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
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There is no report on the heterologous synthesis of L-

Method used

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  • 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

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Experimental program
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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...

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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

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Application Information

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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
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