Recombinant yeast strain and application thereof

A technology for yeast and Saccharomyces cerevisiae, which is applied in the field of genetic engineering, can solve the problems of increasing energy consumption and producing production costs, and achieves the effects of increasing costs, simple operation and easy control.

Active Publication Date: 2019-06-28
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires precise temperature control and low temperature, which will increase energy consumption and generate additional production costs in actual production

Method used

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  • Recombinant yeast strain and application thereof
  • Recombinant yeast strain and application thereof
  • Recombinant yeast strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of Gene Editing System

[0024] In order to facilitate the genetic manipulation of Saccharomyces cerevisiae, the present invention first constructed pHCas9M-gRNA based on the pHCas9-Nours plasmid (Molecular Cloud Cat. No.: MC_0000293) and the pYES2-gRNA-hyg-MCS plasmid (Molecular Cloud Cat. No.: MC_0000294) Plasmid (Cat. No.: MC_0000739). The plasmid contains an HCas9 module, a gRNA expression module, and a G418 module. Specifically, the HCas9 fragment is amplified with primers HCas920170518-F / HCas920170518-R, the G418 fragment is amplified with primers G41820170518-F / G41820170518-R, and the G418 fragment is amplified with primer 2μ20170518 -F / 2μ20170518-R was amplified to obtain a 2μ fragment, and the primer Amp20170518-F / Amp20170518-R was used to amplify the Amp fragment, and then the above fragment was ligated using a recombination kit (ClonExpress MultiS One Step Cloning Kit) to construct pHCas9M-gRNA Plasmid, the pHCas9M-gRNA plasmid such as...

Embodiment 2

[0027] The construction of embodiment 2 growth coupling dynamic regulation bacterial strain

[0028]According to the genome data of Deinococcus gobi, the lycopene synthesis genes crtE, crtB and crtI were synthesized through codon optimization. The nucleotide sequence of the gene crtE is shown in SEQ ID NO.1; the nucleotide sequence of the crtB is shown in SEQ ID NO.2; the nucleotide sequence of the gene crtI is shown in SEQ ID Shown in NO.3. In order to obtain a better expression form, the present invention has carried out the construction of three bacterial strains. 1) Place the crtE, crtB and crtI genes under the HSP26 promoter, and integrate them into the 208a, 720a and 308a sites of the S. and under the HSP82 promoter, integrated into the YPRCt3 site of the Saccharomyces cerevisiae genome in a tandem manner to construct strain BX03. 3) Place the crtB, crtI and crtE genes under the HSP26 promoter and integrate them into the gal80 of the Saccharomyces cerevisiae genome in ...

Embodiment 3

[0048] The dynamic control of embodiment 3 rate-limiting steps

[0049] The HMG1 gene of Saccharomyces cerevisiae is considered to be the rate-limiting step of the entire MVA pathway, because there is a transmembrane region in front of the HMG1 gene, which is subject to strict metabolic regulation. In order to overcome this problem, the present invention overexpresses HMGR derived from myxobacteria, which has the same function as HMG1, but it has no regulatory region. HMGR (OhmgR) was synthesized from the whole gene through codon optimization, and the nucleotide sequence of the HMGR gene is shown in SEQ ID NO.4. In order to obtain a better control mode, the present invention selects Cit 1 , HSP26 and HSP104 promoters were screened and found that Cit 1 The promoter effect is the best, which can reach 2 times of the HSP104 promoter, and their transcriptional characteristics were determined.

[0050] Specifically, based on the BL03 strain constructed in Example 2, use primers ...

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Abstract

The invention discloses a recombinant yeast strain and application thereof. The recombinant yeast strain expresses lycopene synthesizing genes crtE, crtB and crtI, the genes crtE, crtB and crtI come from deinococcus gobiensis, and all the genes are put under a growth-coupling dynamic controlling element HSP26 promoter, and are integrated onto a saccharomyces cerevisiae genome in a series mode; anHMGR gene is dynamically controlled through the overexpression rate-limiting step of the recombinant yeast strain, the HMGR gene is put under a growth-coupling dynamic controlling element Cit1 promoter, a competitive metabolic pathway ERG9 promoter is replaced with a growth-coupling dynamic controlling element PDC1 promoter, an HMG1 gene promoter and a control area of the HMG1 promoter are replaced with the growth-coupling dynamic controlling element Cit1 promoter, and an Ald6 gene is knocked out. The recombinant yeast strain can be applied to synthesis of carotenoid.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant yeast strain and its application. Background technique [0002] Saccharomyces cerevisiae, as a generally recognized as safe microorganism (GRAS), has been widely used in industrial production. As a model eukaryotic system, Saccharomyces cerevisiae has many genetic manipulation tools, and metabolic engineering is convenient and fast; and Saccharomyces cerevisiae shows a high degree of tolerance to harsh industrial production conditions. Therefore, Saccharomyces cerevisiae is often the preferred host for biosynthesis. When performing metabolic engineering, most of the key enzymes in the metabolic pathway are overexpressed through a static control system. The static control system will not only cause the metabolic flow to enter the target metabolic pathway prematurely, affecting the growth of microbial cells; it will also cause the imbalance of the metabo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P23/00C12R1/865
CPCY02E50/10
Inventor 朱红惠苏卜利宋丹丹杨帆
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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