Recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and construction method

A technology of Yarrowia lipolytica and balsamol, which is applied in the biological field to achieve the effect of increasing yield

Inactive Publication Date: 2020-06-05
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the heterologous synthesis of β-amyresinol and oleanolic acid in Yarrowia lipolytica using synthetic biology methods has not been reported

Method used

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  • Recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and construction method
  • Recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and construction method
  • Recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1, Construction of recombinant Yarrowia lipolytica YL-BAS (recombinant bacteria 1) for heterologous synthesis of β-amyresinol

[0071] 1. Module construction

[0072] The gene bAS (beta-arsinol synthase encoding gene) (GenBank: BAA89815.1) derived from Glycyrrhiza glabra was codon optimized and fully chemically synthesized by Wuhan Jinkairui Company for Yarrowia lipolytica , the optimized β-amyresinol synthase coding gene is obtained, and the nucleotide sequence of the optimized β-amyresinol synthase coding gene is shown in SEQ ID NO.1.

[0073] Promoter TEF1 (SEQ ID NO.14), terminator XPR2 (SEQ ID NO.17), rDNA-up (SEQ ID NO.22), rDNA-down (SEQ ID NO.23) are all from Yarrowia lipolytica ATCC 201249 genome, the screening marker gene URA3 (SEQ ID NO.20) sequence comes from Yarrowia lipolytica ATCC 201249, and the gene is synthesized by chemical synthesis by Wuhan Jinkairui Bioengineering Co., Ltd.;

[0074] Using the Yarrowia lipolytica ATCC201249 genome as a te...

Embodiment 2

[0084] Example 2, Construction method of recombinant Yarrowia lipolytica YL-CYP716A12 (recombinant bacteria 2) for heterologous synthesis of β-amyresinol and oleanolic acid

[0085] Gene bAS (GenBank: BAA89815.1) derived from Glycyrrhiza glabra and CYP716A12 (GenBank: CBN88268.1) derived from Medicago truncatula (GenBank: CBN88268.1) were developed by Wuhan Jinkairui Company for Yarrowia lipolytica Codon optimization and total chemical synthesis. The optimized β-amyresinol synthase encoding gene and the optimized oleanolic acid synthase encoding gene are obtained, and the nucleotide sequence of the optimized oleanolic acid synthase encoding gene is shown in SEQ ID NO.2 ;

[0086] The gene encoding cytochrome-NADPH-reductase 1, AtCPR1, was derived from Arabidopsis thaliana. The gene was synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. through chemical synthesis, and the codon was optimized for Yarrowia lipolytica to obtain the optimized The coding gene of cytochrome-NA...

Embodiment 3

[0103] Example 3, gene element cloning and construction of plasmids containing corresponding gene elements

[0104] 1. PCR amplification of genetic elements

[0105] Using the Yarrowia lipolytica ATCC201249 genome as a template, HMG1, tHMG1, ERG1, ERG8, ERG9, ERG10, ERG12, ERG19, ERG20 and IDI were amplified with the primers in Table 1, respectively. The gene fragments obtained after amplification were purified and recovered for future use.

[0106] Table 1 PCR amplification gene primer list

[0107]

[0108]

[0109] The PCR enzyme used in the present invention is from Nanjing Nuoweizan Biotechnology Co., Ltd. Max Super-Fidelity polymerase. The 50 μL PCR amplification system is as follows: DNA template, 1 μL; 2 μL each of the front primer (10 μM) and the back primer (10 μM); dNTP (10 mM), 1 μL; 2×Phanta Max Buffer, 25 μL; Max Super-Fidelity polymerase, 1 μL; finally make up 50 μL with double distilled water. Set up the amplification program on the PCR instrumen...

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Abstract

The invention discloses recombinant Yarrowia lipolytica for heterogeneous synthesis of beta-amyrin and oleanolic acid and a construction method. The construction method comprises the step of introducing an optimized beta-amyrin synthase coding gene, an optimized oleanolic acid synthase coding gene and an optimized cytochrome-NADPH-reductase 1 coding gene into Yarrowia lipolytica, thereby obtaininga recombinant strain 2. Proven by experiments, the recombinant Yarrowia lipolytica capable of yielding the beta-amyrin and the oleanolic acid is obtained through a homologous recombination method, and thus, the yield of the beta-amyrin and the oleanolic acid is increased; and the method disclosed by the invention provides a foundation for artificial synthesis of the beta-amyrin and the oleanolicacid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Yarrowia lipolytica and a construction method for heterologously synthesizing β-amyresinol and oleanolic acid. Background technique [0002] Representative compounds of pentacyclic triterpenes are oleanolic acid (OA) and aglycone ursolic acid (UA) of oleanane-type saponins. The two are isomers of each other. Ursolic acid has various biological effects such as anti-inflammatory and antibacterial. The most important pharmacological activity of oleanolic acid is hepatoprotective effect. In addition, studies have shown that the synergistic administration of ursolic acid and oleanolic acid can mutually enhance the ability of each other to resist malignant cell proliferation. [0003] The biosynthetic pathway of oleanolic acid starts from 2,3-oxidized squalene, which is cyclized to form the pentacyclic triterpene skeleton β-amyresinol, and β-amyresinol is the isomerism of α-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P33/00C12P15/00C12R1/645
CPCC12N9/0042C12N9/90C12N9/93C12P15/00C12P33/00C12Y504/99054
Inventor 卢文玉张传波李大帅魏盼盼
Owner TIANJIN UNIV
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