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Recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and construction method and application of recombinant yarrowia lipolytica strain

A technology of protopanaxadiol and lipolytic Yarrowia, which is applied in the field of recombinant Yarrowia lipolytica strain and construction, can solve the problems of high cost of separation and purification, long growth cycle of ginseng, complex components of ginseng extract, etc., and achieve effective Applied effect

Pending Publication Date: 2020-09-22
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
[0005] However, the use of synthetic biology methods to metabolize xylose for heterologous synthesis of protopanaxadiol in Yarrowia lipolytica has not been reported

Method used

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  • Recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and construction method and application of recombinant yarrowia lipolytica strain
  • Recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and construction method and application of recombinant yarrowia lipolytica strain
  • Recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and construction method and application of recombinant yarrowia lipolytica strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, construction of Yarrowia lipolytica recombinant strain 1

[0031] Introduce the optimized xylose reductase gene XYL1 expression cassette and xylitol dehydrogenase gene XYL2 expression cassette into Yarrowia lipolytica, as well as the endogenous xylulokinase gene XKS expression cassette to obtain recombinant Yarrowia lipolytica Bacteria 1, the nucleotide sequence of the optimized xylose reductase gene XYL1 is shown in SEQ ID NO.1; the nucleotide sequence of the optimized xylitol dehydrogenase gene XYL2 is shown in SEQ ID NO.2 shown; the nucleotide sequence of the xylulokinase gene XKS is shown in SEQ ID NO.3;

[0032] 1. Module construction

[0033] Both xylose reductase gene (XYL1) and xylitol dehydrogenase (xylitoldehydrogenase) gene (XYL2) are derived from Pichia stipitis; Synthesized by chemical synthesis, optimized codons for Yarrowia lipolytica, and mutated XYL1 (K270R / N272D), to obtain optimized xylose reductase gene XYL1 (SEQ ID NO.1) and xylose ...

Embodiment 2

[0044] Embodiment 2, construction of recombinant Yarrowia lipolytica strain 2

[0045] Into the recombinant Yarrowia lipolytica yeast 2, the optimized dammarenediol synthase encoding gene DS expression cassette, the optimized protopanaxadiol synthase encoding gene PPDS expression cassette and the optimized cytochrome-NADPH- The expression cassette of the gene encoding reductase 1 was used to obtain the recombinant Y. lipolytica 2 that utilizes xylose to produce protopanaxadiol.

[0046] The dammarenediol synthase coding gene (DS) is derived from the plant ginseng (Panax ginseng), and the gene is synthesized by chemical synthesis by Wuhan Jinkairui Bioengineering Co., Ltd., and the codons are modified for Yarrowia lipolytica Optimized to obtain the optimized dammarenediol synthase coding gene DS (SEQ ID NO.4);

[0047] The protopanaxadiol synthase coding gene PPDS is derived from the plant ginseng (Panax ginseng), and the gene was synthesized by chemical synthesis by Wuhan Jin...

Embodiment 3

[0059] Embodiment 3, the construction of recombinant Yarrowia lipolytica 3

[0060] Into the recombinant strain 2 that uses xylose to produce protopanaxadiol, introduce the tHMG1 expression cassette of the gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase that is truncated by 1500 nucleotides at the 5' end, The farnesyl pyrophosphate synthase gene ERG20 expression cassette, the squalene synthase coding gene ERG9 expression cassette, obtained the recombinant Y. lipolytica 3 that utilizes xylose to produce protopanaxadiol;

[0061] The nucleotide sequence of the gene tHMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO.7, and the nucleotide sequence of the farnesyl pyrophosphate synthase gene ERG20 It is shown in SEQ ID NO.8; the nucleotide sequence of the squalene synthase gene ERG9 is shown in SEQ ID NO.9.

[0062] 1. Module construction

[0063] Gene POX1A (SEQ ID NO.72), Pfbain (SEQ ID NO.73), xpr2t (SEQ ID NO.12), Pexp1 (SEQ ID NO.11), lip2t ...

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Abstract

The invention discloses a recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and a construction method and application of the recombinant yarrowia lipolytica strain. Yarrowia lipolytica ATCC 201249 is used as an original strain, optimized xylose reductase XYL1, xylitol dehydrogenase XYL2 and an overexpressed endogenous xylulokinase XKS expression cassette areintroduced, and a recombinant strain 1 is obtained; a dammarendiol synthase gene DS, a protopanoxadiol synthase gene PPDS and a cytochrome P450 reductase gene ATR1 are introduced into the recombinantstrain 1 to obtain a recombinant strain 2; a gene tHMG1 for encoding nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase, a gene erg9 for squalene synthase and an erg20 for encoding farnesene pyrophosphate synthase in a truncated mevalonic acid pathway are introduced into the recombinant strain 2 to obtain a recombinant strain 3; and the recombinant yarrowia lipolytica isused for producing the protopanoxadiol by using the xylose, and the shake flask yield reaches 80.88 mg / L.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Yarrowia lipolytica strain for producing protopanaxadiol using xylose, a construction method and application. Background technique [0002] Protopanaxadiol (PPD for short) is an important active ingredient in traditional Chinese medicines such as ginseng and red ginseng, and is the precursor of dammarane-type ginsenosides. Modern medical research shows that PPD has biological activities such as anti-oxidation, anti-tumor, and anti-inflammation, and it also has excellent curative effects in coronary heart disease and anti-depression. [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. Ginseng has a long gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/54C12N15/60C12N15/81C12P33/00C12R1/645
CPCC12N9/0006C12N9/1205C12N9/88C12N9/0038C12N9/1085C12N15/815C12P33/00C12Y101/01307C12Y101/01009C12Y207/01017C12Y402/01125C12Y106/02005C12Y101/01034C12Y205/0101C12Y205/01021C12N2800/22
Inventor 卢文玉张传波武玉芬鞠海燕
Owner TIANJIN UNIV
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