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Recombinant yeast chassis cell transformation for efficiently converting chenodeoxycholic acid and construction and application of recombinant strain

A technology of chenodeoxycholic acid and yeast strains, applied in the field of biosynthesis, can solve problems such as ambiguity

Inactive Publication Date: 2021-04-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, there are only reports about the expression of 7α-HSDH and 7β-HSDH in E. coli, and it is not clear whether the enzyme can achieve the same expression in other strains, and further research is needed for researchers

Method used

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  • Recombinant yeast chassis cell transformation for efficiently converting chenodeoxycholic acid and construction and application of recombinant strain
  • Recombinant yeast chassis cell transformation for efficiently converting chenodeoxycholic acid and construction and application of recombinant strain
  • Recombinant yeast chassis cell transformation for efficiently converting chenodeoxycholic acid and construction and application of recombinant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of mutant yeast strain S.cerevisiae CEN.PK2-1CΔpdc1Δadh1

[0030] Knockout of the PDC1 gene using Cre-LoxP technology:

[0031] Using the plasmid pUG27 with the HIS tag as a template, PDC1-F and PDC1-R as primers (the primer sequences are shown in SEQ.NO.05-SEQ.NO.06), PCR amplification obtained 1544bp for knocking out PDC1 The HIS knockout box of the gene includes the HIS gene and the 45 bp nucleotide homologous sequence between the upstream and downstream of the PDC1 gene. The wild-type Saccharomyces cerevisiae strain CEN.PK2-1C was transformed by LiAc transformation method, coated with SD-His plate, and cultured in a 30°C incubator for 3 days. The obtained transformant was streaked and purified on the SD-His plate for 2-3 days, then inserted into the SD-His liquid medium and cultured overnight to saturation, and the genome was extracted for PCR verification. The primers A (PDC1), BM (PDC1), The corresponding sequences of CM(PDC1) and D(PDC1)...

Embodiment 2

[0037] Example 2 Construction of Transformed Chenodeoxycholic Acid Engineering Yeast

[0038] Using pY15TEF1 and pYX212 as expression plasmids, construct pY15TEF1-7α-HSDH and pYX212-7β-HSDH overexpression vectors. The specific method is as follows: the gene products 7α-HSDH and 7β-HSDH with restriction enzyme sites were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and then the above gene products 7α-HSDH and 7β-HSDH were combined with the plasmid pY15TEF1 , pYX212 were simultaneously digested with XbaI and BamHI, EcoRI and BamHI (primer sequences are shown in SEQ.NO.17-SEQ.NO.20), ligated with T4 DNA ligase, and 5 μL of the ligated product was transformed into 50 μL Escherichia coli competent Cells were spread on LA plates, and the correctness of the obtained transformants was verified by colony PCR and enzyme digestion, double enzyme digestion with XbaI and BamHI, EcoRI and BamHI was used to verify the correct transformants, and the recombinant ...

Embodiment 3

[0042] Embodiment 3 Fermentation of transformed chenodeoxycholic acid engineered yeast

[0043] Saccharomyces cerevisiae culture conditions: Bacteria were taken from the strain preservation tube at -80°C, activated on YPD (transformants containing nutrient marker plasmids in the corresponding SD-deficient medium) plates, and cultured at 30°C for 3 days; pick the activated single colony and inoculate Into a 3mL test tube containing YPD (transformants containing nutrient marker plasmids in the corresponding SD-deficient medium) medium, cultivate overnight at 30°C and 220rpm until saturated.

[0044] Fermentation culture conditions: Pick an activated single colony from the plate and inoculate it into the seed medium, and culture it in a shaker flask at 30°C and 220rpm for 24h until saturated. Transfer to the fermentation medium shake flask with initial OD=0.2, culture at 30°C and 220rpm. The cells were cultured to the stationary phase (48h), collected by centrifugation, resuspen...

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Abstract

The invention discloses recombinant yeast chassis cell transformation for efficiently converting chenodeoxycholic acid and recombinant strain construction and application, a saccharomyces cerevisiae strain S. Cerevisiae CEN.PK21C is used as a chassis cell of a recombinant yeast strain, a mutant yeast strain is obtained by knocking out target genes PDC1 and ADH1, and weakening of an ethanol synthesis path is realized. On the basis, 7 alpha HSDH and 7 beta HSDH coding genes from clostridium are heterologously expressed, and the purpose of biosynthesis of UDCA by taking CDCA as a substrate is achieved. At present, the conversion rate of the substrate CDCA reaches 90%.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis, and in particular relates to the transformation of recombinant yeast chassis cells for efficiently transforming chenodeoxycholic acid and the construction and application of recombinant strains. Background technique [0002] Ursodeoxycholic acid (UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile. It and its corresponding diastereoisomer chenodeoxycholic acid (CDCA) are clinically used to treat various gallstone diseases. A kind of acute and chronic liver disease, has good effect. The yield of extracting UDCA from bear bile of artificially raised bears is low, the source is limited, and it is against animal protection, so artificial synthesis of UDCA is of great significance. The synthesis methods of UDCA mainly include the method of combining total chemical synthesis and chemical enzymatic method, and the starting material is animal-derived...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/60C12N15/81C12P33/02C12R1/865
CPCC12N9/0006C12N9/88C12N15/81C12P33/02C12Y101/01159C12Y101/01201C12Y401/01001C12Y101/01001
Inventor 徐国强史劲松高宇豪许正宏李会龚劲松
Owner JIANGNAN UNIV
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