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Gene Encoding Transcriptional Inducer For Maltase Gene And Maltose Transporter Gene And Use Thereof

Abstract: Abstract GENE ENCODING TRANSCRIPTIONAL INDUCER FOR MALTASE GENE AND MALTOSE TRANSPORTER GENE AND USE THEREOF The present invention relates to a gene encoding a transcriptional inducer for maltase gene and maltose transporter gene and use thereof, in particular, a brewer's yeast with high maltose assimilation ability, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, whose maltose assimilation is enhanced by amplifying expression level of MALR gene encoding MalRp, a maltase and maltose transporter transcription factor in brewer's yeast, especially non-ScMALR gene specific to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc.

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Notices, Deadlines & Correspondence

Patent Information

Application #
Filing Date
29 September 2008
Publication Number
12/2009
Publication Type
INA
Invention Field
BIOTECHNOLOGY
Status
Email
Parent Application

Applicants

SUNTORY LIMITED
1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA 530-8203

Inventors

1. NAKAO, YOSHIHIRO
C/O SUNTORY LIMITED, RESEARCH CENTER, 1-1-1, WAKAYAMADAI, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-8503
2. KODAMA, YUKIKO
C/O SUNTORY LIMITED, RESEARCH CENTER, 1-1-1, WAKAYAMADAI, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-8503
3. SHIMONAGA, TOMOKO
C/O SUNTORY LIMITED, RESEARCH CENTER, 1-1-1, WAKAYAMADAI, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-8503

Specification

DESCRIPTION GENE ENCODING TRANSCRIPTIONAL INDUCER FOR MALTASE GENE AND MALTOSE TRANSPORTER GENE AND USE THEREOF TECHNICAL FIELD The present invention relates to a gene encoding transcriptional inducer for maltase gene and maltose transporter gene, and use thereof, in particular, brewer's yeast with superior maltose fermentability, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast whose maltose assimilation ability is enhanced by amplifying expression level of MALR gene encoding a proteia MaIRp (transcriptional inducer for maltase gene and maltose transporter gene in brewer's yeast), especially non-ScMALR gene specific to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc. BACKGROUND ART In beer production, while wort having about 11% extract concentration is fermented to obtain beer having about 4.5 -5% alcohol concentration, high gravity brewing is sometimes adopted for improvement of beer productivity. The high gravity brewing is a method for producing beer with desired alcohol concentration, by fermenting wort with higher concoitration than conventional wort, followed by diluting the resultant product with water. More specifically, the following measures are considered. (1) A higher temperature than conventional fermentation is adopted; (2) Airflow to wort is increased; (3) Yeast pitching rate is increased; and any combination of these measures. It is said that about 15% original wort extract concentration is maximum in high gravity brewing for conventional bea* production. A first problem in high gravity brewing with higher than 15% extract concentration is remarkable decrease of fermentation speed occurring at the middle to late stage of the fermentation. Main carbohydrates included in wort are maltose, maltolriose, glucose, fiiictose and sucrose. A yeast assimilates glucose, fructose and sucrose initially, then assimilates maltose and maltotriose. Accordingly only maltose and maltotriose exist in fermentation broth, during the middle to late stage of the fermentation. Moreover, there is overwhelmingly a lot of maltose with ratio of 3:1. Maltose is transported into a yeast cell by maltose transporter, hydrolyzed to two glucoses by maltase, followed by conversion to carbon dioxide and ethanol mediated by Embden-Meyerhof pathway. These two enzymes are induced in the presence of maltose, but inhibited in the presence of glucose, at transcriptional level. It is known that transcription factor MalR plays an important role in transcriptional induction of maltase gene and maltose transporter gene in the presence of maltose, and transcription of MalR is also mhibited in the presence of glucose (Mol Cell Biol. 7:2477-2483,1987, Curr Genet28:258-266,1995). Since about 17% of assimilable carbohydrates in wort is glucose, maltose metabolic genes of yeast are inhibited at the early stage of fermentation and causes a significant delay of maltose assimilation. This phenomena becomes more serious ia high gravity brewing, which causes not only delay of fermentation but also remaining large amount of maltose as a residue of sugar at the completion of fermentation. Because of these problems, high gravity brewing (for example, fermentation with double concentration of regular wort) cannot be conducted sufficiently with use of the conventional techniques. Japanese Patent Application Laid-open HI-153082 describes usage of baker's yeast transfected with a plasmid comprising a promoter for alcohol dehydrogenase gene that cannot be inhibited by glucose, maltase gene and maltose transporter gene for improvement of Dough fermentation by baker's yeast. Meanwhile, it is reported that a maltose transporter gene MAL6T of Saccharomyces cerevisiae was highly expressed in brewer's yeast, and high gravity brewing was achieved (Japanese Patent Application Laid-open No. H06-245750). DISCLOSURE OF INVENIION Under the above situations, it is desired to provide a yeast that allows for a high gravity brewiog without imparing fermentation speed and product quality. The present inventors made extensive studies to solve the above problems and as a result, succeeded in identifying and isolatnig a gene encoding a transcriptional inducer for maltase gene and maltose transporter gene from beer yeast. Moreover, the present inventors produced transformed yeast in which the obtained gene was expressed to verify tiiat maltose assimilation ability can be actually improved, thereby completing the present invention. Thus, the present invention relates to a gene encoding a transcriptional inducer for maltase gene and maltose transporter gene existing in brewer's yeast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a method for producing alcoholic beverages by using said transformed yeast in which the expression of said gene is controlled, and 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 tor producing alconolic beverages by using said transtonned yeast, and the J (1) A polynvicleotide selected from the group consisting of: (a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQIDN0:1; (b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ E) NO: 2; (c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino adds thereof are deleted, substituted, inserted and/or added, and having a transcriptional induction activity of maltase and maltose transporter gene; (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 said protein having a transcriptional induction activity of maltase and maltose transporter gene; (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence coroplementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and which encodes a protein having a transcriptional induction activity of maltase and maltose transporter gene; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polyniicleotide encoding the protein having the amino acid sequence of SEQ ID NO: 2 under stringent conditions, and which encodes a protein having a transcriptional induction activity of maltase and maltose transporter gene. (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 encoding the amino acid sequence of SEQ ID NO: 2 in which 1 to 10 amino acids thereof are deleted, substituted, inserted, and/or added, and wherein said protein has a transcriptional induction activity of maltase and maltose transporter gene; (h) a polynudeotide cornprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having a transcriptional induction activity of maltase and maltose transporter gene; and (i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, under high stringent conditions, which encodes a protein having a transcriptional induction activity of maltase and maltose transporter gene. (3) The polynucleotide according to (1) above comprising a polynucleotide consisting o the nucleotide sequence of SEQ ID NO: 1. (4) The polynucleotide according to (1) above comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ E) NO: 2. (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 a RNA 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 flinction in a yeast with respect to transcription termination and polyadenylation of a RNA molecule. (8) A yeast into which the vector according to any one of (7) to (7b) above has been introduced. (9) The yeast according to (8) above, wherein maltose assimilation abihly is increased by introducing the vector according to any one of (7) to (7b) above. (10) The yeast according to (9) above, wherem maltose assimilation ability is increased by increasing an expression level of the protein of (6) above. (11) A method for producing an alcoholic beverage by using the yeast according to any one of Claims (8) to (10) above. (12) The method according to (11) above, wherein the brewed alcoholic beverage is a malt beverage. (13) An alcohoUc beverage produced by the method according to (11) or (12) above. using a primer or probe designed based on the nucleotide sequence of a gene having the nucleotide sequence of SEQ E) NO: 1 and encoding a transcriptional inducer for maltase gene and maltose transporter gene. (14a) A method for selecting a yeast having increased maltose assimilation ability by using the method described in (14) above. (14b) A method for producing an alcoholic beverage (for example, beer) by using the yeast selected with the method described in (14a) above. (15) A method for assessing a test yeast for its high maltose assimilation abiHly, comprising: culturing the test yeast; and measuring the expression level of the gene having tiie nucleotide sequence of SEQ ID NO: 1 and encoding a transcriptional inducer for maltase gene and maltose transporter gene. (15a) A method for selecting a yeast having superior maltose assimilation ability, whidi comprises assessing a test yeast by tiie metiiod described in (15) above and selecting a yeast having a high expression level of gene encoding a transcriptional inducer for maltase gene and maltose transporter gene. (15b) A method fiir producing an alcoholic beverage (for example, beer) by using the yeast selected with the method described in (15a) above. (16) A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein of (6) above or measuring the e}q)Tession level of the gene having the nucleotide seqiience of SEQ ID NO: 1 and encoding a transcriptional mducer for maltase gene and maltose transporter gene; and selecting a test yeast having an amount of the protein or the gene expression level according to desired maltose assimilation ability. (17) The method for selecting a yeast according to (16) above, comprising: culturing a reference yeast and test yeasts; measuring for each yeast the expression level of the gene havmg the nucleotide sequence of SEQ ID NO: 1 and encoding a transcriptional kiducer for maltase gene and maltose transporter gene; and selecting a test yeast having gene e:!q}ression level higher than that in the reference yeast (18) The method for selecting a yeast accordmg to (16) above, conpising: culturing a reference yeast and test yeasts; quantifying the protein according to (6) above in each yeas^ and selecting a test yeast having a larger amount of the protem than that in the reference yeast (19) A method for producing an alcoholic beverage comprismg: conducting fermentation using tiie yeast according to any one of (8) to (10) above or a yeast selected by the method accordii^ to any one of (16) to (18) above. According to the metiiod for producing alcoholic beverages using transformed yeast of the present invention, assimilation of maltose is not inhibited even in the presence of glucose. As a result, a beer brewing with high wort concentration can be achieved since fermentation speed is increased due to maltose assimilation prior to disappearance of glucose. BRIEF DESCRIPllON OF DRAWINGS Figure 1 shows the cell growth with time upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660). Figure 2 shows the extract (sugar) consumption with 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-ScMALR gene in yeasts upon beer fermentation test. The horizontal axis represents fermentation time while the vertical axis represents the intensity of detected signal. BEST MODES FOR CARRYING OXJIIBE INVENTION The present inventors conceived that maltose could be assimilated more efficiently by increasing tiranscriptional induction activity of maltase and maltose transporter gene. The present inventors made extensive studies based on the conception, isolated and identified non-ScMALR gene encoding a transcriptional inducer for maltase gene and maltose transporter gene which is specific to lager brewing yeast,' based on the lager brewing yeast genome information mapped according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-283169. The nucleotide sequence of the gene is represented by SEQ ID NO: 1. Further, an amino acid sequence of a proteui encoded by the gene is represented by SEQ ED NO: 2. 1. Polynucleotide of the invention First of all, the present invention provides (a) a polynucleotide comprising a polynucleotide of tiie nucleotide sequence of SEQ ID NO: 1; and (b) a polynucleotide comprising a polynucleotide encoding a protein of the amino acid sequence of SEQ ID NO: 2. The polynucleotide can be DNA orRNA. The target polynucleotide of the present invention is not limited to the polynucleotide encoding a transcriptional inducer for maltase gene and maltose transporter gene described above, and may include other polynucleotides encoding proteins having equivalent fimctions 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 havmg a transcriptional induction activity of maltase and maltose transporter gene. isucQ proxems mciuae a protein consisung oi an ammo acia sequence or siiy IUINU'.A witn, for example, 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, ItoSS, Ito34,lto33,lto32, Ito31,lto30, Ito29,lto28, lto27, lte26, Ito25,lto24, 1 to23,1 to22,1 to21,1 to20,1 to 19; 1 to 18,1 to 17,1 to 16,1 to 15,1 to 14,1 to 13,1 to 12,1 to 11,1 to 10,1 to 9,1 to 8,1 to 7,1 to 6 (1 to several amino acids), 1 to 5,1 to 4,1 to 3,1 to 2, or 1 amino add residues thereof being deleted, substituted, inserted and/or added and having a transcriptional induction activity of maltase and maltose transporter gene. 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 hi^er, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or hi^er, 85% or higher, 86% or higher, 87% or higjier, 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 hi^r identity with the amino add sequence of SEQ ID NO: 2, and having a transcriptional induction activity of maltase and mlaltose transporter gene. In gaieral, the percentage identity is preferably higher. In addition, transcriptional itidxiction activity of maltase and maltose transporter gene may be measured, by quantification of transcript level of the gene (mRNA). mRNA may be quantified, by Northem hybridization or quantitative RT-PCR (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1994-2003). Furthermore, the present invention also contemplates (e) a polynucleotide comprising a polynucleotide v^hich hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and wtdch encodes a protein having a transaiptional induction activity of maltase and maltose transporter gene and (f) a polynucleotide comprismg a polynucleotide which hybridize to a polynucleotide complementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 under stringent conditions, and vindi encodes a protein having a transcriptional induction activity of maltase and maltose transporter gene. 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 soufliem hybridization technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ED NO: 1 or polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as a probe. The hybridization method may be a method described, for example, in MOLECULAR CLONING 3rd Ed., CUREENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997, and so on. The temi "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringeaicy conditions. "Low stringency conditions" are, for ' example, 5 x SSC, 5 x Denhardt's soMon, 0.5% SDS, 50% forniaraide at 32°C. "Moderate stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C. "High stringency conditions" are, for example, 5 x SSC, 5 x Denhardfs solution, 0.5% SDS, 50% formamide at 50°C. Under these conditions, a polynucleotide, such as a DNA, with higher homology is expected to be obtained efficiently at higher temperature, although ' multiple fectors are involved in hybridization stringency including temperature, 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 example, Alkphos Direct Labehng Reagaits (Amersham Pharmacia) may be used. In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash buffer containing 0.1% (w/v) SDS at 55°C, thereby detecting hybridized polynucleotide, such as DNA. Other polynucleotides that can be hybridized include polynucleotides having about 60% or higher, about 70% or higher, 71 % or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81 % or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher or 99.9% or hi^er identity to polynucleotide encoding the anaino acid sequence of SEQ ID NO: 2 as calculated by homology search software, such as FASTA and BLAST using defeult parametos. Identily between amino acid sequences or nucleotide sequences may be determined using algorithm BLAST by Karlin and Altschul (Proc. Natl Acad Set USA, 87: 2264-2268,1990; Pwc. Natl. Acad Sci. USA, 90:5873,1993). Programs called BLASTN and BLASTS 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 example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed. I. rrotem oi me present mvennon 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 anaino acid sequence of SEQ ID NO: 2 with one or several amino acids thereof being deleted, substituted, inserted and/or added, and having a transcriptional induction activity of maltase and maltose transporter gene. Such protein includes those having an amino acid sequence of SEQ ID NO: 2 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a transcriptional induction activity of maltase and maltose transporter gene. In addition, such protein includes those having homology as described above with the amino add sequence of SEQ ID NO: 2 and having a transcriptional induction activity of maltase and maltose transporter gene. Such proteins may be obtained by employing site-directed mutation described, for example, in MOLECULAR CLONING 3rdEd., CURRENTPROTOCOLSINMOLECULARBIOLOGY,M

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Application Documents

# Name Date
1 5209-CHENP-2008 FORM-6 08-09-2009.pdf 2009-09-08
2 5209-CHENP-2008 FORM-13 08-09-2009.pdf 2009-09-08
3 5209-chenp-2008 pct.pdf 2011-09-04
4 5209-chenp-2008 form-5.pdf 2011-09-04
5 5209-chenp-2008 form-3.pdf 2011-09-04
6 5209-chenp-2008 form-1.pdf 2011-09-04
7 5209-chenp-2008 drawings.pdf 2011-09-04
8 5209-chenp-2008 description (complete).pdf 2011-09-04
9 5209-chenp-2008 correspondence-others.pdf 2011-09-04
10 5209-chenp-2008 correspondence others.pdf 2011-09-04
11 5209-chenp-2008 claims.pdf 2011-09-04
12 5209-chenp-2008 abstract.pdf 2011-09-04