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Ethyl carbamate (EC) low-yield saccharomyces cerevisiae strain obtained by knocking-out CAR1 and over-expressing DUR3 and construction method of saccharomyces cerevisiae strain

The technology of urethane and Saccharomyces cerevisiae strain is applied in the field of bioengineering, and can solve the problems of reduced urease activity, high production cost, cumbersome production process of urease, etc., and achieves the effects of increased expression, broad market prospects and wide application prospects.

Inactive Publication Date: 2016-11-16
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current production process of urease is cumbersome, the production cost is high, and the activity of urease is easily affected by various inhibitory factors in wine and reduced

Method used

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  • Ethyl carbamate (EC) low-yield saccharomyces cerevisiae strain obtained by knocking-out CAR1 and over-expressing DUR3 and construction method of saccharomyces cerevisiae strain
  • Ethyl carbamate (EC) low-yield saccharomyces cerevisiae strain obtained by knocking-out CAR1 and over-expressing DUR3 and construction method of saccharomyces cerevisiae strain
  • Ethyl carbamate (EC) low-yield saccharomyces cerevisiae strain obtained by knocking-out CAR1 and over-expressing DUR3 and construction method of saccharomyces cerevisiae strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of Saccharomyces cerevisiae with low production of urethane

[0036] The starting strain CGMCC No.3373 used in this example. The Escherichia coli DH5a was purchased from Takara Company. The YEPD medium is a general complete medium, and the solid medium contains 2% imported agar powder.

[0037] Based on the yeast genome data in Genebank and the integrated plasmid sequence, the following primers were designed.

[0038] The primers used in the present embodiment of table 1

[0039]

[0040] Note: The underlined part is the restriction site.

[0041] The PCR amplification system used in the present embodiment of table 2

[0042]

[0043] Table 3(a) Solution I ligase ligation method

[0044]

[0045] Note: The reaction conditions are 16°C water bath, overnight connection

[0046] Table 3(b) Infusion ligase ligation method

[0047]

[0048] Note: The reaction conditions are 50°C water bath, connecting for 30min

[0049] (1) Recombi...

Embodiment 2

[0062] Embodiment 2: Fermentation experiment of low-yield EC Saccharomyces cerevisiae strain

[0063] (1) Wine fermentation experiments of recombinant strains and starting strains

[0064] The recombinant strain and the original strain were subjected to wine fermentation experiments at the same time, and the fermentation process roadmap:

[0065] Grape raw materials→cleaning, drying, destemming→crushing→sugar adjustment, acid adjustment→adding sulfurous acid, sterilization→inoculation→pre-fermentation→skin separation→post-fermentation→measurement index

[0066] Process conditions:

[0067] Brix: 20.45Brix; Acidity: pH 3.51; SO 2 Addition amount: 80mg / L; inoculation amount: 3%; pre-fermentation temperature and time: 25°C, 4.5d; post-fermentation temperature and time: 20°C, 9d.

[0068] Pick a ring of yeast cells, put them into a test tube containing 5mL of YEPD medium, culture at 30°C, 180rpm for 12h, then inoculate 10% of the inoculum into a 150mL Erlenmeyer flask containin...

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Abstract

The invention discloses an ethyl carbamate (EC) low-yield saccharomyces cerevisiae strain and a construction method thereof, and belongs to the field of bioengineering technology. The ethyl carbamate low-yield saccharomyces cerevisiae strain is obtained by knocking-out a copy arginase gene CAR1 in a parent saccharomyces cerevisiae strain and by over-expressing a gene DUR3 of urea transporter Dur3p by virtue of a strong promoter PGK1. In a wine brewing experiment, the constructed saccharomyces cerevisiae strain, compared with the parent strain, can avoid other fermenting performances and flavor substances from being affected, and can significantly reduce the contents of urea and EC, wherein the content of the urea is reduced by 45.51% and the content of the EC is reduced by 36.92%. The saccharomyces cerevisiae strain can meet the relatively high requirements of wine related fields on yeast; and the saccharomyces cerevisiae strain has a broad application prospect.

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 Saccharomyces cerevisiae strain with low production of ethyl carbamate and a construction method thereof. Background technique: [0002] Wine is a healthy alcoholic drink with a unique flavor and much-loved. Ethyl Carbamate (EC) is a compound that naturally occurs in fermented food and beverage wine, mainly during fermentation, heating (such as distillation) and storage. EC has a potential carcinogenic effect on the human body and can cause diseases such as lung cancer, lymphoma, liver cancer and skin cancer, and ethanol can promote the carcinogenic effect of EC. In 2007, the International Agency for Research on Cancer reclassified EC from group 2B (or substances that may cause human cancer) to group 2A (substances that may cause human cancer). Since the 1970s, scholars from all over the world have detected EC i...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12G1/022C12R1/865
CPCC12Y305/03001C12G1/0203C12G2200/11
Inventor 肖冬光李园子杜丽平郭学武陈叶福张翠英董健马立娟
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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