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, the genetically engineered bacteria of Saccharomyces cerevisiae with high ester production and its construction field, can solve the problems of poor quality of finished wine, low wine yield of raw materials, and low content of ester aroma substances.

Active Publication Date: 2013-03-27
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

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: Construction of high-yielding Saccharomyces cerevisiae genetically engineered bacteria

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

[0047] Digest the pPGK1 plasmid with Hind III to release a PGK1 fragment of about 1.8 kb in size; digest the vector pUC19 with Hind III; use T 4 DNA ligase ligated PGK1 / Hind III and pUC19 / Hind III to form plasmid pUC-P; BamH I digested IAH1 homologous fragment and plasmid pUC-P respectively; 4 DNA ligase was connected to construct plasmid pUC-PI; Xho I digested ATF1 gene and plasmid pUC-PI respectively; 4 DNA ligase ligated to form plasmid pUC-PIA. Using pUC-PIA as a template, design corresponding primers according to the sequences of PGK1 and ATF1 to verify the direction of ATF1; KpnI digests Kan gene and plasmid pUC-PIA respectively; 4 DNA ligase connection to form plasmid pUC-PIAK, the construction process is as follows figure 1 shown. figure 2 It is the verification electropherogram of the pUC-PIAK plasmid: wher...

Embodiment 2

[0063] Embodiment 2: Simulation rice wine fermentation experiment

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

[0065] 2) Process conditions: rice soaking conditions: 25-30°C, soaking for 72 hours; cooking conditions: steaming at atmospheric pressure for about 30 minutes, with uniform grains and no white inside; pre-fermentation conditions: 28°C, 5 days.

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

[0067] Saccharomyces cerevisiae CGMCC No 2.1525) and its haploids (type a and α) were subjected to semi-solid Fermentation experiment; 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 fraction and main aroma c...

Embodiment 3

[0072] Embodiment 3: simulated liquor fermentation experiment

[0073] The ester-producing properties of alcohol ADY, Saccharomyces cerevisiae receptor strain and S. cerevisiae engineered strain were compared by continuous residue fermentation method. Test method: raw material: distiller's grains=1:2.5, rice husk is 20% of raw material, and the total weight of each pot is about 300g. The added amount of glucoamylase is 250U / ml, and the added amount of alcohol ADY and rice wine yeast is 1.2%. Enter the altar to ferment for 7 days. 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 cerevisiae engineered strain

[00...

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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 ester yield 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 high-ester-yielding Saccharomyces cerevisiae genetically engineered bacterium and a construction method thereof. 【Background technique】 [0002] Ester aroma substances are the main flavor substances in drinking wine. The higher ester content not only imparts an important ester aroma to drinking wine, but also can effectively expand and relax nerves and reduce the side effects caused by drinking. Domestic ordinary liquor and rice wine are mainly fermented by purebred Saccharomyces cerevisiae, which is characterized by a short fermentation cycle and a 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 Difference. The main reason for the high content of ester aroma substances in high-end beverage wine...

Claims

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Application Information

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Patent Type & Authority Patents(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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