Recombinant yeast for fermentation production of chondroitin sulfate with controllable molecular weight and application of recombinant yeast

A chondroitin sulfate and yeast technology, applied in the field of bioengineering, can solve problems such as the inability to regulate the molecular weight of chondroitin sulfate, and achieve the effects of avoiding extraction and purification and post-catalytic processes, controllable molecular weight, and quality and safety assurance.

Active Publication Date: 2021-04-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is that the existing method for preparing chondroitin sulfate cannot regulate the molecular weight of chondroitin sulfate, thereby realizing the synthesis of specific molecular weight chondroitin sulfate

Method used

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  • Recombinant yeast for fermentation production of chondroitin sulfate with controllable molecular weight and application of recombinant yeast
  • Recombinant yeast for fermentation production of chondroitin sulfate with controllable molecular weight and application of recombinant yeast
  • Recombinant yeast for fermentation production of chondroitin sulfate with controllable molecular weight and application of recombinant yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of pGAPZB-C4ST-T2A-kfoC-T2A2-kfoA-T2A3-tuaD plasmid and recombinant strain Pp001

[0044] (1) Using the synthetic genes C4ST, kfoC, kfoA and tuaD (sequences shown in SEQ ID NO: 8, SEQ ID NO.5-7 respectively) as a template, with primers C4-F / C4-R, kfoC-F / Four genes of kfoC-R, kfoA-F / kfoA-R, tuaD-F / tuaD-R were amplified by PCR respectively, and connected to the pGAPZB vector by Gibson assembly to construct pGAPZB-C4ST-T2A-kfoC-T2A2-kfoA -T2A3-tuaD plasmid, wherein T2A, T2A2 and T2A3 amino acid sequences are all SEQ ID NO.2, but have different nucleotide sequences, the nucleotide sequence used by T2A is GAA GGT CGT GGT TCT CTT CTG ACT TGTGGT GAT GTT GAA GAAAAC CCA GGT CCA; the nucleotide sequence used for T2A2 is GAA GGT CGT GGA TCCCTA CTT ACT TGC GGT GAC GTA GAG GAA AAC CCT GGT CCG; the nucleotide sequence used for T2A3 is GAGGGT AGA GGT TCT TTG CTT ACT TGC GGT GAC GTT GAG GAA AAC CCA GGT CCA.

[0045] (2) To prepare competent cells of Pichia pa...

Embodiment 2

[0046] Example 2: Construction of pGAPZB-C6ST-T2A-kfoC-T2A2-kfoA-T2A3-tuaD plasmid and recombinant strain Pp002

[0047] (1) Using the synthetic genes C6ST, kfoC, kfoA and tuaD (sequences shown in SEQ ID NO.9, SEQ ID NO.5-7, respectively) as templates, with primers C6-F / C6-R, kfoC-F / kfoC-R, kfoA-F / kfoA-R, tuaD-F / tuaD-R were respectively amplified by PCR to amplify 4 genes, which were connected to the pGAPZB vector by Gibson assembly to construct pGAPZB-C6ST-T2A-kfoC-T2A2- kfoA-T2A3-tuaD plasmid, wherein T2A, T2A2, T2A3 sequences are designed on the primers.

[0048] (2) To prepare competent cells of Pichia pastoris GS115, the plasmid pGAPZB-C6ST-T2A-kfoC-T2A2-kfoA-T2A3-tuaD obtained above was linearized with fast-cutting enzyme AvrII and then transformed into competent cells. Positive clones were screened on the mycin resistance plate, and the recombinant yeast strain Pp002 integrated with genes C6ST, kfoC, kfoA and tuaD was obtained.

Embodiment 3

[0049] Embodiment 3: Construction of pAO815-ABCI plasmid

[0050] The synthetic gene ABCI (sequence shown in SEQ ID NO.10) was used as a template, and primers ABCI-F / ABCI-R were used for PCR amplification, and one-step cloning enzyme was used to assemble and connect to the pAO815 vector to construct pAO815- ABCI plasmid.

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Abstract

The invention discloses recombinant yeast for fermentation production of chondroitin sulfate with controllable molecular weight and an application of the recombinant yeast, and belongs to the technical field of bioengineering. Pichia pastoris GS115 is used as an original strain by utilizing a synthetic biological technology and a genetic engineering means, and chondroitin sulfate synthetic pathway related proteins including KfoC and KfoA from escherichia coli K4, chondroitin sulfate transferase C4ST or C6ST from mice, UDP-glucose dehydrogenase TuaD from bacillus subtilis and chondroitin sulfate lyase ABCI from proteus vulgaris are heterologously expressed in cells, so that production strains for synthesizing chondroitin sulfate A (CSA) and chondroitin sulfate C (CSC) are obtained, and chondroitin sulfate A and chondroitin sulfate C with specific molecular weights can be obtained by controlling the concentration of an inducer namely methanol and the induction time. According to the recombinant yeast, the chondroitin sulfate with controllable molecular weight and specific configuration is synthesized by utilizing a microbial fermentation carbon source for the first time.

Description

technical field [0001] The invention relates to a recombinant yeast for fermenting and producing chondroitin sulfate with a controllable molecular weight and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (CS) is a linear polysaccharide formed by alternating links of D-glucuronic acid and N-acetylgalactosamine, and is widely distributed in cartilage tissue in the form of proteoglycans. CS has important biological functions. Its skeleton chondroitin sulfate is formed by the sulfation modification of chondroitin by sulfotransferase. According to the position of sulfation modification, chondroitin sulfate can be divided into the following four types: chondroitin sulfate A (4-O-sulfate sulfation), chondroitin sulfate C (6-O-sulfation), chondroitin sulfate D (2,6-di-O-sulfation) and chondroitin sulfate E (4,6-di-O-sulfation). Due to its excellent biocompatibility, CS has a wide range of applications,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12N15/60C12P19/26C12R1/84
CPCC12N15/52C12N9/0006C12N9/13C12N9/88C12N9/2402C12N9/1051C12N15/815C12P19/26C12Y101/01022C12Y208/02005C12Y208/02017C12Y402/02021C12Y402/0202C12Y302/01183C12Y204/01017C12Y204/01226Y02A50/30
Inventor 康振陈坚堵国成金学荣李江华盛靖雨
Owner JIANGNAN UNIV
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