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Gene engineering bacterium for high-yield hyaluronic acid and construction method and application of gene engineering bacteria

A technology of genetically engineered bacteria and hyaluronic acid, which is applied in the field of genetically engineered bacteria with high yield of hyaluronic acid and its construction, can solve the problems of hidden safety hazards, restricted development, and expensive prices for people with protein allergies, and achieve good industrial application prospects Effect

Pending Publication Date: 2017-11-17
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Before the 1980s, the source of hyaluronic acid was mainly biological tissue extraction. The raw material was the comb of a rooster, and the output was very low; at the same time, there would be residual animal protein in the product, which was safe for some people with protein allergies. Hidden danger
Streptococcus fermentation production has high nutritional requirements, the raw materials such as serum and brain heart extract in the culture medium are expensive, and the cost of industrial production is high; The security requirements limit its development

Method used

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  • Gene engineering bacterium for high-yield hyaluronic acid and construction method and application of gene engineering bacteria
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  • Gene engineering bacterium for high-yield hyaluronic acid and construction method and application of gene engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of ldh knockout recombinant strain Cg-△ldh (1) by single exchange homologous recombination

[0053] The construction of the recombinant bacterial strain Cg-△ldh (1) comprises the following steps:

[0054] 1. Construction of Corynebacterium glutamicum suicide plasmid pK18dLDH

[0055] Using the DNA genome of Corynebacterium glutamicum ATCC13032 as a template, PCR was performed with dLDH-F and dLDH-R as primers to obtain a fragment of about 700 bp in the middle of the lactate dehydrogenase gene ldh, and the product was purified as a homology arm;

[0056] Using the plasmid pK18mobsacB (referring to CN105420154A) as a template, and using pK18-F and pK18-R as primers to carry out PCR, amplify a fragment containing the Escherichia coli replication initiation site sequence ori sequence and the kanamycin resistance gene Kan sequence, and purify the product;

[0057] The above two fragments were ligated into a circular plasmid using the Gibson Assembly...

Embodiment 2

[0073] Example 2: Construction of ldh knockout recombinant strain Cg-△ldh by double exchange homologous recombination (2)

[0074] The construction of the recombinant bacterial strain Cg-△ldh (2) comprises the following steps:

[0075] 1. Construction of recombinant plasmid pK18mobmobsacB-dldh

[0076] Pick wild Corynebacterium glutamicum as a template, use dLDH-1 / dLDH-2 and dLDH-3 / dLDH-4 as primers to amplify the sequence of about 700bp upstream and downstream of the ldh gene in the genome, and purify the product ;

[0077] The plasmid pK18mobmobsacB was double digested with EcorI / XbaI, and the product was purified. The digested plasmid, the upstream fragment and the downstream fragment were subjected to Gibson Assembly ligation reaction to obtain the recombinant plasmid pK18mobmobsacB-dldh.

[0078] Primers were synthesized by Platinum Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water and diluted to 10 μM for use. Phanta polymerase Mix used in PCR amplificat...

Embodiment 3

[0090] Example 3: Using Cg-△ldh(1) and Cg-△ldh(2) as hosts to construct recombinant strains producing hyaluronic acid

[0091] Plasmid pXMJ19-A carrying hasA (refer to patent CN103937734A for its construction), plasmid pXMJ19-AB carrying hasA and hasB (refer to patent CN103937734A for its construction), plasmid pXMJ19-AC carrying hasA and hasC, carrying hasA and Plasmid pXMJ19-AD with hasD, plasmid pXMJ19-AE with hasA and hasE, plasmid pXMJ19-ABC with hasA, hasB and hasC (for its construction refer to patent CN103937734A), plasmid pXMJ19-ABD with hasA, hasB and hasD and carrying the plasmid pXMJ19-ABE with hasA, hasB and hasE, the recombinant ldh knockout strains Cg-△ldh (1) and Cg-△ldh (2 )middle.

[0092] Among them, hasA is hyaluronic acid synthase gene, hasB is UDP-glucose dehydrogenase gene, hasC is UDP-glucose pyrophosphorylase gene, hasD is glucosamine uridine transferase gene, hasE is phosphoglucose mutase gene .

[0093] The construction of the above plasmids pXMJ1...

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Abstract

The invention discloses a gene engineering bacterium for high-yield hyaluronic acid and a construction method and application of the gene engineering bacteria, and belongs to the field of gene engineering and biochemical engineering. The gene engineering bacterium for high-yield hyaluronic acid is constructed through the steps that a lactate dehydrogenase gene in corynebacterium glutamicum is inactivated, and then a hyaluronan synthase gene is transferred into the corynebacterium glutamicum. According to the gene engineering bacterium for high-yield hyaluronic acid, a corynebacterium glutamicum host has no pathogenicity to both humans and animals and is a food-grade safe microorganism; and the recombinant corynebacterium glutamicum for high-yield hyaluronic acid is high in hyaluronic acid yield which is up to 22g / L or above and three times the level of existing industrial production, and a new bacterial strain has good industrialization application prospects.

Description

technical field [0001] The invention belongs to the fields of genetic engineering, biochemical industry and microbial fermentation, and specifically relates to a genetically engineered bacterium with high hyaluronic acid production and its construction method and application. Background technique [0002] Hyaluronic acid (Hyaluronic acid, HA) is a kind of acidic mucopolysaccharide widely present in humans and animals, and its molecular structure is composed of D-glucuronic acid (GlcA) and N-acetyl-glucosamine (N-acetyl-D-glucosamine, GlcNAc) alternating linear copolymerization, the molecular weight can reach 10 5 ~10 7 Da. In the human body, hyaluronic acid mainly plays a role in lubrication, promoting wound healing, and regulating the permeability of connective tissue. Clinically, hyaluronic acid is widely used in ophthalmic surgery and arthritis surgery, as well as drug delivery. Since hyaluronic acid can absorb 1000 times its own weight in water, it is often used as a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P19/26C12R1/15
CPCC12N9/0006C12N9/1051C12N9/1241C12N9/90C12N15/77C12P19/26C12Y101/01022C12Y204/01212C12Y207/07009C12Y504/02002
Inventor 于慧敏成方宇沈忠耀
Owner TSINGHUA UNIV
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