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Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria

A technology of urethane and Saccharomyces cerevisiae, applied in the field of yeast metabolic engineering bacteria and its construction, to achieve the effect of reducing the formation of EC and improving the utilization capacity of urea

Inactive Publication Date: 2014-02-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently EC can only be used for scientific research

Method used

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  • Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria
  • Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria
  • Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The effect of the mutation of the regulatory factor Gln3p nuclear localization sequence on the expression of urea metabolism genes in bacterial strains

[0022] The build method is as follows:

[0023] 1) A GLN3 gene fragment ( figure 1 ). The GLN3 gene amplified by PCR was recovered and purified by gel, ligated with pMD19-T Vector (Takara), and transformed into E.coli JM109. The transformed positive clone T-GLN3 was selected by colony PCR on the ampicillin-resistant plate and sent to Shanghai Sangon for sequencing verification. Store the strains with correct sequences verified by sequencing in glycerol tubes at -20°C;

[0024] 2) After activating the strain saved in the previous step, extract the T-GLN3 plasmid. Using the T-GLN3 plasmid as a template and the mutation primers in Table 1 as primers, according to the instructions of the MutanBEST point mutation kit of Bao Bio Company, mutate the 344th, 347th and 355th Ser sites on Gln3p to alanine acid. Th...

Embodiment 2

[0027] Example 2 The effect of deletion of regulatory factor Gln3p nuclear localization regulatory sequence on strain urea metabolism

[0028] The build method is as follows:

[0029] 1) GLN3 with a length of 1959bp was amplified from the genome of Saccharomyces cerevisiae using GLN3-F' and GLN3-R' (see Table 1 for details) primers 1-653 Gene fragment( figure 1 ). GLN3 obtained by PCR amplification 1-653 After the gene was recovered and purified by gel, it was ligated with pMD19-TVector (Takara) and transformed into E.coli JM109. The transformed positive clone T-GLN3 was selected by colony PCR on the ampicillin-resistant plate 1-653 , sent to Shanghai Sangon for sequencing verification. Store the strains with correct sequences verified by sequencing in glycerol tubes at -20°C;

[0030] 2) Extract the constructed T-GLN3 1-653 And the preserved pYX212 plasmid, after Nco I and Sac I double digestion, gel recovery respectively. The recovered GLN3 1-653 The fragment was li...

Embodiment 3

[0032] Embodiment 3 Construction of more optimal low-yield EC yeast engineering bacteria

[0033] On the basis of the positive results of both the mutation of the Gln3p nuclear localization sequence and the nuclear localization regulatory sequence, the two methods were combined to construct a better yeast engineering strain with low EC production. The build method is as follows:

[0034] With the T-GLN3 constructed in embodiment 2 1-653 The plasmid was used as a template, and the mutation primers in Table 1 were used as primers. The Gln3p 1-653 Ser sites 344, 347 and 355 were mutated to alanine. The resulting T-GLN3 1-653,S344A,S347A,S355A The plasmid was transformed into E.coli JM109 and sent to Shanghai Sangon for sequencing verification. The strains with the correct sequence verified by sequencing were stored in glycerol tubes at -20°C.

[0035] In order to test the effect of deletion of the Gln3p nuclear localization regulatory sequence, real-time quantitative PCR was...

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Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate. A regulatory factor G1n3p in saccharomyces cerevisiae is transformed through engineering bacteria. The concrete strategy is that a phosphorylation site on a G1n3p nuclear localization sequence is subjected to mutation to form three phosphorylation sites on the G1n3p nuclear localization sequence; the three phosphorylation sites respectively are the 344th, 347th and 355th serine. In order to obtain a better technical effect, a nuclear localization regulatory region of combining G1n3p with an upstream regulatory factor can be further removed; the G1n3p of a regulatory sequence at the tail end of C is removed by truncated expression, wherein an expression sequence is 1-653. The EC yield of the engineering bacteria disclosed by the invention is reduced by 62% by detection of a yellow rice wine simulation system. In addition, the content of main ingredients is not significantly changed, and the fermentation characteristics and the growth characteristics of a bacterial strain are not affected after the content of other ingredients in a fermentation liquor after fermentation is detected.

Description

technical field [0001] The invention relates to a yeast metabolic engineering bacterium with low production of urethane and a construction method thereof, belonging to the field of microbial genetic engineering. Background technique [0002] my country's traditional fermentation industry has a long history and far-reaching influence. Traditional fermented products are complex and diverse, covering a wide range, such as vinegar, soy sauce, tempeh, fermented bean curd, white wine, rice wine, etc., which have formed a large scale and penetrated into all aspects of people's dietary life. Especially enjoy the rarity in wine, the yellow rice wine consumption market of the title of liquid cake is expanding day by day, is popular in countries in the world gradually, is listed as one of beverage wine of key support and development by the state. However, studies have found that the incomplete metabolism of nitrogen-containing compounds during the fermentation process will produce eth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/865
Inventor 陈坚周景文赵鑫锐堵国成
Owner JIANGNAN UNIV
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