Ammonia transporter gene and use thereof

A transporter and gene technology, applied in the field of ammonia transporter gene and its application, can solve problems such as difficulty in amino acid content

Inactive Publication Date: 2007-08-29
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, as mentioned above, amino acids and ammonia are assimilated by yeast as a nitrogen source during fermentation, so it is extremely difficult to control the content of amino acids at the end of fermentation

Method used

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  • Ammonia transporter gene and use thereof
  • Ammonia transporter gene and use thereof
  • Ammonia transporter gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Cloning of Ammonia Transporter (nonScMEP1)

[0133] As a result of searching using the comparative database described in Japanese Patent Application Laid-Open No. 2004-283169, nonScMEP1 (sequence number 1), an ammonia transporter gene unique to S. cerevisiae, was found. According to the base sequence information obtained, the primers nonScMEP1_F (sequence number 5) / nonScMEP1_R (sequence number 6) used to amplify the full-length gene were designed respectively, and the strain Saccharomyces pastorianus Weihenstepan34 / 70 strain (abbreviated as "W34 / 70 strain) ”) of chromosomal DNA as a template to obtain a DNA fragment including the full-length gene of nonScMEP1.

[0134] The nonScMEP1 gene fragment obtained above was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The base sequence of the nonScMEP1 gene was analyzed and determined by the Sanger method (F. Sanger, Science, 214:1215, 1981).

Embodiment 2

[0135] Example 2: Analysis of nonScMEP1 gene expression in beer brewing

[0136] Brewery yeast Saccharomyces pastorianus W34 / 70 strain was used for trial brewing, and mRNA extracted from brewer's yeast cell during fermentation was analyzed by brewer's yeast DNA microarray.

[0137] Wort extract concentration 12.69%

[0138] Wort volume 70L

[0139] Dissolved oxygen concentration in wort 8.6ppm

[0140] Fermentation temperature 15°C

[0141] The amount of yeast added 12.8×10 6 cells / mL

[0142] The fermented liquid was sampled over time, and the changes over time in the amount of yeast proliferation (Fig. 1) and apparent extract concentration (Fig. 2) were observed. At the same time, the yeast cells were sampled, the prepared mRNA was labeled with biotin, and hybridized with the brewer's yeast DNA microarray described in Japanese Patent Laid-Open No. 2004-283169. Signal detection was performed with GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufa...

Embodiment 3

[0143] Example 3: Construction of nonScMEP1 high expression strain

[0144] The nonScMEP1 / 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. The fragment was ligated to pYCGPYNot treated with restriction enzymes SacI and NotI, thereby constructing nonScMEP1 high-expression vector nonScMEP1 / pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector, and the introduced gene is highly expressed through the promoter of pyruvate kinase gene PYK1. Yeast selectable markers include the aminoglycoside antibiotic (Geneticin) resistance gene G418 r , selectable markers for E. coli include the ampicillin resistance gene Amp r .

[0145]Using the high expression vector prepared by the above method, the Saccharomyces pasteurianus Weihenstepan34 / 70 strain was transformed by the method described in Japanese Patent Laid-Open No. 07-303475. Transformants were selecte...

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Abstract

The present invention relates to ammonia transporter genes and use thereof. The invention relates in particular to a brewer's yeast which shows enhanced ammonia assimilation, alcoholic beverages produced using such yeast, and a method of producing such alcoholic beverages. More specifically, the invention relates to the MEP1 gene which codes for the ammonia transporter Mep1 in brewer's yeast, particularly to a yeast which can control the ammonia assimilation ability by controlling the level of expression of the nonScMEP1 gene or ScMEP1 gene characteristic to beer yeast and to a method of producing alcoholic beverages using such yeast.

Description

technical field [0001] The present invention relates to an ammonia transporter (Ammonia transporter) gene and uses thereof, and particularly relates to brewer's yeast with good ammonia assimilation, an alcoholic beverage produced using the yeast, a method for producing the alcoholic beverage, and the like. More specifically, the present invention relates to the gene MEP1 encoding the ammonia transporter Mep1 of Saccharomyces cerevisiae, in particular to the yeast that controls the ability of ammonia assimilation by controlling the expression level of the characteristic gene nonScMEP1 or gene ScMEP1 of Saccharomyces cerevisiae, and the alcohol using the yeast Beverage manufacturing methods, etc. Background technique [0002] Ammonia and amino acids are known to be nitrogen sources necessary for yeast growth, and ammonia and amino acids contained in raw materials are also assimilated as nitrogen sources for yeast growth during the brewing process. [0003] It is generally bel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/39C12N1/19C12C11/02C12G1/00C12Q1/04
CPCC12C12/006C12C12/004C07K14/705C12G1/0203
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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