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Recombinant Yarrowia lipolytica for producing dammarendiol and protopanoxadiol and application

A technology for producing dammarediol and Yarrowia lipolytica, applied in the biological field, can solve the problems of high cost of separation and purification, complex composition of ginseng extract, pollution and the like

Inactive Publication Date: 2020-04-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ginseng has a long growth cycle and is easily polluted by pesticide residues, resulting in product quality that cannot meet the needs of domestic and foreign markets
2) The components of ginseng extract are complex, and the cost of separation and purification is relatively high
3) The preparation process is often accompanied by high pollution to the environment
However, the heterologous synthesis of dammarenediol and protopanaxadiol in Yarrowia lipolytica using synthetic biology methods has not been reported

Method used

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  • Recombinant Yarrowia lipolytica for producing dammarendiol and protopanoxadiol and application
  • Recombinant Yarrowia lipolytica for producing dammarendiol and protopanoxadiol and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1, Construction of Yarrowia lipolytica Recombinant Bacteria 1

[0057] Introduce the optimized dammarenediol synthase encoding gene DS expression cassette into Yarrowia lipolytica (Yarrowia lipolytica) ATCC 201249 to obtain recombinant Yarrowia lipolytica 1 (abbreviated as recombinant bacterium 1). The nucleotide sequence of the gene DS encoding the mesenediol synthase is shown in SEQ ID NO.1.

[0058] 1. Module construction

[0059] The gene encoding dammarenediol-II synthase (dammarenediol-II synthase, DS, GenBank: AB265170.1) is derived from the plant ginseng (Panaxginseng). Yarrowia lipolytica was codon-optimized to obtain the optimized dammarenediol synthase coding gene DS (SEQ ID NO.1); the following DSs are codon-optimized.

[0060] 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 come...

Embodiment 2

[0071] Example 2, Construction of recombinant Yarrowia lipolytica (abbreviated as recombinant bacteria 2) producing dammarenediol and protopanaxadiol

[0072]Introduce the optimized dammarenediol synthase encoding gene DS expression cassette, the optimized protopanaxadiol synthase encoding gene PPDS expression cassette and the optimized cytochrome-NADPH-reductase 1 encoding gene into Yarrowia lipolytica The expression cassette is obtained from recombinant Yarrowia lipolytica 2, the nucleotide sequence of the optimized dammarenediol synthase coding gene DS is shown in SEQ ID NO.1, and the optimized protopanaxadiol synthase The nucleotide sequence of the coding gene PPDS is shown in SEQ ID NO.2, and the nucleotide sequence of the optimized cytochrome-NADPH-reductase 1 coding gene is shown in SEQ ID NO.3.

[0073] The gene encoding dammarenediol-II synthase (dammarenediol-II synthase, DS, GenBank: AB265170.1) is derived from the plant ginseng (Panaxginseng). codon optimization o...

Embodiment 3

[0098] Example 3, gene element cloning and construction of plasmids containing corresponding gene elements

[0099] 1. PCR amplification of genetic elements

[0100] 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.

[0101] Table 1 PCR amplification gene primer list

[0102]

[0103] 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 instrument. Amplificat...

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Abstract

The invention discloses a recombinant Yarrowia lipolytica for producing dammarendiol and protopanoxadiol and application. The recombinant Yarrowia lipolytica is constructed by the following steps: introducing an optimized dammarendiol synthase encoding gene DS expression cassette, an optimized protopanoxadiol synthase encoding gene PPDS expression cassette and an optimized encoding gene expressioncassette of cytochrome-NADPH-reductase 1 into Yarrowia lipolytica so as to obtain a recombinant yeast 2, and cutting 1500 nucleotides from the 5'end of a gene HMG1 for encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase to obtain a truncated gene tHMG1 for encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase; and introducing the truncated gene tHMG1 into recombinant yeast 2 to obtainrecombinant yeast 3. Experiments prove that the yield of dammarendiol and protopanoxadiol is increased by acquiring the recombinant Yarrowia lipolytica through a homologous recombination method. Themethod disclosed by the invention provides a basis for artificially synthesizing the dammarendiol and the protopanoxadiol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Yarrowia lipolytica for heterologously synthesizing dammarenediol and protopanaxadiol and its use. Background technique [0002] Ginseng, as a precious Chinese herbal medicine, has always been a research and application hotspot no matter in disease treatment or health care. Protopanaxadiol (abbreviated as PPD) and PPD-type ginsenosides are important active ingredients in traditional Chinese medicines such as ginseng and red ginseng. Modern medical research shows that PPD has strong anticancer activity and can effectively kill and kill tumor cells. [0003] Since the PPD content in ginseng is very small, less than 0.05% of the dry weight of ginseng, the production and preparation of PPD mainly comes from the hydrolysis and enzymatic hydrolysis of PPD-type total saponins, but this method has the following disadvantages . 1) There is a shortage of raw materials. Ginsen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/60C12N15/53C12N15/54C12N15/61C12P33/00C12R1/645
CPCC12N9/0006C12N9/0038C12N9/0071C12N9/1029C12N9/1085C12N9/1205C12N9/1229C12N9/88C12N9/90C12P33/00C12Y101/01034C12Y106/02005C12Y114/14C12Y203/01174C12Y205/0101C12Y205/01021C12Y207/01036C12Y207/04002C12Y401/01033C12Y402/01125C12Y503/03002C12Y504/99
Inventor 卢文玉张传波李大帅
Owner TIANJIN UNIV
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