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Recombinant bacteria high in hyaluronic acid yield and construction method and application thereof

A technology of hyaluronic acid and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of streptococcus pathogenicity and production cost increase

Active Publication Date: 2019-10-08
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Foreign countries are also constantly researching the technology of streptococcus producing HA (Pourzardosht, N., Rasaee, M.J., 2017. Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene. Mol.Biotechnol.59,192–199.) But Streptococcus itself has potential pathogenicity, which limits its development to a certain extent
On the other hand, the medium for cultivating streptococcus contains expensive raw materials such as serum and brain heart extract, and its production cost also increases accordingly.

Method used

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  • Recombinant bacteria high in hyaluronic acid yield and construction method and application thereof
  • Recombinant bacteria high in hyaluronic acid yield and construction method and application thereof
  • Recombinant bacteria high in hyaluronic acid yield and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of CghasB enhanced expression strains and comparison of their HA production capacity

[0071]In order to strengthen the expression of the key enzyme gene CghasB in the HA synthesis of Corynebacterium glutamicum, this embodiment carried out transformation on the basis of the recombinant plasmid PXMJ19-AB (see Chinese patent CN103937734A for details), that is, insert an extra gene between the genes shasA and CghasB promoter, so that CghasB can be simultaneously transcribed by the Ptac promoter and this additional promoter. In this embodiment, the additional promoters are selected from PdapB (the promoter of the 4-hydroxytetrahydropyridine reductase gene), PdapA (the promoter of the dihydropicoline synthase gene), Psod (the promoter of the superoxide dismutase gene promoter) and the Ptac promoter. Taking PdapB as an example, the construction method of the recombinant plasmid is as follows:

[0072] Using the genome of Corynebacterium glutamicum as ...

Embodiment 2

[0082] Embodiment 2 constructs fba weakened bacterial strain and compares its HA production capacity

[0083] On the basis of Example 1, this example further uses antisense RNA technology to weaken the reaction catalyzed by the fba gene expression enzyme in glycolysis. The genome of Corynebacterium glutamicum was used as a template, and aF-F and aF-R were used as primers for PCR reaction and purification to amplify the antisense RNA fragment of fba gene. The conditions used for PCR were the same as in Example 1.

[0084] Plasmid pXMJ19 was double digested with SalI / EcorI. The digestion product and the antisense RNA fragment of the fba gene were subjected to a Gibson ligation reaction to obtain an expression cassette plasmid containing the antisense RNA of the fba gene transcribed by the Ptac promoter, and transformed into the E.coli host bacterium TOP10 competent by the method in Example 1 cell. Using the plasmid as a template and using PaFT-F and PaFT-R as primers to carry...

Embodiment 3

[0086] Example 3 Construction of aceE weakened strain and comparison of its HA production capacity

[0087] On the basis of Example 2, this example further uses antisense RNA technology and start codon mutation to weaken the expression of pyruvate dehydrogenase gene aceE, down-regulate the tricarboxylic acid cycle, and strengthen the synthesis of HA. The genome of Corynebacterium glutamicum was used as a template, and aE-F and aE-R were used as primers for PCR reaction and purification to amplify the antisense RNA fragment of aceE gene. Then, with the method in Example 2, using PaET-F and PaET-R as primers for PCR reaction and purification, the aceE gene antisense RNA expression cassette was amplified. Plasmid pXMJ19-A-PdapB-B-aF was digested with XhoI. The digestion product and the expression cassette of antisense RNA of the aceE gene were subjected to Gibson ligation reaction to obtain the plasmid pXMJ19-A-PdapB-B-aF-aE and transformed into E.coli host strain TOP10 competen...

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Abstract

The invention discloses recombinant bacteria high in hyaluronic acid yield in the technical field of genetic engineering, and a construction method and an application thereof. The recombinant bacteriacan be constructed by enhancing the expression of a UDP-glucose dehydrogenase gene in a hyaluronic acid synthesis pathway and weakening a competition pathway. The corynebacterium glutamicum host of the present invention has no pathogenicity to humans and animals, and is a food-grade safe microorganism; the recombinant corynebacterium glutamicum has a high hyaluronic acid yield of up to 28g / L, andthe new strain has a good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant bacterium with high hyaluronic acid production and its construction method and application. Background technique [0002] Hyaluronic acid (Hyaluronic acid, HA) is a type of glycosaminoglycan widely present in humans and animals. Its molecules are linear and its repeating units are composed of D-glucuronic acid (GlcA) and N-acetyl -Glucosamine (N-acetyl-D-glucosamine, GlcNAc) disaccharide unit composition, molecular weight up to 10 4 ~10 7 Da. HA plays a role in soft tissue lubrication, promoting wound healing, and participating in immune response in the human body. The commercial use of HA is also very extensive. High-molecular-weight HA can be used as a filler in clinical operations; medium-molecular-weight HA is used in cosmetics and skin care products; small-molecular HA can be made into oral health products to supplement the sugar ami...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12R1/15
CPCC12N9/0006C12N9/1051C12N15/77C12Y101/01022C12Y204/01212
Inventor 于慧敏成方宇
Owner TSINGHUA UNIV
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