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Branched-chain amino acid aminotransferase gene and use thereof

一种支链氨基酸、氨基转移酶的技术，应用在支链氨基酸氨基转移酶基因及其用途领域

Inactive Publication Date: 2012-01-25
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, although higher alcohols or esters are important aroma components in beer, excess higher alcohols or esters are not welcome because of their organic solvent or ester odor.

Method used

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  • Branched-chain amino acid aminotransferase gene and use thereof
  • Branched-chain amino acid aminotransferase gene and use thereof
  • Branched-chain amino acid aminotransferase gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Cloning of a novel branched-chain amino acid aminotransferase gene (nonScBAT1)

[0113] As a result of searching the comparative database described in JP-A-2004-283169, a novel branched-chain amino acid aminotransferase gene nonScBAT1 (SEQ ID NO: 1) unique to S. cerevisiae was found. According to the base sequence information obtained, primers nonScBAT1_for (SEQ ID NO: 5) / nonScBAT1_rv (SEQ ID NO: 6) for amplifying the full-length gene were respectively designed, and the strain Saccharomyces pastorianus Weihenstephaner34 / 70 strain (sometimes abbreviated as " The chromosomal DNA of W34 / 70 strain ") is the PCR of template, has obtained the DNA fragment (about 1.2kb) that contains nonScBAT1 full-length gene.

[0114] The nonScBAT1 gene fragment obtained by the above method was inserted into pCR2.1-TOPO (manufactured by Invitrogen) vector by TA cloning. The base sequence of the nonScBAT1 gene was analyzed and determined by the dideoxy sequencing method (F. Sanger...

Embodiment 2

[0115] Example 2: Analysis of nonScBAT1 gene expression in beer trial brewing

[0116] Saccharomyces pastorianus (Saccharomyces pastorianus) W34 / 70 strain was used for trial brewing, and mRNA extracted from Saccharomyces pastorianus during fermentation was detected by Saccharomyces DNA microarray.

[0117] Wort extract concentration 12.69%

[0118] Wort volume 70L

[0119] Dissolved oxygen concentration in wort 8.6ppm

[0120] Fermentation temperature 15°C

[0121] The amount of yeast added 12.8×10 6 cells / mL

[0122] 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. At the same time, the yeast cells were sampled, the prepared mRNA was labeled with biotin, and hybridized on the brewer's yeast DNA microarray. Signal detection was performed using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix). exist image...

Embodiment 3

[0123] Example 3: Disruption of the nonScBAT1 gene

[0124] According to the method described in the literature (Goldstein et al., yeast. 151541 (1999)), a fragment for gene disruption was prepared by PCR using a plasmid (pFA6a(G418r)) containing a drug resistance marker as a template. Use nonScBAT1_delta_for (SEQ ID NO: 9) and nonScBAT1_delta_rv (SEQ ID NO: 10) as PCR primers.

[0125] A spore clone (W34 / 70-2) from Saccharomyces pastorianus strain W34 / 70 was transformed with the fragment for gene disruption prepared by the above method. Transformation adopts the method described in Japanese Patent Application Hei 07-303475, with the YPD plate medium (1% yeast extract, 2% polypeptone, 2% glucose, 2% polypeptone containing aminoglycoside antibiotics (Geneticin) 300mg / L agar) to select transformants.

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Abstract

The present invention relates to a branched-chain amino acid aminotransferase gene and a use thereof, particularly, to a brewery yeast for producing alcoholic drinks with enhanced flavor, to alcoholic drinks produced with said yeast and to a method for producing said drinks. More particularly, the present invention relates to a yeast whose capacity of producing amyl alcohol and / or isobutanol and / or isoamyl acetate that contribute to the product flavor is controlled by controlling the expression levels of BAT1 and BAT2 genes coding for brewery yeast branched-chain amino acid aminotransferases Bat1p and Bat2p, respectively, particularly nonScBAT1 or nonScBAT2 gene specific to lager brewing yeast, and to a method for producing alcoholic drinks using said yeast.

Description

technical field [0001] The present invention relates to a branched-chain amino acid aminotransferase 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 controlling the expression level of the gene BAT1 encoding the branched-chain amino acid aminotransferase Bat1p of Saccharomyces cerevisiae, or the gene BAT2 encoding Bat2p, especially the characteristic gene nonScBAT1 or nonScBAT2 in Saccharomyces cerevisiae, thereby controlling Yeast contributing to the ability to produce amyl alcohol and / or isobutanol and / or isoamyl acetate, which contributes to the flavor of a product, and a method for producing alcoholic beverages using the yeast. Background technique [0002] Higher alcohols such as amyl alcohol and isobutanol, and esters such as isoamyl acetate are one of the important aroma componen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12C11/00C12N1/19C12N15/09C12N9/10C12N15/54C12G1/00
CPCC12N9/1096C12R1/865C12P7/62C12P7/16Y02E50/10C12R2001/865C12N1/185C12N9/10C07K14/395C12N15/52
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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