Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and construction method

A technology for recombining Saccharomyces cerevisiae and L-pimamate acid, which is applied in the biological field to achieve the effect of increasing yield

Pending Publication Date: 2020-04-21
TIANJIN UNIV
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is no report on the heterologous synthesis of L-pimaric adiene and L-pimaric acid in 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
  • Recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and construction method
  • Recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and construction method
  • Recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, source of each fragment and plasmid preparation method

[0026] (1) The gene element LPS sequence (levopimaradiene synthase LPS, GenPept: Q947C4.1) used in the present invention is derived from plant Ginkgo biloba (Ginkgo biloba); P450 s depend on cytochrome P450reductase CPR) is derived from the plant Taxus cuspidate, synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. through chemical synthesis, and codon-optimized for Saccharomyces cerevisiae, and connected to the E. coli plasmid , preserved in E. coli. The nucleotide sequence of the optimized cytochrome P450 enzyme coding gene CYP720B1 is shown in SEQ ID NO.2 and the nucleotide sequence of the optimized cytochrome P450 reductase coding gene TcCPR is shown in SEQ ID NO.3.

[0027] (2) The method of transforming the gene LPS encoding L-pimarone diene synthase into TΔLPS of truncation mutation is as follows:

[0028] TΔLPS (the N-terminal truncation of 79 amino acids of the amino acid sequence encod...

Embodiment 2

[0054] Embodiment 2, the construction of Saccharomyces cerevisiae recombinant strain 1

[0055] Into Saccharomyces cerevisiaeW303-1a, USA ATCC208352, the transformed levopipine diene synthase encoding gene TΔLPS, the optimized cytochrome P450 enzyme encoding gene CYP720B1 and the optimized cytochrome P450 reductase encoding gene TcCPR were introduced to obtain the recombinant strain 1. The nucleotide sequence of the transformed levopipine diene synthase coding gene TΔLPS is shown in SEQ ID NO.1, and the nucleotide sequence of the optimized cytochrome P450 enzyme coding gene CYP720B1 is shown in SEQ ID NO.2 The nucleotide sequence of the optimized cytochrome P450 reductase coding gene TcCPR is shown in SEQ ID NO.3.

[0056] 1. Module construction

[0057] rDNA-up (SEQ ID NO.13), promoter TEF1 (SEQ ID NO.14), terminator ADH2 (SEQ ID NO.15), promoter PGK1 (SEQ ID NO.16), terminator ADH1 (SEQ ID NO. .17), promoter TDH3 (SEQ ID NO.18), terminator TDH2 (SEQ ID NO.19), rDNA-down (...

Embodiment 3

[0090] Embodiment 3, the construction of Saccharomyces cerevisiae recombinant strain 2

[0091] Introducing the 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into the recombinant bacterium 1 to obtain the recombinant bacterium 2, the 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 The nucleotide sequence of is shown in SEQ ID NO.4.

[0092] 1. Preparation method of plasmid pRS304-tHMG1

[0093] According to Table 6, using the Saccharomyces cerevisiae genome as a template, ApaI-Tdh3p-F (SEQ ID NO.46) as a front primer, and Tdh3p-R-tHMG1 (SEQ ID NO.47) as a back primer, PCR amplified fragment P Tdh3 ; Using the Saccharomyces cerevisiae genome as a template, Tdh3p-tHMG1-F (SEQ ID NO.48) as the front primer, and tHMG1-R-Cyc1t (SEQ ID NO.49) as the back primer, PCR amplifies the fragment tHMG1, (encoding The amino acid sequence at the N-terminal of HMGR1 is truncated, and only the sequence of 503 amino acids encoding the C-terminal of the HMGR1 protein...

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 recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and a construction method. The method comprises the following steps: introducing a modified L-pimaric diene synthase encoding gene T delta LPS, an optimized cytochrome P450 enzyme encoding gene CYP720B1 and an optimized cytochrome P450 reductase encoding gene TcCPR into saccharomyces cerevisiae to obtain a recombinant bacterium 1; introducing a 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into the recombinant bacterium 1 to obtain a recombinant bacterium 2; introducing a farnesyl pyrophosphate synthase encoding gene Erg20 into the recombinant bacterium 2 to obtain a recombinant bacterium 3; and replacing a wild squalene synthase promoter in the recombinant bacterium 3 with a Met3 promoter to obtain a recombinant bacterium 4; experiments prove that the fermentation of the recombinant bacteria disclosed by the invention can improve the yields of the levopimaric diene and the levopimaric acid; and a foundation is laid for artificial cell synthesis of the levopimaric diene and the levopimaric acid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Saccharomyces cerevisiae producing L-pimaric adiene and L-pimaric acid and a construction method thereof. Background technique [0002] Levopimaradiene is a prerequisite substance for the synthesis of ginkgolides, which have pharmacological effects such as anti-platelet aggregation, anti-inflammation, and anti-thrombosis. Levopimaric acid (levopimaric acid) is an important rosin resin acid. Levopimaric acid has a homocyclic conjugated double bond structure, and can undergo Diels-Alder addition reaction with maleic anhydride at room temperature to generate maleopimaric acid. Widely used in coatings, printing and dyeing, ink and other industries, it can be used as a plasticizer. Amides and imides of maleopimaric acid have anti-veterinary hepatitis effects, and its derivatives have anti-inflammatory and anti-ulcer effects. L-pimaric acid and 2-(2,4-dioxo-5-thiazolidinedi...

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): C12N15/81C12N1/19C12R1/865
CPCC12N15/81C12Y402/03032C12N9/0042C12Y106/02004C12Y101/01088C12N9/88C12N9/0006C12N9/001C12N9/1085
Inventor 卢文玉张传波刘婷鞠海燕
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com