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Recombinant saccharomycetes for producing ursolic acid and oleanolic acid and construction method and application of recombinant saccharomycetes

A technology of recombinant bacteria and homologous recombination, applied in the biological field, can solve the problems of low yields of ursolic acid and oleanolic acid

Active Publication Date: 2020-05-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the studies on the simultaneous synthesis of ursolic acid and oleanolic acid by microorganisms reported so far, the yields of both ursolic acid and oleanolic acid are very low

Method used

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  • Recombinant saccharomycetes for producing ursolic acid and oleanolic acid and construction method and application of recombinant saccharomycetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the construction of synthetic α-amyresinol and β-amyresinol Saccharomyces cerevisiae recombinant bacteria

[0036] 1. Module construction

[0037] According to the gene sequence of the multifunctional balsamol synthase CrAS (Catharanthus roseus, GenBank: JN991165) in periwinkle, the codon optimization for Saccharomyces cerevisiae was obtained by chemical synthesis (Nanjing GenScript Biotechnology Co., Ltd.) Gene CrAS (SEQ ID NO.1) encoding the multifunctional balsamol synthase.

[0038] Squalene epoxidase coding gene ERG1 (SEQ ID NO.2), promoter P GAL1 (SEQ ID NO.8), P TEF1 (SEQID NO.9) and terminator T CYC1 (SEQ ID NO. 10), T ADH1 (SEQ ID NO.11) are all from Saccharomyces cerevisiae W303-1a genome; Selection marker gene HIS3 is from plasmid pxp320 (purchased from Addgene, Inc. www.addgene.org ).

[0039] Using the genome of Saccharomyces cerevisiae W303-1a (USA, ATCC: 208352) as a template, P GAL1 -CrAS-F (SEQ ID NO.12) and P GAL1 -CrAS-R (SEQ ID...

Embodiment 2

[0048] Embodiment 2, the construction of Saccharomyces cerevisiae strain Sc310LCZ02

[0049] Saccharomyces cerevisiae endogenous tHMG1 (SEQ ID NO.3), ERG20 (SEQ ID NO.4), and promoter P PGK1 (SEQ ID NO. 30), P TDH3 (SEQ ID NO.31) and terminator T CYC1 (SEQ ID NO.10) are all from the Saccharomyces cerevisiae W303-1a genome. PRS304 plasmid was purchased from BioVector (http: / / www.biovector.net) and PRS405 plasmid was purchased from Biofeng (www.biofeng.com).

[0050] 1. Construction of plasmid ptHMG1

[0051] Using the genome of Saccharomyces cerevisiae W303-1a as a template, the primers P TDH3 -F-ApaI (SEQ ID NO.32), P TDH3 -R-tHMG1 (SEQ ID NO.33) and tHMG1-T CYC1 -F (SEQ ID NO. 34), T CYC1 -R-PstI (SEQ ID NO.35) is primer, amplifies P TDH3 (SEQ ID NO.31) promoter and T CYC1 (SEQ ID NO.10) terminator, with P TDH3 -tHMG1-F (SEQ ID NO.36) and P TDH3 -R-tHMG1 (SEQ ID NO.37) is a primer for amplifying the tHMG1 gene (SEQ ID NO.3). By the fusion PCR condition fusion P in...

Embodiment 3

[0055] Embodiment 3, the construction of synthetic ursolic acid and oleanolic acid Saccharomyces cerevisiae recombinant bacteria

[0056] According to vinca C-28 oxidase CYP716AL1 (Catharanthus roseus, GenBank: JN565975), C-28 oxidase CYP716A12 (Medicagotruncatula, GenBank: ABC59076.1) and Arabidopsis The gene sequence of cytochrome-NADPH-reductase 1 gene AtCPR1 (Arabidopsis thaliana, GenBank: BT008426.1) in mustard was optimized for Saccharomyces cerevisiae, and then chemically synthesized (Nanjing KingScript Synthesis) to obtain the coding gene CYP716AL1 (SEQ ID NO.7) encoding the C-28 oxidase of vinca argentinol and CYP716A12 (SEQ ID NO.5) and Arabidopsis cytochrome-NADPH-reductase 1 encoding gene AtCPR1 (SEQ ID NO.6) in Arabidopsis thaliana. promoter P TDH3 (SEQ ID NO. 31), P PGK1 (SEQ ID NO.30) and terminator T CYC1 (SEQ ID NO. 10), T ADH3 (SEQ ID NO.44) are all from Saccharomyces cerevisiae w303-1a genome; the selection marker gene ura3 is from plasmid pxp218 (Addge...

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Abstract

The invention discloses recombinant saccharomycetes for producing ursolic acid and oleanolic acid and a construction method and application of the recombinant saccharomycetes. The construction methodcomprises the following steps: by using a homologous recombination method, introducing an oxidase encoding gene CYP716A12 at a C-28 locus of a-amyrin and an encoding gene AtCPR1 of arabidopsis cytochrome-NADPH-reductase 1 into a recombinant bacterium Sc310LCZ02 so as to obtain a recombinant strain Sc310LCZ03; or introducing an oxidase encoding gene CYP716A1 at the C-28 locus of a-amyrin and the encoding gene AtCPR1 of arabidopsis cytochrome-NADPH-reductase 1 into the recombinant bacterium Sc310LCZ02 so as to obtain a recombinant strain Sc310LCZ04; and producing a mixture of ursolic acid and oleanolic acid of different ratios by using the recombinant strains. The invention makes bases for artificial synthesis of ursolic acid and oleanolic acid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant yeast for producing ursolic acid and oleanolic acid, a construction method and application. Background technique [0002] Both ursolic acid and oleanolic acid are pentacyclic triterpene compounds with various biological activities that can be detected in a variety of medicinal plants. Anti-diabetic, anti-cancer and other biological effects. In addition, although ursolic acid and oleanolic acid each have good anticancer activity, studies have shown that the synergistic use of ursolic acid and oleanolic acid can mutually enhance the ability of each other to resist malignant cell proliferation. The synergistic activity of ursolic acid and oleanolic acid was also verified by in vivo experiments in mice with skin cancer. Simultaneous synthesis of ursolic acid and oleanolic acid using microbial cell factories is an effective way to realize their industrial production. It is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/61C12N15/53C12N15/54C12P33/00C12R1/865
CPCC12N9/0006C12N9/0042C12N9/0071C12N9/0073C12N9/1085C12N9/90C12N15/81C12N15/905C12N2800/22C12P33/00C12Y101/01C12Y106/02004C12Y114/13C12Y114/14C12Y205/0101C12Y504/99
Inventor 卢文玉张传波陆春哲
Owner TIANJIN UNIV
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