Method for simultaneously enhancing and inhibiting multiple key genes in synthesis of saccharomyces cerevisiae 7-dehydrocholesterol

A technology of dehydrocholesterol and Saccharomyces cerevisiae, applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc.

Active Publication Date: 2022-06-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the microbial metabolic engineering synthesis methods of 7-DHC are carried out based on strains containing natural sterol synthesis pathways, because the metabolic flow of sterol synthesis pathways accounts for the total amount of bacteria The proportion of metabolic flow is relatively low, mainly focusing on the enhancement of genes in known synthetic pathways and the blocking of branched pathways. For example, Chinese patent CN104988168A increases the synthesis of acetyl-CoA, a precursor of natural sterol synthesis, to increase the production of 7-DHC. Yield, but also through the traditional replacement of constitutive strong promoters to enhance the expression of genes (acs, adh2, acl and ald6), but this will cause excessive metabolic pressure on the bacteria
Chinese patent CN104988168A and Chinese patent CN103275997A further enhance the synthesis of 7-DHC by directly knocking out the key genes in the ergosterol synthesis pathway: erg5 and erg6, but completely blocking the synthesis of ergosterol will have an irreversible effect on the growth of the strain, Not conducive to industrial mass production
The chemical synthesis method needs to use more expensive lanolin cholesterol as raw material, after multi-step group protection and deprotection, to obtain 7-dehydrocholesterol, and then to obtain VD3 through light reaction, ring opening and isomerization, due to the use of various Organic reagents, the separation and purification process of the product is complicated, and the product yield is low

Method used

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  • Method for simultaneously enhancing and inhibiting multiple key genes in synthesis of saccharomyces cerevisiae 7-dehydrocholesterol
  • Method for simultaneously enhancing and inhibiting multiple key genes in synthesis of saccharomyces cerevisiae 7-dehydrocholesterol
  • Method for simultaneously enhancing and inhibiting multiple key genes in synthesis of saccharomyces cerevisiae 7-dehydrocholesterol

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Embodiment 1

[0045] Example 1 Construction of Saccharomyces cerevisiae producing 7-DHC and related genes in the enhanced pathway

[0046] (a) Using the S288C genome of Saccharomyces cerevisiae as a template, use primers ADH-F and ADH-R to amplify the gene fragment ADH2, use primers IDI-F and IDI-R to amplify the gene fragment IDI1, and use primers E1-F , E1-R amplified to obtain gene fragment ERG1, using primers E11-F, E11-R to amplify to obtain gene fragment ERG11, using primers E24-F, E24-R to amplify to obtain gene fragment ERG24, using primers E25-F, E25 -R to amplify the gene fragment ERG25, use primers E26-F and E26-R to amplify the gene fragment ERG26, use primers E27-F and E27-R to amplify the gene fragment ERG27, use primers GAL1-F, GAL1-R The promoter gene fragment gal1p was amplified, and the promoter gene fragment gal7p was amplified by using primers GAL7-F and GAL7-R. Through gene synthesis, fragments dhcr24 and thmg1 were obtained. Using primers 208UP-F and 208UP-R to ampli...

Embodiment 2

[0101] Construction and application of embodiment 2dCpf1-VP activation system

[0102] (a) Using the plasmid pCSN068 purchased from Addgene as a template, Cpf1 was mutated into dCpf1 using primers dCpf1Tu-F and dCpf1Tu-R. The activation domain VP was gene synthesized, and the activation domain VP was connected with pCSN068 containing dCpf1 using seamless cloning to obtain the plasmid pCSN068-VP. Using the plasmid pML104 purchased from Addgene as a template, primers 52p-F and 52p-R were used to amplify the promoter gene fragment SNR52p. The fragment gal1p23-1 was synthesized directly by overlapping primers gal1p23-F1 and gal1p23-R1, and the fragment gal7p23-1 was synthesized directly by overlapping primers gal7p23-F1 and gal7p23-R1. The fragment gal1p23-2 was synthesized directly by overlapping primers gal1p23-F2 and gal1p23-R2, and the fragment gal7p23-2 was synthesized directly by overlapping primers gal7p23-F2 and gal7p23-R2. The fragment gal1p23-3 was synthesized directly...

Embodiment 3

[0145] Construction and application of embodiment 3dCas9-RD suppression system

[0146] (a) Using the plasmid pML104 purchased from Addgene as a template, Cas9 was mutated into dCas9 using primers dCas-F1, dCas-R1, dCas-F2, and dCas-R2. Gene synthesis inhibitory domain RD, use seamless cloning to connect activation domain RD and pML104 to obtain plasmid pML104-RD, use the above constructed plasmid as a template, and use CIT-F and CIT-R as primer loop P to obtain plasmid pML104 -RD-CIT, using MLS1-F and MLS1-R as primer loop P to obtain plasmid pML104-RD-MLS, using ERG6-F and ERG6-R as primer loop P to obtain plasmid pML104-RD-ERG6. The fragments URA-dCas9-RD-CIT, URA-dCas9-RD-MLS, and URA-dCas9-RD-ERG6 were amplified by using URA-104-F and URA-104-R as primers, respectively. Using the plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene as a template, the fragment pFA6a-TRP1-GFP was amplified using primers pFA6a-F and pFA6a-R, and using the genome of Saccharomyces cerevisiae S...

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Abstract

The invention provides a method for simultaneously enhancing and inhibiting multiple key genes in synthesis of saccharomyces cerevisiae 7-dehydrocholesterol, and belongs to the technical field of bioengineering. Through a dCpf1-VP activation system and a dCas9-RD inhibition system, the purpose of simultaneous expression and inhibition of 13 P450 enzymes is achieved, and the engineering yeast capable of producing 7-dehydrocholesterol at high yield is constructed, so that the yield of 7-DHC reaches 464mg / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae. Background technique [0002] Vitamin D3 (Cholecalciferol, VD3) is a hormone precursor in the human body. It can regulate cell differentiation and promote bone calcification. VD3 is hydroxylated in the human liver to form 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 , 25-OH-VD3), which is converted into 1,25-dihydroxyvitamin D3 (1,25-dihydroxyvitamin D3, 1,25-(OH)2-VD3) in the kidney. The main source of vitamin D3 in the human body is through ultraviolet light It is transformed from 7-dehydrocholesterol (7-dehydrocholestero, 7-DHC) in the bottom layer of the irradiated skin, and part of it can also be obtained through food, but due to the different dietary structure of each person, the effect of supplementing VD3 through diet is not go...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/04C12N9/90C12N9/02C12N9/10C12N15/53C12N15/54C12N15/61C12N15/81C12P33/02C12R1/865
CPCC12N9/0006C12N9/90C12N9/0071C12N9/001C12N9/1007C12N9/1025C12N15/81C12P33/02C12Y101/01001C12Y101/01034C12Y503/03002C12Y103/0107C12Y101/0117C12Y101/0127C12Y201/01041C12Y103/01072C12Y203/03009C12N2800/22C12N2800/102
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰修翔
Owner JIANGNAN UNIV
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