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Recombinant yarrowia lipolytica for heterogeneous synthesis of alpha-amyrin and ursolic acid and construction method

A technology of Yarrowia lipolytica and balsamic resin 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 α-amyresinol and ursolic 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 alpha-amyrin and ursolic acid and construction method
  • Recombinant yarrowia lipolytica for heterogeneous synthesis of alpha-amyrin and ursolic acid and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Construction of Heterologous Synthesis of α-Amyrin Recombinant Yarrowia lipolytica YL-AAS (Recombinant Bacteria 1)

[0046] 1. Module construction

[0047] The mixedAS gene (GenBank: AFJ19235.1) from the plant Vinca (Catharanthus roseus) was codon optimized and fully chemically synthesized by Wuhan Jinkairui Company for Yarrowia lipolytica, and the optimized mixed arysin alcohol synthesis Enzyme encoding gene, the nucleotide sequence of the optimized mixed balsam alcohol synthase encoding gene is shown in SEQ ID NO.1;

[0048] 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 is from Yarrowia lipolytica ATCC 201249, which was synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. through chemical synthesis;

[0049] Using Yarrowia lipolytica ATCC201249 genome as a template,

[0050] Use rD...

Embodiment 2

[0056] Example 2, Construction of recombinant Yarrowia lipolytica YL-UA (recombinant bacteria 2) for heterologous synthesis of α-amyresinol and ursolic acid

[0057] The mixedAS gene (GenBank: AFJ19235.1) from the plant Vinca (Catharanthus roseus) was codon optimized and fully chemically synthesized by Wuhan Jinkairui Company for Yarrowia lipolytica, and the optimized mixed arysin alcohol synthesis Enzyme encoding gene, the nucleotide sequence of the optimized mixed balsam alcohol synthase encoding gene is shown in SEQ ID NO.1;

[0058] CYP716A12 (GenBank: CBN88268.1) derived from Medicago truncatula (GenBank: CBN88268.1) was codon optimized and fully chemically synthesized by Wuhan Jinkairui Company for Yarrowia lipolytica, and the optimized ursolic acid synthase encoding gene was obtained. The optimized nucleotide sequence of the gene encoding ursolic acid synthase is shown in SEQ ID NO.2.

[0059] The gene encoding cytochrome-NADPH-reductase 1, AtCPR1, was derived from Ara...

Embodiment 3

[0068] Example 3, gene element cloning and construction of plasmids containing corresponding gene elements

[0069] 1. PCR amplification of genetic elements

[0070] 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.

[0071] Table 1 PCR amplification gene primer list

[0072]

[0073] 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 machine. Amplificati...

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Abstract

The invention discloses recombinant yarrowia lipolytica for heterogeneous synthesis of alpha-amyrin and ursolic acid and a construction method. The construction method comprises steps as follows: an optimized mixed amyrin synthase encoded gene, an optimized ursolic acid synthase encoded gene and an optimized cytochrome-NADPH-reductase encoded gene are imported into yarrowia lipolytica, and recombinant bacteria 2 are obtained; and experiments prove that the recombinant yarrowia lipolytica for producing alpha-amyrin and ursolic acid is obtained with a homologous recombination method, so that theyield of alpha-amyrin and ursolic acid is improved. the method provides a basis for artificial synthesis of alpha-amyrin and ursolic acid.

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 α-amyresinol and ursolic acid. Background technique [0002] Ursolic acid (Ursolic acid, UA), is a pentacyclic triterpene, the molecular formula is C 30 h 48 o 3 , with a molecular weight of 456.7, acidic, white needle-like crystals, a melting point of 283-285°C, and a maximum ultraviolet absorption wavelength of about 450nm. Ursolic acid is a triterpenoid composed of C-30 isoprenoid compounds. Insoluble in water, soluble in hot ethyl acetate and ethanol sodium hydroxide solution. The biosynthesis of ursolic acid is mainly through the folding and cyclization of squalene to form a dammarane cation, further undergoing ring expansion and additional cyclization to form a fifth ring. Because ursolic acid contains three oxygen atoms, it can act as a double or triple ligand, which can easily tra...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/61C12N15/55C12N15/53C12N15/54C12N15/60C12P33/00C12R1/645
CPCC12N9/0006C12N9/0038C12N9/1085C12N9/1205C12N9/1229C12N9/16C12N9/88C12N9/90C12P33/00C12Y101/01034C12Y106/02C12Y205/0101C12Y205/01021C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y504/99007C12Y504/99039
Inventor 卢文玉魏盼盼李大帅张传波
Owner TIANJIN UNIV
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