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Genetically-engineered bacterium realizing high production of hyaluronic acid and application thereof

A technology of genetically engineered bacteria and hyaluronic acid, which is applied in the field of genetically engineered bacteria with high hyaluronic acid production, can solve problems such as unseen

Active Publication Date: 2014-07-23
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no report of using Corynebacterium glutamicum as a host to produce hyaluronic acid

Method used

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  • Genetically-engineered bacterium realizing high production of hyaluronic acid and application thereof
  • Genetically-engineered bacterium realizing high production of hyaluronic acid and application thereof
  • Genetically-engineered bacterium realizing high production of hyaluronic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of Corynebacterium glutamicum-Escherichia coli shuttle plasmid carrying hyaluronan synthase sHasA gene and CgHasB and CgHasC genes

[0046] Construction of plasmid pXMJ19-A: Based on the codon preference of Corynebacterium glutamicum and Escherichia coli, the gene sequence shasA of hyaluronan synthase sHasA was designed and chemically synthesized, as shown in SEQ ID No:1. The codon usage frequency of this gene and the protococcal Streptococcus equi hyaluronan synthase gene in Corynebacterium glutamicum is analyzed, and it is found that SEQ ID No:1 does not contain the highly rare codon (frequency of use) of Corynebacterium glutamicum 0-10%), and the number of common rare codons is only 29, which is also greatly reduced compared to 63 in the original gene. The codon usage frequency of SEQ ID No:1 in Escherichia coli was also analyzed, and it was found that SEQ ID No:1 is also suitable for expression in Escherichia coli, and does not contain highly rare codon...

Embodiment 2

[0059] Construction of genetically engineered bacteria expressing hyaluronan synthase gene shasA and overexpressing CgHasB and CgHasB

[0060] The recombinant plasmid pXMJ19-A carrying hyaluronan synthase sHasA, the recombinant plasmid pXMJ19-AB overexpressing sHasA and CgHasB, and the recombinant plasmid pXMJ19-ABC overexpressing sHasA, CgHasB and CgHasC constructed in Example 1 were used as The competent cells of Corynebacterium glutamicum ATCC13032 are transformed by electroporation, and genetically engineered bacteria C.glu A, C.glu AB and C.glu ABC producing hyaluronic acid can be obtained. Add 5 μL of recombinant plasmid and 100 μL of Corynebacterium glutamicum ATCC13032 competent cells to a 1.5mL centrifuge tube, mix well, add a 0.1cm electroporation cup, and ice-bath for 30min; adjust the voltage of the electroporator to 1.8kV, and put the electroporation cup into Press the electric shock button on the electroporator; after the electric shock, add 1mL of SOC recovery m...

Embodiment 3

[0062] Production of Hyaluronic Acid Using Genetically Engineered Bacteria C.glu A, C.glu AB and C.glu ABC

[0063] The genetically engineered bacteria C.glu A, C.glu AB and C.glu ABC that will be transferred into the Corynebacterium glutamicum C.glu P of original plasmid pXMJ19 and embodiment 2 are inoculated in LB liquid culture medium (containing 5 μ g / mL Chloramphenicol), cultured at 37°C and 200rpm for 16 hours, inserted into LB liquid medium with a glucose concentration of 40g / L at a ratio of 5%, cultivated at 37°C and 200rpm for 3h, added IPTG (final concentration 1mM) , continue culturing for 24 hours, and centrifuge at 8000 rpm at room temperature to obtain a hyaluronic acid-containing fermentation broth. Take 1 mL of the obtained fermentation broth and add 1 mL of 0.1% w / v SDS solution to mix, incubate at room temperature for 20 minutes; centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant to a new 10 mL EP tube, add 2 times the volume of ice ethanol, 4 ...

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Abstract

The invention discloses genetically-engineered bacterium realizing high production of hyaluronic acid and application of the genetically-engineered bacterium, belonging to the field of biotechnology and biochemical industry. The genetically-engineered bacterium is formed by transforming corynebacterium glutamicum by use of a recombinant vector containing a hyaluronic acid synthetase gene, and the nucleotide sequence of the hyaluronic acid synthetase gene is as shown in a sequence table SEQ ID No:1. The genetically-engineered bacterium is used for producing hyaluronic acid, high-yield hyaluronic acid can be obtained by independently expressing hyaluronic acid synthetase, and CgHasB and CgHasC do not become choke points which limit the high production of recombined bacterium. According to the method provided by the invention, a corynebacterium glutamicum host is free of pathogenicity to both humans and animals and is food-grade safe microorganism, the yield of the synthesized hyaluronic acid is high and above 6.0g / L, so the genetically-engineered bacterium has good industrial application prospect.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biochemical industry, and in particular relates to a genetic engineering bacterium with high hyaluronic acid production and its application. Background technique [0002] Hyaluronic acid (Hyaluronic acid, HA) is an acidic mucopolysaccharide widely present in humans and animals. It is composed of D-glucuronic acid (GlcA) and N-acetyl-glucosamine (N- acetyl-D-glucosamine, GlcNAc) alternately constituted straight-chain polysaccharides, and its molecular weight can reach 105-107 Daltons. Hyaluronic acid is soluble in water and insoluble in organic solvents. Hyaluronic acid has a variety of important physiological functions in the human body, mainly including lubricating joints, promoting wound healing, regulating the permeability of blood vessel walls, regulating the diffusion and operation of protein / water electrolytes, and affecting the maturity and function of human skin. Aging and more. Clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/15
Inventor 于慧敏龚倩莹王君婷成方宇沈忠耀
Owner TSINGHUA UNIV
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