Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach

A technology for Saccharomyces cerevisiae and yeast, applied in the fields of microbial genetics and molecular biology, can solve the problems of lack of Dal81p activation and decreased expression, and achieve the effects of less urea accumulation, reduced urea accumulation, and reduced urea accumulation.

Inactive Publication Date: 2016-01-27
JIANGNAN UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

In the absence of Dal81p and Dal82p activation, the expression of DUR1, 2 and DUR3 will be significantly reduced

Method used

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  • Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach
  • Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach
  • Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach

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Experimental program
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Embodiment 1

[0033] Elimination of the Dal81p ubiquitination site: use the MutanBESTkit site-directed mutagenesis kit to mutate the 69th and 918th lysine (K) sites on the Dal81p sequence of SEQ ID NO.1 to alanine (A ), see Table 1 for references used.

[0034] Table 1 Primers for site-directed mutagenesis of ubiquitination sites on Dal81p

[0035]

[0036] The mutant product was connected to the cloning vector pMD-19TSimple, and transformed into Escherichia coli JM109. Positive transformants were picked and verified by Sanger sequencing for overexpression in the next step.

[0037] Dal81p K69A,K918A and Dal82p expression vector construction: design primers to amplify DAL81 K69A,K918A And amino acid sequence such as the DAL82 gene of SEQIDNO.2 (forward primer adds SmaI or NotI restriction endonuclease cutting site; Reverse primer adds HindIII or BglII restriction endonuclease cutting site) (seeing table 2) . By enzyme cleavage-ligation, the DAL81 K69A, K918A and DAL82 were success...

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Abstract

The invention discloses a method for reducing saccharomyces cerevisiae urea accumulation by modifying a urea metabolism regulation approach and belongs to the field of microbial genetics and molecular biology. According to the method, related activating factors of a urea metabolic pathway are modified, and particularly, on the basis of eliminating ubiquitination regulatory sites of Dal81p, Dal81p<K69A>, <K918A> and Dal82p are subjected to overexpression. After the related activating factors of the saccharomyces cerevisiae urea metabolic pathway are subjected to genetic modification according to the method, urea accumulation during saccharomyces cerevisiae fermentation can be reduced fundamentally, and urea accumulation quantity is reduced by 55.7%.

Description

technical field [0001] The invention relates to a method for reducing urea accumulation in Saccharomyces cerevisiae by transforming a urea metabolism regulation pathway, and belongs to the field of microbial genetics and molecular biology. Background technique [0002] Urethane is considered a potential carcinogen to humans, and was classified as a Class 2B carcinogenic compound by the International Agency for Research on Cancer (IARC) in 1974. Since then, researchers have discovered that urethane can also directly induce human liver cancer, which further prompted IARC to raise the carcinogenic level of urethane from 2B to 2A (same level as formaldehyde) in 2007. The ethyl carbamate in rice wine has become an important hidden danger that endangers the health of consumers and affects its market competitiveness. [0003] In sake and rice wine, urea produced by the metabolism of Saccharomyces cerevisiae is the most important precursor of ethyl carbamate. During the fermentati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/865
Inventor 周景文陈坚堵国成吕永坤赵鑫锐
Owner JIANGNAN UNIV
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