JPH05192155A - Gene capable of suppressing production of hydrogen sulfide with yeast and yeast for brewing containing the same gene transduced thereinto - Google Patents

Abstract

PURPOSE:To suppress the ability to produce hydrogen sulfide and enable obtaining of beer in a short time by transducing a gene (NHS5) capable of exhibiting a prescribed base sequence suppressing the production of the hydrogen sulfide with a yeast. CONSTITUTION:A plasmid (pHS5), capable of suppressing H2S and having a well-known gene capable of suppressing H2S is subcloned to prepare a gene (NHS5) that is a protein having an amino acid sequence composed of a sequence of bases, etc., expressed by the formula in which the interval between prescribed positions is coded in the base sequence. The resultant NHS5 is then treated with a restriction enzyme to afford a plasmid (e.g. YCpAG-5SS), which is subsequently transduced into a yeast of the genus Saccharomyces for brewing to provide a transformant. The obtained transformant is then inoculated into a wort containing a hop added thereto and fermented to reduce the amount of the produced hydrogen sulfide from the parent strain by, 20-40%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene that suppresses hydrogen sulfide production in yeast, a protein having a specific nucleotide sequence of this gene, and a brewing yeast into which the gene has been introduced.

[0002]

2. Description of the Related Art It is unfavorable in terms of quality to sense the smell of hydrogen sulfide (H ₂ S) in Pilsner type light beer which is mainly sold in Japan. However, brewer's yeast (bottom yeast) used in the same type of beer production has a characteristic of producing this hydrogen sulfide in the main fermentation step. Therefore, controlling the amount of hydrogen sulfide in beer below a threshold value in post-fermentation and subsequent steps is an important issue in beer production.
The solution was to extend the storage period.

Therefore, studies on factors affecting the production of hydrogen sulfide and breeding studies on low-hydrogen sulfide-producing yeasts by the mutation method and cell fusion method have been reported. A yeast suitable for use as a brewing yeast has not been obtained so far because it not only reduces the amount of hydrogen sulfide produced but also affects other brewing characteristics (fermentation rate, beer flavor) of the yeast.

[0004]

However, recently, a new trial using the gene manipulation technology developed in recent years was reported by Tezuka et al. (Proceedings of the Japan Fermentation Engineering Society Conference, page 5 (1988). ), Biotechnology in brewing, Industrial Publishing (1990), p. 82). It is the isolation of three DNA fragments with hydrogen sulfide-suppressing activity from the chromosome of yeast (x-2180), which does not normally produce hydrogen sulfide outside the cells, and each DNA fragment is introduced into bottom brewer's yeast to reduce hydrogen sulfide. This is an attempt to breed the producing yeast.

This report attempts to transfer only the property of yeast that does not produce hydrogen sulfide to the bottom yeast by using genetic engineering technology. In particular, hydrogen sulfide production without changing the brewing characteristics of the bottom yeast. It is considered that it is superior to the conventional breeding method by mutation or cell fusion in that only the ability can be reduced. However, in this report, it is unknown where the hydrogen sulfide suppressor gene exists on the isolated DNA fragment, the amino acid sequence of the protein encoded in that part, and the decrease in the amount of hydrogen sulfide is due to the flavor of beer. However, it is not possible to practically produce beer only with the reported findings.

An object of the present invention is to find a gene that can practically produce beer by suppressing only hydrogen sulfide production without changing the brewing characteristics of yeast, and to introduce this gene into yeast.

[0007]

The present invention is a gene (NHS5) which suppresses the production of hydrogen sulfide in yeast and whose nucleotide sequence is represented by Chemical formulas 5-8. In addition, the present invention includes the following chemical formulas
In the nucleotide sequence of the gene (NHS5) shown by
It is a protein with an amino acid sequence encoded by Furthermore, the present invention is a brewer's yeast of the genus Saccharomyces into which the gene (NHS5) has been introduced to reduce the amount of hydrogen sulfide produced. The present invention also provides a D having a nucleotide sequence that allows the protein having the amino acid sequence to be expressed in yeast.
It is a brewer's yeast of the genus Saccharomyces in which NA is introduced to reduce the amount of hydrogen sulfide produced. (Base sequence)

[0008]

[Chemical 5]

[0009]

[Chemical 6]

[0010]

[Chemical 7]

[0011]

[Chemical 8]

[0012] Specifically, first, based on the previously reported plasmid containing the hydrogen sulfide suppressor gene, it was found by subcloning experiments where the hydrogen sulfide suppressor gene is present on this plasmid. That is, about −12 Kb of x-2180 on a plasmid (hereinafter referred to as pHS5)
The strain-derived DNA fragment was cleaved with various restriction enzymes, and it was examined which of a plurality of generated fragments contained the hydrogen sulfide suppressor gene. As a result, it was found that the hydrogen sulfide suppressor gene was present at the positions shown in Chemical formulas 5 to 8 above, and this gene was identified as NHS.
It was named 5. Therefore, the structure of NHS5 was examined in more detail by examining the DNA base sequence of this portion.
The nucleotide sequence obtained as a result and the amino acid sequence deduced therefrom are shown in Chemical formulas 5-8.

From the above results, the location of NHS5 was identified. Therefore, as will be shown in the next Example, three types of vectors, that is, a YEp type vector having a replication origin derived from 2 μDNA and a centromere portion of the yeast chromosome are included. YC
NHS5 for p-type and YIp-type for integrating yeast-derived DNA into yeast chromosome without NHS5
Were prepared, and a fermentation test was performed using yeast for brewing into which these plasmids were introduced.

As a result of the fermentation test, the amount of hydrogen sulfide in the fermentation gas of the strain having these three plasmids was 20 to 40 compared with the parent strain.
%, And NHS5 was found to suppress hydrogen sulfide production in the parent strain using any type of vector. In addition, as a result of a sensory test on the aroma of the fermentation broth (young beer) at the end of fermentation, it was confirmed that the hydrogen sulfide odor was reduced in the NHS5-introduced strain compared to the parent strain,
No off-flavor other than hydrogen sulfide was felt. The above results make it possible to control the hydrogen sulfide odor by using the technique of the present invention, which has been the only countermeasure for extending the aging period in the past, and as a result, compared with the conventional technique, the odor of immature odor can be reduced in a shorter period of time. It shows that no beer can be produced.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, a gene capable of practically producing beer by suppressing hydrogen sulfide production in yeast can be obtained. Therefore, by using a yeast into which this gene has been introduced, the brewing characteristics of yeast can be changed. It is possible to produce beer in a short period of time without the ability to generate sulfurization.

[0016]

[Examples] With respect to the present invention, the determination of the DNA base sequence and amino acid sequence of NHS5 will be described below in Example 1.
The method of constructing the plasmid used to introduce 5 into yeast was carried out in Example 2, and the introduction of the constructed plasmid into yeast was carried out in Example 3, and the fermentation test was carried out by yeast in which hydrogen sulfide production was reduced by the gene. In Example 4,
Each will be described in more detail.

The base sequence shown in Example 1 is any base sequence as long as it encodes the amino acid sequence shown in Chemical formulas 5 to 8 above and a protein having the amino acid sequence is expressed in yeast. May be. In particular, known base sequence portions that control transcription and translation of genes may be used in appropriate combination. The amino acid sequences shown in Chemical Formulas 5 to 8 may have deletions, substitutions, additions, etc. to some of the amino acids as long as the gene has a hydrogen sulfide suppressing action in yeast.

Next, the plasmid used for introducing the gene into yeast in Example 2 is not limited to any of the three exemplified types, as long as the gene is introduced into yeast and stably retained. In addition, when the gene is integrated into the yeast chromosome, any integration method may be used. For example, a known yeast gene may be added to both ends of the gene, or only the gene itself may be incorporated. G418 shown in Example 2 was used as a selection marker for transformation, and as a resistance gene for the drug, yeast alcohol dehydrogenase (AD) was added before and after the structural gene part in the G418 resistance gene derived from Escherichia coli.
H) It is preferable to use a gene in which a transcription promoter of the gene and a termination signal are linked, from the viewpoint of transformation frequency and stability of the plasmid, but other selectable marker and its resistance gene may be used in combination. Needless to say, a nutritional requirement marker may be used.

Although it was first confirmed by the present inventors that the production amount of hydrogen sulfide in yeast is reduced by using the hydrogen sulfide suppressor gene, the method for producing the used plasmid, the transformation method, etc. The genetic engineering method of the above is a molecular biology, a conventional method used in biology, for example, molecular cloning (Molecular Cloning, Col
d Spring Harbor Laboratory, 1982).

Example 1 The plasmid pHSG5 (literature name: already reported) has been reported.
Proceedings of the Japan Society for Fermentation Engineering, page 5 (1988),
Biotechnology in brewing Industry-style publication (1990)
In order to investigate where the hydrogen sulfide suppressor gene is present on the yeast-derived DNA fragment contained in page 82), more detailed restriction enzyme map construction and subcloning experiments were performed. The results are shown in FIG.

As shown in the restriction map of FIG. 1, NHS5
Was found to reside on the Xba I- Hpa I fragment of approximately 1.5 Kb. Therefore, the nucleotide sequence around the Bgl II cleavage site in the DNA fragment derived from the x-2180 strain was determined by the dideoxy method (Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. USA 74 , 5463).
(1977)).
An open reading frame consisting of p was found. Amino acids by this open reading frame
It was found that a protein with a molecular weight of 49,588 consisting of 448 residues was encoded.

Example 2 NHS5 was introduced into yeast using three types of vectors and a fermentation test was conducted to confirm the inhibitory action of NHS5 on hydrogen sulfide.
The three types of vectors used are Bam HI and YEp24, YCp11, and pBR322, which are known plasmid vectors, respectively.
And digested with Sal I, vector-derived Bam HI of about 290 bp
-Sal I fragment was removed and reported by Ogata et al. Instead (Proceedings of the Japan Society for Fermentation Engineering, p. 109 (Showa 63)
)) G418 resistance gene (the G418 resistance gene derived from the transposon of E. coli, the promoter portion of the yeast alcohol dehydrogenase gene and the terminator of the yeast cytochrome C gene were ligated at both ends of approximately 2.5 Kb, Bam HI and Sal I digestion. It was inserted into Sal I cleavage site - a DNA fragment) with the site Bam HI vector.

The DNA fragment containing NHS5 is shown in FIG.
Cleavage of pHS5 shown in the above with the restriction enzyme Bam HI, followed by addition of a Bam HI linker to the resulting end obtained by digesting the cleaved end with the exonuclease Bal 31 for about 2 Kb, and then adding H
2.5 Kb D containing NHS5 obtained by cutting again with pa I
The NA fragment, YEp24 the Bam HI - the Eco RV cleavage site, Bam HI in the case of pBR322 - Eco RV respectively cleavage site inserted to YEpAG-5SS and 2 pHSI5
The seed plasmid was generated.

In the case of the YCp type having a centromere, Bam H is also present at the HpaI cleavage site of its 2.5 Kb fragment.
An I linker was added, and the vector was inserted into the Bam HI cleavage site of the vector to prepare YCpAG-5SS. The three types of plasmids thus obtained are shown in FIGS. 2 and 3. Example 3 For introduction of these three types of plasmids into yeast, 100 ml of the parent strain was used.
After culturing up to 2-4 × 10 ⁷ cells / ml in a Sakaguchi flask, wash with cold water, suspend in 0.2 ml of 1 mol sorbitol solution, and add 50 μl of this suspension with 1 μg (5 μl) of plasmid.
Transfer to a 0.2 cm cuvette and use the electroporation method (Nucleic Acid Research Nucl. Acids. Res., 16, 6) using Gene Pulser (manufactured by Bio-Rad).
127 (1988)) and transformation was carried out under the conditions of 3.5 KV / cm and 25 μF. For selection of transformants, YPD containing G418 (50 μg / ml) (1% yeast extract, 1 peptone) was selected.
%, Glucose 2%) agar medium was used.

Example 4 Using the three transformants thus obtained and the parent strain, a total of 4 strains were used to measure the sugar content.
A fermentation test was conducted using 11 added hop worts. The pre-culture was carried out by statically culturing the plate-transformed transformant at a scale of 10 ml at 25 ° C. for 2 days and then at 15 ° C. for 4 days. Fermentation test Using a 500 ml triangular flask, it was carried out at 15 ° C for 8 days. In addition, the added hop wort used in the fermentation test was performed without adding the selective drug G418, including preculture. For the determination of hydrogen sulfide, hydrogen sulfide in the fermentation gas was once trapped with a zinc acetate solution, and then colorimetrically determined by the methylene blue method. The fermentation process was followed by the weight loss of the fermented liquor accompanying the production of carbon dioxide.

As a result, as shown in FIG. 4, the amount of hydrogen sulfide produced in the NHS5-introduced transformant for 8 days was 20 to 40% lower than that of the parent strain. On the other hand, the fermentation process at that time showed almost no difference as shown in FIG. Finally, a sensory test was conducted to examine the aroma of the fermentation liquid (young beer) thus obtained. A sensory test was conducted by a panel of 10 persons, and a 5-level evaluation was conducted on two items, that is, the intensity of the hydrogen sulfide odor and the presence or absence of a strange odor. As a result, as shown in Table 1, N
All the strains introduced with HS5 had a functionally reduced hydrogen sulfide odor compared to the parent strain, and no offensive odor due to the introduction of NHS5 was observed. It was expressed as the average value of the scores of five panelists. (5 strong-1
(Weak) From the above results, it was confirmed that the present invention is effective in reducing hydrogen sulfide generated in the fermentation process and, as a result, improving beer quality and shortening the production period.

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map prepared in Example 1,

FIG. 2 is a diagram showing a restriction enzyme map of YEpAG-5SS and pHSI5 among the three types of plasmids prepared in Example 2,

FIG. 3 is a diagram showing a restriction map of YCpAG-5SS among the three types of plasmids prepared in Example 2,

FIG. 4 is a graph showing changes in the amount of hydrogen sulfide produced in the NHS5-introduced transformant over 8 days in Example 4, where ○ is a control, ● is YEpAG-5SS, and black Δ is YCpAG-5SS, black. □ is the case of pHSI5.

FIG. 5 is a graph showing the decrease in extract obtained by following the 8-day fermentation process of the NHS5-introduced transformant every 24 hours in Example 4, where ○ is a control and ● is a Y.
EpAG-5SS, black Δ is YCpAG-5SS, and black □ is pHSI5.

Front page continuation (51) Int.Cl. ⁵ Identification code Office reference number FI Technology display area // (C12N 15/31 C12R 1: 645) (C12N 1/19 C12R 1: 865) (C12P 21/02 C12R 1: 865)

Claims (4)

[Claims] 1. A gene (NHS5) which suppresses hydrogen sulfide production in yeast and is represented by the following nucleotide sequence. (Base sequence) [Chemical 2] [Chemical 3] [Chemical 4] 2. A protein having an amino acid sequence encoded by A to B in the nucleotide sequence of the gene (NHS5) according to claim 1. 3. A brewing yeast of the genus Saccharomyces in which the gene (NHS5) according to claim 1 has been introduced to reduce the amount of hydrogen sulfide produced. 4. A brewer's yeast of the genus Saccharomyces, in which a DNA having a nucleotide sequence for expressing the protein having the amino acid sequence according to claim 2 in yeast is introduced to reduce hydrogen sulfide production.