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Recombinant Corynebacterium glutamicum high in hyaluronic acid yield and preparation method and application thereof

A technology of Corynebacterium glutamicum and hyaluronic acid, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of product application limitations and extremely high requirements for the separation process

Active Publication Date: 2016-12-07
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because these strains secrete endotoxin, the isolation process is extremely demanding and the product application is limited

Method used

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  • Recombinant Corynebacterium glutamicum high in hyaluronic acid yield and preparation method and application thereof
  • Recombinant Corynebacterium glutamicum high in hyaluronic acid yield and preparation method and application thereof
  • Recombinant Corynebacterium glutamicum high in hyaluronic acid yield and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Knockout of Corynebacterium glutamicum glucose 6-phosphate dehydrogenase gene zwf

[0068]1. Using the genomic DNA of Corynebacterium glutamicum MB001 as a template and using zwf-up-F and zwf-up-R as primers to perform PCR to obtain a homologous fragment zwf about 1000 bp upstream of the glucose 6-phosphate dehydrogenase gene zwf -up and perform PCR product purification;

[0069] 2. Use the genomic DNA of Corynebacterium glutamicum MB001 as a template, and use zwf-down-F and zwf-down-R as primers to perform PCR to obtain a homologous fragment zwf-down about 1000 bp downstream of zwf and purify the PCR product;

[0070] 3. The Corynebacterium glutamicum suicide plasmid pK18mobsacB (Journal of Biotechnology 104 (2003) 287-299) was double digested with EcoRI / XbaI, and the zwf-up and zwf-down fragments were zwf-up and zwf-down with Gibson Assembly kit (NEB) One-step ligation to the digested pK18mobsacB, and the obtained recombinant plasmid was named pK18-zwf;

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Embodiment 2

[0077] Example 2 Verifying the impact of the knockout of the glucose 6-phosphate dehydrogenase gene zwf on the production of hyaluronic acid

[0078] Based on the amino acid sequence of hyaluronan synthase from Streptococcus zooepidemicus, a codon-optimized hyaluronan synthase gene hasA (SEQ ID NO: 7) was designed and entrusted to Wuxi Qinglan Biotechnology Co., Ltd. for gene synthesis; The hasA gene fragment was used as a template, and hasA-F (5'-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3') and hasA-R (5'-gcctgcaggtcgactcgtctcggttggcagtgac-3') were used as primers for PCR to obtain a hasA fragment of about 1400 bp, and the PCR product was purified . The plasmid pXYJ-12 (purchased from Addgene) with the lacY gene (SEQ ID NO: 8) was double digested with EcoRI / XbaI, and the hasA fragment was ligated to pXYJ-12 using the Gibson Assembly kit (NEB) to obtain The recombinant plasmid was named pXYJ-hasA. The plasmid pXYJ-hasA was transformed into wild-type Corynebacterium glutamicu...

Embodiment 3

[0082] Example 3 Construction of recombinant Corynebacterium glutamicum with high production of medium molecular weight hyaluronic acid

[0083] 1. Construct the recombinant plasmid pXYJ-hasA, the method is as described in Example 2;

[0084] 2. Using the genome of Corynebacterium glutamicum MB001 as a template and using udgA-F and udgA-R as primers to perform PCR, obtain a fragment of UDP-glucose dehydrogenase gene udgA of about 1200 bp, and purify the PCR product;

[0085] 3. The plasmid pXYJ-hasA was single-digested with SalI, and the udgA fragment was connected to the plasmid pXYJ-hasA using the Gibson Assembly kit, and the obtained recombinant plasmid was named pXYJ-hasA-udgA;

[0086] 4. Transform pXYJ-hasA-udgA into the recombinant Corynebacterium glutamicum Δzwf of Example 1 by electroporation (the conditions of electroporation are the same as those described in Example 1), screen positive clones, and name the obtained recombinant bacteria CgΔzwf / pXYJ-hasA-udgA.

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Abstract

The invention provides recombinant Corynebacterium glutamicum high in hyaluronic acid yield and a preparation method and application thereof. The recombinant Corynebacterium glutamicum high in hyaluronic acid yield is constructed by genetic engineering technology. The invention also provides application of the recombinant Corynebacterium glutamicum high in hyaluronic acid yield in the fermentative production of hyaluronic acid. The microbial Corynebacterium glutamicum of food safety grade is utilized which never secretes endotoxin, and post-extraction process is simplified; the recombinant Corynebacterium glutamicum high in hyaluronic acid yield constructed by genetic engineering technology can produce hyaluronic acid of different molecular weights (3000 Da to 2000000 Da), the yield of the hyaluronic acid may reach 7 to 20 g / L according to the different molecular weights, and a product may satisfy the needs of foods, cosmetics and pharmaceuticals without complex separation.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial fermentation, in particular to a recombinant Corynebacterium glutamicum with high hyaluronic acid production and a preparation method and application thereof. Background technique [0002] Hyaluronic acid is a polymer polysaccharide composed of D-glucuronic acid and N-acetylglucosamine connected by β-1,3 and β-1,4 glycosidic bonds, also known as hyaluronic acid. Hyaluronic acid widely exists in various parts of the human body and has extremely important physiological functions, such as assisting the diffusion and transport of water and electrolytes, lubricating joints, regulating the permeability of blood vessel walls, and promoting wound healing. In addition, hyaluronic acid has a strong moisturizing effect and is known as an ideal natural moisturizing factor. It is the substance with the best moisturizing properties for cosmetics found in nature. Hyaluronic acid has different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P19/26C12R1/15
CPCC12N9/0006C12N9/1051C12N15/77C12N2800/22C12P19/26C12Y101/01022C12Y204/01212
Inventor 陈振刘德华
Owner TSINGHUA UNIV
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