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Recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression and construction method thereof

A technology for Saccharomyces cerevisiae and a construction method, which is applied in the field of bioengineering and can solve the problems of increasing the cost of raw materials, the complexity of fermentation control, and the disadvantages.

Inactive Publication Date: 2019-12-31
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the fermentation process, this method of inhibiting gene expression needs to add a large amount of methionine to achieve better results, which increases the cost of raw materials for fermentation and the complexity of fermentation control, which is not conducive to the actual production process.

Method used

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  • Recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression and construction method thereof
  • Recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression and construction method thereof
  • Recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0035] The ERG7p promoter mutant was constructed by using TetR and TetO regulatory elements, and the promoter strength was evaluated by using the green fluorescent protein GFP, whose gene sequence is shown in SEQ ID NO.2.

[0036] GFP biological source: Aequorea Victoria (codon-optimized for Saccharomyces cerevisiae)

[0037] 1. Construct the TetR gene expression cassette ACS1p-TetR-Cyc1t, and transform the gene expression cassette into the genome of Saccharomyces cerevisiae (W303-1a). The nucleotide sequence of the TetR is shown in SEQ ID NO.1.

[0038] Using the Saccharomyces cerevisiae W303-1a genome as a template, using ACS1p-F (SEQ ID NO.3) and ACS1p-R (SEQ ID NO.4) as primers to amplify the ACS1p promoter;

[0039] The Cyc1t terminator was amplified using the Saccharomyces cerevisiae W303-1a genome as a template and Cyc1t-F (SEQ ID NO.7) and Cyc1t-R (SEQ ID NO.8) as primers.

[0040] The TetR (SEQ ID NO.1) gene synthesized by Suzhou Jinweizhi Co., Ltd. was used as a te...

Embodiment 2

[0111] A method for constructing a recombinant bacterium that finely regulates the expression of Saccharomyces cerevisiae ERG7, comprising the steps of:

[0112] 1. Introducing the ACS1p-TetR-Cyc1t gene expression cassette into the original panaxadiol synthetic strain (W3a-HU)

[0113] W3a-HU is obtained by recovering the URA3 and HIS3 marker genes of W3a through plasmid pSH63.

[0114] The strain W3a comes from the authorized patent ZL201410735927.X, and the specific construction process of the W3a strain refers to this patent.

[0115] In the W3a strain, the URA3 and HIS3 markers have been utilized, but these two markers are flanked by two loxP sites. These two marker genes were first knocked out with the plasmid pSH63 expressing Cre protein (purchased from Biowind) to generate W3a-HU strain. The specific operation adopts lithium acetate transformation: the preparation of Saccharomyces cerevisiae competent state is the same as that in Example 1, and the transformation mixe...

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Abstract

The invention discloses recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression and a construction method thereof. The construction method comprises the following steps of: (1) constructing a TetR gene expression cassette ACS1p-TetR-Cy1t, and transferring the cassette into saccharomyces cerevisiae; (2) inserting a TetO sequence into places from an initial codon to -78 bp of a saccharomyces cerevisiae ERG7p promoter so as to obtain a saccharomyces cerevisiae ERG7p promoter mutant ERG7Cp; and (3) replacing the endogenous ERG7p promoter in the saccharomyces cerevisiae obtained in the step (1) by the ERG7Cp to obtain the recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression. According to the invention, a tetracycline repressor protein TetR and a TetO sequence control element are utilized to realize fine control on ERG7 expression. The constructed recombinant bacteria for fine regulation and control of saccharomyces cerevisiae ERG7 expression can inhibit the expression of the ERG7 gene in different degrees and improve the yield of engineering saccharomyces cerevisiae protopanaxadiol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant bacterium and a construction method for finely regulating the expression of Saccharomyces cerevisiae ERG7. Background technique [0002] Triterpenoids, such as protopanaxadiol, oleanolic acid, glycyrrhetinic acid, betulinic acid, ursolic acid, etc., are the main components of many Chinese herbal medicines to exert their medicinal activities, and are widely used in medicine, health products and food fields . At present, such compound products are mainly obtained through plant extraction. Taking protopanaxadiol as an example, ginsenosides in ginseng are extracted, and then obtained through acid-base hydrolysis or enzymatic hydrolysis. Traditional production methods have low extraction efficiency, long production cycle, high cost and great damage to the environment, which are far from meeting the demand for triterpenoids in the chemical and pharmace...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P33/00C12R1/865
CPCC12N15/81C07K14/00C12P33/00
Inventor 卢文玉赵方龙南伟华张传波
Owner TIANJIN UNIV
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