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Gene capable of enhancing low temperature fermentation ability and/or freezing stress resistance and use thereof

A technology of low-temperature performance and genetics, applied in beer fermentation methods, methods of using fungi, and beer brewing, etc., can solve problems such as unclear genes, and achieve the effects of improving low-temperature fermentability, good freezing resistance, and shortening fermentation time

Inactive Publication Date: 2008-08-27
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low-fermentation yeast used in the production of lager has good low-temperature fermentability, but the genes related to low-temperature fermentability have not yet been elucidated

Method used

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  • Gene capable of enhancing low temperature fermentation ability and/or freezing stress resistance and use thereof
  • Gene capable of enhancing low temperature fermentation ability and/or freezing stress resistance and use thereof
  • Gene capable of enhancing low temperature fermentation ability and/or freezing stress resistance and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Cloning of a novel gene (nonScDLT1) that enhances low-temperature fermentability

[0093] As a result of searching the comparison database described in JP 2004-283169, a novel gene nonScDLT1 (sequence number 1) unique to brewer's yeast that enhances low-temperature fermentability was found. According to the obtained base sequence information, the primers nonScDLT1_F (SEQ ID NO: 3) / nonScDLT1_R (SEQ ID NO: 4) used to amplify the full-length gene were respectively designed, and PCR was performed using the chromosomal DNA of the genome reading strain Saccharomyces pastorianus Weihenstephan34 / 70 as a template , to obtain a DNA fragment including the full-length gene of nonScDLT1.

[0094] The nonScDLT1 gene fragment obtained above was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The base sequence of the nonScDLT1 gene was analyzed and determined by the Sanger method (F. Sanger, Science, 214, 1215, 1981).

Embodiment 2

[0095] Example 2: Analysis of nonScDLT1 gene expression in beer trial brewing

[0096] The brewer's yeast Saccharomyces pastorianus W34 / 70 strain was used for beer trial brewing, and the mRNA extracted from the brewer's yeast cell during fermentation was detected by the brewer's yeast DNA microarray.

[0097] Wort extract concentration 12.69%

[0098] Wort volume 70L

[0099] Dissolved oxygen concentration in wort 8.6ppm

[0100] Fermentation temperature 15°C

[0101] The amount of yeast added 12.8×10 6 cells / mL

[0102] The fermentation broth was sampled over time to observe the yeast proliferation ( figure 1 ), the apparent concentration of the extract ( figure 2 ) changes over time. Simultaneously, 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. Carry out signal detection with GeneChip Operating System (GCOS; GeneChip Operating Soft...

Embodiment 3

[0103] Example 3 Preparation of nonScDLT1 high-expression strain

[0104] The nonScDLT1 / pCR2.1-TOPO described in Example 1 was digested with restriction enzymes SacI and NotI to prepare a DNA fragment including the full-length protein coding region. This fragment was ligated to pYCGPYNot treated with restriction enzymes SacI and NotI to construct nonScDLT1 high expression vector nonScDLT1 / pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector, and the introduced gene is highly expressed under the control of the promoter of pyruvate kinase gene PYK1. A selectable marker for yeast containing the geneticin resistance gene G418 r , in addition, the selectable marker for E. coli contains the ampicillin resistance gene Amp r .

[0105] Using the high expression vector made by the above method, adopt the method described in Japanese Patent Laid-Open 07-303475 to transform the AJL4004 strain, use the YPD plate medium (1% yeast extract, 2% polypeptone, 2% polypeptone) containing...

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Abstract

Disclosed are a gene capable of enhancing low temperature properties (low temperature fermentation ability and / or resistance to freezing stress), and use of the gene. Particularly, disclosed are a brewing yeast having excellent low temperature properties, an alcoholic beverage produced using the yeast, a method for production of the alcoholic beverage, and others. More specifically, disclosed are a gene DLT1 encoding DLT1p which can enhance the low temperature properties of a brewing yeast, an yeast which is improved in low temperature properties by increasing the expression amount of a nonScDLT1 gene that is characteristic of a brewer's yeast, a method for production of an alcoholic beverage using the yeast, and others.

Description

technical field [0001] The present invention relates to a gene that enhances low-temperature fermentability and / or freezing resistance (hereinafter referred to as "low-temperature performance") and its use, and particularly relates to brewing yeast with good low-temperature performance, alcohol produced using the yeast, and its production method. More specifically, the present invention relates to yeast with improved low-temperature performance by increasing the expression level of the gene DLT1 encoding the protein Dlt1p that enhances low-temperature performance in brewer's yeast, especially the nonScDLT1 characteristic gene of brewer's yeast, and wines using the yeast. manufacturing method, etc. Background technique [0002] The characteristic of lager beer storage is that it is fermented at low temperature (10-15°C), has no off-flavors, and has a refreshing taste. The bottom-fermenting yeast used in the production of lager has good low-temperature fermentability, but the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/395C12C11/02C12G1/00C12G3/02C12N1/19C12Q1/04C12Q1/68
CPCC12G2200/11C12R1/85C12C12/006C12C12/004C12C11/003C12G1/0203C07K14/395C12R2001/85C12N1/185C12N15/09
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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