Method for increasing yield of 7-dehydrocholesterol in saccharomycetes by using compartmentalization

A dehydrocholesterol and yeast technology, applied in the field of metabolic engineering, can solve the problems of acetyl-CoA deficiency, reduce the feedback inhibition of metabolic pathways, increase the storage space of the final product, etc., achieve reduced losses, reduce feedback effects, and improve conversion efficiency Effect

Active Publication Date: 2021-05-18
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

[0006] In order to solve the current problem of insufficient acetyl-CoA in the synthesis of 7-DHC in the yeast cytoplasm, the present invention uses the acetyl-CoA in peroxisomes and mitochondria to pass the enzymes in the 7-DHC synthesis pathway through peroxidase Somatosome and mitochondrial localized protein tags are respectively located in mitochondria and peroxisomes, so that yeast can not only synthesize important 7-DHC in the cytoplasm, but also synthesize in peroxisomes and mitochondria, achieving improved metabolism The transformation efficiency of the substrate in the pathway increases the storage space of the final product and reduces the feedback inhibition in the metabolic pathway. Finally, the yield of 7-DHC in yeast is 52.31mg / L through this method

Method used

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  • Method for increasing yield of 7-dehydrocholesterol in saccharomycetes by using compartmentalization
  • Method for increasing yield of 7-dehydrocholesterol in saccharomycetes by using compartmentalization
  • Method for increasing yield of 7-dehydrocholesterol in saccharomycetes by using compartmentalization

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Construction of Saccharomyces cerevisiae CDHC-2

[0026] (a) Artificially synthesized gene fragment P TEF1 -DHCR24-T cyc1 -P GAP -DIC-T ADH1 , using the Saccharomyces cerevisiae S228C genome as a template, using primers 208F-F, 208F-R to amplify the gene fragment 208-F, using primers 208R-F, 208R-R to amplify the gene fragment 208-R, using pMHyLp-trp plasmid As a template, use primers loxT-F and loxT-R to amplify the loxT fragment of the gene fragment.

[0027] (b) the four fragments P obtained in step (a) TEF1 -DHCR24-T cyc1 -P GAP -DIC-T ADH1, 208F-F, 208-R and locxT gene fragments were subjected to overlap extension PCR, and after 1% agarose gel electrophoresis was verified to be correct, the fragments were recovered by cutting the gel to obtain the fusion gene fragment 208-F-loxT-P TEF1 -DHCR24-T cyc1 -P GAP -DIC-T ADH1 -208-R gene fragment.

[0028] (c) Transform the gene fragment obtained in step (b) into the competent strain of Saccharomyce...

Embodiment 2

[0039] Example 2: Construction of Saccharomyces cerevisiae mitochondrial compartmentalization

[0040] (a) Using the S228C genome of Saccharomyces cerevisiae as a template, the mitochondrial localization signal peptide MMF1 was selected. The gene fragment ERG12-MMF1 was amplified by primers ERG12-F and ERG12-MMF1-R, the gene fragment ERG13-MMF1 was amplified by primers ERG13-F1 and ERG13-MMF1-R1, and the gene fragment ERG13-MMF1 was amplified by primers GAL-F1 and GAL-R1. The GAL1 promoter and GAL10 promoter gene fragment GAL1-10 were amplified, the gene fragment Lox-Trp was amplified by primers loxT-GAL-F and LoxT-GAL-R, and the gene fragment was amplified by primers GAL80F1-F and GAL80F1-R Fragment GAL80F1, use primers GAL80R1-F, GAL80R1-R to amplify the gene fragment to obtain GAL80R1, use CYC1-S-F, CYC1-S-R to amplify the gene fragment to obtain CYC1, use primers ADH1-S-F, ADH1-S-R to amplify the gene fragment to obtain ADH1, Using MMF1-F, MMF1-R, MMF2-F, MMF2-R as primer...

Embodiment 3

[0067] Example 3: Construction of Saccharomyces cerevisiae peroxisome compartmentalization

[0068] (a) Using the S228C genome of Saccharomyces cerevisiae as a template, the peroxisome localization signal peptide PTS1 was selected. The gene fragment ERG12-PTS1 was amplified by primers ERG12-F and ERG12-PTS1-R, the gene fragment ERG13-PTS1 was amplified by primers ERG13-F and ERG13-PTS1-R, and the gene fragment ERG13-PTS1 was amplified by primers GAL-F and GAL-R. The GAL1 promoter and GAL10 promoter gene fragment GAL1-10 were amplified, the gene fragment Lox-Trp was amplified by primers loxT-GAL-F and LoxT-GAL-R, and the gene fragment was amplified by primers GAL80F3-F and GAL80F3-R Fragment GAL80F3, use primers GAL80R3-F, GAL80R3-R to amplify the gene fragment to obtain GAL80R3, use primers CYC1-F, CYC1-13-R to amplify the gene fragment to obtain CYC1, use primers ADH1-10-F, ADH1-R to amplify The gene fragment was obtained as ADH1.

[0069] (b) The CDHC-10 strain obtained in...

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Abstract

The invention relates to a method for increasing the yield of 7-dehydrocholesterol in saccharomycetes by using compartmentalization. The method comprises the following steps of: by taking saccharomycetes as an original strain, expressing heterologous sterol delta 24-reductase and delta-cholestenase, wherein the yield of 7-DHC in saccharomyces cerevisiae S288C is detected to be 10.15mg / L. According to the method disclosed by the invention, partial enzymes in a 7-DHC synthesis path are positioned in each compartmentalization in the saccharomyces cerevisiae by using an oxidase and a mitochondrial positioning tag, and a relatively independent 7-DHC synthesis path is formed. Meanwhile, the storage space of precursor substances needed by 7-DHC synthesis is increased, the feedback effect is reduced, the conversion efficiency between enzymes is improved in the same compartment, the loss of the acting substrate is reduced, and finally the yield of the 7-DHC is improved by 4 times and reaches 53.31mg / L.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a method for increasing the yield of 7-dehydrocholesterol in yeast by using compartmentalization. Background technique [0002] 7-dehydrocholesterol is a sterol with high added value. The 7-dehydrocholesterol (7-DHC) synthesized in the human body is directly converted into vitamin D3 after being irradiated by sunlight in the skin tissue. Vitamin D3 is not only a fat-soluble vitamin necessary for the growth and development of human skeletal muscles, but also reduces the risk of immune system disorders and various cancers, and also has a preventive effect on cardiovascular diseases. The lack of vitamin D has been recognized as a global problem. Health problems, which also increase the annual global demand for vitamin D3 or its immediate precursor 7-DHC. 7-DHC is an important precursor for the synthesis of sterol drugs such as androstenedione and 9α-hydrox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/02C12N1/19C12N15/81C12N15/53C12N15/61C12R1/865
CPCC12P33/02C12N9/001C12N9/90C12N15/81C12Y103/01072C12Y503/03005
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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