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Gene encoding acetolactate synthase and use thereof

A technology of acetolactate and synthase, applied in the direction of enzyme, lyase, beer brewing, etc.

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

[0008] In European Brewery Convention, Proceedings of the 26th EBCcongress, Maastricht, 455-460 (1997), Dulieu et al. proposed that the precursor of DA, α-acetolactate, be rapidly converted into 3-hydroxy by using α-acetolactate decarboxylase -2-butanone method, but since α-acetolactate decarboxylase can only be produced by DNA recombinant technology, Japan's tax system stipulates that the enzyme cannot be added to the fermentation broth during fermentation

Method used

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  • Gene encoding acetolactate synthase and use thereof
  • Gene encoding acetolactate synthase and use thereof
  • Gene encoding acetolactate synthase and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Embodiment 1: Cloning of novel acetolactate synthase gene (non-ScILV2)

[0113] Using the search results of the comparison database described in JP 2004-283169, a novel acetolactate synthase gene non-ScILV2 (serial number: 1) unique to S. cerevisiae was discovered. Based on the obtained nucleotide sequence information, primers non-ScILV2_F (sequence number: 3) / non-ScILV2_R (sequence number: 4) were designed to amplify their respective full-length genes, and the strain Saccharomyces pastorianus Weihenstephan was read from the genome. The chromosomal DNA of the W34 / 70 strain was used as a template, and a DNA fragment containing the full-length gene of nonScILV2 was obtained by PCR.

[0114] The non-ScILV2 gene fragment obtained as described above was inserted into pCR2.1-TOPO vector [manufactured by Invitrogen] by TA cloning. The base sequence of the non-ScILV2 gene was analyzed by the Sanger method (F. Sanger, Science, 214:1215, 1981) to confirm the base sequence.

Embodiment 2

[0115] Example 2: Analysis of non-ScILV2 gene expression in beer test brewing

[0116] The brewer's yeast Saccharomyces pastorianus W34 / 70 strain was used for the experimental brewing of beer, and the mRNA extracted from the fermented brewer's yeast cell was detected by yeast DNA microarray.

[0117] Wort extract concentration 12.69%

[0118] Wort capacity 70L

[0119] Wort dissolved oxygen concentration 8.6ppm

[0120] Fermentation temperature 15°C

[0121] Yeast input amount 12.8×10 6 cells / mL

[0122] The fermentation broth was sampled over time, and the changes over time of yeast growth (Figure 1) and apparent extract concentration (Figure 2) were observed. At the same time, the yeast cells were sampled to prepare mRNA, and the prepared mRNA was biotin-labeled to be hybridized with S. cerevisiae DNA microarray. Signal detection was performed using GeneChip Operating System [GCOS; GeneChip Operating Software 1.0, manufactured by AFFYMETRIX Corporation]. The gene expr...

Embodiment 3

[0123] Example 3: Complementary testing of nonScILV2 using laboratory-designed yeast strains

[0124] A laboratory-engineered yeast strain in which the endogenous ILV2 gene was disrupted confirmed that the nonScILV2 gene product functions as an acetolactate synthase.

[0125] According to the method of the literature [Goldstein et al., yeast.151541 (1999)], using the plasmid [pAG25(natl)] containing the drug resistance marker as a template, a fragment for destroying the ILV2 gene was prepared by PCR. The sequences of the primers used are shown in SEQ ID NOs: 5 and 6. Using this fragment, the S.cerevisiae X2180-1A strain was transformed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and the YPD plate medium containing nourseothricin 50 mg / L (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) for selection. The resulting ILV2 disrupted strain was treated with SC plate medium [0.67% yeast nitrogen source without amino acid, 0.2% amino acid m...

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Abstract

The present invention relates to an acetolactate synthase 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 vicinal diketones, especially diacetyl, that are responsible for off-flavors in products, is reduced by repressing expression level of ILV2 gene encoding an acetolactate synthase (Ilv2p), especially non-ScILV2 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 a gene encoding acetolactate synthase 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 the alcoholic beverages. More specifically, the present invention relates to inhibiting the expression of the gene ILV2 encoding the acetolactate synthase of Saccharomyces cerevisiae, especially the characteristic nonScILV2 gene in S. Yeast with reduced production of diacetyl and method for producing alcoholic beverages using the yeast. Background technique [0002] Among the taste components of alcoholic beverages, diacetyl (hereinafter referred to as DA) is one of the representative bad tastes in alcoholic beverages brewed from beer, sake and wine. DA taste (appeared as rancid rice or butter in beer, and nauseating in sake) is when diketones (hereinafter referred to as VDK) mainly DA are present in the pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N1/19C12C11/02C12G1/00C12Q1/68
CPCC12N9/88
Inventor 中尾嘉宏儿玉由纪子下永朋子
Owner SUNTORY HLDG LTD
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