CN1920044A - O-acetylhomoserinesulfhydorelace gene and use thereof - Google Patents

Abstract

The present invention relates to an o-acetylhomoserinesulfhydorelace gene and use thereof, especially a brewery yeast having controlled hydrogen sulfide-producing capability, a process for producing alcoholic beverages with controlled hydrogen sulfide amount. More particularly, the present invention relates to a yeast whose hydrogen sulfide-producing capability that increases the product flavor is controlled by enhancing the expression level of MET17 gene encoding brewery yeast O-acetylhomoserinesulfhydorelace Met17p, particularly non-ScMET17 gene specific to lager brewing yeast, and to a method for producing alcoholic beverages with said yeast.

Description

O-acetylhomoserine sulfydryl enzyme gene and uses thereof

Technical field

The present invention relates to O-acetylhomoserine sulfydryl enzyme (O-acetyl homoserine sulfhydrylase) gene and uses thereof, particularly relate to the brewer's yeast of the drinks of making the fragrance excellence, the drinks that uses this yeast manufacturing and manufacture method thereof etc.Further specifically, the present invention relates to by improving the gene M ET17 of coding brewer's yeast O-acetylhomoserine sulfydryl enzyme Met17p, distinctive non-ScMET17 expression of gene amount in the cereuisiae fermentum particularly improves the yeast of product fragrance and uses the manufacture method etc. of this zymic drinks.

Background technology

The cereuisiae fermentum that uses in the pale beer manufacturing of commercially available pilsner type has the characteristic that generates hydrogen sulfide in the Primary Fermentation process.This hydrogen sulfide is one of reason that causes unwelcome bright beer flavor on the beer quality, is to carry out secondary fermentation or prolong the storage wine phase with its countermeasure that is reduced under the threshold value.

Be to reduce the hydrogen sulfide in the beer, in the past promptly relevant for research (Jangaard, N.O., Gress, H.S.and Coe, the R.W. of the factor that influence the hydrogen sulfide generation; Amer.Soc.Brew.Chem.Proc., p46,1973; Kuroiwa, Y.and Hashimoto, N.; Brew.Dig., 45,44,1970; Hysert, D.W.and Morrison, N.M.; J.Amer.Soc.Brew.Chem., 34,25,1976), use sudden change method or cytogamy method to carry out hydrogen sulfide and generate few zymic breeding research (Molzahm, S.W.; J.Amer.Soc.Brew.Chem., report 35,54,1977).

These methods have all not only reduced zymic hydrogen sulfide growing amount, have also influenced other brewing characteristics of zymic (fermenting speed, beer fragrance), so, also do not obtain being suitable as brewage zymic yeast so far.In recent years, also utilize gene manipulation techniques to brewage and use the zymic breeding, disclose the cereuisiae fermentum of the dna fragmentation that imports the coding cystathionine in the Japanese kokai publication hei 5-244955 communique, can reduce the generation of hydrogen sulfide.But its minimizing degree is very little, and the hydrogen sulfide growing amount of shape matter transformant still is about 60～80% of a parental plant growing amount.

In the zymic substance metabolism, hydrogen sulfide is the sulfate ion (SO that is absorbing from substratum ₄ ^2-) reduction process in generate.This metabolic system is the biosynthetic pathway of sulfur-containing amino acid such as methionine(Met), halfcystine, and relevant for play-by-play (Tabor, H.and Tabor, the C.W. (eds.) of the enzyme and the gene (MET17 gene) thereof in each stage; Methods in Enzymology Vol.17B, Academic Press, London, 1971; Jakoby, W.B.and Griffith, O.W. (eds.); Methods in EnzymologyVol.143, AcademicPress, London, 1987).

O-acetylhomoserine sulfydryl enzyme is that sulphur atom is transferred to the enzyme of O-acetylhomoserine (O-acetyl homoserine) from hydrogen sulfide, by the MET17 genes encoding.This enzyme also has the activity that shifts sulphur atom to O-acetylserine.The cereuisiae fermentum strain that has report to point out to adopt the MET17 genome that derives from SaccharomycescerevisiaeX2180-1A to become second nature and express, the growing amount of hydrogen sulfide has reduced to about 2% (Japanese kokai publication hei 7-303475 communiques) of parental plant.

Summary of the invention

As mentioned above, the hydrogen sulfide growing amount reduces in the product in order to make, and has obtained mutant strain, but its result has shown that the fermentation of not expected postpones and the increase of unwelcome flavour ingredient, so, also exist problem as the practicality yeast.Thus, expecting to have very much neither influences fermenting speed, does not damage product quality, can reduce the zymic breeding method of hydrogen sulfide growing amount again.

Present inventors etc. successfully identify the novel gene of having isolated coding O-acetylhomoserine sulfydryl enzyme for solving the result that above-mentioned problem is constantly studied with keen determination from cereuisiae fermentum.And prepared the shape matter transformed yeast that the gained gene is imported yeast and make its expression, confirmed the minimizing of hydrogen sulfide growing amount, thereby finished the present invention.

That is, the present invention relates to New O-acetylhomoserinesulfgenerelace generelace that characteristic in the cereuisiae fermentum exists, this genes encoding protein, regulated the transformed yeast of this genetic expression, the shape matter transformed yeast of having regulated this genetic expression by use is with the method for hydrogen sulfide growing amount in the control product etc.Specifically, the invention provides polynucleotide as follows, contain these polynucleotide carrier, imported this carrier shape matter transformed yeast, use the manufacture method etc. of the drinks of this shape matter transformed yeast.

(1) polynucleotide of from following (a)～(f) a group, selecting:

(a) contain by sequence number: the polynucleotide of the polynucleotide that 1 base sequence is formed;

(b) contain handlebar by sequence number: the protein that 2 aminoacid sequence is formed is the polynucleotide of polynucleotide encoding in addition;

(c) at sequence number: in 2 the aminoacid sequence, the aminoacid sequence that contains one or more aminoacid deletion of handlebar, replacement, insertion and/or added is formed, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity;

(d) contain having and sequence number: 2 aminoacid sequence has the aminoacid sequence that is equal to or greater than 60% identity, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity;

(e) contain and sequence number: the polynucleotide that the complementary base sequence of the base sequence in 1 is formed, hybridize under stringent condition, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity; And

(f) contain the handlebar sequence number: the protein that 2 aminoacid sequence is formed carries out the polynucleotide of the complementary base sequence composition of polynucleotide encoding base sequence, hybridize under stringent condition, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity.

(2) above-mentioned (1) described polynucleotide, it is selected from following (g)～(i) one group:

(g) at sequence number: 2 aminoacid sequence or sequence number: in 2 the aminoacid sequence, 1～10 aminoacid deletion, replacement, insertion and/or the aminoacid sequence that added are formed, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity;

(h) contain that handlebar has and sequence number: 2 aminoacid sequence has the aminoacid sequence that is equal to or greater than 90% identity, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity; And

(i) contain and sequence number: polynucleotide or sequence number that 1 base sequence is formed: the polynucleotide that the complementary base sequence of 1 base sequence is formed, hybridize under stringent condition, and the polynucleotide of polynucleotide encoding in addition of the protein with O-acetylhomoserine sulfydryl enzymic activity.

(3) polynucleotide described in above-mentioned (1), it contains by sequence number: the polynucleotide that 1 base sequence is formed.

(4) polynucleotide described in above-mentioned (1), it contains handlebar by sequence number: the protein that 2 aminoacid sequence is formed is polynucleotide encoding in addition.

(5) each described polynucleotide in above-mentioned (1)～(4), it is DNA.

(6) protein, it is by each described polynucleotide encoding in above-mentioned (1)～(5).

(7) carrier, it contains each described polynucleotide in above-mentioned (1)～(5).

(7a) carrier described in above-mentioned (7), it contains the expression framework with following (x)～(y) element:

(x) promotor that in yeast cell, can transcribe;

(y) each described polynucleotide in above-mentioned (1)～(5), its with this promotor to combine forward or backwards; And

(z) with the Transcription Termination and the polyadenylic acidization signal relevant, that in yeast, work of RNA molecule.

(8) yeast has wherein imported the carrier described in above-mentioned (7).

(9) yeast described in above-mentioned (8), it reduces hydrogen sulfide generation energy by importing the carrier described in above-mentioned (7).

(10) yeast described in above-mentioned (9), it reduces hydrogen sulfide and generates energy by increasing the protein expression amount described in above-mentioned (6).

(11) manufacture method of drinks, each described yeast in its use above-mentioned (8)～(10).

(12) manufacture method of the drinks described in above-mentioned (11), its drinks of brewageing is a malt beverage.

(13) manufacture method of the drinks described in above-mentioned (11), its drinks of brewageing is a grape wine.

(14) drinks, each described method manufacturing in its use above-mentioned (11)～(13).

(15) evaluation method, it is to use according to having sequence number: the primer or the probe of the O-acetylhomoserine sulfydryl enzyme gene base sequence design of 1 base sequence, estimate the method that tested zymic hydrogen sulfide generates energy.

(15a) according to the method described in above-mentioned (15), sort hydrogen sulfide and generate the yeast method that to have reduced.

(15b) use the yeast that sorts by method described in (15a) to make the method for drinks (for example beer).

(16) evaluation method, it is to have sequence number by cultivating tested yeast, measuring: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, to estimate the method that tested zymic hydrogen sulfide generates energy.

(16a) use the method described in above-mentioned (16) to estimate tested yeast, sort the high yeast of O-acetylhomoserine sulfydryl enzyme gene expression amount, sort hydrogen sulfide and generate the yeast method that can reduce.

(16b) use the yeast that sorts according to method described in above-mentioned (16a), make the method for drinks (for example beer).

(17) zymic system of selection, it comprises cultivates tested yeast, quantification of protein or mensuration described in above-mentioned (6) is had sequence number: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, select to generate the corresponding aforementioned protein mass of energy or the tested yeast of aforementioned gene expression amount with purpose hydrogen sulfide.

(17a) cultivate tested yeast, mensuration hydrogen sulfide generates energy or O-acetylhomoserine sulfydryl enzymic activity, selects purpose hydrogen sulfide to generate the tested zymic yeast system of selection of energy or O-acetylhomoserine sulfydryl enzymic activity.

(18) the yeast system of selection described in above-mentioned (17), it comprises cultivation standard yeast and tested yeast, mensuration has sequence number: the expression amount of O-acetylhomoserine sulfydryl enzyme gene in each yeast of 1 base sequence, select the tested yeast of this gene than standard yeast high expression level.

(19) the yeast system of selection described in above-mentioned (17), it is cultivation standard yeast and tested yeast, to the quantification of protein described in above-mentioned in each yeast (6), the tested yeast of selecting this protein mass to Duo than the standard yeast.That is, cultivate multiple yeast, the protein described in above-mentioned in each yeast (6) is carried out quantitatively, select the wherein many tested yeast of this protein mass.

(20) manufacture method of drinks, it is characterized in that, use the yeast described in above-mentioned (8)～(10) and, be used for the fermentation that drinks is made, regulate the hydrogen sulfide growing amount by any yeast in the yeast of the selection of the method described in above-mentioned (17)～(19).

Drinks manufacture method according to use of the present invention shape matter transformed yeast can consume hydrogen sulfide rapidly by O-acetylhomoserine sulfydryl enzyme, the concentration of hydrogen sulfide in brewage and the product can be suppressed to very low-level, thereby produce the drinks of fragrance excellence.

Description of drawings

Fig. 1 be during beer test is brewageed the Yeast proliferation amount through the time synoptic diagram that changes.X-coordinate is represented fermentation time, and ordinate zou is represented the value of OD660.

Fig. 2 be during beer test is brewageed the extract consumption through the time synoptic diagram that changes.X-coordinate is represented fermentation time, and ordinate zou is represented apparent extract concentration (w/w%).

Fig. 3 is the synoptic diagram of zymic nonScMET17 genetic expression change during beer test is brewageed.X-coordinate is represented fermentation time, and ordinate zou is represented detected signal briliancy.

Fig. 4 be beer test brewage middle parental plant, nonScMET17 high expression level strain Yeast proliferation amount through the time synoptic diagram that changes.X-coordinate is represented fermentation time, and ordinate zou is represented the value of OD660.

Fig. 5 be beer test brewage middle parental plant, nonScMET17 high expression level strain extract consumption through the time synoptic diagram that changes.X-coordinate is represented fermentation time, and ordinate zou is represented apparent extract concentration (w/w%).

Embodiment

Present inventors etc. think, increase by making zymic O-acetylhomoserine sulfydryl enzymic activity, can reduce hydrogen sulfide more efficiently.Study repeatedly based on this imagination, based on the beer yeast gene group information of understanding with disclosed method among the TOHKEMY 2004-283169, isolation identification the non-ScMET17 gene of the distinctive O-acetylhomoserine sulfydryl of the cereuisiae fermentum enzyme of encoding.This base sequence is as sequence number: shown in 1.In addition, by the proteinic aminoacid sequence of this genes encoding as sequence number: shown in 2.

1. polynucleotide of the present invention

At first, the invention provides (a) contains by sequence number: the polynucleotide of the polynucleotide that 1 base sequence is formed; Reach and (b) contain the handlebar sequence number: the protein that 2 aminoacid sequence is formed carries out the polynucleotide of polynucleotide encoding.

As the polynucleotide of object of the present invention, be not only limited to the polynucleotide of coding from the O-acetylhomoserine sulfydryl enzyme of above-mentioned cereuisiae fermentum, also comprise proteinic other polynucleotide of protein tool same function therewith of encoding.As the identical protein of function, for example, can exemplify for (c) by sequence number: one or more aminoacid deletion, replacement, insertion and/or the aminoacid sequence that added are formed in 2 the aminoacid sequence, and the protein with O-acetylhomoserine sulfydryl enzymic activity.

This kind protein, in the aminoacid sequence of sequence number 2, for example, can be by 1～100,1～90,1～80,1～70,1～60,1～50,1～40,1～39,1～38,1～37,1～36,1～35,1～34,1～33,1～32,1～31,1～30,1～29,1～28,1～27,1～26,1～25,1～24,1～23,1～22,1～21,1～20,1～19,1～18,1～17,1～16,1～15,1～14,1～13,1～12,1～11,1～10,1～9,1～8,1～7,1～6 (1～several), 1～5,1～4,1～3,1～2,1 amino-acid residue disappearance, replace, the aminoacid sequence that has inserted and/or added is formed, and the protein with O-acetylhomoserine sulfydryl enzymic activity.In addition, above-mentioned amino-acid residue disappearance, replacement, insertion and/or pair quantity that adds, general preferred less quantity.This kind protein can exemplify into, have (d) and sequence number: 2 aminoacid sequence is equal to or greater than about 60%, be equal to or greater than about 70%, be equal to or greater than 71%, be equal to or greater than 72%, be equal to or greater than 73%, be equal to or greater than 74%, be equal to or greater than 75%, be equal to or greater than 76%, be equal to or greater than 77%, be equal to or greater than 78%, be equal to or greater than 79%, be equal to or greater than 80%, be equal to or greater than 81%, be equal to or greater than 82%, be equal to or greater than 83%, be equal to or greater than 84%, be equal to or greater than 85%, be equal to or greater than 86%, be equal to or greater than 87%, be equal to or greater than 88%, be equal to or greater than 89%, be equal to or greater than 90%, be equal to or greater than 91%, be equal to or greater than 92%, be equal to or greater than 93%, be equal to or greater than 94%, be equal to or greater than 95%, be equal to or greater than 96%, be equal to or greater than 97%, be equal to or greater than 98%, be equal to or greater than 99%, be equal to or greater than 99.1%, be equal to or greater than 99.2%, be equal to or greater than 99.3%, be equal to or greater than 99.4%, be equal to or greater than 99.5%, be equal to or greater than 99.6%, be equal to or greater than 99.7%, be equal to or greater than 99.8%, be equal to or greater than the aminoacid sequence of 99.9% identity, and have the protein of O-acetylhomoserine sulfydryl enzymic activity.The numerical value of above-mentioned homology generally is the bigger the better.

Also have, O-acetylhomoserine sulfydryl enzymic activity for example can be according to Yeast9 (12): 1335-42, and the method described in 1993 is measured.

In addition, the present invention also comprises (e) and contains and sequence number: polynucleotide hybridize under stringent condition that the complementary base sequence of 1 base sequence is formed and coding have the polynucleotide of the proteinic polynucleotide of O-acetylhomoserine sulfydryl enzymic activity; And (f) contain with coding by sequence number: the polynucleotide of polynucleotide hybridize under stringent condition that the complementary base sequence of the proteinic polynucleotide base sequence that 2 aminoacid sequence is formed is formed and the proteinic polynucleotide of encoding with O-acetylhomoserine sulfydryl enzymic activity.

" polynucleotide of hybridize under stringent condition " herein, be meant with sequence number: polynucleotide that the complementary base sequence of 1 base sequence is formed or encoding sequence number: all or part of of the polynucleotide of 2 aminoacid sequence be probe, the polynucleotide dna that obtains by use colony hybridization method, plaque hybridization method or Southern hybrid method etc.Hybridizing method for example can utilize Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley ﹠amp; Method described in the Sons 1987-1997 etc.

" stringent condition " described in this specification sheets means in the middle and high stringent condition of low stringency condition any." low stringency condition " for example, is 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 32 ℃ condition." middle stringent condition " for example, is 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 42 ℃ condition." high stringent condition " for example, is 5 * SSC, 5 * Denhardt solution, 0.5%SDS, 50% methane amide, 50 ℃ condition.In above-mentioned condition, improve temperature more, can expect the polynucleotide dna that acquisition efficiently has high homology more.The factor of influence hybridization severity is multiple factors such as temperature, concentration and probe concentration, probe length, ionic strength, time, salt concn, and those skilled in the art all can realize same stringent condition by suitable these factors of selecting.

In addition, when using the test kit of market sale in the hybridization, for example, can use Alkphos DirectLabelling Reagents (Amersham Pharmacia corporate system).At this moment, according to incidental explanation scheme in the test kit, with mark probe carry out cultivating a night after, film under 55 ℃ condition, after 1 washing of the lavation buffer solution that contains 0.1% (w/v) SDS, can be detected the polynucleotide dna after the hybridization.

In addition, interfertile polynucleotide, pass through FASTA, homology search softwares such as BLAST, Use Defaults (default) when calculating, can be and encoding sequence number: the polynucleotide of 2 aminoacid sequence are equal to or greater than about 60%, be equal to or greater than about 70%, be equal to or greater than 71%, be equal to or greater than 72%, be equal to or greater than 73%, be equal to or greater than 74%, be equal to or greater than 75%, be equal to or greater than 76%, be equal to or greater than 77%, be equal to or greater than 78%, be equal to or greater than 79%, be equal to or greater than 80%, be equal to or greater than 81%, be equal to or greater than 82%, be equal to or greater than 83%, be equal to or greater than 84%, be equal to or greater than 85%, be equal to or greater than 86%, be equal to or greater than 87%, be equal to or greater than 88%, be equal to or greater than 89%, be equal to or greater than 90%, be equal to or greater than 91%, be equal to or greater than 92%, be equal to or greater than 93%, be equal to or greater than 94%, be equal to or greater than 95%, be equal to or greater than 96%, be equal to or greater than 97%, be equal to or greater than 98%, be equal to or greater than 99%, be equal to or greater than 99.1%, be equal to or greater than 99.2%, be equal to or greater than 99.3%, be equal to or greater than 99.4%, be equal to or greater than 99.5%, be equal to or greater than 99.6%, be equal to or greater than 99.7%, be equal to or greater than 99.8%, be equal to or greater than the polynucleotide of 99.9% identity.

In addition, the identity of aminoacid sequence, base sequence can be used BLAST algorithm (Proc.Natl.Acad.Sci.USA 872264-2268,1990 according to Karlin and Altschul; Proc Natl AcadSci USA 90:5873,1993) decision.Developed the program that is called as BLASTN, BLASTX (Altschul SF, et al:J Mol Biol 215:403,1990) based on the BLAST algorithm.When using BLASTN to analyze base sequence, parameter for example is score=100, wordlength=12.In addition, when using the BLASTX analysis of amino acid sequence, parameter for example is score=50, wordlength=3.When using BLAST and Gapped blast program, use default value (default) parameter of each program.

2. protein of the present invention

The present invention also provides by any encoded protein matter in the above-mentioned polynucleotide (a)～(i).Preferred protein of the present invention, by sequence number: one or more aminoacid deletion, replacement, insertion and/or the aminoacid sequence that added are formed in the aminoacid sequence in 2, and the protein with O-acetylhomoserine sulfydryl enzymic activity.

This kind protein for example, can exemplify that the aminoacid sequence that lacked, replace, insert and/or added for amino-acid residue by above-mentioned quantity in the aminoacid sequence of sequence number 2 forms, and the protein with O-acetylhomoserine sulfydryl enzymic activity.In addition, this kind protein can exemplify to containing and sequence number: 2 aminoacid sequence has as the aminoacid sequence of above-mentioned homology and protein with O-acetylhomoserine sulfydryl enzymic activity.

This kind protein, can use the site-directed mutagenesis method described in (" molecular cloning " the 3rd edition), " Current Protocols in MolecularBiology ", " Nuc.Acids.Res., 10,6487 (1982) ", " Proc.Natl.Acad.Sci.USA; 79; 6409 (1982) ", " Gene, 34,315 (1985) ", " Nuc.Acids.Res.; 13; 4431 (1985) ", " Proc.Natl.Acad.Sci.USA, 82,488 (1985) " etc. to obtain.

The amino-acid residue that is equal to or greater than 1 in the protein amino acid sequence of the present invention is lacked, is replaced, is inserted and/or added, be meant in same sequence arbitrarily and on the position of one or more aminoacid sequence, one or more amino-acid residues disappearance is arranged, replace, insert and/or additional meaning, disappearance, replace, insert and add in 2 kinds or also can take place simultaneously greater than 2 kinds.Below, represent the amino-acid residue that can replace mutually for example.The amino-acid residue that comprises in same group can replace mutually.

A group: leucine, Isoleucine, nor-leucine, Xie Ansuan, norvaline, L-Ala, 2-aminobutyric acid, methionine(Met), neighbour-methyl Serine (o-methylserine), tertiary butyl glycine (t-butylglycine), tertiary butyl L-Ala (t-butyl alanine), Cyclohexylalanine (cyclohexyl alanine); B group: aspartic acid, L-glutamic acid, different aspartic acid, isoglutamic acid (isoglutamic acid), 2-aminoadipic acid, the amino suberic acid (2-aminosuberic acid) of 2-; C group: l-asparagine, glutamine; D group: Methionin, arginine, ornithine, 2,4-diamino-butanoic, 2,3-diaminopropionic acid; E group: proline(Pro), 3-oxyproline, 4-oxyproline; F group: Serine, Threonine, homoserine; G group: phenylalanine, tyrosine.

Protein of the present invention can pass through the chemosynthesis manufactured of Fmoc method (fluorenylmethoxycarbonyl), tBoc method (t-Butyl Oxy Carbonyl) etc.Also can utilize the peptide synthesizer of Advanced Chem Tech corporate system, Perkin Elmer corporate system, Farumashia corporate system, Protein TechnologiesInstruments corporate system, Synthecell-Vega corporate system, Perceptive corporate system, Shimadzu Seisakusho Ltd.'s corporate system etc. to carry out chemosynthesis.

3. carrier of the present invention and imported the shape matter transformed yeast of this carrier

Secondly, the invention provides the carrier that contains above-mentioned polynucleotide.Carrier of the present invention relates to contain the carrier of each described polynucleotide (DNA) in above-mentioned (a)～(i).The common following formation of carrier of the present invention, it contains the promotor that (x) can transcribe in yeast cell; (y) with this promotor with each described polynucleotide (DNA) among bonded, above-mentioned (a)～(i) forward or backwards; And (z) contain with the Transcription Termination of RNA molecule and polyadenylic acidization signal relevant, that in yeast, work as the expression framework that constitutes the factor.Among the present invention, in the brewage described later, when making the protein high expression level of the invention described above, the expression of each described polynucleotide (DNA) in above-mentioned for promoting (a)～(i) imports these polynucleotide at this promotor forward.

The carrier that uses when importing yeast can utilize in multiple copied type (YEp type), single copy type (YCp type), the chromosomal integration type (YIp type) any.For example, the YEp24 in the YEp type carrier (J.R.Broach et al., Experimental Manipulation of Gene Expression, Academic Press, New York, 83,1983), YCp50 (M.D.Rose et al., gene, 60 in the YCp type carrier, 237,1987), YIp5 (K.Struhl et al., the Proc.Natl.Acad.Sci.USA in the YIp type carrier, 76,1035,1979) all known, and easily obtain.

Be used for regulating the promotor/terminator of yeast genetic expression, if can in brewageing, work, be not subjected to the influence of composition in the mash simultaneously again with yeast, but arbitrary combination.For example can utilize the promotor of glyceraldehyde 3-phosphate dehydro-genase gene (TDH3), the promotor of 3-phoshoglyceric acid kinase gene (PGK1) etc.These genes are cloned, for example can pass through M.F.Tuite et al., EMBO J., and the currently known methods of 1,603 (1982) middle write up obtains at an easy rate.

Can not utilize nutrient defect type mark because of brewageing with yeast, so, the selected marker of using when shape matter transforms can be utilized aminoglycoside-resistant microbiotic (geneticin) gene (G418r), anti-copper gene (CUP1) (Marin et al., Proc.Natl.Acad.Sci.USA, 81,337 1984), the gene of anti-cerulenin the (fas2m, PDR4) (by pure heir of Pigs Kidney etc., biochemical respectively, 64,660,1992; Hussain et al., gene, 101,149,1991) etc.

The carrier of Gou Jianing is imported into host's yeast as mentioned above.Host's yeast can exemplify the yeast arbitrarily for can be used for brewageing, and for example beer, grape wine, pure mellow wine etc. brewages with yeast etc.Specifically, though can exemplify yeast into yeast belong (Saccharomyces) etc., but in the present invention, can use for example Saccharomyces pastorianus W34/70 etc. of cereuisiae fermentum, Saccharomyces carlsbergensisNCYC453, NCYC456 etc., Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954 etc.And, can use the whisky yeast, for example Saccharomycescerevisiae NCYC90 etc.; Also can use association's grape wine for example with No. 1, with No. 3, with No. 4 etc. wine yeasts; For example association's yeast, pure mellow wine with No. 7, with No. 9 etc. saccharomyces sakes, but be not limited thereto.Among the present invention, cereuisiae fermentum for example can preferably use Saccharomyces pastorianus.

Zymic shape matter method for transformation can utilize general spendable known method.For example, can use electroporation " Meth.Enzym.; 194; p182 (1990) ", spheroplast method (Spheroplast method) " Proc.Natl.Acad.Sci.USA; 75 p1929 (1978) ", Lithium Acetate method " J.Bacteriology; 153; p163 (1983) ", Proc.Natl.Acad.Sci.USA, 75 p1929 (1978), Methods in yeastgenetics, method described in the 2000 Edition:A Cold Spring Harbor Laboratory Course Manual etc. is implemented, but is not limited thereto.

More particularly, host's yeast placed in the standard yeast nutrition substratum (for example YEPD substratum " Genetic Engineering.Vol.1, Plenum Press, New York, 117 (1979) " etc.) cultivate, the value when making OD600nm is 1～6.To collect after this culturing yeast centrifugation, wash, the alkalimetal ion, the preferred lithium ion that are about 1～2M with concentration carry out pre-treatment.With this cell in about 30 ℃ of conditions, leave standstill about 60 minutes after, with the DNA (about 1～20 μ g) that imports simultaneously in about 30 ℃ of conditions, left standstill about 60 minutes.Add polyoxyethylene glycol, preferably add approximately 4, the polyoxyethylene glycol of 000Dalton makes ultimate density be about 20%～50%.About 30 ℃, leave standstill about 30 minutes after, with this cell about 5 minutes of about 42 ℃ of conditions, heat treated.Preferably with this cell suspending liquid with the washing of standard yeast nutrition substratum after, put into quantitative fresh standard yeast nutrition substratum, about 30 ℃, left standstill about 60 minutes.Afterwards, be transplanted in the standard nutrient agar that contains the microbiotic that uses as selected marker etc., obtain shape matter transformant.

Other relevant general clone technologies, can be with reference to (" molecular cloning " the 3rd edition), " Methodsin Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) " etc.

4. drinks manufacture method of the present invention and the drinks that obtains according to its method for making

The carrier of the invention described above is imported in the yeast that is fit to brewage as the drinks of manufacturing object, and can be, make needed and reduced hydrogen sulfide content, increased the drinks of fragrance by using its yeast.In addition, the yeast of selecting by following zymic evaluation method of the present invention also can use equally.Drinks as manufacturing object is not limited to this, for example can exemplify the beer taste beverage into beer, sparkling wine etc., grape wine, whisky, pure mellow wine etc.

When making above-mentioned drinks, resulting brewer's yeast replaces can utilizing known gimmick the parental plant in using the present invention.Therefore, raw material, producing apparatus, manufacturing management etc. can not increase cost for making the drinks that reduces hydrogen sulfide content with identical in the past.That is,, can produce the drinks of fragrance excellence using existing facility, not increasing under the condition of cost according to the present invention.

5. zymic evaluation method of the present invention

The present invention relates to, use according to having sequence number: the primer or the probe of the O-acetylhomoserine sulfydryl enzyme gene base sequence design of 1 base sequence, estimate the method that tested zymic hydrogen sulfide generates energy.It is known using the general gimmick of the evaluation method of primer or probe, for example, and as the method for record in No. 01/040514 communique of WO, the Japanese kokai publication hei 8-205900 communique etc.Below, carry out simple declaration with regard to this evaluation method.

At first, prepare tested zymic genome.The preparation method can use known any methods [for example, Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, p130 (1990)] such as Hereford method, Potassium ethanoate method.With resulting genome is object, uses the primer or the probe of base sequence (the preferred ORF sequence) design according to O-acetylhomoserine sulfydryl enzyme gene, measures the specific sequence that whether has its gene or its gene in the tested zymic genome.The design of primer or probe can use known gimmick to carry out.

The detection of gene or specific sequence can use known gimmick to carry out.For example, to contain comprise specific sequence part or all polynucleotide or the polynucleotide of the complementary base sequence of its base sequence use as a primer, another primer use contain the upstream that comprises this sequence or downstream sequence part or all polynucleotide or the polynucleotide of the complementary base sequence of its base sequence, by PCR method amplification zymic nucleic acid, and measure the size etc. of the having or not of amplified material, amplified material molecular weight.The base number that is used for the polynucleotide of primer is generally and is equal to or greater than 10bp, is preferably 15～25bp.In addition, be clipped in the base number between two primers, it is more suitable to be generally 300～2000bp.

The reaction conditions of PCR method is not particularly limited, and for example, can use denaturation temperature: 90～95 ℃, annealing temperature: 40～60 ℃, elongating temperature: 60～75 ℃, cycle number: be equal to or greater than 10 semiprecious conditions.Resulting resultant of reaction can use the electrophoretic method of sepharose etc. etc. to separate, and measures the molecular weight of amplified production.According to this method, whether the molecular weight by amplified production is the size that contains the dna molecular of special part, comes its zymic hydrogen sulfide of prediction and evaluation to generate energy.And the base sequence by analysing amplified thing, can be further the above-mentioned performance of prediction and evaluation more correctly.

Among the present invention, can have sequence number by cultivating tested yeast, measuring: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, estimate tested zymic hydrogen sulfide and generate energy.In addition, the mensuration of O-acetylhomoserine sulfydryl enzyme expression of gene amount can be by cultivating tested yeast, and the product mRNA or the quantification of protein of O-acetylhomoserine sulfydryl enzyme gene carried out.MRNA or proteinic quantitative can use known gimmick.MRNA quantitatively for example can pass through Northern hybrid method, quantitative RT-PCR, proteinic (CurrentProtocols in Molecular Biology, the John Wiley ﹠amp of quantitatively for example can being undertaken by the Western blotting; Sons 1994-2003).

In addition,, by cultivating tested yeast, mensuration has sequence number: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, select to generate the yeast of the corresponding aforementioned gene expression amount of energy, can select to be fit to the yeast that zythepsary needs drinks with purpose hydrogen sulfide.Also can cultivate standard yeast and tested yeast, measure aforementioned expression of gene amount in each yeast, the aforementioned expression of gene amount of standard of comparison yeast and tested zymic is to select required yeast.Specifically, for example, can be by cultivation standard yeast and tested yeast, mensuration has sequence number: the expression amount in each yeast of the O-acetylhomoserine sulfydryl enzyme gene of 1 base sequence, select the tested yeast of this gene, select to be fit to the yeast that required drinks is brewageed than standard yeast high expression level.

Perhaps, can be by cultivating tested yeast, select hydrogen sulfide generate can be low or the high or low yeast of O-acetylhomoserine sulfydryl enzymic activity, to select to be fit to the tested yeast that required drinks is brewageed.

At this moment, tested yeast or standard yeast for example can use the above-mentioned yeast that imports carrier of the present invention, suppress the yeast of polynucleotide (DNA) expression of the invention described above, the yeast of having implemented the sudden change processing, the yeast of spontaneous mutation etc.The hydrogen sulfide growing amount, any the described method that for example can pass through among Brauwissenschaft.31.1 (1978), Applied.Environm.Microbiol.66:4421-4426 (2000), the J.Am.Soc.Brew.Chem.53:58-62 (1995) is measured.O-acetylhomoserine sulfydryl enzymic activity for example can be passed through Yeast 9 (12): 1335-42, and the method described in 1993 is measured.Sudden change is handled, for example can use the physical method of uviolizing, radiation exposure etc., any method of the chemical process by ethyl methane sulfonate (EMS:Ethylmethanesulfonate), N-methyl-N-nitrosoguanidine chemicals treatment such as (N-methyl-N-nitrosoguanidine) etc. carry out (for example, with reference to big island Thailand control write, Biochemistry Experiment method 39 molecular genetics in yeast laboratory methods, p67-75, association publishing centre etc.).

In addition, can be used as the yeast that standard yeast, tested yeast use, can exemplify spendable yeast arbitrarily when brewageing, for example beer, grape wine, pure mellow wine etc. brewages with yeast etc.Specifically, can exemplify yeast into yeast belong (Saccharomyces) etc., in the present invention, can use for example Saccharomyces pastorianus W34/70 etc. of cereuisiae fermentum, Saccharomyces carlsbergensisNCYC453, NCYC456 etc., Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954 etc.But also can use association's grape wine for example with No. 1, with No. 3, with No. 4 etc. wine yeasts; For example association's yeast, pure mellow wine with No. 7, with No. 9 etc. saccharomyces sakes, but be not limited thereto.Among the present invention, cereuisiae fermentum for example can preferably use Saccharomyces pastorianus.Standard yeast, tested yeast can be selected with arbitrary combination from above-mentioned yeast.

Embodiment

Below, be described in detail the present invention by embodiment, but the invention is not restricted to following examples.

Embodiment 1: the clone of New O-acetylhomoserinesulfgenerelace generelace (nonScMET17)

Use the result of the comparative data library searching of putting down in writing among the TOHKEMY 2004-283169, found distinctive New O in the cereuisiae fermentum-acetylhomoserinesulfgenerelace generelace nonScMET17 (sequence number: 1).Based on resulting base sequence information, 3)/nonScMET17_rv (sequence number: 4) be designed for the primer nonScMET17_for of full-length gene that increases separately (sequence number:, the chromosomal DNA of understanding strain Saccharomyces pastorianus Weihenstephaner 34/70 strain with genome is a template, by PCR, obtained to contain the dna fragmentation (about 1.3kb) of nonScMET17 full-length gene.

The nonScMET17 gene fragment that obtains as mentioned above is inserted into pCR2.1-TOPO carrier (invitrogen corporate system) by the TA clone.The base sequence of nonScMET17 gene is analyzed with Sanger method (F.Sanger, Science, 214,1215,1981), to confirm base sequence.

Embodiment 2: beer test is brewageed middle nonScMET17 gene expression analysis

Use cereuisiae fermentum Saccharomyces pastorianus W34/70 strain to carry out beer test and brewage, the mRNA that will extract from the cereuisiae fermentum thalline the fermentation detects with the cereuisiae fermentum dna microarray.

Wheat juice extract concentration 12.69%

Wheat juice capacity 70L

Wheat juice dissolved oxygen concentration 8.6ppm

15 ℃ of leavening temperatures

Yeast input amount 12.8 * 10 ⁶Cells/mL

To fermented liquid through the time sample, observe Yeast proliferation amount (Fig. 1), apparent extract concentration (Fig. 2) through the time change.Meanwhile, the mRNA after the preparation is carried out biotin labeling, it is hybridized at the cereuisiae fermentum dna microarray the sampling of yeast thalline.Signal detection is used GCOS; Gene Chip OperatingSoftware 1.0 (AFFYMETRIX corporate system) carries out.The nonScMET17 gene expression pattern as shown in Figure 3.The result can confirm that the nonScMET17 gene is expressed in the common beer fermentation thus.

The making of embodiment 3:nonScMET17 gene high expression strain

NonScMET17/pCR2.1-TOPO described in the embodiment 1 is cut with restriction enzyme SacI and NotI enzyme, and preparation comprises the dna fragmentation of protein coding zone total length.This fragment is connected with the pYCGPYNot that handles with restriction enzyme SacI and NotI, has made up nonScMET17 high-expression vector nonScMET17/pYCGPYNot.PYCGPYNot is the Yeast expression carrier of YCp type, and the gene of importing is by the promotor high expression level of pyruvate kinase gene PYK1.Selected marker in the yeast comprises aminoglycoside-resistant microbiotic (geneticin) gene G418 ^r, the selected marker in the intestinal bacteria comprises ampicillin resistance gene Amp ^r

Use transforms Saccharomyces pastorianus Weihenstephaner 34/70 strain by the high-expression vector of method for preparing with the method described in the Japanese kokai publication hei 07-303475.Select shape matter transformant with the YPD plate culture medium that contains aminoglycoside antibiotics (geneticin) 300mg/L (1% yeast extract, 2% polyprotein peptone, 2% glucose, 2% agar).

Embodiment 4: the parsing of hydrogen sulfide growing amount during beer test is brewageed

Use parental plant and carried out fermentation test by following condition with the nonScMET17 high expression level strain that embodiment 3 obtains.

Wheat juice extract concentration 12%

Wheat juice capacity 1L

The about 8ppm of wheat juice dissolved oxygen concentration

15 ℃ of leavening temperatures are certain

The yeast input amount 5g yeast thalline/L wheat juice that wets

To fermentation liquid through the time sample, observe Yeast proliferation amount (OD660) (with reference to Fig. 4), extract consumption through the time change (with reference to Fig. 5).To hydrogen sulfide in the fermentation quantitatively, with reference to the gimmick of [Brauwissenschaft.31.1 (1978)] such as Takahashi.Mensuration contains the sample of the hydrogen sulfide of predicting concentration, make the calibration curve of hydrogen sulfide from detected hydrogen sulfide peak area, condition mensuration fermentation liquid with same with the analysis condition of standard test specimen carries out quantitatively the hydrogen sulfide amount from the area of detected hydrogen sulfide and the relation of calibration curve.

Hydrogen sulfide amount when table 1. fermentation stops in the fermentation liquid

	Parental plant	The strain of nonScMET17 high expression level
			H 2S(ppb)	22.1	-

(notes)-; Limit of detection is following (not to detect H ₂The peak of S)

By table 1 as seen, the growing amount of hydrogen sulfide when fermentation stops, for the 22.1ppb of parental plant, its nonScMET17 high expression level strain is below the limit of detection.Thus the result as can be known, by the nonScMET17 high expression level, the growing amount of hydrogen sulfide has reduced significantly.

According to drinks manufacturing process of the present invention, the concentration of hydrogen sulfide can be suppressed to very low level in brewage and the product, so, can produce the drinks of fragrance excellence.

The right of priority of the application advocates Japanese patent application 2005-240351 number (application on August 22nd, 2005) and Japan's patent application 2006-47564 number (application on February 23rd, 2006), the application is all included in the record of its application in.And the application is also all included in the record of other citing document in.

Sequence table (SEQUENCE LISTING)

＜110〉Suntory Ltd (Suntory Limited)

＜120〉O-acetylhomoserine sulfydryl enzyme gene and uses thereof

(O-acetylhomoserinesulfhydorelace gene and its use)

<130>PCT06-0076

<150>JP2005-240351

<151>2005-08-22

<150>JP2006-47564

<151>2006-02-23

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＜213〉yeast (yeast)

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gcctctttcc tactactaca aggtgtcgaa acattatctt tgagagctga aagacacggt 900

gaaaatgcct tgaagttggc caaatggttg gagcagtctc cttacgtatc ctgggtatcc 960

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ttcggtggtg tcttatcctt cggtgttaag gacttgccaa acgctgacaa ggaaactgat 1080

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35 40 45

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50 55 60

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65 70 75 80

Leu Ala Val Ser Ser Gly Gln Ala Ala Gln Thr Leu Ala Ile Gln Gly

85 90 95

Leu Ala His Thr Gly Asp Asn Ile Val Ser Thr Ser Tyr Leu Tyr Gly

100 105 110

Gly Thr Tyr Asn Gln Phe Lys Ile Ser Phe Lys Arg Phe Gly Ile Glu

115 120 125

Ala Arg Phe Val Glu Gly Asp Asn Pro Glu Asp Phe Glu Lys Val Phe

130 135 140

Asp Glu Arg Thr Lys Ala Val Tyr Leu Glu Thr Ile Gly Asn Pro Ser

145 150 155 160

Tyr Asn Val Pro Asp Phe Glu LysIle Val Ala Ile Ala His Lys His

165 170 175

Gly Ile Pro Val Val Val Asp Asn Thr Phe Gly Ala Gly Gly Phe Phe

180 185 190

Cys Gln Pro Ile Lys Cys Gly Ala Asp Ile Val Thr His Ser Ala Thr

195 200 205

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225 230 235 240

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245 250 255

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260 265 270

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Claims (20)

1. polynucleotide of from following (a)～(f) a group, selecting, it is:

(a) contain by sequence number: the polynucleotide of the polynucleotide that 1 base sequence is formed;

(b) contain handlebar by sequence number: the protein that 2 aminoacid sequence is formed carries out the polynucleotide of polynucleotide encoding;

(c) contain handlebar by sequence number: one or more aminoacid deletion, replacement, insertion and/or the aminoacid sequence that added are formed in 2 the aminoacid sequence, and the protein with O-acetylhomoserine sulfydryl enzymic activity carries out the polynucleotide of polynucleotide encoding;

(d) contain that handlebar has and sequence number: 2 aminoacid sequence has the aminoacid sequence that is equal to or greater than 60% identity, and the protein with O-acetylhomoserine sulfydryl enzymic activity carries out the polynucleotide of polynucleotide encoding;

(e) contain and sequence number: the polynucleotide hybridize under stringent condition that the complementary base sequence of 1 base sequence is formed, and have the polynucleotide that the protein of O-acetylhomoserine sulfydryl enzymic activity carried out polynucleotide encoding; And

(f) contain the handlebar sequence number: the protein that 2 aminoacid sequence is formed carries out the polynucleotide hybridize under stringent condition of the complementary base sequence composition of polynucleotide encoding base sequence, and the protein with O-acetylhomoserine sulfydryl enzymic activity is carried out the polynucleotide of polynucleotide encoding.

2. according to the polynucleotide described in the claim 1, it is selected from following (g)～(i) one group:

(g) contain by sequence number: 2 aminoacid sequence or sequence number: 1～10 aminoacid deletion, replacement, insertion and/or the aminoacid sequence that added are formed in 2 the aminoacid sequence, and the protein with O-acetylhomoserine sulfydryl enzymic activity carries out the polynucleotide of polynucleotide encoding;

(h) contain and have and sequence number: 2 aminoacid sequence has the aminoacid sequence that is equal to or greater than 90% identity, and the protein with O-acetylhomoserine sulfydryl enzymic activity carries out the polynucleotide of polynucleotide encoding; And

(i) contain and sequence number: polynucleotide or sequence number that 1 base sequence is formed: the polynucleotide hybridize under stringent condition that the complementary base sequence of 1 base sequence is formed, and the protein with O-acetylhomoserine sulfydryl enzymic activity is carried out the polynucleotide of polynucleotide encoding.

3. according to the polynucleotide described in the claim 1, it contains by sequence number: the polynucleotide that 1 base sequence is formed.

4. according to the polynucleotide described in the claim 1, it contains handlebar by sequence number: the protein that 2 aminoacid sequence is formed carries out polynucleotide encoding.

5. according to each described polynucleotide in the claim 1～4, it is DNA.

6. protein, it is by each described polynucleotide encoding in the claim 1～5.

7. carrier, it contains each described polynucleotide in the claim 1～5.

8. a primary yeast has wherein imported the carrier described in the claim 7.

9. according to the yeast described in the claim 8, it reduces hydrogen sulfide generation energy by importing the carrier described in the claim 7.

10. according to the yeast described in the claim 9, it increases by making the protein expression amount described in the claim 6, reduces hydrogen sulfide and generates energy.

11. the manufacture method of a drinks, it uses each described yeast in the claim 8～10.

12. according to the manufacture method of the drinks described in the claim 11, its drinks of brewageing is a malt beverage.

13. according to the manufacture method of the drinks described in the claim 11, its drinks of brewageing is a grape wine.

14. a drinks, it uses each described method manufacturing in the claim 11～13.

15. an evaluation method, it uses according to having sequence number: the primer or the probe of the base sequence design of the O-acetylhomoserine sulfydryl enzyme gene of 1 base sequence, and estimate tested zymic hydrogen sulfide and generate energy.

16. an evaluation method, it has sequence number by cultivating tested yeast, measuring: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, and estimate tested zymic hydrogen sulfide and generate energy.

17. zymic system of selection, it comprises cultivates tested yeast, quantification of protein described in the claim 6 or mensuration are had sequence number: the O-acetylhomoserine sulfydryl enzyme expression of gene amount of 1 base sequence, select tested yeast with purpose hydrogen sulfide generation corresponding aforementioned protein mass of energy or aforementioned gene expression amount.

18. according to the zymic system of selection described in the claim 17, it comprises cultivation standard yeast and tested yeast, mensuration has sequence number: the expression amount of O-acetylhomoserine sulfydryl enzyme gene in each yeast of 1 base sequence, select the tested yeast of this gene than standard yeast high expression level.

19. according to the zymic system of selection described in the claim 17, it comprises cultivation standard yeast and tested yeast, to the quantification of protein described in the claim 6 in each yeast, and the tested yeast of selecting this protein mass to Duo than the standard yeast.

20. the manufacture method of a drinks, it is characterized in that, use the yeast described in the claim 8～10 and, be used for the fermentation that drinks is made, regulate the growing amount of hydrogen sulfide by any yeast in the yeast of the selection of method described in the claim 17～19.

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