Fusion protein capable of improving dammarendiol conversion efficiency and construction method

A technology for fusion protein and dammarenediol, which is applied in the field of fusion protein and construction, and can solve the problem of low conversion efficiency of dammarenediol

Inactive Publication Date: 2015-04-08
TIANJIN UNIV
View PDF3 Cites 0 Cited by
  • 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 synthesis pathway of Saccharomyces cerevisiae,

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fusion protein capable of improving dammarendiol conversion efficiency and construction method
  • Fusion protein capable of improving dammarendiol conversion efficiency and construction method
  • Fusion protein capable of improving dammarendiol conversion efficiency and construction method

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, 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.) as 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 the terminator CYC1t (SEQ ID NO.10), ADH1t (SEQ ID NO.11), ADH3t (SEQ ID NO.12) are all from Saccharomyces cerevisiae w303-1a genome; selection marker gene leu2 is from Plasmid prs405 (ATCC, USA), his3 was 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 PGK1p-DS-R (SEQ ID NO...

Embodiment 2

[0048] Example 2, the construction of synthetic protopanaxadiol Saccharomyces cerevisiae cells W3 and W3plus

[0049] 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).

[0050] 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

[0054] Example 3. Construction of a fusion protein that can improve the conversion efficiency of dammarenediol

[0055] According to the amino acid sequence of the protopanaxadiol synthase gene PPDS and the cytochrome-NADPH-reductase 1 gene AtCPR1 in Arabidopsis thaliana, codon optimization for Saccharomyces cerevisiae was carried out, and then chemically synthesized (Jinweizhi Biotechnology Co., Ltd. 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 selection marker gene ura3 is from the plasmid pxp218.

[0056]The two proteins are linked by a base sequence encoding the polypeptide GGG. In this example, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a fusion protein capable of improving the dammarendiol conversion efficiency and a construction method thereof. The construction method comprises the following steps: (1) cutting off the front 138 bases at the 5' end of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain the sequence shown by SEQ ID No.2; (2) removing the termination codon TAA at the 3' end of the protopanoxadiol synthase gene PPDS in ginseng shown by SEQ ID No.3, and connecting the termination codon TAA with the base sequence of the coding polypeptide GGG at the 5' end of the sequence shown by SEQ ID No.2 to construct a gene element of the fusion protein; and (3) connecting the gene element of the fusion protein with the endogenous promoter and terminator of saccharomyces cerevisiae cells to construct a gene expression box of the fusion protein, and converting into the saccharomyces cerevisiae cells for expression. The fusion protein disclosed by the invention can improve the conversion rate of dammarendiol to protopanoxadiol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein and a construction method capable of improving the conversion efficiency of dammarenediol. 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 Saccharomyces cerevisiae 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12P33/00C12N15/70C12N9/02C12R1/865
Inventor 卢文玉赵方龙
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap