Acyl-CoA: ethanol O-acyltransferase/esterase gene and use thereof

A technology of acyltransferase and acyl group, applied in the direction of transferase, genetic engineering, plant genetic improvement, etc., can solve the problem of unpopularity of esters

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

On the other hand, for beer, although ester is an important aroma component, excess ester will be unwelcome due to ester smell

Method used

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  • Acyl-CoA: ethanol O-acyltransferase/esterase gene and use thereof
  • Acyl-CoA: ethanol O-acyltransferase/esterase gene and use thereof
  • Acyl-CoA: ethanol O-acyltransferase/esterase gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Cloning of a novel acyl-CoA:ethanol O-acyltransferase / esterase gene (nonScEHT1 )

[0108] As a result of searching using the comparative database described in Japanese Patent Application Laid-Open No. 2004-283169, a novel acyl-CoA:ethanol O-acyltransferase / esterase gene nonScEHT1 (sequence number 1) unique to S. cerevisiae was found. According to the base sequence information obtained, the primers nonScEHT1_for (sequence number 3) / nonScEHT1_rv (sequence number 4) for amplifying the full-length gene were designed respectively, and the strain Saccharomyces pastorianus Weihenstepan34 / 70 strain (abbreviated as "W34 / 70 Strain") chromosomal DNA was used as a template for PCR to obtain a DNA fragment (about 1.4 kb) including the full-length gene of nonScEHT1.

[0109] The nonScEHT1 gene fragment obtained by the above method was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The base sequence of the nonScEHT1 gene was analyzed and deter...

Embodiment 2

[0110] Example 2: Analysis of nonScEHT1 gene expression in beer trial brewing

[0111] Brewer's yeast Saccharomyces pastorianus W34 / 70 strain was used for trial brewing, and mRNA extracted from brewer's yeast cell during fermentation was detected by brewer's yeast DNA microarray.

[0112] Wort extract concentration 12.69%

[0113] Wort volume 70L

[0114] Dissolved oxygen concentration in wort 8.6ppm

[0115] Fermentation temperature 15°C

[0116] The amount of yeast added 12.8×10 6 cells / mL

[0117] 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, and the prepared mRNA was labeled with biotin to be hybridized with the brewer's yeast DNA microarray. Signal detection was performed using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix Corporation), and the expr...

Embodiment 3

[0118] Example 3: Production of nonScEHT1 high-expression strain

[0119] The nonScEHT1 / pCR2.1-TOPO described in Example 1 was digested with restriction enzymes SacI and NotI to prepare a DNA fragment including the full length of the protein coding region. The fragment was connected to pYCGPYNot treated with restriction enzymes SacI and NotI to construct nonScEHT1 high expression vector nonScEHT1 / 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 resistance (Geneticin) gene G418 r , selectable markers for E. coli include the ampicillin resistance gene Amp r . Saccharomyces pastorianus Weihenstepan 34 / 70 strain was transformed using the high expression vector prepared by the above method using the method described in Japanese Patent Application Laid-Open No. 07-303475. Transformants were selected using YPD pla...

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Abstract

The present invention relates to an acyl-CoA: ethanol O-acyltransferase / esterase gene and use thereof, in particular, a brewery yeast for producing alcoholic beverages with superior flavor, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, whose capability of producing ester, which contribute to aroma and flavor of products, is controlled by regulating expression level of EHT1 gene encoding a protein (Eht1p) that is an acyl-CoA: ethanol O-acyltransferase / esterase in a brewery yeast, especially the nonScEHT1 gene specific to a lager brewing yeast, and to a method for producing alcoholic beverages with said yeast.

Description

technical field [0001] The present invention relates to an acyl-CoA:ethanol O-acyltransferase / esterase gene and uses thereof, and particularly relates to a brewer's yeast for producing alcoholic beverages with a good aroma, alcoholic beverages produced using the yeast, and a production method thereof. More specifically, the present invention relates to the gene EHT1 encoding the acyl-CoA:ethanol O-acyltransferase / esterase Ehtlp of Saccharomyces cerevisiae, and in particular to controlling the ester that imparts flavor to the product by controlling the expression level of the characteristic nonScEHT1 gene of Saccharomyces cerevisiae A yeast capable of producing alcohol, a method for producing alcoholic beverages using the yeast, and the like. Background technique [0002] Esters in wine are one of the important aroma components. It is known that the increase of esters in sake, wine, whiskey, etc. will make the wine rich in aroma and improve the sensory evaluation. On the oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/54C12N15/55C12N9/00C12N9/10C12N9/16C12N1/19C12C11/02C12G1/00C12Q1/68
CPCC12N9/1029C12N9/18C12N1/18C12N9/10C12N15/52
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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