Genetically engineered bacterium with high yield of squalene, construction method and application thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of high-yield squalene genetically engineered bacteria and its construction, to achieve efficient transformation and integration, shorten the construction cycle, and increase production

Pending Publication Date: 2022-02-18
湖北冠众通科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many studies on transforming Saccharomyces cerevisiae to produce squalene, but there is no microbial fermentation of squalene that has been mass-produced and put into the market. Therefore, the construction of high-yield and stable squalene-producing Saccharomyces cerevisiae production strains is of great significance for its production and application. important meaning

Method used

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  • Genetically engineered bacterium with high yield of squalene, construction method and application thereof
  • Genetically engineered bacterium with high yield of squalene, construction method and application thereof
  • Genetically engineered bacterium with high yield of squalene, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Construction of high-yield squalene genetically engineered strain GS-A3-S4:

[0064] Step S1, construction of overexpressed tHMG1 module

[0065] tCYC1_tHMG1_pGAL10pGAL1

[0066] 1) Using Saccharomyces cerevisiae 3000B genomic DNA as a template, PCR reactions were performed with primers tCYC1-F and tCYC1-R, tHMG1-F and tHMG1-R, pGAL1pGAL10-F and pGAL1pGAL10-R, respectively, to obtain DNA fragments tCYC1, tHMG1 and pGAL10pGAL1;

[0067] 2) The three DNA fragments tCYC1, tHMG1 and pGAL10pGAL1 obtained in step S1 were connected together by overlapping extension PCR reaction with primers tCYC1-F and pGAL1pGAL10-R to obtain the overexpressed tHMG1 module, namely the tCYC1_tHMG1_pGAL10pGAL1 module.

[0068] Step S2, construction of fusion expression ERG20-Linker-ERG9 module

[0069] ERG20_Linker_ERG9_tERG20

[0070] 1) Using Saccharomyces cerevisiae 3000B genomic DNA as a template, PCR reactions were performed with ERG20-F and ERG20-Linker-S-R primers respectively, and the...

Embodiment 2

[0093] Construction of genetically engineered bacteria GS-A3-S5:

[0094] The construction method is the same as in Example 1, wherein the Linker selected in step S3 is connected to Linker2, and the constructed DNA fragment for knocking out the expression of the galactose regulatory protein GAL80 gene is:

[0095] GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_ERG9_tERG20_GAL80right;

[0096] The constructed recombinant plasmid vector pSZ200 is:

[0097] pCZ200ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_ERG9_tERG20;

[0098] The constructed engineered bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene is GS-A3-S2.

Embodiment 3

[0100] Construction of genetically engineered bacteria GS-A3-S6:

[0101] The construction method is the same as in Example 1, wherein the Linker selected in step S3 is Linker3, and the constructed DNA fragment for knocking out the expression of the galactose regulatory protein GAL80 gene is:

[0102] GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_ERG9_tERG20_GAL80right;

[0103] The constructed recombinant plasmid vector pSZ300 is:

[0104] pCZ300ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_ERG9_tERG20;

[0105] The engineered bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene is GS-A3-S3.

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Abstract

The invention provides a construction method of a genetically engineered bacterium with high yield of squalene, an MVA pathway rate-limiting enzyme-truncated HMG-CoA reductase encoding gene (tHMG1) is integrated and expressed, and an FPP synthase encoding gene (ERG20) and a saccharomyces cerevisiae endogenous squalene synthase encoding gene (ERG9) is integrated and expressed in saccharomyces cerevisiae in a homologous recombination manner; the MVA pathway metabolism intensity is enhanced, and the squalene expression is enhanced. Meanwhile, a squalene monooxygenase encoding gene ERG1 promoter is replaced with a copper ion induced promoter pCUP1, the expression level of the squalene monooxygenase encoding gene ERG1 promoter is lowered, ergosterol synthesized by squalene epoxidation is reduced, and the squalene yield is increased; and finally the genetically engineered strain with high squalene yield can be obtained. The shake flask fermentation yield of squalene of the genetically engineered bacterium can reach about 57mg / L, the fermentation tank yield can reach about 7g / L, and the genetically engineered bacterium completely has a commercial production level and has a good industrial application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a high-yielding squalene genetically engineered bacterium and its construction method and application. Background technique [0002] Molecular formula of squalene: C 30 h 60 , also known as squalene, is an acyclic triterpene composed of 6 isoprenes. Widely present in animals, plants and microorganisms, it is widely used in the fields of cosmetics, food, health products and medicine because of its strong biological activity. [0003] Squalene can be extracted from the liver oil of deep-sea sharks, which is expensive and the number of sharks is decreasing sharply. Sharks are unsustainable as a source of squalene; it can also be extracted and separated from various oil crops, but the content is extremely low and the raw material The pretreatment is complicated and the output is unstable, which makes it difficult to carry out the industrial production of squalene. In ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12N15/31C12P5/02C12R1/865
CPCC12N15/81C12N9/0006C12N9/1085C07K14/395C12N1/18C12P5/007C12Y101/01034C12Y205/0101C12Y205/01021
Inventor 陈强刘登辉向景刘传春
Owner 湖北冠众通科技有限公司
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