Specification
GENE ENCODING GLYCOGEN SYNTHASE AND USE THEREOF
TECHNICAL FIELD
The present invention relates to a gene encoding glycogen synthase and use thereof in particular, a yeast for practical use with superior resistance property to dryness and/or resistance property to low-temperature storage, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, whose resistance property to dryness and/or resistance property to low-temperature storage is enhanced by amplifying expression level of GSY1 or GSY2 gens encoding Gsylp or Gsy2p which is a glycogen synthase in brewer's yeast, especially non-ScGSYl gene or non-ScG5Y2 gene spsrific to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc. Further, the yeast of the present invention is useful as a baker's yeast or an industrial yeast as well
BACKGROUND ART
Beer brewing is characterized by a process recovering yeasts after fermentation and using the recovered yeasts at the subsequent fermentation, which is called tcRenjo". 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 storage, not only the next fermentation process is interfered, but also constituents of the yeast cells released by cell lysis may impart unfavorable taste to product. Therefore, it is very important for allowing some variance to design production process and for stable production of quality products to use yeasts with superior resistant property to iow-temperafure storage.
"Renjo" may be terminated at a certain times of fermenrarion is carried out The number of times of "Renjo" may vary according to ferrnentation conditions or properties of yeasts used in the., process. A process to develop yeasts for fermentation freshly is called propagation. Yeasts are subcultured several times enlarging scales of culture successively during the propagation process. Because propagation process requires from several days to several weeks, it brings great advantages in production efficiency if term of the process is shortened or yeast cells which are large-scale pre-cultured are able to be stored stably for extended period of time at low temperature or under dry condition.
Concerning a method for producing dry yeast maintaining high viable cell ratio, improvement of drying equipment, or improvement of manufacturing conditions such as temperature or addition of emulsifiers, etc. have been made. For example, L-drying method is not
practical to be used at industrial production scale because, though it can. maintain extremely high viable call ratio, but at the same time it takes a lot of time and cost.
Regarding low-temperature resistance of yeast, some experiments designed to improve refrigeration-resistant property mainly of baker's yeast were reported. This is because
Saccharomyces cersvisiae, which is a baker's yeast, has poor low-temperature storage property in comparison with brewer's yeast for beer or sake, which can ferment at low temperature. For example, baker's yeasts having refrigeration-resistant property and drying-resistant property were found out mainly by screening methods in Japanese Patent Application Laid-open No. HI 1-155559 and Japanese Patent Application Laid-open No. 2003-304864. Further, regarding examples
utilizing genetic engineering techniques, trehalose highly accumulating strains by disruption of NTH1, which is a trehalase gene, is reported in Japanese Patent Application Laid-open No. HI 0-117771 and a strain highly accumulating specific amino acids such as arginine by disruption of CAR1, which is an arginase gene, is reported in Japanese Patent Application Laid-open No. 2001-238665.
DISCLOSURE OF INVENTION
Under the above situations, there has been a need to make high-efficiency production of alcoholic beverages or useful materials possible by using a gene encoding a protein responsible for drying and/or low-temperature storage-resistant property of brewery yeast and said protein.
The present inventors made extensive studies to solve the above problems and as. a result, succeeded in identifying and isolating a gene encoding glycogen synthase from beer yeast Moreover, the present inventors produced transformed yeast in which the obtained gene was expressed to verify mat drying-resistant property and/or low-temperature storage-resistant property can be actually improved, thereby completing the present invention.
Thus, the present invention relates to a gene eiKodins a glycogen synthase ofbrewery ysast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a method for enhancing drying-resistant property and/or low-temperature storage-resistant property of yeast using a yeast in which the expression of said gene is controlled, or the like. More specifically, the present invention provides the following polynucleotides, a vector comprising said polynucleotide, a transformed yeast introduced with said vector, a method for producing alcoholic beverages by using said transformed yeast, and the like.
(1) A polynucleotide selected from the group consisting of
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDNO: 1 orSEQIDNO: 3;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SBQ ID NO: 4;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which one or more amino acids thereof are deleted, substituted, inserted and/or added, and having a glycogen synthase 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 or SEQ ID NO: 4, and said protein having a glycogen synthase activity;
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and which encodes a protein having a glycogen synthase 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 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions, and which encodes a protein having a glycogen synthase activity.
(2) The polynucleotide according to (1) above selected from the group consisting of:
(g) a polynucleotide comprising a polynucleotide encoding a protein consisting of the
amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or encoding the amino acid sequence of
SEQ ID NO: 2 or SEQ ID NO: 4 in which 1 to 10 amino acids thereof are deleted, substituted,
inserted, and/or added, and wherein said protein has a glycogen synthase activity;
(h) a porynucleoude comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and having a glycogen synthase activity; and
© a polynucleotide comprising a polynucleotide winch hybridizes to a polyiEicleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: lor SEQ ID NO: 3, under high stringent conditions, which encodes a protein having a glycogen synthase activity.
(3) The polynucleotide according to (1) above comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
(4) The polynucleotide according to (1) above comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4.
(5) The polynucleotide according to any one of (1) to (4) above, wherein the
polynucleotide is DNA.
(6) A protein encoded by the polynucleotide according to any one of (1) to (5) above.
(7) A vector containing the polynucleotide according to any one of (1) to (5) above.
(7a) The vector of (7) above, which comprises the expression cassette comprising the following components:
(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 function in a yeast with respect to transcription termination and polyadenylation of aRNA molecule.
(7b) The vector of (7) above, which comprises the expression cassette comprising the following components:
(x) a promoter that can be transcribed in a yeast cell;
(y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense direction; and
(z) a signal that can function in a yeast with respect to transcription termination and polyadenylation of aRNA molecule.
(S) A yeast into which the vector according 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
property is increased. The "yeast for practical use" means that a yeast which possesses practical
value such as brewer's (brewery) yeast, baker1 s yeast or industrial yeast etc.
(10) The yeast according to (S) above, wherein iow-temperature storage-resistant
property is increased.
(11) The yeast according to (9) above, wherein the dryihg-resistani property is increased
by increasing an expression level of the protein of (6) above.
(12) The yeast according to (10) above, "wherein the low-temperature storage-resistant
property is increased by increasing an expression level of the protein of (6) above
(12a) The yeast according to any one of (9) to (12) above, wherein the yeast is a brewery
yeast.
(13) A method for producing an alcoholic beverage by using the yeast according to any
one of (S) to (12a) above.
(14) The method according to (13) above, wherein the brewed alcoholic beverage is a
malt beverage.
(15) The method according to (13) above, wherein the brewed alcoholic beverage is
wine.
< (16) An alcoholic beverage produced by the method according to any one of (13) to (15)
above.
(17) A method for assessing a test yeast for its drying-resistant property and/or
iow-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 or SEQ ID NO:
3 and encoding a protein having a glycogen synthase activity.
(17a) A method for selecting a yeast having an increased drying-resistant property and/or low-temperature storage-resistant property by using the method described in (17) above.
(17b) A method for producing an alcoholic beverage (for example, beer or alcohol for industrial use, etc.) by using the yeast selected with the method described in (17a) above.
(17c) A method for producing an useful materials (for example, protein) by using the yeast selected with the method described in (17a) above.
(18) A method far assessing a test yeast for its drying-resistant property and/or
low-temperature storage-resistant property, comprising: culturing the test yeast; and measuring the
expression level of the geme having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and
encoding a protein having a glycogen synthase activity.
(18a) A method for selecting a yeast having a Mgh drying-resistant property and/or low-temperature storage-resistant property, which comprises assessing a test yeast by the method described in (18) above and selecting a yeast having a high expression level of gene encoding a protein having a glycogen synthase activity
(1 Sb) A method for producing an alcoholic beverage (for example, beer) by using the yeast selected with the method described in (18a) above.
(18c) A method for producing an useful material (for example, protein) by using the yeast selected with the method described in (18a) above.
(19) A method for selecting a yeast, comprising: cumiring test yeasts; quantifying the protein of (6) above or measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having a glycogen synthase activity; and selecting a test yeast having an amount of the protein or the gene expression level according to favorable drying-resistant property and/or low4emperature storage-resistant property.
(20) The method for selecting a yeast according to (19) above, comprising: culturing a reference yeast and test yeasts; measuring for each yeast the expression level of the gene haying the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having a glycogen synthase 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 (19) above, comprising: culturing a
reference yeast and test yeasts: quantifying the proton according to (6) above in each yeast; and. selecting a test yeast having a larger amount of the protein than that in the reference 5^ast.
(22) A method ior producing an alcoholic beverage comprising: conducting fermentation using the yeast according 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-temperature storage. Therefore, when it is used for brewing and so on, painfullness of conserving yeast can be eliminated. Further, it is expected to contribute to quality | stabilization Moreover dry yeast is suitable ibr long-storage, and it is very advantageous to distribution or transportation due to its reduced weight It is also useful as microorganisms for industrial application such 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 am (QD560).
Figure 2 shows the extract (sugar) consumption with time upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 3 shows the expression profile of non-ScGSYl gene in yeasts upon beer fermentation test The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal..
Figure 4 shows the result of drying-resistant property -test of parent strain and non-ScGSYl highly expressed strain.
Figure 5 shows the cell growth with time upon best fermentation test The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).
Figure 6 shows the extract (sugar) consumption with time upon beer fermentation test The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).
Figure 7 shows the expression profile of non-ScGSY2 gene in yeasts upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal. BESTMODES FOR CARRYING OCT THE INVENTION
The present inventors isolated and identified non-ScGSYl gene and non-ScGSY2 gene
' encoding glycogen synthases of brewery yeast based on the lager brewing yeast genome information napped according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-283169. The nucleotide sequences of the genes are represented by SEQ ID NO: 1 and SEQ ID NO: 3. Further, amino acid sequences of proteins encoded by the genes are represented by SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
1. Polynucleotide of the invention
First of all, the present invention provides (a) a polynucleotide comprising a polynucleotide of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and (b) a polynucleotide comprising a polynucleotide encoding a protein of the amino add sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The polynucleotide can be DNA or RNA,
The target polynucleotide of the present invention is not limited to the polynucleotide encoding a protein having a glycogen synthase activity described above and may include other polynucleotides encoding proteins having equivalent functions to said protein. Proteins with equivalent functions include, for example, (c) a protein of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 with one or more amino acids thereof being deleted, substituted, inserted and/or added and having a glycogen synthase activity.
having a glycogen synthase activity. In general, the number of deletions, substitutions, insertions, and/or additions is preferably smaller. In addition, such proteins include (d) a protein having an amino acid sequence with about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 7-4% or higher, 75% or higher, 76% or higher, 77% or higher, 7S% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88%- or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and having a glycogen synthase activity. In general, the percentage identity is preferably higher.
Glycogen synthase activity may be measured, for example, by a method described in
Cheng et al. Mot. Cell. Biol, 15(12), 6632-6640 (1995).
Furthermore, the present invention also contemplates (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under stringent
i conditions and which encodes a glycogen synthase; and (i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide complementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions, and which encodes a glycogen synthase.
Herein, "a polynucleotide that hybridizes under stringent conditions" refers to nucleotide
i sequence, such as a DNA, obtained by a colony hybridization technique, a plaque hybridization technique, a southern hybridisation Technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or polynucleotide encoding the ammo acid sequence of SEQ ID NO; 2 or SEQ ID NO: 4 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd
■ Ed., CURRENTPROTOCOLS IKM^LECULAR BIOLOGY, John Wiley & Sons 1987-1997, and so on.
The term "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 32X. "Moderate stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50%
1 formamide at 42°C. "High, stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50°C. Underthese conditions, a polynucleotide, such as a DNA, with higher homology is expected to be obtained efficiently at higher temperature, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe lajgrh. ionic strength, time, salt concentration and others, and one skilled in the art may appropriatery select -these factors to realize similar stringency.
When a commercially available kit is used for hybridization, for example, ABqphos Direct Labeling Reagents (Amersham Pharmacia) may be used In this case, acairding to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash buffer cortaining 0.1% (w/v) SDS at 55DC, thereby detecting hybridized polynucleotide, such
' as DNA
Identity between amino add sequences or nucleotide sequences may be determined using algorithm BLAST by Karlin and Altschul (Proc. Nail. Acad Sci USA, 87: 2264-2268, 1990; Proc. Natl Acad Sci USA, 90: 5873,1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et al, J. Mot Biol 215: 403, 1990). .When a nucleotide sequence is sequenced using BLASTN, the parameters are, &r example, score = 3 00 and word length = 12. When an amino acid sequence is sequenced using BLAS TX, the parameters are, for example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed.
2. Proton of the present invention
The present invention also provides proteins encoded by any of the polynucleotides (a) to (i) above. A preferred protein of the present invention comprises an amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 4 with one or several amino acids thereof being deleted, substituted, inserted and/or added, and having a glycogen synthase activity.
Such protein includes tiiose having an amino acid sequence of SEQ ED NO: 2 or SEQ ID NO: 4 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a glycogen synthase activity. In addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and having a glycogen synthase activity.
Such proteins may be obtained by employing she-directed mutation described, for example,
inMoi®2UL4RC!J3^G3rdEd,CURl^ Res.,
10: 6487 (1982), Proc. Nail Acad Sci USA 79: 6409 (1982), Gene 34:315 (19B5), Nuc. Acids. Pes., 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, examples 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, norleucine, valine, norvalhie, alanine, 2-aminobutanoic acid, methionine, o-methyiserine, t-butylelycine, t-butylalanine, cyclohsxyialanine; Group B: ascaraiic acid, glutamic acid, isoasparatic acid, isoghitamic acid, 2-aminoadipic acid, 2-arninosuberic acid; Group C: asparaginic glutamine; Group Di lysine, argraine, ornithine, 2,4-diarumobutanoic acid,
> 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyprofine, 4-hydroxyproiine; Group F: serine, threonine, homoserine; and Group G: phenylalanine, tyrosine.
The protein of the present invention may also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). In addition, peptide synthesizers available from, for example, Advanced ChemTecb,
l PerkkEVmer, Pharmacia, Protein Technology" Instrument, Synthecell-Vega, PsrSsprive, Shimazu Corp. can also be used for chemical synthesis.
3. Vector of the Invention and yeast transformed with the 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 0) above or any of the polynucleotides described in (j) to'(rn) above. Generally, the vector of the present invention comprises an expression cassette including as components (x) a promoter that can transcribe in a yeast cell; (y) a polynucleotide described in any of (a) to (i) above that is linked to the promoter in sense or andsense direction; and (z) a signal that functions in the yeast with respect to transcription termination and polyadenylation of KNA molecule. Further, in order to highly express the protein of the invention, these polynucleotides are preferably introduced in the sense direction to the promoter to promote expression of the polynucleotide (DNA) described 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 typeX or a chromosome integration type (Yip type). For example. YEp24 Q. R Broach at aL SK?ERltvM3T,John'WTley& Sons 1994-2003).
Furthermore, test yeasts are cultured and expression levels of the gene encoding a protein having a glycogen synthase activity having the nucleotide sequence of SEQ K> NO: 1 or SEQ ID NO: 3 are measured to select a test yeast with the gene expression level according to the target glycogen-producing ability, thereby a yeast favorable for brewing desired alcoholic beverages can be selected 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 a favorable test yeast can be selected. More specifically, for example, a reference yeast and one or more test yeasts are cultured and an expression level of the gene encoding a protein having a glycogen synthase activity having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is measured in each yeast By
selecting a test yeast with the gene expressed higher than that in the reference yeast, a ysast suitable for brewing desired alcoholic beverages or production of useful materials can be selected.
Alternatively, test yeasts are cultured and a yeast with a high glycogen synthase activity 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 glycogen synthase activity can be measured by, for example, a method described in Cheng et aL Mol. Cell. Biol., 15(12), 6632-6640 (1995). The mutation treatment may employ, any-methods including, for example, physical methods such as ultraviolet irradiation and radiation irradiation, and chemical methods associated with treatments with drugs such as EMS (ethylmethane sulphonate) ami N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima Ed, BIOCH3**BIR.Y EXPERIMENTS vol. 39, Yeast Molecular Genetic Experiments, pp. 67-75, JSSP).
In addition, examples of yeasts used as the reference yeast or the test yeasts include any yeasts (yeasts for practical use), for example, brewery yeasts for beer, wine, sake and the like or baker's yeast, yeast for producing industrial alcohol or yeast for producing useful proteins, etc. More specifically, yeasts such as genus Saccharomyces may be used {e.g., S pastorianus, S. cerevisiae, and S. carlsbergensis). According to the present invention, a lager brewing yeast, for example, Saccharomyces pastorianus W34/70;' Saccharomyces carlsbergensis NCYC453 or NCYC456; or Saccharoiryces cerevisiae TttRC195l,'NBRCl952, NBRC1953 orNBRC1954, etc., may be used. Further, 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, baker's yeast such as NBRC0555, NBRC1346 and NBRC2043, etc, may also be used hot not limited thereto. In the present invention, lager brewing yeasts such as Saccharomyces pasturianus may preferably be used. The reference yeast and the test yeasts may be selected from the above yeasts in any combmalicn.
EXAMPLES
Hereinafter, the present invention will be described in more detail with reference to working examples. The present invention, however, is not limited to the examples described below.
Example 1: Cloning of Gene Encoding Glycogen synthase (non-ScGSYl)
A gene encoding a glycogen synthase of lager brewing yeast (non-ScGSYl) (SEQ JX> NO: 1) was 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-ScG5Yl_for (SEQ E> NO: 5) and non-ScGSYl_rv (SEQ ID NO: 6) were designed to amplify the Ball-length of the gene. PCR was earned out using chromosomal DNA of a genome sequencing strain, Saccharomyces pastorianus Weihenstephan 34/70 (sometimes abbreviaied as "W34/70 strain"), as a template to obtain DNA fragments including the full-length gene of non-ScGSYl.
The non-ScGSYl gene fragments thus obtained were inserted into pCR2.1-TOPO vector (Invitrogen) by TA cloning. The nucleotide sequences of the non-ScGSYl gene were analyzed by Sanger's method (F.Sanger, Science, 214:1215, 1981)toconfirm the nucleotide sequence. •
Example 2: Analysis of Expression of non-ScGSYl Gene during Beer Fermentation
A beer fermentation test was conducted using a lager brewing yeast, Saccharomyces pastorianus W34/70, and mRNA extracted from the lager brewing yeast during fermentation was detected by a beer yeast DNA microairay.
Wort extract concentration 12.69%
Wortcontent 70L
Wort dissolved oxygen concentration 8.6 ppm
Fermentation temperature 15°C
Yeast pitching rate 12.8x10s cells/raL
The fermentation liquor was sampled over time, and the time-course changes in amount of yeast cell growth (Fig. 1) and apparent extract concentration (Fig. 2) were observed. Simultaneously, yeast cells were sampled to prepare mRNA, and the prepared rriRNA was labeled with biotin and was hybridized to a beer yeast DNA microairay. The signal was detected using GeneChip Operating system (GCOS; GeneChip Operating Software 1.0, manmactured by Asymetrix Co). Expression pattern of the non-ScGSYl gene is shown in Figure 3. This result confirmed the expression ofthenon-ScGSYl gene in the general beer fermentation
Example 3: Construction of non-ScGSYl Highly Expressed Strain
The non-ScGSYl/pCR2.1-TOP0 described in Example 1 was digested with the restriction enzymes Sad and Not! to prepare a DNA fragment containing the entire length of the protein-encoding region. This fragment was ligated to pYCGPYNot treated with the restriction enzymes Sad and NoH thereby constructing the non-ScGSYl high expression vector non-ScGSYl/pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector. A gene
inserted is highly expressed by the pyruvate kinase gene PYK1 promoter. The geneticin-resistant gene G418r is included as the selectable marker in the yeast and the ampicillin-resistant aene Amp1 as the selectable marker in Escherichia coll
Using the high expression vector prepared by the above method, an AJL4004 strain was transformed by the method described in Japanese Patent Application Laid-open No. H07-303475. The transformants were selected on a YPD plate medium (1% yeast extract, 2% polypeptone, 2% glucose and 2% agar) containing 300 mg/L of geneticin.
Example 4: Evaluation of Drying-resistant Property of non-ScGSYI Highly Expressed
Strain
Drying-resistant properties of the parent strain (AJL40Q4 strain) and the non-ScGSYI highly expressed Strain 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 mg/L of geneticin, and stirred at 30DC overnight (precultivalion). The precuitivation liquid was inoculated into 10 mL wort containing 100 mg/L of geneticin to make its OD660 *= 0.5, then main culture was initiated The culture was continued for 2 days until the growth of the yeast reached stationary phase. Turbidity of the culture was measured at the completion of the culture, then the culture liquid was suspended in sterile water to make its OD = 2. One hundred microliter (100 uL) of the suspension thus obtained was dispensed into a 1.5 mL microtube, then the yeast cells were dried by evaporation for 1 hour- using a reduced-pressure concentrator (DNA110 SpeedVac (registered trademark), manufactured by ThermoSavant).
"Viable cell ratio was measured by a method described below. The dried yeast cells obtained above were resuspended in 50 uL of sterile water, then 50 uL of 0.02% methylene bhie solution (pH 4.5) was added to the suspension. Blue-stsined yeast cells winch had lost reducing power were considered as dead yeast sells. Then the suspension was observed under a microscope, and viable cell ratio was measured using a Cell Vital Analyzer System (DA cell counter, manufactured by Yamato Scientific Co., Ltd.). The cells were counteduntil the population reached more than 2000 cells to -minimize experimental error.
As indicated in Figure 4, viable cell ratio of the highly-expressed strain was 27.6%, though viable, cell ratio of the parent strain was 19.9%. It was demonstrated by the results that drying-resistant property of yeast was increased by high expression of non-ScGSYI.
Example 5: Evaluation of Low-temperature Resistant Property of non-ScGSYI Highly Expressed Strain
Low-temperature resistant property of the parent strain (AJL4004 strain) and the non-ScGSYl highly expressed strain obtained by the method described in Example 3 are evaluated by the mehtod described below. Nine hundred microliter (900 pL) of the yeast suspensions cultured by the method described in Example 4 and prepared as OD660=2 are dispensed into two microtubes, respectively. One hundred microliter (100 uL) of sterile water is added to one of the microtubes, on the other hand, 100 uL of 99.5 % ethanol is added to another ore (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 Example 4.
Example 6: nnninp nf Gene Encoding Glycogen synthase : TvaJuation of Low-temperature Resistant Property of non-SeGSVl Htohh; "Expressed Strain
Low-temperature resistant property of the parent strain (AJL4004 strain) and the nan-ScGSY2 highly expressed strain obtained by me method described in Example S are evaluated by the same method as Example 5.
INDUSTRIAL APPLICABILITY
According to the present invention, yeast can be stored stably for extended period of time, because drying-resistant properly and/or low-temperature storage-resistant property can be enhanced by the present invention. Accordingly, efficiency of brewing alcoholic beverages (such as beer), production of bread, or inanufacturing useful materials such as industrial alcohol production or
production of useful proteins, etc., can be improved by the present invention.
CLAIMS
i. A. polynucleotide selected from the group consisting of.
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: lor SEQ ID NO: 3;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2orSEQIDNO: 4;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which, one or more amino acids thereof are deleted, substituted, inserted and/or added, and having a glycogen synthase 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 or SEQ ID NO: 4, and said protein having a glycogen synthase activity;
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQIDNO: 1 or SEQ ID NO: 3 under stringent conditions, and which encodes a protein having a glycogen synthase activity; and
(f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of die polynucleotide encoding the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions, and which encodes a protein having a glycogen synthase activity.
2. The porynudectide according to Claim 1 selected from the group consisting of
(g) a polynucleotide comprising a polynucleotide encoding a protein consisting of the
amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or encoding the mm acid sequence of
SEQ ID NO: 2 or SEQ ID NO: 4 in which 1 to 10 amino adds thereof are deleted, substituted,
inserted and/or added, and wherein said protein has a glycogen synthase activity;
(h) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and having a glycogen synthase activity; and
(i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, under high stringent conditions, which encodes a protein having a glycogen synthase activity.
3. The polynucleotide according to Claim 1 comprising E polynucleotide consisting of the nucleotide sequence of SEQ ID NO; 1 or SEQ ID NO: 3.
i 4. The polynucleotide according to Claim 1 comprising a polynucleotide encoding a
protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
5. The polynucleotide according to any one of Claims 1 to 4, wherein the polynucleotide isDNA
6. A protein encoded by the polynucleotide according to any one of Claims 1 to 5.
7. A vector containing the polynucleotide according to any one of Claims 1 to 5.
8. A yeast into which the vector according to Claim 7 has been introduced,
9. The yeast accordingto Claim 8, wherein drying-resistant property is increased.
10. The yeast accordingto Claim 8, wherein low-temperature storage-resistant property is increased.
11. The yeast according to Claim 9, wherein the drying-resistant property is increased by increasing an expression level of the proteia of Claim 6.
12. The yeast according to Claim 10, -wherein the low-temperature storage-resistant property is increased bv increasing an expression, level of the protem of Claim 5.
13. A method for producing an alcoholic beverage by using the yeast according to any one of Claims 8 to 12.
14. The method according to Claim 13, wherein the brewed alcoholic beverage is a malt beverage.
15. The method according to Claim 13, wherein the brewed alcoholic beverage is wine.
16. An alcoholic beverage produced by the method according to any one of Claims 13 to
17. A method for assessing a test yeast for its drying-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 or SEQ ID NO:
3 and encoding a protein having a glycogen synthase activity.
18. A method fbi assessing a test yeast for its drying-resistant property and/or
low-temperature storage-resistant property, comprising-, culturing the test yeast; and measuring the
expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having a glycogen synthase activity.
19. A method for selecting a yeast, comprising: culturing test yeasts; quantifying the
protein of Claim 6 or measuring the expression level of the gene having the nucleotide sequence of
SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having a glycogen synthase activity; and
selecting a test yeast having an amount of the protein or the gene expression level according to
favorable drying-resistant property and/or low-temperature storage-resistant property,
20. The method for selecting a yeast according to Claim 19, comprising: culturing a
reference yeast and test yeasts; measuring for each yeast the expression level of the gene having the
nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoomg a protein havmg a glycogen
synthase 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. comprising culturing a
reference yeast and test yeasts; quantifying the protein according to Claim 6 in each yeast; and
selecting a test yeast having a larger amount of the proteh than that m the reference yeast.