JP2011223951A - Brewing of beer yeast highly discharging sulfurous acid - Google Patents

Abstract

A self-cloning yeast that discharges sulfurous acid with high efficiency and produces little hydrogen sulfide. More specifically, the present invention provides a self-cloning brewer's yeast having high sulfite high-efficiency and low hydrogen sulfide production ability.
A self-cloning yeast strain that highly expresses a gene SSU1 encoding a protein that excretes sulfite, particularly a self-cloning beer yeast strain that highly expresses SSU1.
[Selection figure] None

Description

  The present invention relates to a yeast having enhanced sulfite excretion ability and suppressed hydrogen sulfide production and a method for producing such yeast. In particular, the present invention relates to a brewer's yeast having enhanced sulfite discharging ability and suppressed hydrogen sulfide production and a method for producing such a brewer's yeast.

Hydrogen sulfide that can be produced by yeast in the production of fruit and other alcoholic beverages, bread and the like has been a problem in that it gives an unfavorable aroma to the product. For example, some sulfur-containing compounds reduce the quality of alcoholic beverages such as brewed sake. Such sulfur-containing compounds include sun odor causing substance MBT (3-methyl-2-butene-1-thiol) and leek odor causing substance 2M3MB (2-mercapto-3-methyl-1-butanol). It is. These sulfur-containing compounds are thought to be related to the production of hydrogen sulfide produced during brewing. Therefore, breeding of low hydrogen sulfide producing yeast has been desired.
Several reports have been made on low hydrogen sulfide producing yeast and its breeding. For example, it has been reported that yeasts that suppress hydrogen sulfide production have been created by enhancing expression of enzyme genes involved in sulfide metabolism (Patent Documents 1 to 3). On the other hand, these methods depend on the yeast strain, and it has been reported that the effect cannot be obtained depending on the yeast strain (Non-patent Document 1). In addition, it has been reported that a low hydrogen sulfide-producing yeast was isolated from a sulfur-containing amino acid analog resistant strain (Patent Document 4). Furthermore, a method for selecting a yeast having a low hydrogen sulfide-producing ability by using a specific primer set has been reported (Patent Document 5).
On the other hand, since sulfite has an antioxidant effect on beer and the like, the amount of sulfite is increased by increasing the amount discharged from yeast (Non-patent Documents 2 and 3) or by enhancing the expression of enzyme genes involved in sulfide metabolism. Discharging (Patent Document 6) has been performed.

Japanese Patent Laid-Open No. 5-192155 Japanese Unexamined Patent Publication No. H5-244955 JP 7-303475 A JP-A-8-214869 JP2007-319095 WO2007 / 08659

Applied Environmental Microbiology Vol.66, p4421-4426 (2000) Journal of Biotechnology Vol.50, p75-87 (1996) Nature Biotechnology Vol.14, p1587-1591 (1996)

As described above, although there are reports on yeasts that excrete sulfurous acid at a high rate, yeasts with low hydrogen sulfide production ability, and methods for breeding these yeasts, they are not necessarily effective. For example, the production of yeast with low hydrogen sulfide production often has different effects depending on the yeast strain and medium used. Specifically, Patent Document 3 reports that the amount of hydrogen sulfide produced can be reduced by strongly expressing the MET25 (MET17) gene that metabolizes hydrogen sulfide, but depending on the yeast strain, the effect can be obtained at all. Not reported (Non-patent Document 1).
These methods are achieved by a gene recombination technique using a heterologous gene or by a mutation treatment. When breeding the target yeast by gene recombination technology using such heterologous genes, it is doubtful whether those breeding methods can be implemented immediately by regulations such as the Cartagena method. Moreover, when producing the target yeast by the mutation treatment, a practically useful yeast, for example, a yeast strain suitable for alcohol fermentation is not always obtained. In particular, when mutating the lower surface brewer's yeast, the lower surface brewer's yeast is a higher-order polyploid, so that it is expected that it will be very difficult to obtain a desired yeast strain.
The present invention provides a self-cloning yeast that excretes sulfite with high efficiency and produces little hydrogen sulfide, that is, a self-cloning yeast strain that has a high ability to excite sulfite and has a low ability to produce hydrogen sulfide. More specifically, the present invention provides a self-cloning brewer's yeast having a high sulfite high-efficiency and a low hydrogen sulfide-producing ability, particularly a self-cloning bottom brewer's yeast having such properties.

  The present invention provides a self-cloning yeast strain that highly expresses the gene SSU1 encoding a protein that excretes sulfite, particularly a self-cloning brewer yeast strain that highly expresses SSU1, particularly a self-cloning bottom brewer yeast strain that highly expresses SSU1. .

FIG. 1 is a diagram schematically showing a procedure for producing a brewer's yeast spore isolate that highly expresses SSU1 by transforming a brewer's yeast spore isolate using a PCR product amplified using plasmid pST106 as a template DNA. is there. SC-URA3 represents the SC type URA3 marker, and P _TDH3 represents the TDH3 promoter. FIG. 2 schematically shows a procedure for producing a brewery yeast transformant that highly expresses SSU1 by transforming brewer's yeast with a PCR product amplified using the genome of a brewer's spore isolate that highly expresses SSU1 as a template. FIG. SC-URA3 represents the SC type URA3 marker, and P _TDH3 represents the TDH3 promoter. FIG. 3 is a diagram showing temporal changes in hydrogen sulfide generation in a 5 L scale fermentation test using homo-recombinant brewer's yeast. ◆ is hydrogen sulfide production of FY-2, a non-transformant, ■ is a self-cloning recombinant brewer's yeast homozygous for the highly expressed SC-SSU1 gene, and ▲ is self-cloning homologous for the highly expressed SB-SSU1 gene Represents hydrogen sulfide production of recombinant beer yeast.

The present invention is a self-cloning yeast strain that highly expresses SSU1, which is a gene encoding a protein that excretes sulfite, particularly a self-cloning brewer's yeast strain that highly expresses SSU1. In this specification, “self-cloning” refers to performing genetic manipulation using only the same type of nucleic acid, for example, self-cloning brewer's yeast refers to performing genetic manipulation using only nucleic acid derived from brewer's yeast. . In the present invention, “same species” means that the target species to be genetically manipulated and the species of the source of all nucleic acids used in the genetic manipulation are the same in the biological classification. The brewer's yeast is roughly classified into upper brewer's yeast and lower brewer's yeast, and the upper brewer's yeast is mainly classified into S. cerevisiae. ) And is classified as Saccharomyces pastorianus (S. pastorianus). Thus, for example, S. pastorianus that has been genetically modified using only nucleic acids derived from S. pastorianus so as to highly express the SSU1 gene is a “self-cloning bottom brewer's yeast”. More specifically, the bottom yeast (S. pastorianus) is a hybrid of S. cerevisiae and S. bayanus and retains both genomes. The lower surface yeast genetically modified using is also a “self-cloning lower surface yeast”. For example, a bottom beer yeast into which a nucleic acid molecule containing only a nucleic acid sequence derived from S. cerevisiae or S. bayanus or a hybrid nucleic acid sequence of both is introduced into the genome is a “self-cloning bottom yeast”.
High expression of the SSU1 gene can be achieved at the translational or transcriptional level, but typically it will be convenient to achieve at the transcriptional level. For example, a self-cloning yeast strain that highly expresses the SSU1 gene can be produced by introducing a gene construct containing a DNA fragment having a homologous high expression promoter connected upstream of the SSU1 gene into the same kind of yeast. If necessary, homologous regulatory sequences that enhance transcription such as homologous enhancers can be further connected to the gene construct. The nucleotide sequence of the SSU cerevisiae type SSU1 structural gene of the bottom brewer's yeast is shown in SEQ ID NO: 1, and the amino acid sequence of the SSU1 protein is shown in SEQ ID NO: 2. Further, the nucleotide sequence of the S. bayanus type SSU1 structural gene of the lower surface brewer's yeast is shown in SEQ ID NO: 25, and the amino acid sequence of the SSU1 protein is shown in SEQ ID NO: 26.

As used herein, “gene construct” refers to a nucleic acid molecule that can be inherited in some form to offspring, and typically includes a nucleic acid molecule that encodes a polypeptide or RNA. The gene highly expressed in the present invention is a gene identical to or “homologous” to the gene encoding the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 26. These genes are collectively referred to as the SSU1 gene. The gene “homologous” to the gene encoding the amino acid sequence shown in SEQ ID NO: 2 is a gene having high sequence homology with the nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID NO: 2, for example, 70% or more, preferably Is a gene having a sequence homology of 80% or more, particularly preferably 90% or more. The same applies to a gene that is “homologous” to the gene encoding the amino acid sequence set forth in SEQ ID NO: 26. The polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is encoded by nucleotide numbers 1016-2389 of the nucleic acid sequence set forth in SEQ ID NO: 1.
Homology can be calculated using programs well known to those skilled in the art, such as FASTA, with standard parameters. Programs for calculating sequence homology include the World Institute of Genetics Bioinformatics / DDBJ Research Center (DDBJ / CIB) (http://www.ddbj.nig.ac.jp/Welcome-j.html) FASTA Ver.2.0, 3.0, 3.2, 3.3 etc. are provided with standard parameters from public institutions in each country. In addition, such highly homologous nucleic acid molecules include nucleic acid molecules that can hybridize with a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 26 under stringent conditions.

Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, the washing conditions in Southern hybridization include conditions corresponding to 50 ° C., 2 × SSC, 0.1% SDS, preferably 1 × SSC, 0.1% SDS, more preferably 0.1 × SSC, 0.1% SDS. Such conditions are well known to those skilled in the art, such as Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. Are listed.
In the present invention, the SSU1 gene and the high expression promoter that are highly expressed are derived from the same species as the yeast to be introduced, respectively, but may be independently derived from the same strain or different strains.

  A gene construct for highly expressing the SSU1 gene used in the present invention can be prepared using methods well known to those skilled in the art. For example, the promoter used in the gene construct capable of expressing the SSU1 gene may be a promoter that can function in yeast cells. For example, promoters such as ADH1, GAL1, and GAL10 can be used. The promoter is preferably a high expression promoter, and such promoters include promoters such as ADH1 promoter, phosphoglycerate kinase (PGK), enolase, glyceraldehyde-3′-phosphate dehydrogenase (TDH3). For molecular biological means including methods for designing, producing and isolating gene constructs as described above, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) and FM Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). Further, transformation of yeast and yeast spore isolate may be performed by any method well known to those skilled in the art, and these include lithium acetate method, protoplast method, electroporation method, particle gun method and the like.

  The yeast with reduced hydrogen sulfide production of the present invention can be typically produced as follows (see FIG. 1). First, DNA fragments having the SSU1 gene sequence at the 3 ′ end and 5 ′ end of a DNA fragment containing a high expression promoter such as TDH3 and an appropriate selection marker are prepared. Such a DNA fragment can be prepared for both SC-type SSU1 (SC-SSU1) and SB-type SSU1 (SB-SSU1). Such fragments can be prepared by methods well known to those skilled in the art. Although the marker for selection is not particularly limited, for example, an auxotrophic marker can be used. Such markers include uracil requirement (URA3), leucine requirement (LEU2), tryptophan requirement (TRP1), histidine requirement (HIS3) markers. This fragment can be used to transform beer yeast spore isolates. The genome insertion state of the resulting transformant can be confirmed by performing PCR using appropriate primers and amplifying a DNA fragment of the expected length. In the obtained spore isolate transformant, the promoter upstream of the SSU1 gene on the genome is replaced with a highly expressed promoter (FIG. 1). Therefore, the obtained transformed spore isolate highly expresses the SSU1 gene.

  Next, PCR can be performed using the obtained spore isolate transformant genomic sequence as a template and appropriate primers to obtain a DNA fragment in which a high expression promoter is connected upstream of the SSU1 gene. The primer to be used is selected so that the DNA fragment flanked by the SSU1 gene and its upstream sequence is amplified as a high expression promoter (FIG. 2). The beer yeast can be transformed using the obtained DNA fragment. A transformant can be selected using, for example, sulfite resistance as an index. Specifically, for example, in a YPD agar medium (1% Yeast extract, 2% Peptone, 2% Glucose, 2% agar) adjusted to pH 3.5 with a final concentration of 75 mM tartaric acid, the final concentration of sodium sulfite is 5 mM. The transformed yeast can be selected by selecting a colony formed by applying at 8 mM and culturing at 25 ° C. Furthermore, by performing PCR using appropriate primers and confirming that a DNA fragment of the expected size is amplified, homologous recombination in which the promoter upstream of the SSU1 gene on the genome was replaced with a highly expressed promoter. Can be confirmed. In the transformed brewer's yeast, the promoter upstream of the SC-SSU1 gene and / or SB-SSU1 gene on the genome is replaced with a high expression promoter (FIG. 2).

The obtained brewer's yeast can be evaluated for the amount of SSU1 gene product (protein), the amount of SSU1 mRNA, sulfite tolerance as an index of sulfite excretion ability, and the ability to produce hydrogen sulfide. The amount of SSU1 protein can be evaluated by, for example, Western blotting, and the amount of mRNA can be evaluated by, for example, Northern blotting, quantitative RT-PCR, or the like. Sulfite resistance can be performed, for example, by the method of Uzuka et al. (J. Ferment. Technol. Vol. 63, p107-114, 1985). In short, 2% glucose, 0.67% Yeast nitrogenbase w / o amino acids (Difco), 0.15% tartaric acid, pH 3 to 3.5 medium, the corresponding yeast is initially cultured at about 2 × 10 ⁶ cells / ml When sodium bisulfite is added from the beginning of the culture and cultured at about 25 ° C. for 1 to 7 days, and the yeast is clearly grown even in the presence of sulfite (for example, 10 ⁷ It can be considered that the yeast is resistant to sulfite when it has grown to the number of yeasts of cells / ml or more.
The concentration of free sulfite in the culture solution can be measured using Hennig's method (JAKOB Untersuchungsmethoden fuer Wein und aehnliche Getraenke p87-89 (1973)). More simply, F-kit sulfurous acid manufactured by JK International Co., Ltd. can be used. This method is a method of quantifying sulfite by reacting dissolved sulfite with sulfite oxidase, reacting the produced hydrogen peroxide with NADH peroxidase, and determining the decreasing NADH by absorbance change at 340 nm. . The sulfite excretion capacity of brewer's yeast is enhanced by the present invention, and generally becomes resistant to sulfite of higher than 0 to 50 ppm, preferably 10 to 50 ppm, particularly preferably 20 to 50 ppm, and the concentration of sulfite in the medium is not transformed. Increase at least 1.5 times more than the body.

  The ability of yeast to produce hydrogen sulfide is determined by, for example, culturing yeast in a static culture medium in SD medium (2% glucose, 0.67% Yeast nitrogen base w / o amino acids (Difco)) at about 25 ° C. for 1 day. It can be evaluated by measuring the amount of hydrogen sulfide dissolved in the slag. The measurement of hydrogen sulfide may be performed by any general method. For example, a certain amount of culture solution is collected, and the culture solution from which the yeast has been separated by centrifugation at 3000 rpm for 10 minutes is placed in a vial containing a stirrer bar, a predetermined amount of sodium chloride is added, 3N hydrochloric acid, an internal standard solution (ethyl methyl sulfide) 10 mg / mL), seal it with an aluminum cap, rotate the stirrer bar at room temperature for 10 minutes to dissolve sodium chloride, and dissolve it in the culture solution from the internal standard ratio by headspace GC-FPD. This can be done by quantifying the hydrogen concentration.

  Yeast introduced with a gene construct capable of increasing SSU1 gene expression according to the present invention is about 80% or less, preferably about 70% or less, particularly preferably compared to the corresponding non-transformant yeast of the same species, preferably the same strain. Hydrogen sulfide production per unit time is reduced to about 60% or less. More specifically, the yeast of the present invention, particularly the brewer's yeast, is at least about 45% to 80%, preferably at least about 40% to 70% relative to the corresponding homologous, preferably non-transformant yeast of the same strain, Particularly preferably, the hydrogen sulfide production is reduced to at least about 45% to 60%. The decrease in the amount of hydrogen sulfide produced can be evaluated using as an indicator the decrease in the amount of hydrogen sulfide dissolved in the culture supernatant after culturing the yeast for about 1 to 7 days under the conditions described above.

  The high sulfite high emission and / or low hydrogen sulfide production yeast obtained by the present invention can be used for equivalent applications under the same conditions as the corresponding non-transformant yeast of the same species or the same strain. Further, it can be used under the condition where the concentration of sulfurous acid is increased, or a part or all of the measures for removing hydrogen sulfide can be omitted.

Preparation of SSU1 high-expressing bottom beer yeast spore isolate by self-cloning (1) Preparation of gene construct for constructing SSU1 high expression system A gene construct for high expression of SSU1 gene of bottom beer yeast was prepared. Since the bottom brewer's yeast has S. cerevisiae (SC) type and S. bayanus (SB) type genomes, PCR amplified fragments are obtained using the following primers, and these are obtained for each of the SC type and SB type. Used as a gene construct for high expression of SSU1 gene.

Primer for constructing gene construct for constructing SC type SSU1 gene high expression system
SC-SSU1f-pST: 5'-TACAGCTTTCCCCTAGTAACGATTGTTGATTGAGCTCAGAAGCTTTTCAATTCAATTCATC-3 'SEQ ID NO: 3
SC-SSU1r-pST: 5'-CAAGTACCCAATTGGCAACCATTTTTTTCTTGTACTTGTCTTTATTCGAAACTAAGTTCTTGG-3 'SEQ ID NO: 4

Primer for preparing gene construct for constructing SB type SSU1 gene high expression system
SB-SSU1f-pST: 5'-CGCTATAACAACGACTACTGATCGAATTCAGGATACAAACAGCTTTTCAATTCAATTCATC-3 'SEQ ID NO: 5
SB-SSU1r-pST: 5'-TGAGCATCCAACTAGCGACCATTCTCTGCTATGTTACCTGTTTATTCGAAACTAAGTTCTTGG-3 'SEQ ID NO: 6

PCR conditions are as follows.
Each primer final concentration 0.2μM
Template DNA Plasmid pST106
Total reaction solution 50μL
35 cycles of 98 ° C for 10 seconds, 55 ° C for 15 seconds, 68 ° C for 3 minutes
68 ℃ for 7 minutes Enzyme PrimeStar (Takara Bio)

Plasmid pST106, which is a template DNA, contains the TDH3 (glyceraldehyde-3′-phosphate dehydrogenase) promoter derived from S. cerevisiae, which is a high expression promoter, and the URA3 gene of S. cerevisiae, which is a selectable marker gene (FIG. 1). The nucleotide sequence of the PCR target is shown as SEQ ID NO: 7.
After the PCR was completed, unreacted primers were removed with QIAquick PCR Purification Kit (manufactured by QIAGEN) to obtain a PCR amplification product. PCR amplification products amplified using plasmid pST106 as a template DNA and primers SC-SSU1f-pST and SC-SSU1r-pST or SB-SSU1f-pST and SB-SSU1r-pST The coding sequence of the SSU1 gene or the upstream sequence thereof (FIG. 1). Therefore, when introduced into the lower brewer's yeast that is the host, homologous recombination with the genomic DNA occurs, and as a result, the URA3 marker and the TDH3 promoter are expected to be inserted into the genomic DNA of the yeast.

(2) Transformation of beer yeast spore isolate The bottom brewer yeast spore isolate was transformed using this PCR amplification product. The lower brewer's yeast spore isolate used for transformation is the URA3 gene variant W-1B-ura3 of the spore isolate W-1B of Weihenstephan34 / 70. After collecting and washing the yeast cells in the logarithmic growth phase, 0.1 mL of 0.2 M lithium acetate solution was added to 0.1 mL of yeast cell suspension suspended at 10 ⁹ cells / ml, and reacted at room temperature for 1 hour. Thereafter, 0.1 mL of a 70% polyethylene glycol 4000 solution was added to 0.1 mL of the reaction solution, suspended, and about 20 μg of the DNA fragment amplified by PCR was added and reacted at room temperature for 1 hour. After treatment in a water bath at 42 ° C for 5 minutes, the cells are collected, washed, and applied to SD medium (2% glucose, 0.67% Yeast nitorogenbase w / o amino acids (Difco), 2% agar), which is a selective medium. Then, a transformant was selected from the formed colonies. The genome insertion state of the obtained transformant was examined by PCR using the following primers. PCR conditions are the same as described above.

Primer for analysis of yeast strains with high expression of SC type SSU1 gene
SC-SSU1-f603: 5'-TTTTGTATGTCACTGGATGTATAC-3 'SEQ ID NO: 9
SC-SSU1-r1001: 5'-TAGTACGACAGGATTGAAACGACAG-3 'SEQ ID NO: 10

Primer for analysis of yeast strain with high expression of SB type SSU1 gene
SB-SSU1-f206: 5'-AAAGACTCATCGTTGTATTTGGCAC-3 'SEQ ID NO: 11
SB-SSU1-r248: 5'-TATTCATTGAAGTAATCTCTAAAGC-3 'SEQ ID NO: 12

For transformants where the expected PCR product of the fragment length was confirmed, the DNA base sequence was also confirmed, and the PCR amplified fragment used for transformation was inserted at the position of the SC-type SSU1 gene or SB-type SSU1 gene It was confirmed. The brewer's spore isolate transformed with the highly expressed TDH3 promoter connected to the SC-type SSU1 gene was designated as W-1B / P _TDH3- SC-SSU1, and the highly expressed TDH3 promoter was connected to the SB-type SSU1 gene. The transformant of the brewer's yeast spore isolate was _designated as W-1B / P _TDH3- SB-SSU1 (FIG. 1).

(3) Fermentation test of transformed lower surface brewer's yeast spore isolate SC-1S1 gene highly expressed lower surface brewer's yeast spore isolate W-1B / P _{TDH3 -SC} -SSU1 or W- 1B / P _TDH3 -SB-SSU1 was transferred to 100 mL of wort, and the fermentation was started at 15 ° C. with an initial cell concentration of 2 × 10 ⁷ cells / mL. The amount of hydrogen sulfide produced and the amount of sulfurous acid discharged were examined. Hydrogen sulfide and sulfurous acid dissolved in the fermentation broth after the fermentation was measured. For the measurement of hydrogen sulfide, about 7 mL of the culture solution was collected, and 5 mL of the culture solution from which the yeast was separated by centrifugation at 3000 rpm for 10 minutes was placed in a vial containing a stirrer bar, 1.0 g of sodium chloride was added, An internal standard solution (ethyl methyl sulfide 10 mg / mL) 50 μL was added, sealed with an aluminum cap, and the stirrer bar was rotated at room temperature for 10 minutes to dissolve sodium chloride. The dissolved hydrogen sulfide concentration in the culture solution was quantified from the internal standard ratio by headspace GC-FPD. For the measurement of sulfurous acid, F-kit sulfurous acid manufactured by JK International Co., Ltd. was used. In this method, dissolved sulfite is reacted with sulfite oxidase, the produced hydrogen peroxide is reacted with NADH peroxidase, and the decreased NADH is determined by determining the absorbance at 340 nm. Table 1 shows the hydrogen sulfide production amount and sulfite discharge amount of each yeast strain.

Table 1. Hydrogen sulfide production and sulfite emissions from bottom beer yeast spore isolates
It was confirmed that the lower brewer's yeast spore isolate with high expression of SC- or SB-SSU1 gene has reduced hydrogen sulfide production ability and increased sulfite excretion ability under wort fermentation conditions.

Preparation of SSU1 gene high expression bottom brewer's yeast by self-cloning (1) Transformation of bottom brewer's yeast A SSU1 gene high expression transformant of spore isolate of bottom brewer's yeast was obtained. Based on this genomic DNA sequence, We tried to obtain a SSU1 gene high expression transformant of brewer's yeast. The genomic DNAs of transformants W-1B / P _{TDH3 -SC} -SSU1 and W-1B / P _TDH3 -SB-SSU1 of the lower brewer's yeast spore isolate were extracted, respectively. Genomic DNA was extracted by Qiagen Genomic-Tip 20 / G (Qiagen). A PCR amplified fragment containing the SSU1 gene connected to the highly expressed TDH3 promoter was obtained using each genomic DNA as a template (FIG. 2). The primers used are shown below. PCR conditions are the same as those in Example 1 (1).

PCR primers for DNA fragment amplification for the preparation of yeast strains with high expression of SC type SSU1 gene
SC-SSU1-P _TDH3 f: 5'-ATTTAGACAACACACAAATTACAGCTTTCCCCTAGTAACGATTGTTGATTGAGCTCAGACATTATCAATACTGCCATTTC-3 'SEQ ID NO: 13
SC-SSU1r557: 5'-GGAATGATATCCAGTAGCAATACAC-3 'SEQ ID NO: 14

PCR primers for DNA fragment amplification for the production of yeast strains with high expression of SB type SSU1 gene
SB-SSU1-P _TDH3 f: 5'-AGTATACAATCTATATTCACGCTATAACAACGACTACTGATCGAATTCAGGATACAAACCATTATCAATACTGCCATTTC-3 'SEQ ID NO: 15
SB-SSU1-r525: 5'-GTGGTCGGAAGCCATTCGTGAACTG-3 'SEQ ID NO: 16

  After the PCR was completed, unreacted primers were removed with QIAquick PCR Purification Kit (manufactured by QIAGEN) to obtain a PCR amplification product. The upstream of the TDH3 promoter in the obtained PCR amplification product is a 60 bp or 59 bp sequence upstream of the translation start point of the SC- or SB-SSU1 gene (FIG. 2). On the other hand, the SC- or SB-SSU1 gene translation region is connected downstream of the TDH3 promoter. Therefore, this PCR-amplified fragment is homologous to the genomic DNA of the lower brewer's yeast because the 5 'and 3' terminal DNA sequences are homologous to the SC- or SB-SSU1 gene DNA sequence of the lower brewer's yeast. It is expected that the recombination will occur and homologous integration into the genomic DNA of the lower brewer's yeast (FIG. 2). The nucleotide sequence of the SC-SSU1 gene expected to undergo homologous recombination is shown in SEQ ID NO: 17, the amino acid sequence is shown in SEQ ID NO: 18, the nucleotide sequence of the SB-SSU1 gene is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20. . The lower surface brewer's yeast used for transformation is FY-2, and the transformation method is the same as that described in Example 1 (2), but before applying directly to the selective medium, it is YPD which is a nutrient medium. The treated yeast cells were suspended in a medium (1% Yeast extract, 2% Peptone, 2% Glucose) and shaken at 25 ° C. all day and night. Transformants were selected using sulfite resistance as an index. To obtain a sulfite resistant transformant, apply sodium sulfite to a YPD agar medium (1% Yeast extract, 2% Peptone, 2% Glucose, 2% agar) adjusted to pH 3.5 with a final concentration of 75 mM tartaric acid, This was performed by selecting colonies formed by culturing at 25 ° C. The final concentration of sulfurous acid was 5 mM to 8 mM. It was examined by PCR whether the obtained colonies were generated by homologous recombination in the expected SC- or SB-SSU1 gene region. In addition, the lower brewer's yeast has both S. cerevisiae and S. bayanus genomes, and thus has two SC-SSU1 genes and two SB-SSU1 genes. This PCR is one in which homologous recombination has occurred for one allele (hetero-recombinant transformant) for each of the SC type SSU1 gene and the SB type SSU1 gene, or both of the two alleles. Whether homologous recombination has occurred (homo-recombinant transformant) can also be determined. The following PCR primers were used.

Primer for analysis of yeast strain with high expression of SC type SSU1 gene
SC-SSU1-f603: 5'-TTTTGTATGTCACTGGATGTATAC-3 'SEQ ID NO: 21
SC-SSU1-r1001: 5'-TAGTACGACAGGATTGAAACGACAG-3 'SEQ ID NO: 22

Primer for analysis of yeast strain with high expression of SB type SSU1 gene
SB-SSU1-f206: 5'-AAAGACTCATCGTTGTATTTGGCAC-3 SEQ ID NO: 23
SB-SSU1-r942: 5'-TGCCAAAGCAAATGTCATGCCCAG-3 'SEQ ID NO: 24

For transformants in which the amplification product of the expected fragment length was confirmed by PCR, the DNA base sequence was also confirmed, and the PCR-amplified fragment used for transformation was inserted at the position of the SC-type SSU1 gene or SB-type SSU1 gene. Confirmed that. Among the transformants of the lower surface brewer's yeast in which the highly expressed TDH3 promoter is connected to the SC-type SSU1 gene on the genome, the heterorecombinant transformant is FY-2 / P _TDH3- SC-SSU1 / SC-SSU1, The transformant was _designated as FY-2 / P _TDH3- SC-SSU1 / P _TDH3- SC-SSU1. Among transformants beer yeast spores isolates the high expression TDH3 promoter was connected to the SB type SSU1 gene on the genome, a hetero transformant _{FY-2 / P TDH3 -SB-} SSU1 / SB-SSU1, homo trait The transformant was FY-2 / P _TDH3 -SB-SSU1 / P _TDH3 -SB-SSU1.

(2) Fermentation test of transformed bottom brewer's yeast (comparison between hetero-recombinant bottom brewer's yeast and homo-recombinant bottom brewer's yeast)
As described above, there are two SC-SSU1 genes and two SB-SSU1 genes in the bottom brewer's yeast. Therefore, fermentation tests of SC-SSU1 gene hetero-recombinant brewer's yeast and homo-recombinant brewer's yeast were conducted, and hydrogen sulfide production and sulfite emission were measured in the same manner as described in Example 1 (3). And compared. The results are shown in Table 2.

Table 2. Comparison of hetero recombinant beer yeast and homo recombinant beer yeast
Compared with the hetero-recombinant underside brewer's yeast, the homo-recombinant underside brewer's yeast showed a lower hydrogen sulfide production and a higher sulfite discharge.

(3) Fermentation test of homo-transformed bottom beer yeast (5L fermentation test using homo-recombinant bottom beer yeast)
SC-SSU1 high expression underside beer yeast, homo recombinant W / P _TDH3- SC-SSU1 / P _TDH3- SC-SSU1, and SB-SSU1 high expression underside beer yeast , Homo-recombinant W / P _TDH3- SB-SSU1 / P _TDH3- SB-SSU1 were each transferred to 5 L of wort, and the initial cell concentration was 1.5 × 10 ⁷ cells / mL at 12 ° C. Fermentation was started and hydrogen sulfide production under beer fermentation conditions was examined. The fermentation liquor was collected at regular intervals, and hydrogen sulfide and sulfurous acid dissolved in the fermentation liquor were measured. The method for measuring hydrogen sulfide is the same as that described in Example 1 (3). The measurement results are shown in FIG. The amount of hydrogen sulfide produced by the SSU1 gene highly expressing brewer's yeast was significantly suppressed. Table 3 shows the amount of sulfurous acid produced on the seventh day of fermentation. The method for measuring the amount of sulfurous acid is the same as the method shown in Example 1 (3).

Table 3. Amount of sulfurous acid discharged from bottom beer yeast transformants

  Furthermore, the causative substance MBT (3-methyl-2-butene-1-thiol) causing sunlight odor and the causative substance 2M3MB (2-mercapto-3-methyl-1-butanol) causing leek odor were also measured. In 500 mL of sample, 25 mL of 2 mM p-HMB (p-hydroxymercurybenzoic acid) dissolved in 0.1 M Tris and 500 μL of 20 mM tert-butyl-4-methoxyphenol (BHA) ethanol solution, 500 ng / mL as an internal standard solution 4 -Methoxy-2-methyl-2-mercaptobutane (4M2M2MB) in ethanol (100 mL) was added, sealed, and stirred vigorously with a stir bar for 10 minutes at room temperature. Sulfur-containing compounds adsorb to p-HMB, a mercury compound. After this reaction product was adsorbed on a Dowex-1 (strong anion exchange resin) column, the column was washed with 100 mL of 0.1 M sodium acetate buffer (pH 6) containing 0.2 mM tert-butyl-4-methoxyphenol. The sulfur-containing compound is eluted from the column with 100 mL of 0.1 M sodium acetate buffer (pH 6) containing 10 mg / ml L-cysteine hydrochloride monohydrate. The eluate was extracted twice with 0.5 mL of ethyl acetate and 5 mL of dichloromethane, and the organic solvent layer was dehydrated with anhydrous sodium sulfate, concentrated to 100 μL with nitrogen gas, and subjected to GC analysis equipped with a mass spectrometer. The amount of sun odor causative substance MBT and leek odor causative substance 2M3MB was quantified from the internal standard ratio. The results are shown in Table 4.

Table 4. Content of S-type odorous substances under wort fermentation conditions using bottom brewer's yeast transformants
The bottom brewer's yeast in which the SC-SSU1 or SB-SSU1 gene was highly expressed also produced a small amount of MBT and 2M3MB.

Claims (5)

  A gene construct capable of expressing a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 26 or a gene having a sequence capable of hybridizing under stringent conditions with a nucleic acid molecule encoding the polypeptide. Self-cloning brewer's yeast introduced.   A self-cloning brewer's yeast introduced with a gene construct capable of expressing a gene encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 26.   3. The self-cloning brewer's yeast according to claim 1 or 2, wherein the production amount of hydrogen sulfide is reduced to 60% or less with respect to a corresponding non-transformant yeast of the same kind in which no gene construct is introduced.   The self-cloning brewer's yeast according to any one of claims 1 to 3, wherein the amount of sulfite excretion is increased by 60% or more relative to a corresponding non-transformant yeast of the same kind in which no gene construct is introduced.   The self-cloning beer yeast according to any one of claims 1 to 4, wherein the yeast is a bottom beer yeast.