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Saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol and construction method and use

A technology of protopanaxadiol and Saccharomyces cerevisiae, which is applied in the field of high-yielding protopanaxadiol Saccharomyces cerevisiae engineering strains and construction, which can solve the problems of cell growth toxicity and efficient synthesis of unfavorable products

Inactive Publication Date: 2019-12-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the release of reactive oxygen species (ROS) during the catalytic process by the P450 monooxygenase present in the synthesis pathway, it is toxic to cell growth and is not conducive to the efficient synthesis of products.

Method used

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  • Saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol and construction method and use
  • Saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol and construction method and use
  • Saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol and construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of the starting strain Saccharomyces cerevisiae engineered strain W3a-HU with high protopanaxadiol production:

[0024] W3a-HU is obtained by recovering the URA3 and HIS3 marker genes of W3a through plasmid pSH63.

[0025] Strains W3a and W2 come from the authorized patent ZL201410735927.X. The specific construction process of the strain can refer to this patent.

[0026] In the W3a strain, the URA3 and HIS3 markers have been utilized, but these two markers are flanked by two loxP sites. These two marker genes were first knocked out with the plasmid pSH63 expressing Cre protein (purchased from Biowind) to generate W3a-HU strain.

[0027] The specific operation adopts lithium acetate transformation, and the lithium acetate transformation method is as follows: after cultivating Saccharomyces cerevisiae W3a in 2mLYPD medium for 18 hours, inoculate it into a new YPD medium at an inoculation ratio of 10%, and continue culturing for 4 hours. Then, co...

Embodiment 2

[0038] Example 2 On the basis of the W3a-HU strain, the YBP1 gene was highly expressed (YBP1 is an oxidative stress regulator in Saccharomyces cerevisiae BY4741, the nucleotide sequence of the gene is shown in SEQ ID NO.11, from Saccharomyces cerevisiae Yeast BY4741 (USA, Number: 201388)), construct strain W3a-Py.

[0039] Construct gene Pgk1p-YBP1-Cyc1t gene expression cassette:

[0040] Using the Saccharomyces cerevisiae BY4741 genome as a template, using sequence 1 (SEQ ID NO.1) and sequence 2 (SEQ ID NO.2) as primers to amplify the Pgk1p promoter; using sequence 3 (SEQ ID NO.3) and sequence 4 (SEQ ID NO.4) was used as a primer to amplify the YBP1 gene; and sequence 5 (SEQ ID NO.5) and sequence 6 (SEQ ID NO.6) were used as primers to amplify the Cyc1t terminator. By fusion PCR method, the Pgk1p promoter, YBP1 gene and Cyc1t terminator were fused into a Pgk1p-YBP1-Cyc1t gene expression cassette.

[0041] Construction of the accessory fragments URA3r-Pgk1p and Cyc1t-URA3f...

Embodiment 3

[0047] The shake flask feeding fermentation process of embodiment 3 engineering bacteria

[0048] For fed-batch fermentation in shake flasks, each engineered strain was inoculated in a shake flask (250 mL) containing 30 mL of YPD medium, and cultured at 30° C. and 220 rpm. In the 48h of fermentation, add 5mL feeding solution, the composition of feeding solution is 500g / L glucose, 9g / L KH2PO4, 5.12g / L MgSO4 7H2O, 3.5g / LK2SO4, 0.28g / LNa2SO4, 0.5g / L adenine, 0.6g / L uracil, 1.2g / L lysine, 10mL / L trace element solution, 12mL / L vitamin solution. The composition of trace elements and vitamins is as follows:

[0049] Among them, the trace element composition and ratio are ( / L): EDTA 15g, ZnSO4 7H2O 10.2g, MnCl2 4H2O 0.50g, anhydrous CuSO40.5g, CoCl2 6H2O 0.86g, Na2MoO4 2H2O 0.56g, CaCl2 2H2O 3.84g and FeSO4.7H2O 5.12g.

[0050] Vitamin ingredients and ratios are ( / L): biotin 0.05g, calcium pantothenate 1g, nicotinic acid (nicotinic acid) 1g, inositol 25g, vitamin B11g, pyridoxine h...

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Abstract

The invention discloses a saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol and a construction method and use. The construction method of the saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol comprises the following steps: constructing a Pgk1p-YBP1-Cyc1t gene expression cassette, and performing introduction into a ura3 site of a saccharomycescerevisiae engineering strain to obtain the saccharomyces cerevisiae engineering strain W3a-Py for high yield of protopanaxadiol. The experimental results show that the saccharomyces cerevisiae engineering strain for high yield of protopanaxadiol can reduce an intracellular ROS level of the strain by 62.3% (84 h of fermentation), and the yield of protopanaxadiol in a shake flask is increased from1.40 g / L to 1.67 g / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yield protopanaxadiol Saccharomyces cerevisiae engineering strain, a construction method and application. Background technique [0002] Protopanaxadiol is the active ingredient in ginseng, which has strong anti-tumor activity and can effectively kill and kill tumor cells. Moreover, protopanaxadiol basically has no toxic and side effects on the human body, and can also enhance the body's immunity, and is suitable for postoperative treatment after tumor surgery or chemotherapy. In addition, protopanaxadiol has excellent curative effect and effect in the treatment of coronary heart disease and depression, improving the body's immunity and learning ability. [0003] In the production of protopanaxadiol, the traditional ginseng extraction method is restricted by the source of raw materials, pesticide residues and environmental protection issues, the producti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P33/00C12R1/865
CPCC12N15/81C12P33/00
Inventor 卢文玉南伟华赵方龙张传波
Owner TIANJIN UNIV
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