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Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, which is applied in the breeding of industrial microorganisms, high-yielding Saccharomyces cerevisiae genetically engineered bacteria and its construction, can solve the problems of poor quality of finished wine, low yield of raw materials, low content of ester aroma substances, etc.

Active Publication Date: 2011-09-28
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, rice wine and baijiu are special wines in my country. Ordinary wine has a high yield of raw materials, but the content of ester aroma substances is low, and the quality of finished wine is poor; high-end wine has high ester aroma substances and good quality, but the raw materials yield Low alcohol, high production costs
At present, there are still many problems in the domestic method of increasing the content of ester aroma substances in beverage alcohol

Method used

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  • Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof
  • Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof
  • Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Construction of genetically engineered strains of high ester-producing Saccharomyces cerevisiae

[0046] 1) Construction of pUC19-PIAK plasmid:

[0047] Hind III digested pPGK1 plasmid, releasing a PGK1 fragment of about 1.8 kb; digested vector pUC19 with Hind III; used T 4 DNA ligase connects PGK1 / Hind III and pUC19 / Hind III to form plasmid pUC-P; BamH I digests the homologous fragment of IAH1 and plasmid pUC-P respectively; 4 DNA ligase is ligated to form the pUC-PI; Xho I digests the ATF1 gene and the plasmid pUC-PI respectively; use T 4 DNA ligase ligation constitutes plasmid pUC-PIA. Using pUC-PIA as a template, corresponding primers were designed according to the sequence of PGK1 and ATF1 to verify the direction of ATF1; KpnI digested Kan gene and plasmid pUC-PIA respectively; using T 4 DNA ligase ligation to form plasmid pUC-PIAK, the construction process is as follows figure 1 Shown. figure 2 It is the verification electropherogram of pUC-PIAK plasmid: Lan...

Embodiment 2

[0063] Example 2: Simulated rice wine fermentation experiment

[0064] 1) See the fermentation process route Figure 7 .

[0065] 2) Process conditions: rice soaking conditions: 25-30℃, 72h immersion; cooking conditions: steaming at atmospheric pressure for about 30 minutes, uniform particles and no white inside; pre-fermentation conditions: 28℃, 5 days.

[0066] 3) Ingredients: japonica rice: 100g; cooked wheat koji: 10g; water: 105ml, the water includes 60ml of clear water and 45ml of slurry water, excluding water absorption for soaking rice and water absorption for steaming rice; inoculation amount: 10% (20mL).

[0067] According to the above simulation process, the Saccharomyces cerevisiae engineering strain EY-13 and its haploid (type a and α) and the starting strain (Saccharomyces cerevisiae CGMCC No 2.1525) and its haploid (type a and α) were semi-solid respectively. Fermentation experiment; shake and weigh every 12h during the fermentation, record the weight loss; after the f...

Embodiment 3

[0072] Example 3: Simulated liquor fermentation experiment

[0073] The slag fermentation method compares the ester production performance of three kinds of yeasts: alcohol ADY, Saccharomyces cerevisiae acceptor strain and Saccharomyces cerevisiae engineering strain. Test method: raw material: distillers grains = 1:2.5, rice husk is 20% of the raw material, and the total weight of each jar is about 300g. The addition amount of saccharification enzyme is 250U / ml, and the addition amount of alcohol ADY and rice wine yeast is 1.2%. Ferment for 7 days in the altar. After the fermentation, the residual starch, alcohol volume fraction and main aroma component content of each jar were measured, and its comprehensive performance was characterized by fermentation capacity, residual sugar concentration and product production. The results are shown in Table 2.

[0074] Table 2 Liquor fermentation performance of alcohol ADY, Saccharomyces cerevisiae receptor strain and Saccharomyces cerevisi...

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Abstract

The invention discloses Saccharomyces cerevisiae genetic engineering bacteria with high ester yield. The Saccharomyces cerevisiae genetic engineering bacteria EY-13 were collected in China General Microbiological Culture Collection Center on November 17, 2010, the collection number is CGMCC NO.4350, and the Saccharomyces cerevisiae genetic engineering bacteria are recommended to be named Saccharomyces cerevisiae. PGK1 derived from the Saccharomyces cerevisiae is selected as a promoter; and the construction method of the Saccharomyces cerevisiae genetic engineering bacteria with the high esteryield comprises a step of knocking-out an IAH1 gene for encoding ester hydrolase in a Saccharomyces cerevisiae genome when an ATF1 gene which is derived from the Saccharomyces cerevisiae and is used for encoding alcohol acetyltransferase is overexpressed. Compared with initial recipient bacteria, the Saccharomyces cerevisiae genetic engineering bacteria have the advantages that: after rice wine fermentation is simulated, the content of isoamylol is about one half, the content of ethyl acetate is improved by 20 times, the content of isoamyl acetate is 100mg / L, and the content of isobutyl acetate is 5 to 7mg / L; and after liquor fermentation is simulated, the content of total esters is improved by 4 times and the content of the ethyl acetate is improved by 35 times, so an excellent strain is provided for the production of brewing industry.

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 genetically engineered strain of high-ester producing Saccharomyces cerevisiae and a construction method thereof. 【Background technique】 [0002] Ester aroma substances are the main flavor substances in beverage wines. The higher ester content not only imparts important ester aroma to beverage wines, but also can effectively expand and relax the nerves and reduce the side effects caused by drinking. Domestic ordinary liquor and rice wine are mainly fermented by pure-bred Saccharomyces cerevisiae, which is characterized by short fermentation period and high yield of raw materials. However, due to the extremely low ability of Saccharomyces cerevisiae to produce ester aroma substances, the quality of the finished wine is relatively low. difference. High-end beverage wines, such as rice wine, white wine, etc., are mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/55C12N15/54C12N15/63C12G3/02C12R1/865
Inventor 肖冬光张翠英郭学武张建炜孙溪王文阳葛枭雄
Owner TIANJIN UNIV OF SCI & TECH
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