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Fusion protein capable of improving dammarenediol conversion efficiency, construction method and application

A technology for fusion protein and dammarene diol, which is applied in the field of fusion protein and construction, can solve the problem of low conversion efficiency of dammarene diol, and achieve the effect of being conducive to efficient synthesis and improving conversion efficiency

Active Publication Date: 2015-05-13
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have found that PPDS is a rate-limiting step in the artificially constructed protopanaxadiol yeast synthesis pathway, and simply increasing the copies of PPDS and its reductase cannot solve the problem, resulting in the conversion efficiency of dammarenediol relatively low

Method used

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  • Fusion protein capable of improving dammarenediol conversion efficiency, construction method and application
  • Fusion protein capable of improving dammarenediol conversion efficiency, construction method and application
  • Fusion protein capable of improving dammarenediol conversion efficiency, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of Saccharomyces cerevisiae cells for the synthesis of dammarenediol

[0035] 1. Module construction

[0036] According to the amino acid sequence of dammarenediol synthase in ginseng, codon optimization for Saccharomyces cerevisiae was carried out, and then the gene DS encoding dammarenediol synthase was obtained by chemical synthesis method (synthesized by Jinweizhi Biotechnology Co., Ltd.) It is SEQ ID NO.4; Saccharomyces cerevisiae endogenous tHMG1 (SEQ ID NO.5), erg1 (SEQ ID NO.6), and promoters PGK1p (SEQ ID NO.7), TEF1p (SEQ ID NO.8) , TDH3p (SEQ ID NO.9) and terminator CYC1t (SEQ ID NO.10), ADH1t (SEQ ID NO.11), ADH3t (SEQ ID NO.12) are all from Saccharomyces cerevisiae w303-1a genome; screening marker gene leu2 comes from plasmid prs405 (ATCC, USA), and his3 comes from plasmid pxp320 (purchased from Addgene, Inc.WWW.addgene.org).

[0037] Using the Saccharomyces cerevisiae W303-1a (USA, ATCC) genome as a template, PGK1p-DS-F (SEQ ID NO.13) (and P...

Embodiment 2

[0047] Example 2, the construction of synthetic protopanaxadiol Saccharomyces cerevisiae cells W3 and W3plus

[0048] According to the amino acid sequences of panaxadiol synthase in ginseng and cytochrome-NADPH-reductase 1 in Arabidopsis, codon optimization for Saccharomyces cerevisiae was carried out, and then obtained by chemical synthesis (synthesized by Jinweizhi Biotechnology Co., Ltd.) The gene PPDS encoding the original panaxadiol synthase in Panax ginseng is SEQ ID NO.3 in the sequence listing, and the gene AtCPR1 in Arabidopsis thaliana is the gene sequence SEQ ID NO.1 in the sequence listing. TDH3p, PGK1p and terminator CYC1t, ADH3t are all from S. cerevisiae w303-1a genome; selection marker gene ura3 is from plasmid pxp218 (Addgene, Inc.WWW.addgene.org).

[0049] Using the Saccharomyces cerevisiae W303-1a genome as a template, TDH3p-AtCPR1-F (SEQ ID NO.43) and TDH3p-AtCPR1-R (SEQ ID NO.44) and AtCPR1-ADH3T-F (SEQ ID NO.47) and AtCPR1-ADH3T-R (SEQ ID NO.48) was used...

Embodiment 3

[0053] Example 3. Construction of a fusion protein that can improve the conversion efficiency of dammarenediol

[0054] According to the gene sequence of Panaxadiol synthase gene PPDS in Panax ginseng and Cytochrome-NADPH-reductase 1 gene AtCPR1 in Arabidopsis, the codon optimization for Saccharomyces cerevisiae was carried out, and then chemically synthesized (Jinweizhi Biotechnology Co., Ltd. company synthesis) to obtain gene fragments. The PPDS gene in ginseng is shown in SEQ ID NO.3, and the AtCPR1 gene in Arabidopsis is shown in SEQ ID NO.1; the first 138 bases at the 5' end of the AtCPR1 gene in Arabidopsis are excised to obtain SEQ ID NO. 2 sequence shown. Both the endogenous promoter PGK1p (SEQ ID NO.7) and the endogenous terminator ADH3t (SEQ ID NO.12) are from the genome of Saccharomyces cerevisiae w303-1a; the screening marker gene ura3 is from the plasmid pxp218.

[0055]The two proteins are linked by a base sequence encoding the polypeptide GSTSSGSG. In this exa...

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Abstract

The invention discloses fusion protein capable of improving dammarenediol conversion efficiency, a construction method and application. The construction steps comprise: excising first 138 bases at 5' end of arabidopsis thaliana cytochrome-NADPH-reductase 1 gene AtCPR1, so as to obtain a sequence shown as SEQ ID NO. 2; (2) removing a termination codon TAA at 3' end of ginseng protopanaxadiol synthase gene PPDS with a sequence shown as SEQ ID NO. 3, and connecting with the base sequence which at 5' end of the sequence shown as SEQ ID NO. 2 and used for coding polypeptide GSTSSGSG, so as to construct a gene member of fusion protein; and (3) connecting the gene member of the fusion protein with saccharomyces cerevisiae cell endogenous promoter and terminator, so as to construct a fusion protein gene expression cassette, and transforming into saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method is capable of improving the conversion rate of dammarenediol to protopanaxadiol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein capable of improving the conversion efficiency of dammarenediol, its construction method and its application. Background technique [0002] Ginsenoside is the main active ingredient of traditional Chinese medicine ginseng, which has the pharmacological effects of protecting cardiovascular, anti-fatigue, anti-aging and anti-cancer. Traditional ginseng cultivation, tissue culture and downstream plant tissue extraction have been difficult to meet the market demand for ginsenosides. [0003] As a triterpenoid, the endogenous metabolic pathway of yeast can provide the precursor 2,3-oxysqualene for the synthesis of ginsenosides. Subsequent epoxidation, oxidation, and glycosyl addition are accomplished by dammarenediol synthase, protopanaxadiol synthase, and glycosyltransferase, respectively. The dammarenediol synthase gene was discovered in 2006. Tansakul et al. reporte...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/81C12P33/20C12R1/865
CPCC12N9/0081C12P33/20C12Y114/15006
Inventor 卢文玉赵方龙
Owner TIANJIN UNIV
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