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Yeast engineering bacteria for fermentation production of chondroitin sulfate and application of yeast engineering bacteria

A kind of chondroitin sulfate, chondroitin technology, applied in the field of bioengineering

Active Publication Date: 2021-04-27
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the synthesis of chondroitin sulfate directly by microorganisms using carbon sources in microbial cells at home and abroad.

Method used

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  • Yeast engineering bacteria for fermentation production of chondroitin sulfate and application of yeast engineering bacteria
  • Yeast engineering bacteria for fermentation production of chondroitin sulfate and application of yeast engineering bacteria
  • Yeast engineering bacteria for fermentation production of chondroitin sulfate and application of yeast engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Construction of pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid and bacterial strain GS115 / CAD

[0080] (1) Take the synthetic genes kfoC, kfoA and tuaD (sequences shown in SEQ ID NO.1-3 respectively) as templates, and use primers kfoC-F / kfoC-R, kfoA-F / kfoA-R, tuaD-F / tuaD-R PCR amplified 3 genes respectively, used Gibson to assemble and connect to pGAPZB vector, and constructed pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid, in which T2A and T2A2 sequences were designed on the primers, and the complete sequence is SEQ ID NO.6, SEQ ID NO.7;

[0081] (2) Prepare Pichia pastoris GS115 competent cells, transform the plasmid pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD obtained above into competent cells after linearization with fast cutting enzyme AvrII, and pass bleomycin resistance The positive clones were screened on the plate, and the integrated genes kfoC, kfoA and tuaD were obtained, and the strain was named GS115 / CAD.

Embodiment 2

[0082] Embodiment 2: Construction of pGAPHyg-MET13 plasmid

[0083] The genome of Saccharomyces cerevisiae GS115 / CAD was extracted, and the primers MET13-F / MET13-R were designed to amplify the endogenous gene MET13, which was connected to the modified vector GAPHyg by GAPZB by one-step cloning assembly to construct the GAPHyg-MET13 plasmid.

Embodiment 3

[0084] Embodiment 3: Construction of GS115 / CADM bacterial strain

[0085] Prepare GS115 / CAD recombinant bacterial competent cells, transform the GAPHyg-MET13 plasmid obtained in Example 2 into GS115 / CAD recombinant bacterial competent cells after linearization with fast-cutting enzyme AvrII, and obtain positive results by screening with hygromycin resistance plates The clone is GS115 / CADM recombinant strain.

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Abstract

The invention discloses yeast engineering bacteria for fermentation production of chondroitin sulfate and an application of the yeast engineering bacteria, and belongs to the technical field of bioengineering. Pichia pastoris GS115 and S.cerevisiae CEN.PK2-1C are used as starting strains by utilizing a synthetic biological technology and a genetic engineering means, and chondroitin sulfate synthetic pathway related genes including genes kfoC and kfoA from escherichia coli K4, chondroitin sulfate transferase genes C4ST and C6ST from mice and UDP-glucose dehydrogenase genes tuaD from bacillus subtilis are heterologously expressed in cells. Production strains for synthesizing chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and chondroitin sulfate E (CSE) are obtained from ATP sulfatase gene MET13 of S.cerevisiae. According to the invention, chondroitin sulfate with different configurations is synthesized by utilizing a microbial fermentation carbon source for the first time.

Description

technical field [0001] The invention relates to a yeast engineering bacterium for fermenting and producing chondroitin sulfate and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (CS) is a proteoglycan widely distributed in cartilage tissue and has important biological functions. Its backbone is a linear polysaccharide formed by alternating links of D-glucuronic acid and N-acetylgalactosamine. Chondroitin sulfate is formed by sulfation modification of chondroitin through sulfotransferase. According to the position of sulfation modification, chondroitin sulfate can be divided into the following four types: Chondroitin sulfate A (4-O-sulfation) , Chondroitin Sulfate C (6-O-sulfated), Chondroitin Sulfate D (2,6-di-O-sulfated), and Chondroitin Sulfate E (4,6-di-O-sulfated). Due to its excellent biocompatibility, CS has a wide range of applications, such as medical health, health care products, food a...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P19/26C12R1/865C12R1/84
CPCC12P19/26C12N9/90C12N9/0006C12N9/13C12N9/1241C12Y208/02005C12Y208/02017C12Y207/07004C12N15/81C12N15/815
Inventor 康振金学荣陈坚堵国成李江华张天萌
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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