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Recombinant saccharomyces cerevisiaes for producing 7-dehydrocholesterol and construction method thereof

A technology of dehydrocholesterol and recombinant strains, applied in the field of microbial metabolism, to achieve the effect of increasing production

Active Publication Date: 2021-07-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, no one has done so far to improve the production of 7-dehydrocholesterol by rearranging the distribution of enzymes in the 7-dehydrocholesterol synthesis pathway in different organelles

Method used

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  • Recombinant saccharomyces cerevisiaes for producing 7-dehydrocholesterol and construction method thereof
  • Recombinant saccharomyces cerevisiaes for producing 7-dehydrocholesterol and construction method thereof
  • Recombinant saccharomyces cerevisiaes for producing 7-dehydrocholesterol and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Rearrange the organelle distribution of enzymes downstream of yeast sterols to increase the yield of 7-dehydrocholesterol The preliminary construction method of the recombinant Saccharomyces cerevisiae strain producing 7-dehydrocholesterol of the present invention is as follows:

[0082] 1. The high-yielding 7-dehydrocholesterol Saccharomyces cerevisiae was provided by Yuan Yingjin's research group, and the strain number is SyBE_Sc0125X001 (Guo, X.J., Xiao, W.H., Wang, Y., Yao, M.D., Zeng, B.X., Liu, H. , Zhao, G.R., and Yuan, Y.J. (2018). Metabolic engineering of Saccharomyces cerevisiae for 7-dehydrocholesterol overproduction. Biotechnol Biofuels 11, 192).

[0083] 2. Acquisition of exogenous functional gene elements

[0084] The exogenous gene is the C24 reductase gene DHCR24 (as shown in SEQ ID No.1) used for the synthesis of 7-dehydrocholesterol, and the source is chicken source (gallus). After circumventing common restriction enzyme cutting sites, 5' en...

Embodiment 2

[0107] Example 2 Rearranging the organelle distribution of yeast sterol upstream enzymes to improve 7-dehydrocholesterol production

[0108] 1. Construction of modular integration plasmids

[0109] First, the applicant designed to divide the enzymes present in the endoplasmic reticulum upstream of zymosterol in the post-squalene pathway into two modules: ERG1-ERG11-ERG24 is the first module, and ERG25-ERG26-ERG27 is the second module. In order to ensure the stable existence of the module, a method of integrating the module into the genome is selected. Since repeated sequences in Saccharomyces cerevisiae are easy to produce repeated sequences and are lost from the genome by homologous recombination, it is necessary to avoid the integration of two modules containing AAM-B sequences at the same site, so this experiment is designed to The first two genes of the three genes in the module were fused, and the Oleosin sequence was placed between the two genes for positioning; the thi...

Embodiment 3

[0123] Example 3 Combining the organelle distribution of the upstream and downstream enzymes of zymosterol to further improve the production of 7-dehydrocholesterol

[0124] 1. Construction of modular integration plasmids

[0125] In order to further reduce the by-products in the strain, the authors introduced the downstream genes ERG2 and ERG3 in the post-squalene pathway into the tau3 site in the high-yield 7-dehydrocholesterol strain. The specific construction process is to use the genome as a template to amplify the 700bp fragment upstream and downstream of tau3 and the terminators TDH2t and PGK1t. The upstream homology arm tau3_L was obtained by linking tau3_up700 and TDH2t by OE-PCR, and the downstream homology arm tau3_R was obtained by linking PGK1t and tau3_down700.

[0126] 2. Modular integration construction of recombinant Saccharomyces cerevisiae strains producing 7-dehydrocholesterol

[0127] First, cut the previously constructed expression module TDH2t-gal1p-ER...

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Abstract

The invention relates to the field of microbial metabolism, and particularly relates to saccharomyces cerevisiaes for producing 7-dehydrocholesterol and a construction method thereof. The invention discloses two recombinant saccharomyces cerevisiaes for producing 7-dehydrocholesterol, named as saccharomyces cerevisiae SyBE_Sc01250043 and saccharomyces cerevisiae SyBE_Sc01250045, or SyBE_Sc01250043 and SyBE_Sc01250045 for short, as well as a construction method thereof. Compared with the existing reported strains, the recombinant saccharomyces cerevisiaes of the present invention has the advantage of significantly improving yield of 7-dehydrocholesterol.

Description

technical field [0001] The invention relates to the field of microbial metabolism, in particular to a saccharomyces cerevisiae strain producing 7-dehydrocholesterol and a construction method thereof. Background technique [0002] 7-Dehydrocholesterol is a steroid with high added value and is widely used in industry and medicine. It can be used in liquid crystal manufacturing, electromagnetic detection, biomedicine and other fields. 7-dehydrocholesterol can be converted into vitamin D3 by ultraviolet radiation, and 7-dehydrocholesterol in human skin can be converted into vitamin D by sunlight ultraviolet radiation. Vitamin D can be used to treat vitamin D3 deficiency, rickets, etc., familial hypophosphatemia, hypoparathyroidism, etc., and vitamin D deficiency may lead to cardiovascular diseases, metabolic syndrome, cancer, autoimmune diseases, etc. At present, vitamin D3 is mainly obtained from tuna liver oil, or synthesized by photochemical reaction of 7-dehydrocholesterol....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P33/02C12R1/865
CPCC12N9/00C12N15/81C12P33/02
Inventor 元英进郭晓静王颖肖文海姚明东
Owner TIANJIN UNIV
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