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A xylitol-producing industrial Saccharomyces cerevisiae strain and its construction method

A technology of Saccharomyces cerevisiae strain and xylitol, which is applied in the field of microbial genetic engineering, can solve the problems of low expression level, high xylitol yield, loss of exogenous genes, etc., achieve high yield, superior performance, and avoid exogenous The effect of gene loss

Active Publication Date: 2019-01-04
SICHUAN UNIV
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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide an industrial Saccharomyces cerevisiae strain producing xylitol, which not only overcomes the problems of exogenous gene loss and low expression level during the expression of existing Saccharomyces cerevisiae strains; high

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  • A xylitol-producing industrial Saccharomyces cerevisiae strain and its construction method
  • A xylitol-producing industrial Saccharomyces cerevisiae strain and its construction method
  • A xylitol-producing industrial Saccharomyces cerevisiae strain and its construction method

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Embodiment

[0047] A method for constructing a xylitol-producing industrial Saccharomyces cerevisiae strain, comprising the following steps: (1) Design and amplify primers containing a target gene: According to the published δ sequence in the NCBI database, design and synthesize two pairs of primers: one A pair of primers is δ-pB-F (SEQ ID NO.2), δ-pB-R (SEQ ID NO.3); another pair of primers is δ-XR-XDH-F (SEQ ID NO.4), δ- pB-R (SEQ ID NO. 3);

[0048] (2) Preparation of pK-XR-Ct plasmid;

[0049] (3) Amplification of the target gene: using the pK-XR-Ct plasmid as a template and δ-pB-F and δ-pB-R as primers, amplify xyl1 containing xylose reductase gene and G418 resistance screening gene Linearized DNA fragment A of KanMX, and purify and recover fragment A obtained by PCR amplification; the promoter of gene xyl1 is pGAPDH, and the terminator is tGAPDH; the promoter of KanMX gene is pTEF1, and the terminator is tTEF1; PCR amplification reaction system (50 μl) is composed as follows: 5×Pr...

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Abstract

The present invention relates to a xylitol-producing industrial Saccharomyces cerevisiae strain and a construction method, the preservation number of the Saccharomyces cerevisiae strain is: CGMCC11326, and the construction method comprises the following steps: using pK-XR-Ct plasmid as a template, δ-pB- F, δ-pB-R are primers, PCR amplification obtains DNA fragment A, takes DNA fragment A as template, amplifies and obtains DNA fragment B; the G418 optimal tolerance concentration screening of Saccharomyces cerevisiae fusion strain; Saccharomyces cerevisiae fusion strain Transformation; single-cell transformants were screened to obtain Saccharomyces cerevisiae strains. The Saccharomyces cerevisiae SEB6 strain prepared by the construction method of the present invention has superior performance in producing xylitol by xylose fermentation, and the yield of xylitol is high; at the same time, it avoids the loss of exogenous genes and low expression level when expressing the existing Saccharomyces cerevisiae strains The problem.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a xylitol-producing industrial Saccharomyces cerevisiae strain and a construction method. Background technique [0002] Agricultural wastes such as straws, husks, and cores are rich in crude fibers (pentanose), but these agricultural wastes are mostly disposed of by waste or incineration, resulting in a great waste of resources and environmental pollution. And xylitol can be obtained by hydrolysis of crude fiber (pentose). Using agricultural wastes as raw materials to prepare xylitol is an effective way to turn agricultural and sideline products into treasures, protect the environment, and save resources. As a functional sweetener, xylitol is an adjuvant therapeutic agent for diabetics. It has functions such as weight loss, intestinal regulation, and liver function improvement. It is widely used in the food industry. [0003] The methods for producing xyli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/865
Inventor 汤岳琴杨白雪苟敏孙照勇木田建次夏子渊
Owner SICHUAN UNIV
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