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Recombinant corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and oligosaccharides thereof

A technology of Corynebacterium glutamicum and hyaluronic acid, which is applied in the field of bioengineering, can solve the problems of reducing the quality of hyaluronic acid products and increasing the difficulty of downstream hyaluronic acid purification, so as to achieve the goal of increasing yield and purity and improving synthesis capacity Effect

Active Publication Date: 2020-08-11
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These polysaccharides not only compete for the substrates of the hyaluronic acid synthesis pathway, but also increase the difficulty of downstream hyaluronic acid purification and reduce the quality of hyaluronic acid products

Method used

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  • Recombinant corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and oligosaccharides thereof
  • Recombinant corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and oligosaccharides thereof
  • Recombinant corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and oligosaccharides thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the construction of recombinant Corynebacterium glutamicum

[0039] (1) Knockout of genes cg0420 and cg0424

[0040] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and using 0420-up-F and 0420-up-R as primers to perform PCR, obtain a fragment 0420-up about 500 bp upstream of cg0420 and perform PCR product purification;

[0041] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and using 0420-down-F and 0420-down-R as primers, PCR was carried out to obtain a fragment 0420-down about 500 bp downstream of cg0420 and purify the PCR product;

[0042] The Corynebacterium glutamicum suicide plasmid pK18mobsacB was double-digested with EcoRI / BamhI, and the 0420-up and 0420-down fragments were ligated to the pK18mobsacB after digestion with the GibsonAssembly kit in one step, and the recombinant plasmid obtained was named pK18- 0420;

[0043] The plasmid pK18-0420 was transformed into Corynebacterium glutami...

Embodiment 2

[0067] Embodiment 2: recombinant Corynebacterium glutamicum produces hyaluronic acid

[0068] The constructed recombinant Corynebacterium glutamicum strains: WT, △0420, △0424 and △0420&△0424 were single-cloned in 5ml BHI medium, 200rpm, 30°C overnight culture. After 10 hours, transfer to 250ml Erlenmeyer shake flask (filling liquid volume is 25ml fermentation medium) according to 1% inoculum amount. Placed at 200rpm, cultivated at 28°C, cultivated for three hours, and added IPTG with a final concentration of 0.25Mm to induce gene expression. The fermentation period was 48 hours. After the fermentation, take the supernatant and add four times the volume of ethanol for alcohol precipitation to remove some Impurities, after repeated alcohol precipitation twice, the content of hyaluronic acid was determined by the sulfuric acid carbazole method, from figure 2 It can be seen that by knocking out cg0420, the yield and purity of hyaluronic acid in cg0424 have been improved to a cer...

Embodiment 3

[0069] Embodiment 3: the 5L fermenter fermentation culture of recombinant Corynebacterium glutamicum

[0070] The recombinant Corynebacterium glutamicum bacterial strain that will construct: Corynebacterium glutamicum HasA-VGB / pXMJ19-pgm-ugd-galU, pECX99E-glmU-glmM-glmS (knockout cg0420, cg0424) receive single clone in 5mlBHI medium, 200rpm, Incubate overnight at 30°C. After 10 hours, the inoculum was transferred to a 250 ml Erlenmeyer shaker flask (filling liquid volume is 25 ml fermentation medium) according to the inoculum amount of 1%. Placed at 200rpm, cultured at 28°C for 10h, inoculated in the fermenter with 10% inoculation amount, maintained the glucose content in the fermenter at about 10g / L by feeding glucose during the fermentation process, and controlled the pH to be neutral by feeding NaOH properties, and exogenously added hyaluronan hydrolase with a final concentration of 6000U / mL at 20h of fermentation, the fermentation period was 72h, from image 3 It can be ...

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Abstract

The invention discloses recombinant corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and oligosaccharides thereof, and belongs to the technical field of bioengineering. The hyaluronic acid generated by the recombinant corynebacterium glutamicum constructed by the invention achieves yield of 40g / L and achieves crude product purity of 95%. a hyaluronic acid hydrolytic enzyme is added by an external source and fermentation conditions are optimized, so that the hyaluronic acid oligosaccharides with a specific molecular weight can be obtained, further increase ofthe yield of the hyaluronic acid is implemented, and the yield achieves 72g / L. The invention lays the solid foundation for microbial efficient synthesis of the high-purity hyaluronic acid, and the constructed recombinant corynebacterium glutamicum is suitable for industrial production application.

Description

technical field [0001] The invention relates to a recombinant coryneform bacterium glutamicum capable of efficiently synthesizing high-purity hyaluronic acid and its oligosaccharides, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronic acid is a linear acidic mucopolysaccharide polymerized from disaccharide units composed of N-acetylglucosamine and glucuronic acid. Ultra-high molecular weight hyaluronic acid has good viscoelasticity, moisture retention and anti-inflammatory functions, and can be used as a viscoelastic agent in ophthalmic surgery and for intra-articular injection therapy. High molecular weight hyaluronic acid has good moisturizing and lubricating effects and can be used in the field of cosmetics. High-purity hyaluronic acid and hyaluronic acid oligosaccharides have the functions of anti-tumor, promoting wound healing, promoting bone and angiogenesis, and immune regulation. In 2016, the global hyaluronic acid market w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/10C12N15/31C12P19/26C12R1/15
CPCC12N9/1051C07K14/195C12P19/26C12Y204/01212C12N15/77C12R2001/15C12N15/52C07K14/805C12N1/20C12N1/205C12N9/2474C12N2800/101C12P19/04C12Y302/01035
Inventor 康振陈坚胡立涛堵国成王阳李江华李佳莲张天萌
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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