Gene encoding trehalose-6-phosphate phosphatase and use thereof

A technology of phosphate dephosphorylation and trehalose, applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve the problems of poor low-temperature storage of baker's yeast, and achieve the effect of eliminating difficult problems and reducing weight

Inactive Publication Date: 2007-09-26
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because baker's yeast Saccharomyces cerevisiae has poor low-temperature preservation compared to brewing yeast such as beer and sake fermented at low temperature.

Method used

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  • Gene encoding trehalose-6-phosphate phosphatase and use thereof
  • Gene encoding trehalose-6-phosphate phosphatase and use thereof
  • Gene encoding trehalose-6-phosphate phosphatase and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 The gene encoding trehalose-6-phosphate phosphatase (non ScTPS2 ) clone

[0101] As a result of searching using the comparative database described in JP-A-2004-283169, the non-ScTPS2 gene (SEQ ID NO: 1) encoding trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae was found. According to the obtained nucleotide sequence information, the primers non ScTPS2_for (SEQ ID NO: 3) / non ScTPS2_rv (SEQ ID NO: 4) used to amplify the full-length gene were respectively designed, and the strain Saccharomycespastorianus Weihenstepan34 / 70 strain (referred to as "W34 / 70 strain ") chromosomal DNA as template PCR, obtain the DNA fragment that comprises non-ScTPS2 full-length gene.

[0102] The non-ScTPS2 gene fragment obtained above was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The nucleotide sequence of the non-ScTPS2 gene was analyzed and determined by the Sanger method (F. Sanger, Science, 214, 1215, 1981).

Embodiment 2

[0103] Example 2 Expression analysis of non-ScTPS2 gene in beer trial brewing

[0104] Saccharomyces pastorianus W34 / 70 strain was used for beer trial brewing, and the mRNA extracted from the fermenting Saccharomyces pastorianus was detected by Saccharomyces DNA microarray.

[0105] Wort extract concentration 12.69%

[0106] Wort volume 70L

[0107] Dissolved oxygen concentration in wort 8.6ppm

[0108] Fermentation temperature 15°C

[0109] The amount of yeast added 12.8×10 6 cells / mL

[0110] The fermented liquid was sampled over time to observe the changes in the amount of yeast proliferation (Figure 1) and the apparent concentration of the extract (Figure 2). At the same time, the yeast cells were sampled to prepare mRNA, and the prepared mRNA was labeled with biotin to be hybridized with the brewer's yeast DNA microarray. Signal detection was performed using the GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix Corporation)...

Embodiment 3

[0111] Example 3 Construction of non-ScTPS2 high expression strain

[0112] The non-ScTPS2 / pCR2.1-TOPO obtained by the method described in Example 1 was digested with restriction enzymes SacI and NotI to prepare a DNA fragment containing the non-ScTPS2 gene. The fragment was connected to pYCGPYNot treated with restriction enzymes SacI and NotI to construct non ScTPS2 high expression vector non ScTPS2 / 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 .

[0113] Using the high expression vector obtained by the above method, adopt the method described in JP 07-303475 to transform AJL4004 strain, adopt YPD plate medium (1% yeast extract, 2% yeast extract) containing aminoglycoside antibiotics (Genetici...

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Abstract

The present invention relates to a gene encoding trehalose-6-phosphate phosphatase and use thereof, in particular, a yeast for practical use with superior resistance property to dryness and / or low-temperature storage, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, whose resistance property to dryness and / or resistance property to low-temperature storage is enhanced by amplifying expression level of TPS2 gene encoding a trehalose-6-phosphate phosphatase Tps2p in brewer's yeast, especially non-ScTPS2 gene specific to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc.

Description

technical field [0001] The present invention relates to a gene encoding trehalose-6-phosphate phosphatase and its use, in particular to a practical yeast with good desiccation resistance and / or low temperature storage resistance, an alcoholic beverage produced using the yeast and a production method thereof. More specifically, the present invention relates to improving desiccation resistance and Tps2p protein gene TPS2 with trehalose-6-phosphate phosphatase activity of coding brewer's yeast, especially the expression level of characteristic gene non-ScTPS2 of brewer's yeast. and / or yeast with improved low-temperature storage stability, a method for producing alcoholic beverages using the yeast, and the like. In addition, the yeast of the present invention can also be used as baker's yeast or industrial yeast. Background technique [0002] The beer brewing process is characterized in that the yeast after fermentation is recovered and used in the next fermentation (called con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/63C12N1/19C12C11/02C12G1/00C12Q1/68C12Q1/04A23L11/20
CPCC12Q1/42G01N2333/39C12Y301/03012C12Q1/02C12R1/85C12N9/16C12C12/006C12C12/004C12G1/0203C12R2001/85C12N1/185
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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