Cysteine synthase gene and use thereof

A cysteine ​​and synthetase technology, applied in the direction of enzymes, lyases, enzymes, etc., can solve problems such as unclear relationship between hydrogen sulfide and unexperimental confirmation of functions

Inactive Publication Date: 2007-02-28
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of this gene has not been confirmed experimentally, and the relationship with the amount of hydrogen sulfide produced is unclear.

Method used

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  • Cysteine synthase gene and use thereof
  • Cysteine synthase gene and use thereof
  • Cysteine synthase gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1: Cloning of novel cysteine ​​synthase gene (nonScYGR012W)

[0106] As a result of searching the comparative database described in JP-A-2004-283169, a novel cysteine ​​synthase gene nonScYGR012W (sequence number: 1) unique to S. cerevisiae was discovered. Based on the obtained base sequence information, primers nonScYGR012W_for (sequence number: 3) / nonScYGR012W_rv (sequence number: 4) were designed for amplifying the respective full-length genes, and the strain Saccharomyces pastorianus Weihenstephaner 34 / 70 strain ( Chromosomal DNA (sometimes abbreviated as "W34 / 70 strain") was used as a template, and a DNA fragment (about 1.2 kb) containing the full-length nonScYGR012W gene was obtained by PCR.

[0107] The nonScYGR012W gene fragment obtained as described above was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The base sequence of the nonScYGR012W gene was analyzed by the Sanger method (F. Sanger, Science, 214, 1215, 1981) to c...

Embodiment 2

[0108] Example 2: Analysis of nonScYGR012W gene expression in beer test brewing

[0109] The beer yeast Saccharomyces pastorianus W34 / 70 strain was used for the experimental brewing of beer, and the mRNA extracted from the fermentation yeast cell was detected by the Saccharomyces DNA microarray.

[0110] Wort extract concentration 12.69%

[0111] Wort capacity 70L

[0112] Wort dissolved oxygen concentration 8.6ppm

[0113] Fermentation temperature 15°C

[0114] Yeast input amount 12.8×10 6 cells / mL

[0115]The fermented liquid was sampled over time to observe the changes in the amount of yeast proliferation (Fig. 1) and apparent extract concentration (Fig. 2) over time. At the same time, the yeast cells were sampled, and the prepared mRNA was biotin-labeled to be hybridized on the brewer's yeast DNA microarray. Signal detection was performed using GCOS; Gene Chip Operating Software 1.0 (manufactured by AFFYMETRIX). The nonScYGR012W gene expression pattern is shown in F...

Embodiment 3

[0116] Example 3: Preparation of nonScYGR012W gene high expression strain

[0117] NonScYGR012W / pCR2.1-TOPO described in Example 1 was digested with restriction endonucleases SacI and NotI to prepare a DNA fragment including the full length of the protein coding region. This fragment was ligated with pYCGPYNot treated with restriction enzymes SacI and NotI to construct a nonScYGR012W high-expression vector nonScYGR012W / pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector, and the introduced gene is highly expressed through the promoter of pyruvate kinase gene PYK1. Selectable markers in yeast include aminoglycoside antibiotic resistance (geneticin) gene G418 r , the selectable marker in Escherichia coli contains the ampicillin resistance gene Amp r .

[0118] Using the high expression vector prepared by the above method, Saccharomyces pastorianus Weihenstephaner 34 / 70 strain was transformed by the method described in JP-A-07-303475. Transformants were selected with Y...

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Abstract

The present invention relates to a cysteine synthase 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 YGR012W gene encoding brewery yeast cysteine synthase Ygr012wp, particularly non-ScYGR012W gene specific to lager brewing yeast, and to a method for producing alcoholic beverages with said yeast.

Description

technical field [0001] The present invention relates to a cysteine ​​synthase gene and its use, and in particular to brewer's yeast for producing alcoholic beverages with excellent aroma, alcoholic beverages produced using the yeast, and methods for producing them. More specifically, the present invention relates to a yeast that improves product aroma and uses the yeast by increasing the expression level of the gene YGR012W encoding brewer's yeast cysteine ​​synthase Ygr012wp, especially the characteristic nonScYGR012W gene or ScYGR012W gene in brewer's yeast. The production method of alcoholic beverages, etc. Background technique [0002] Brewer's yeast used in the manufacture of commercially available pilsner type pale beer has the property of producing hydrogen sulfide during the main fermentation process. This hydrogen sulfide is one of the causes of the unfavorable tender beer taste in terms of beer quality, and measures to reduce it below a threshold value include pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N1/19C12G1/00C12C7/00C12Q1/68C12Q1/02
CPCC12N9/88C12C12/006C12C12/004C12G3/02C12G1/0203C12C11/00
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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