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High-yield acetate brewing yeast engineering bacteria

A technology of Saccharomyces cerevisiae and acetate, applied in the field of bioengineering, can solve the problem of low ester production capacity of Saccharomyces cerevisiae

Active Publication Date: 2012-07-25
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of low ester production capacity of Saccharomyces cerevisiae, and provide a high-yield acetate Saccharomyces cerevisiae engineering strain

Method used

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  • High-yield acetate brewing yeast engineering bacteria
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  • High-yield acetate brewing yeast engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of high-yielding Saccharomyces cerevisiae genetically engineered bacteria

[0029] (1) Construction of genetic engineering strains

[0030] 1) Ligate the PGK1 promoter and terminator with the pUC19 plasmid to obtain pUC-PGK1;

[0031] 2) Ligate the homologous fragment IAH derived from Saccharomyces cerevisiae to the pUC-PGK1 obtained in step 1) to obtain pUC-PGK1-IAH;

[0032] 3) inserting the gene encoding alcohol acetyltransferase ATF2 into the pUC-PGK1-IAH obtained in step 2) between the PGK1 promoter and the terminator to obtain pUC-PGK1-IAH-ATF2;

[0033] 4) connecting the kan gene to the pUC-PGK1-IAH-ATF2 obtained in step 3) to obtain the plasmid pUC-PGK1-IAH-ATF2-kan (hereinafter referred to as pUC-PIA2K);

[0034] figure 1 It is the verification electropherogram of the pUC-PIA2K plasmid: where lane 1 is the 5000bp DNALadder Marker; lane 2 is the homologous fragment IAH amplified by the genome of the recipient strain; lane 3 is the h...

Embodiment 2

[0043] Example 2: Research on the fermentation performance of high-yielding Saccharomyces cerevisiae engineering bacteria and starting strains

[0044] The engineering bacteria and acceptor bacteria obtained in Example 1 were respectively inserted into 5 mL of wort culture solution, and cultured overnight at 30° C. for 12 hours; all the bacterial liquids were transferred to 20 mL of fresh wort culture solution, and cultivated at 30° C. for 24 hours. Take 100g of japonica rice and place it in water at 25-30°C for 72 hours; take it out, wash it, and cook it under normal pressure for 30 minutes. Cool the rice, put it into a 500mL conical flask, add 10g of cooked wheat koji and 105mL of water. Finally, 25 mL of yeast wort culture liquid was inserted and fermented at 28° C. for 5 days. Oscillate and weigh every 12 hours during the fermentation period, and record the weight loss; after the fermentation is over, stop the cultivation and weigh; measure the residual sugar concentratio...

Embodiment 3

[0048] Example 3: Research on the Fermentation Performance of High Ester Yield Saccharomyces cerevisiae Engineering Haploids and Haploids of Starting Strains

[0049] Put the engineering haploid (type a / α) and the haploid of the recipient bacteria (type a / α) into 5mL wort culture solution, culture overnight at 30°C for 12h; transfer all the bacteria solution to 20mL fresh wort Incubate at 30°C for 24 hours in the culture medium. Take 100g of japonica rice and place it in water at 25-30°C for 72 hours; take it out, wash it, and cook it under normal pressure for 30 minutes. Cool the rice, put it into a 500mL conical flask, add 10g of cooked wheat koji and 105mL of water. Finally, 25 mL of yeast wort culture liquid was inserted and fermented at 28° C. for 5 days. Oscillate and weigh every 12 hours during the fermentation period, and record the weight loss; after the fermentation is over, stop the cultivation and weigh; measure the residual sugar concentration, alcohol volume fr...

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Abstract

The invention relates to high-yield acetate brewing yeast engineering bacteria; a method for preparing the brewing yeast bacteria provided by The invention comprises the following steps: selecting strong promoters PGK1 so as to overexpress ATF2 gene coding alcohol acetyl transferase, thereby obtaining high-yield acetate brewing yeast engineering bacteria SaccharomycescerevisiaeEY-14, wherein a preservation number is CGMCC (China General Microbiological Culture Collection Center) No.5635. According to The invention, on a condition that other fermenting performance is not influenced, transformant bacterial strains are compared with parent strains (SaccharomycescerevisiaeCGMCC No 2.1525); after simulation of semi-solid fermentation, the content of ethyl acetate is increased by 3.6 times; thecontent of isoamyl acetate is increased to 55.2mg / L; the content of isobutyl acetate is increased to 33.8mg / L; the sifted engineering bacteria has no specific demand for fermentation equipment and conditions; the equipment and conditions of normal white sprits factories can be used; therefore, the engineering bacteria has wide application propose and brings marked economic benefit to industrial production of the white sprits factories.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the breeding of industrial microorganisms, in particular to a high-yielding acetate-producing Saccharomyces cerevisiae engineered bacterium. Background technique [0002] Rice wine and baijiu are the special wines in my country. Domestic common baijiu and rice wine are mainly fermented by purebred S. The capacity is extremely low, resulting in poor quality of finished wine. The main reason for the high content of ester aroma substances in high-grade beverage wine (yellow wine, white wine) is that natural koji-making microorganisms are used to make koji fermentation. Increase the ester content in wine, and the existence of these wild yeasts seriously affects the yield of raw materials, and its alcohol fermentation efficiency is less than one-third of that of Saccharomyces cerevisiae, which leads to high grain consumption, long production cycle and low efficiency of my country'...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/865
Inventor 肖冬光张翠英郭学武张建炜戴隆海
Owner TIANJIN UNIV OF SCI & TECH
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