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Reorganized corynebacterium glutamicum producing chondroitin and application of reorganized corynebacterium glutamicum

A technology of Corynebacterium glutamicum and chondroitin synthase, which is applied in the field of bioengineering, can solve the problems of increasing downstream product processing, difficulty in purification, lack of key enzyme transformation for chondroitin synthesis, and low chondroitin yield, and avoids fructosaccharification. Effects of modification, improved association, and low cost of cultivation

Active Publication Date: 2018-11-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, the synthesis process of fructosylated chondroitin is subject to complex regulation, resulting in low yields
These shortcomings will not only cause high production costs, but also increase the difficulty of processing and purification of downstream products
In other microbial cells such as Escherichia coli BL21 and Bacillus subtilis, the recombinant chondroitin synthesis focuses on optimizing the metabolic pathway, and lacks the transformation of key enzymes for chondroitin synthesis, resulting in low efficiency of the pathway and low production of chondroitin

Method used

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  • Reorganized corynebacterium glutamicum producing chondroitin and application of reorganized corynebacterium glutamicum
  • Reorganized corynebacterium glutamicum producing chondroitin and application of reorganized corynebacterium glutamicum
  • Reorganized corynebacterium glutamicum producing chondroitin and application of reorganized corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: P trc -kfoA-kfoC integrated into the C. glutamicum genome

[0018] Ptrc-F and Ptrc-R were used as primers, and pEC-XK99E was used as template to amplify P trc , with kfoA-F and kfoA-R as primers, kfoA was amplified with the Escherichia coli K4 genome as a template; with kfoC-F and kfoC-R as primers, kfoC was amplified with the Escherichia coli K4 genome as a template; the obtained P trc , kfoA and kfoC used Ptrc-F and kfoC-R as primers for overlap extension PCR to obtain P trc -kfoA-kfoC expression cassette. With glgA up-F and glgA down-R as primers, with P trc -kfoA-kfoC and Corynebacterium glutamicum glgA gene upper and lower 500bp homology arms (two 500bp homology arms respectively through primers glgA up-F and glgAup-R, glgA down-F and glgA down-R with glutamine Acid Corynebacterium ATCC13032 genome as a template for PCR) as a template for overlapping extension PCR again to obtain H1-P trc-kfoA-kfoC-H2.

[0019] H1-P trc -kfoA-kfoC-H2 was ligated i...

Embodiment 2

[0020] Embodiment 2: the construction of recombinant bacteria C.glutamicum Chon1

[0021] Corynebacterium glutamicum ATCC 13032 was inoculated in 5 mL of BHIS liquid medium, cultured at 30°C and 200 rpm for 24 hours, the cells were collected, and genomic DNA was extracted using a cell genome extraction kit. Step 1: Construction of pXMJ19-glmU-galU. Primers Ppyc-F / Ppyc-R, glmU-F / glmU-R, galU-F / galU-R were designed, using the extracted C. glutamicum ATCC 13032 genomic DNA as a template, using standard PCR amplification system and procedures to amplify Add Ppyc, glmU, galU genes. The amplified glmU and galU were used as templates and glmU-F and galU-R were used as primers to perform overlap extension PCR to obtain connected glmU-galU fragments. Using pXMJ19-F / pXMJ19-R as primers to amplify the linear pXMJ19 plasmid with deletion of Ptac and lacIQ regions. The glmU-galU fragment and the linearized plasmid pXMJ19 were subjected to one-step cloning and splicing reactions, respect...

Embodiment 3

[0025] Example 3: Construction and screening of KfoC (H357G / N363S) mutants

[0026] According to the operation manual of the kit, using kfoC-F and kfoC-R as primers, use Diversify TM PCR RandomMutagenesis Kit PCR Random Mutagenesis Kit performs random mutation on the kfoC gene. According to the description in Example 1, P trc Promoter and kfoA gene assembly to form P trc -kfoA-kfoC (random mutant) expression cassette, and according to the description in Example 1, the P trc -kfoA-kfoC (random mutant) expression cassette integrated into the C. glutamicum genome. Contains P trc The recombinant Corynebacterium glutamicum single colony library of -kfoA-kfoC (random mutant) was fermented in a 96-well plate. After the glucose was exhausted (after 40 hours), the bacterial cells were collected by centrifugation in a 96-well plate, and the bacterial cells were washed twice with deionized water. After the bacteria were diluted by appropriate multiples, the content of chondroitin w...

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Abstract

The invention discloses reorganized corynebacterium glutamicum producing chondroitin and an application of the reorganized corynebacterium glutamicum, and belongs to the technical field of bioengineering. According to the reorganized corynebacterium glutamicum disclosed by the invention, firstly UDP-N-acetylglucosamine C4 isomerase coding genes kfoA and chondroitin synthetase coding genes kfoC aresubjected to heterologous expression in corynebacterium glutamicum, glmU, galU, ugd, glmS and glmM are subjected to overexpression, and a synthesis route of chondroitin is preliminarily constructed;and then random mutation is performed on chondroitin synthetase (KfoC), and the situation that the yield of chondroitin can be increased by 200% through a KfoC (H357G N363S) mutant is found. Through aprotein fusion technique, kfoA, kfoC (mutants) and ugd genes (coding UDP-glucose dehydrogenase) are fused, so that the expressed UDP-N-acetylglucosamine C4 isomerase, the chondroitin synthetase and the UDP-glucose dehydrogenase form an artificial compounds, and the yield of the chondroitin is further increased.

Description

technical field [0001] The invention relates to a chondroitin-producing recombinant Corynebacterium glutamicum and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate is an important glycosaminoglycan, which consists of disaccharide units composed of glucuronic acid and N-acetylgalactosamine connected alternately by β1-3 and β1-4 glycosidic bonds. Chondroitin sulfate is widely used in fields such as healthcare, cosmetics and food additives. At present, chondroitin sulfate is mainly obtained from animal tissue through chemical extraction. Because the sulfation degree of chondroitin sulfate in different animal tissues is quite different, it is difficult to obtain a product with high purity and uniform quality. The recent rise of biological enzymatic catalysis represents an important development direction of chondroitin sulfate and production methods. In the method, chondroitin is used as a substrate t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/15
CPCC12N9/0006C12N9/88C12N9/90C12P19/26C12Y101/01022C12Y402/02004C12Y501/03007
Inventor 康振王阳胡立涛陈坚堵国成
Owner JIANGNAN UNIV
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