDter in sense or antisense direction; and (z) a signal that functions in the yeast with respect to transcr5)tion termination and polyadenylation of RNA molecule. According to the present invention, in order to highly express the protein of the invention described abov6 upon brewing alcohohc bev^ages (e.g., beer) described below, these polynucleotides are introduced into the promoter in the sense direction to promote expression of the polynucleotide (DNA) described in any of (a) to (i) above. In orda: to rq>ress the above protein of the invention, the polynucleotide may be introduced such that the polynucleotide of any of the (j) to (m) is expressed According to the present invention, the target- gene (DNA) may be disn^ted to repress the e?q>ression of the DNA or the protein. A gene may be disrupted by adding or deleting one or more bases to or fix)m a region involved in expression of the gene product in the target gene, for example, a coding region or a promoter region, or by deleting these regions entirely. Such disnqjtion of gene naay be found in known publications (see, e.g., Proc. Natl. Acad. Sci. USA, 76, 4951(1979) , Methods in Enzymology, 101, 202(1983), J^anese Patent Application Laid-Open . No.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 example, YEp24 (I R. Broach et al., E?(pmiMENrrAL MANIPULATION OF GENE EXPRESSION, Academic Press, New York, 83,1983) is known as a YEp type vector, YCp50 (M. D. Rose et aL, Gem 60: 237, 1987) is known as a YCp type vector, and YIp5 (KL Stnihl et al., Pwa Natl. Acad. Sd. USA, 76: 1035; 1979) is known as a Yip type vector, all of v^hich are readily available.
. Promotors/temiinators for adjusting gene expression in yeast may be in any combination as long as they function in the brewery yeast and they have'no influence, on the concentration of amino acid, sugar, higher alcohol or ester in fermentation broth ' tor exan:5)le, a promoter of glyceraldehydes 3-phosphate dehydrograase gene (TDH3), or a promoter of 3-phosphoglycerate kinase gene (PGKl) may be used These genes have previously been cloned, desaibed in detail, for exa33:5)le, in M. F. Tuite e^al., EMBO J., 1, 603 (1982), and are .-readily .available by known methods.
Since an auxotrophy marker cannot be used as a s^ective marker upon transformation for a hrew^ yeast, for •exan5)le, a geneticin-resistant gone. (G418r), a cSopper-resistant gene (CUPl) (Marin et al, Proc. Natl Acad. ScL USA, 81,337 1984)'^r a CQiilenin-resistant gene (fas2m, PDR4) (Junji Inokoshi et al., Bihchemistry, 64, 660, 1992; and Hussain et al., Gene, 101: 149, 1991, respectively) may be used.

A vector constructed as described above is introduced into a host yeast. Examples of the host yeast include any yeast that can be used for brewing, for exair^le, brewery yeasts for beer, wine and sake. Specifically, yeasts such as. genus Saccharotnyces may be used According to the present invention, a lager brewing' yeast, for example, Saccharomyces pastonanus W34/70, Saccharomyces carlsbet^gmsis NCYC453 or NCYC456j or Saccharomyces cerevisiae NBRC1951, NBRG1952, NBRC1953 or >ffiRC1954 may be used In addition, whisky yeasts such as Saccharomyces cere\nsiae NCYC90, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japan, and sake yeasts such as sake yeast #7 and 9 from the Brewing Society of Japan may also be used but not limited thereto. In the present invention, lager brewing yeasts such as Saccharomyces pastonanus may be used preferably.
A yeast transformation method may be a generally used known method. For Qxsraph, methods that can be used include but not bmited to an electroporation method {Metk EyTzynu, 194: . 182 (1990)), aspheroplast metiiod (Proa Natl Acad Set USA, 75: 1929(1978)), a hthium acetate method (J. Bacteriology^ 153: 163 (1983)), and methods described inProc. Natl Acad. Sci USA, 75: 1929 (1978), METHODS IN YEAST GRNIETICS, 2000' Edition: A Cold Spring Hai-bor Laboratory Course Manual
More specifically, a host yeast is cultured in a standard yeast nutrition medium (e.g., YEPD medium (Genetic Engineering. Vol. 1, Plenum Press, New York, 117(1979)), etc.) such that OD600 nm will be 1 to 6. This culture yeast is collected by centrifugatiqn, washed and pre-treated with
Ok
alkah ion mdtal ion, preferably lithium ion at a. concentration of about 1 to 2 M. Aft^- 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
r
20 ^g) 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 niitrition medium, added to a predetermined amount of fresh standard yeast nutrition medium and left to stand at aix)ut 30°C for about 60 minutes. Thereafter, it is seeded to a standard agar medium containing an antibiotic or the like as a selective marker to obtain a transformant.
Other general cloning techniques may be found, for example, ki'MOLECULAR CLONING 3rd Ed, and METHODS IN YEAST GENETICS, A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
"' 4. Method of producing alcoholic beverages accordipg to the present iavention and alcoholic beverages produced bv t^e 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 bevferage with enhanced flavor with an increased content of sulfite. In addition, yeasts to be selected by the yeast assessraent method of the present invention can also be used. The target alcoholic beverages include, for cxanaph, but not limited to beer, wine, whisky, sake and the like.
In order to produce these alcoholic beverages, a known technique can be used except that a breway yeast obtained according to the present invention is used in the place of a parent strain. Since materials, manufecttaring equipment, manufecturing control and the like may be exactly the same as the conventional ones, liiere is no need of increasing the cost for podiicing alcoholic bevCTages with an increased content of sulfite. Thus, according to the present invention, alcoholic beverages with enh^iced flavor can be produced using the existing fecility without increasing the cost.
Further, since in a yeast A^erein said gene is highly expressed, a su^hate ion in the culture
medium is efBciently incorporated, well growth of yeast and/or alcohohc feamentation may be possible when a raw naaterial containing low sulfur source, e.g., a wort having low malt ratio in the case of beer.
Alternatively, in a yeast wherein the function of synthetic s>^em for suUur-containing amino acid is too active, sulfiir-containing conpounds including, hydrogen sulfide as an intermediate-naetabolite in the pathway, whici\ cause undesirable ojQf-flavor for alcoholic beverages, " are sometimes generated in large amounts and accumulated. By si5)pressing or disrupting said gene function of such yeast, incorporation of sulphate ion as a starting n^atmal may be suppressed. As a result, an alcoholic beverage v^iierein the oflF-flavor is reduced, can be produced.
5> Yeast assessment method of the invention
The present invention relates to a method for assessing a test yeast for its sulfite-producir^ capabiUty by using a primer or a probe designed based on a nucleotide sequence of a sulfite adenyltransfemse gene having the nucleotide sequence of SEQ ID N0;1. General techniques for such assessment method is khown and is described in, for exanple, 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 beVused (e.g., METHODS IN YEAST GENETICS, Cold Spring Harbor Laboratory Press, 130 (1990)). Using a primer or aprobe designed based on a

/
nucleotide sequence (preferably, ORF sequence) of the sulfate adenyltransferase gene, the existence of the gene or a sequence specific to the gene is determined in the test yeast genome obtained. The primer or the probe may be designed according to a known technique.
Detectbn of the gene or the specific sequence may be carried out by employing a known technique. For exanple, a polynucleotide including part or all of the specific sequence or a polynucleotide including a nucleotide sequmce conplementary to said nucleotide sequence is used as one primer, while a polynucleotide including part or all of the sequence upstream or downstream from this sequence or a polynucleotide including a nucleotide sequence con:plementary to said nucleotide sequence, is used as another primer to amplify a nucleic acid of the yeast by a PCR method, thereby determining the existence of amplified products and molecular weight of the an:5)lified products. The number of bases of polynucleotide used for a primer is generally 10 base pairs (bp) or more, and preferably 15 to 25 bp. In general^ the number of bases between die primers is suitably 300 to 2000 bp.
The reaction conditions, for PCR are not particularly liinited but may be, for exanple, a
, denaturation tenq^erature of 90 to 95^C, an annealing temperature of 40 to 60°Q an elongation temperature of 60 to 75°C, and the number of cycle of 10 or more. The resulting reaction product
. may be separated, for exan^le, by electi'ophoresis using agarose gel to determine the moleculai* weight of the amplified product. This method allows prediction and assessment of the capability of the yeast to produce sulfite as determined by whether the molecular weight of the an:5)lified product is a size that contains the DNA molecule of the specific part. In addition, by analyzing the nucleotide sequence of the amplified product, the capability may be predicted and/or assessed more precisely.
Moreover, in the present invention, a test yeast is cultured to measure an expression level of the sulfete adenyltransferase gene having the nucleotide sequence of SEQ E) NO: 1 to assess the test yeast for its sulfite-producing capability., In this case^ the test yeast is cultured and then mRNA or a protein resulting firom the sulfate adenyltransferase gene is ciiiantified. The quantification of mRNA or protein may be carried out by employing a krfown technique. For example, mRNA may be quantified, by Northern hybridization or quantitative RT-PCR, while protein may be quantified, for example, by Western blotting (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1994-2003).
Furthermore, test yeasts are cultured and expression levels of the sulfate adenyltransferase gene Imving the nucleotide sequence of SEQ ID NO:. 1 are measured'to select a test j^east with the
' gene expression level according to the target capability of producing sulfite, thereby selecting a yeast fevdrable for brewing desired alcoholic beverages. In addition, a reference yeast and a test yeast may be cultured so as to measure and compare the expression level of the gene in each of the yeasts.

thereby selecting a fevorable test yeast. More specifically, for ^xsmaple, a referoice yeast and one or more test yeasts' are cultured and an e^qwession level of the sulfete adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured in each yeast. By selecting a test yeast with the gene expressed highea: than that in the reference yeast, a yeast suitable for brewing alcoholic beverages can be selected.
^- Alternatively, test yeasts are cultured and a yeast with a higher sulfite-producing capability or a higher sulfete adenyltransferase activity is selected, thereby selecting a yeast suitable for brewing desired alcoholic beverages.
In these cases, the test yeasts or the reference yeast may be, for cxaraph, a yeast introduced with the vector of the invention, a yeast in which an repression of the polynucleotide (DNA) of the present invention is enhanced or rq)ressed, an artificially mutated, yeast oi* a naturally mutated yeast. Sulfete adenyltransferase activity may be measured, for exanple, by a method of Klonus et aL as described in Plant J. 6(1): 105-12, 1994 Jul. The mutation treatment niay enqjloy any methods including, for exanple, physical methods such as ultraviolet irradiation and radiation irradiation, and chemical methods associated with treatments with drugs such as EMS (ethyhnethane sulphonate) and N-methyl-N-nitrosoguanidine {see, e.g., Yasuji Oshima Ed, BIOCHEMISTRY EXPHUMENTS voL - 39, Yeast Molecular Genetic E:xperiments^ pp. 67-75, JSSP).
In addition, examples of yeasts used as the reference yeast or the test yeasts include any yeasts that can be used for brewing, for cxaxraple, brewery yeasts for beer, wine, sake and the like. More specifically, yeasts such as genus Saccharomyces may be used (e.g., 51 pastorianus, S cerevisiae, and S, carlsbergensis). According to the present invention, a lager brewing yeast, for exan5)le, Saccharomyces pastorianus W34/70; SaccHaromyces carlsbergensis NCYC453 or NCYC456; or Saccharomyces cerei^iszfleNBRC1951, NBRC1952,NBRC1953 orNBRC1954 may be used.. Further, wine yeasts such as wine yeasts #1, 3 and 4 fi^m the Brewing Society of Japan; and sake yeasts such as sake yeast #7 and 9 fiom the Brewing Society of Japan may also be used but not limited thereto. In the present invention, lager brewing'yeasts such as SaccJmromyces pastorianus may preferably be used. The reference ydhst and the test yeasts may be selected fi^om the above yeasts in any combination.
EXAMPLES
Hereinafter, the present invention will be desctibed in more detail with reference to working examples. The present invention, howev^, is not limited to the examples described below. ^
Example 1: rinninyr nf Sulfate Adenyltransferase (non-ScMET3) Gene

A.specific novel sulfate adenyltransferase gene (non-ScMET3) gene (SEQ ID NO: 1) from a lager brewing yeast were found, as a result of a search utilizing the comparison database described in Japanese Patent Application Laid-Open No. 2004-283169. Based on the acquired nucleotide sequence information, primers non-ScMET3__for (SEQ ID NO: 3) and non-ScMET3_rv (SEQ ID NO: 4) were designed to amplify the full-length genes, respectively. PCR was carried out using chromosomal DNA of a genome sequencing strain, Saccharomyces pastoiianus Weihenstephan 34/70 strain, as atemplateto obtainDNA fragments including the full-length gene of non-ScMET3.
The thus-obtained non-ScMET3 gene fragment was inserted into pCR2.1-TOPO vector (Invitrogen) by TA cloning. The nucleotide sequences of non-ScMET3 gene were analyzed according to Sanger's method (F. Sanger, Science, 214: 1215, 1981) to confirm the nucleotide sequence.
Example 2: Analysis of Expression of iion-ScMET3 Gene during Beer Brewing Testing
A beer brewing testing was conducted using a lager brewing yeast, Saccharom)>ces
pastoiianus Weihenstephan 34/70 strain and then mRNA extracted ^om a beer yeast fiingal body during fermentation was detected by a DNA microarray.
Wort extract concentration 12.69%
Wort content 70 L
Wort dissolved oxygen concentration *' 8.6 ppm
Fermentation tenperature 15°C
Yeast input 12.8x 10^ cells/mL
Sampling of fermentation liquor was perfonned with time, and variation with time of j^east growth amouil (Fig. l) and apparentextract concentration (Fig. 2) was observed. Simultaneously, sampling of a yeast fiingal body was performed, and the prepared mRNA was subjected to be biotin-labeled and was hybridized to a beer yeast DNA microarray as described in Japanese Patent Application Laid-Open No. 2004-283169. The signal was detected using GCOS; GeneChip Operating Software 1.0 (manufectured by AfiEymetrix Co). Expression pattan of non-SclV[ET3 gene is shown in Figure 3. As a result, it was confirmed that non-ScMET3 gene was expressed in the general beer fermentation.
', ' ■ '
Example 3: Constitutive Expression of noD-ScMET3 Genes
The non-Sc]S'lET3/pCR2.1-TOPO described in Example 1 was digested with restriction

enzymes Sad and NotI to prepare a DNA fragment including non-ScMET3 gene. This fragtnent was linked to plJP3GLP2 treated with restriction enzymes Sad aiid NotI, thei-eby constructing a non-ScMET3 constitutive expression vector, pL)P-nonScMET3. The yeast expression vector, plJP3GLP25 is a Yip type (chromosome integration type) yeast expression vector liaving orotidine-5-phosphoric acid decarboxylase gene LIRA3 at the homologous recombinant site. The introduced gene was constitutively expressed by the promoter and terminator of glycerylaldehyde-3-phosphoric acid dehydrogenase gene, TDH3. Drug-resistant gene YAPl as a selective mai'ker for yeast was introduced undo- the control of the promotei* and tenninator of galactokinase GALl, whereby the expression is induced in a culture media conprising galactose. Annpicillin-resistant gene Amp"^ as a selective marker for £, coli was also included.
The constitutive expression vector prq)ared by the method above was used to transform Saccharomyces pastorianus Weihenstephan 34/70 strain according to the metliod described in Japanese Patent Application Laid-Open No. 07-303475. Right assessment on the non-ScMET3 gene cannot be conducted if sulfite is accumulated within the fimgal body since the yeast itself is damaged by sulfite. Thus, first, a strain in which non-ScSSUl gene encoding a sulfite efflux pump
k
is highly expressed, was prepared according to the method described in Japanese Patent Application -Laid-open No. 2004-283169. Then, .the obtained strain was used as a parent strain and transfomied by pLiP-non-ScMET3. Cemlenin-resistant strains were selected in a YPGal plate medium (1% yeast extract, 2% polypeptone, 2% galactose, 2% agar) containing 1.0 mg/L cerulenin.
Example 4: Analysis of Amount of Sulfite Produced during Beer Brewing Testing
The pai-ent strain, and non-ScMET3-highly expressed strains (two strains) obtained in Example 3, were used to cany, out beer brewing testing under the following conditions.
Wort extract concenti'ation 11.96% ?
Wort content 1 L
Wort dissolved oxygen concentration about 10 ppm
Fermentation tenrperature 15^C constantly
Yeast input 6 g of wet yeast cells/L of wort
The fermentation brotli was sampled with time to observe the cell growth (OD660) (Fig. 4)
and sugar consumption with time (Fig. 5). Quantification of the sulfite content upon completion of
feraientation was carried out by coUecting sulfite in hydrogen pei"Oxide solution by distillation undei'
acidic condition, and titration with alkali (Revised BCOJ Beer Analysis Method by the Brewing
' Society of Japan).
With respect to the amount of sulfite produced upon conpletion of fermentation, wliile the

pa^ent strain produced 25.4 ppnx the non-ScMET3-high]y expressed strains produced 32.3 ppni and 33.8 ppm, respectively (Fig. 6). Thus, it was found that about 30% of the amount of sulfite produced can be increased by high expression of the non-ScMET3. In these cases, differences in the growth rates and the extract consumption rates were little between the pai'ent sti'ain and the constitutively expressed strains.
As can be appreciated from the above results, by constitutively expressing sulfate adenyltransferase unit[ue to a lager brewing yeast as described herein in the yeast with enlianced sulfite-producing capability, it became possible to specifically increase production amount of sulfite fanctioning as anti-oxidant for alcoholic beverages such as beer without altering the fermentation procedure or time. Thus, alcoholic beverages with enhanced flavor and long shelf life (with good quaUty), can be produced.
Example 5: Beer Brewing Testing using Wort Containing Low Sulftir Source
Saccbaromyces pastorianus Weihenstephan 34/70 strain is transfonned with the liigh expression vector prepared in Example 3 to obtain Sc and n6n-ScMET3 (sole) highly expressed strains, respectively. Then, a wort containing 24% of malt ratio is prepared as a wort containing low sulfur source. Subsequently, using parent and the highly expressed strains obtained, under the following conditions beer brewing testing is carried out.
Wort extract concentration 13%
Wort content 2L
Wort dissolved oxygen concentration about 8 ppm
Fennentation temperature 15°C constantly
Yeast input 10.5 g of wet j^east cells/2 L of wort
The fermentation broth is sampled with time tp observe the cell growth (OD660) and the sugai' consumption with time.
Example 6: Disruption of MET3 Gene
According to the publication (Goldstein et al, yeast. 15 1541 (1999)), PCR using a plasmid including a-drug-resistant marker (pFA6a {G41S) or pAG25 (natl)) as a template is conducted to prepai'e a fragment for KdETS gene dismption.
^ With the fragment for gene disruption prepared, W34/70 ?train or spore cloning sti'ain (W34/70-2) is transfomied. The transfonnation is pprfonned in accordance with the method
described in Japanese Patent Application Laid-Open No. H07-303475. The concentrations of the
♦
drugs for selection ai*e 300 mg/L for geneticin and 50 mg/L of noui'seothricin, respectively.

Example 7; Analysis of Amounts of Sulfur-Containing Compound Produced upon Beer Brewing Testing
Using parent strain and the gene-disrupted strain obtained in Example 6, under the following conditions, beer brewing testing is canied out.
Wort extract concentration 13%
Wort content 2L
Wort dissolved oxygen concentration about 8 ppm
Fermentation temperature 15°C constantly
Yeast input 10.5 g of wet yeast ceUs/2 L of wort
The fermentation broth is sampled with time to observe the cell growth (OD660) and the sugar consunption with time. Analysis of sulftir-containing confounds in broth is performed by enploying head-space gas chromatography.
Industrial Applicability
According to the method for producing alcoholic beverages of the present invaition,
' because of increase in content of sailfite having anti-oxidative action in a product, alcoholic
beverages with enhanced flavor and long shelf life (with good quality), can be produced Also,
since the yeast of the present invention can efficiently reduce a sulphate ion as a sulfur source to
synthesize a sulfur-containing compound necessary for growth, desirable alcoholic fermentation can
be performed by using raw mataials with low contents of sulfur-containing amino acid, e.g.,
sparkling liquor (happoushu) worL. Moreover, by suppressing an expression of said gene in yeast
wherein sulfur-containing compounds as an off-flavor are highly generated, an alcoholic beverage
having desirable flavor can be produced.
i' ,
This application claims benefit of Japanese Patent Application Nos. 2005-235011 filed
August 12,2005 and 2006-84289 filed March 23, 2006, which are herein incorporated by reference
in their entirety for all purposes. All other references cited above are also incorporated herein in
their entirety for all purposes.

CLAIMS
1. A polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID N0:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a sulfate adenyltransferase activity;
(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO;2, and having a sulfate adenyltransferase activity;
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence con^lementary to the nucleotide sequence of SEQ ID N0:1 under stringent conditions, and which encodes a protein having a sul&te adenyltransferase activity;
- and
(f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide
consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide
encoding the protein of the amino acid sequence of SEQ ID N0:2 under stringent conditions, and
which encodes a protein having a sulfate adenyltransferase activity.
2. The polynucleotide 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 acids thereof is
deleted, substituted, inserted, and/or added, and wherein said protein has sulfate adenyltransferase
activity;
(h) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having sulfate adenyltransferase activity; 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 stringent conditions, and which encodes a protein having sulfate adenyltransferase activity.
3. The polynucleotide of Claim 1 comprising a polynucleotide consisting of SEQ ID NO:

4. The polynucleotide of Claim 1 comprising a polynucleotide encoding a protein consisting of SEQ ID NO; 2.
5. The polynucleotide of any one of Claims 1 to 4, wherein the polynucleotide is DNA.
6. A polynucleotide selected from the group consisting of
(j) a polynucleotide encoding RNA of a nucleotide sequence corrplementary to a transcript of the polynucleotide (DNA) according to Claim 5;
(k) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to Claim 5 through KNAi effect;
(1) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to Claim 5; and
(m) a polynucleotide encoding RNA that represses expression of the polynucleotide (DNA) according to Claim 5 through co-supression effct
7. A protein encoded by the polynucleotide of any one of Claims 1 to 5.
8. A vector comprising the polynucleotide of any one of Claims 1 to 5.
9. A vector comprising the polynucleotide of Claim 6.
.10:' A yeast conprising the vector of Claim 8 or 9.
11. The yeast of Claim 10, wherein a sulfite-producing ability is enhanced by introducing the vectorx of Claim 8.
12. A yeast, wherein an expression of the polynucleotide (DNA) of Claim 5 is suppressed
by introducing the vector of Claim 9, or by disrupting a gene related to -the polynucleotide (DNA) of
Claims.
13. The yeast of Claim 10, wherein a sulfite-producing ability is elevated by increasing an
expression level of the protein of Claim 7.
-14. A method for producing an alcoholic beverage comprising culturing the yeast of anv

one of Claims 10 to 13.
15. The method for producing an alcoholic beverage of Claim 14, wherein the brewed alcoholic beverage is a malt beverage.
16. The method for producing an alcoholic beverage of Claim 14, wherein the brewed alcoholic beverage is wine.'
17. An alcoholic beverage produced by the method of any one of Claims 14 to 16.
18. A method for assessing a test yeast for its sulfiterproducing capability, comprising using a primer or a probe designed based on a nucleotide sequence of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1.
19. A. method for assessing a test yeast" for its sulfite-producing capability, comprising:
culturing a test yeast; and measuring an expression level of a sulfete adenyltransferase gene having
. the nucleotide sequence of SEQ ID NO: 1.
20. A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein according to Claim 7 or measuring an expression level of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: t; and selecting a test yeast having said protein amount or said gene expression level according to a target capability of producing sulfite.
21. The method for selecting a yeast according to Claim 20, comprising: culturing a reference yeast and test yeasts; measuring an e?q3ressioii level of a sulfete adenyltransferase 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 protein according to Claim 7 in each yeast; and selecting a test yeast having said protein for a larger or smaller amount than that in the reference
yeast.
23. A method /or producing an alcoholic beverage conprising: 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 sulfite.