Saccharomyces cerevisiae strain with high yield of flavor ethyl ester and construction method of saccharomyces cerevisiae strain

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

Inactive Publication Date: 2016-05-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of low ester production ability of Saccharomyces cerevisiae, and provide a high-yield flavor ethyl ester Saccharomyces cerevisiae strain and its construction method, the method is to overexpress the fatty acid synthase gene β subunit in the Saccharomyces cerevisiae starting strain The encoding gene FAS1 of the base, and the negative regulatory gene OPI1 mediated by inositol is knocked out at the same time, the steps are as follows:

Method used

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  • Saccharomyces cerevisiae strain with high yield of flavor ethyl ester and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain with high yield of flavor ethyl ester and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain with high yield of flavor ethyl ester and construction method of saccharomyces cerevisiae strain

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Embodiment 1

[0029] Embodiment 1: Construction of high-yield flavor ethyl ester Saccharomyces cerevisiae genetically engineered strain

[0030] (1) Construction of genetic engineering strains

[0031] 1) Construction of Yep-PF1K plasmid

[0032] Use Yep352 as the base plasmid to construct the recombinant plasmid Yep-PF1K, the construction process is as follows figure 1 As shown, the strong promoter PGK1 was ligated into the Yep352 plasmid by BamHI / salI double enzyme digestion to obtain the plasmid Yep-P; the FAS1 gene was obtained by PCR amplification using the haploid α5 of AY15 as a template, and the Infusion ligase was used to pass the XhoI single enzyme Cut between the promoter PGK1p and the terminator PGK1t inserted on the Yep-P plasmid to obtain the plasmid Yep-PF1; use pUG6 as a template to PCR amplify the 1613bp KanMX gene, and use SmaI to single-digest the plasmid Yep-PF1 and the fragment respectively KanMX was ligated with SolutionI ligase to form the recombinant plasmid Yep-PF...

Embodiment 2

[0044] Embodiment 2: Fermentation experiment of simulated corn raw material liquid liquor

[0045] Fermentation process route:

[0046] Corn flour→soaking→liquefaction→saccharification→cooling→inoculation→fermentation→steaming wine→measurement index

[0047] Process conditions: soaking conditions: 60-70°C, immersion for 20 minutes; liquefaction conditions: 85-90°C, add high-temperature-resistant α-amylase, liquefy for 90 minutes; saccharification conditions: 55-60°C, add glucoamylase, and saccharify for 20 minutes Fermentation conditions: 30°C, 5 days. When steaming wine, take 100mL mash, add 100mL water, and steam 100mL wine sample.

[0048] Ingredients: corn flour: 60g; add water 130mL; high temperature resistant α-amylase: 30μL; glucoamylase: 90μL; acid protease: 1.2mL; nutrient salt: 1mL;

[0049] According to the above-mentioned simulation process, the recombinant strain α5-FAS1 of Saccharomyces cerevisiae and the starting strain α5 were respectively subjected to the f...

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Abstract

The invention provides a saccharomyces cerevisiae strain with high yield of flavor ethyl ester and a construction method of the saccharomyces cerevisiae strain. An encoding gene FAS1 of a strong promoter PGK1 over-expressed fatty acid synthetase beta subunit is selected, an inositol-mediated controlling gene OPI1 is knocked out, and the saccharomyces cerevisiae strain with high yield of flavor ethyl ester is obtained. Compared with original strains, the strain has the following advantages under the condition that other fermentation performance of the strain is basically not affected: the content of ethyl acetate reaches 52.49 mg/L and is increased by 2.61 times than that of the original strains after 5 days of simulated liquid-state liquor fermentation of a corn material; the content of medium-chain fatty acid ethyl ester ethyl caproate, the content of ethyl caprylate, the content of ethyl caprate and the content of ethyl laurate are increased by 1.21 times, 2.66 times, 2.53 times and 1.27 times respectively than those of the original strains; the content of long-chain fatty acid ethyl ester ethyl myristate and the content of ethyl palmitate are increased by 1 time and 56.7% respectively.

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-yield flavor ethyl ester Saccharomyces cerevisiae strain and a construction method thereof. Background technique [0002] The esters produced by Saccharomyces cerevisiae during the fermentation process have a fruity aroma and are the characteristic flavor substances of alcoholic beverages. Increasing the content of esters can improve their flavor quality. For Chinese liquor, ethyl acetate, ethyl caproate, ethyl lactate and ethyl butyrate are the four main aroma components of Chinese liquor, which directly determine the quality of liquor products. However, in traditional fermentation, ordinary liquor produced mainly by pure Saccharomyces cerevisiae has low content of ester aroma substances and poor quality of finished wine. However, the high content of esters in high-grade liquor is mainly produced by the mixe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/865
CPCC07K14/395C12N9/1029C12Y203/01085
Inventor 陈叶福肖冬光罗伟伟钱泓龚瑞郭学武
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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