Fusion protein capable of increasing conversion efficiency of dammarendiol and construction method

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: 2015-03-25
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 increasing conversion efficiency of dammarendiol and construction method
  • Fusion protein capable of increasing conversion efficiency of dammarendiol and construction method
  • Fusion protein capable of increasing conversion efficiency of dammarendiol and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of Saccharomyces cerevisiae cells for the synthesis of dammarenediol

[0034] 1. Module construction

[0035] 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).

[0036] 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 sequence of protopanaxadiol synthase and cytochrome-NADPH-reductase 1 in Arabidopsis, codon optimization for Saccharomyces cerevisiae was carried out, and then the code was obtained by chemical synthesis (synthesized by Jinweizhi Biotechnology Co., Ltd.) The gene PPDS gene of Panaxadiol synthase in Panax ginseng is SEQ ID NO.3 in the sequence listing, and the AtCPR1 gene 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 as a pr...

Embodiment 3

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

[0054] According to the amino acid sequence of the ginseng ginseng ginseng PPDS and the Arabidopsis cytochrome-NADPH-reductase 1 gene AtCPR1, codon optimization for S. 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 method of fusion PCR is used to realize the connection of two gene fragments. The specific implementation process is: using the PPDS gene (SEQ ID NO.3) as a template, using the PPDS-F (SEQ ID NO.51) and PPD...

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Abstract

The invention discloses a fusion protein capable of increasing the conversion efficiency of dammarendiol and a construction method. The construction method comprises the following steps: (1) excising the front 138 basic groups of the end 5' of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain a sequence shown in SEQ ID NO. 2; (2) removing the termination codon TAA of the end 3' of the synthase gene PPDS of protopanoxadiol in ginseng shown in SEQ ID NO. 3, and connecting with a sequence at the end 5' of the sequence shown in SEQ ID NO. 2 to construct a genetic element of the fusion protein; (3) connecting the genetic element of the fusion protein with the internal promoter and the terminator of a saccharomyces cerevisiae cell to construct a genetic expression kit of the fusion protein, and transforming to enter the saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method disclosed by the invention is capable of increasing the conversion efficiency of converting from dammarendiol to protopanoxadiol.

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 and a construction method. 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. reported that PNA (DDBJ da...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/62C12N9/02C12N9/10C12P33/20
Inventor 卢文玉赵方龙
Owner TIANJIN UNIV
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