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Genetically engineered bacterium with high yield of valarene as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and construction methods, applied in the field of high-yield valencene genetically engineered bacteria and its construction, can solve the problems of not reaching industrial production, poor separation, low yield, etc., and achieve increased yield, efficient transformation and integration, The effect of enhancing metabolic strength

Pending Publication Date: 2022-03-01
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

[0004] At present, the strains for biofermentation to produce valencene mainly use Escherichia coli engineered bacteria and Saccharomyces cerevisiae engineered bacteria transformed with valencene synthase, but the yields are all low, around 10mg / L, far from the level of industrial production
Moreover, Escherichia coli will produce endotoxin during fermentation and production, and it is not easy to separate in the later stage. The production of valencene, which is used in close contact with people, is at a disadvantage

Method used

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  • Genetically engineered bacterium with high yield of valarene as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium with high yield of valarene as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium with high yield of valarene as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Construction of high valencene-producing genetically engineered strain GS-A3-W4:

[0065] Step S1, construction of overexpressed tHMG1 module

[0066] tCYC1_tHMG1_pGAL10pGAL1

[0067] 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;

[0068] 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.

[0069] Step S2, construction of fusion expression ERG20-Linker-SmVS module

[0070] ERG20_Linker_SmVS_tERG20

[0071] 1) Using Saccharomyces cerevisiae 3000B genomic DNA as a template, PCR reactions were performed with ERG20-F and ERG20-Linker-R primers respectively, and ...

Embodiment 2

[0095] Construction of genetically engineered bacteria GS-A3-W5:

[0096] The construction method is the same as in Example 1, wherein the Linker selected in step S3 is Linker1, and the constructed DNA fragment for knocking out the expression of the galactose regulatory protein GAL80 gene is:

[0097] GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_SmVS_tERG20_GAL80right;

[0098] The constructed recombinant plasmid vector pWZ200 is:

[0099] pWZ200ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_SmVS_tERG20;

[0100] The engineered bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene is GS-A3-W1.

Embodiment 3

[0102] Construction of genetically engineered bacteria GS-A3-W6:

[0103] 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:

[0104] GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_SmVS_tERG20_GAL80right;

[0105] The constructed recombinant plasmid vector pWZ100 is:

[0106] pWZ100ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_SmVS_tERG20;

[0107] The engineered bacteria for knocking out the expression of the galactose regulatory protein GAL80 gene is GS-A3-W2.

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Abstract

The invention provides a construction method of a high-yield valarene gene engineering bacterium, which comprises the following steps: integrating and expressing an MVA pathway rate-limiting enzyme-truncated HMG-CoA reductase coding gene (tHMG1) and integrating and fusing and expressing an FPP synthase coding gene (ERG20) and a valarene synthase coding gene (SmVS) in saccharomyces cerevisiae in a homologous recombination manner, so as to enhance the metabolic intensity of an MVA pathway; and the expression of the varrenene is enhanced. Meanwhile, a squalene synthase encoding gene ERG9 promoter is replaced with a copper ion induced promoter pCUP1, the ergosterol competitive pathway is reduced, and the yield of the valarene is increased; and finally, obtaining the gene engineering strain with high yield of the varrenene. The shake flask fermentation yield of the valarene of the genetically engineered bacterium can reach about 117mg / L, the fermentation tank yield can reach about 8g / 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 genetically engineered bacterium with high valencene production and its construction method and application. Background technique [0002] Valencene (Valencene), molecular formula: C 15 h 24 , also known as valenciene, valenciene, valencene, is a sesquiterpene, which is the main aroma component of citrus fruits and citrus flavors, mainly obtained by cold pressing citrus fruit peels . As a spice, valencene is widely used in the food and cosmetic industries, and at the same time, it can be used as a precursor molecule to produce Nootkatone. Nootkatone is usually extracted from grapefruit by chemical or biochemical oxidation of valencene Yes, it has high economic value and medicinal value. [0003] Valencene can be isolated and extracted from plants, but the extraction steps are cumbersome, and the quality is closely related to the raw materials, and because the sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/54C12N15/53C12N15/60C12N15/31C12N15/81C12P5/00C12R1/865
CPCC07K14/395C12N9/1085C12N9/88C12N9/0006C12N15/81C12P5/002C12Y205/0101C12Y402/03073C12Y205/01021C12Y101/01034Y02A50/30
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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