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Recombinant expression vectors pQHK and pHK producing hyaluronic acid and construction method thereof

A technique for hyaluronic acid and expression vectors, which is applied in the field of recombinant expression vectors pQHK and pHK for high-efficiency hyaluronic acid-producing engineering bacteria and their construction, which can solve the problems of low safety and shortage of hyaluronic acid resources, etc.

Active Publication Date: 2012-12-05
MICROBIAL FERMENTATION ENG RES CENT CO LTD OF YUNNAN PROVINCE
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  • Abstract
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  • Claims
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Problems solved by technology

[0010] The purpose of the present invention is to provide a kind of high-efficiency hyaluronic acid producing engineered Escherichia coli recombinant expression vector pQHK and pHK and construction method thereof, overcome the resource shortage, safety problem of producing hyaluronic acid in the prior art and the comparatively low efficiency in engineering Escherichia coli. Problems with low hyaluronic acid synthesis

Method used

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  • Recombinant expression vectors pQHK and pHK producing hyaluronic acid and construction method thereof
  • Recombinant expression vectors pQHK and pHK producing hyaluronic acid and construction method thereof
  • Recombinant expression vectors pQHK and pHK producing hyaluronic acid and construction method thereof

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Embodiment 1

[0057] Example 1: (Construction of recombinant expression vector pQHK of engineering Escherichia coli for high-efficiency hyaluronic acid production)

[0058] from Pasteurella multocida ( Pasteurella multocida subsp. Multocida ) in the hyaluronan synthase gene pmHas clone and containing the hyaluronan synthase gene pmHas The construction specific steps of the expression vector pQEpmHas are as follows:

[0059] (1) Construct the expression vector pQEpmHas containing the hyaluronic acid synthase gene, and clone it from Pasteurella multocida ( Pasteurella multocida subsp. Multocida ) in the hyaluronan synthase gene ( P Asteurella m ultocida h Yaluronic a cid synthase PmHas) , the primers used for its PCR amplification clone are designed as follows:

[0060] Upstream primer pmHas P1 is 5'-TA GGATCC ATG AACACATTATCACAAGCAAT-3', (SEQ ID No:3 in the sequence listing) contains Bam HI site GGATCC and initiation codon ATG;

[0061] The downstream prime...

Embodiment 2

[0086] Construction of engineering Escherichia coli recombinant expression vector (broad-spectrum Gram-negative bacteria host) pHK for high-efficiency hyaluronic acid production:

[0087] To make pmHas and kfi D can be expressed in other Gram-negative bacteria including Escherichia coli, and primers were designed according to the DNA sequence (GenBank No. Y14439) of the Gram-negative bacteria host broad-spectrum plasmid pBBR122 (GenBank No. Y14439) of MoBiTec, Germany:

[0088] Upstream primer PBBR P1:5'-TTTGGT GTC GAC CTTGCCAGCCCGTGGATATGTGG-3' (SEQ ID NO: 10 in the sequence listing); contains Sal I restriction site, GTCGAC

[0089] Downstream primer PBBR P1: 5'-TTAGGT GTC GAC TCTGTGATGGCTTCCATGTCGGCAG-3' (SEQ ID NO: 11 in the sequence listing), containing Sal I restriction site, GTCGAC);

[0090] Use the upper and lower primers above to amplify the fragment of plasmid pBBR122 including its replication site and kanamycin resistance gene. The parameters of PCR ampl...

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Abstract

The invention discloses recombinant expression vectors pQHK and pHK producing hyaluronic acid and a construction method thereof. The pQHK and pHK disclosed by the invention are both constructed by a vector pQE8L initially, and contain hyaluronic acid synzyme pmHas and uridine diphosphate glucose dehydrogenase kfiD genes; and T5 drives the co-expression of pmHas and kfiD. As pHK contains necessaryfragments which are derived from the plasmid pBBR122 and can be replicated in gram negative bacteria in a broad-spectrum manner, the pHK has relatively good activity in most gram negative bacteria, and can be used for producing hyaluronic acid by the gram negative bacteria (other bacteria except escherichia coli). Each liter of the engineering escherichia coli constructed by using the recombinantexpression vector disclosed by the invention can produce 2-2.5 g of hyaluronic acid, and the yield is increased by more than 10 times in comparison with that produced through the engineering escherichia coli currently.

Description

technical field [0001] The invention relates to the technical field of genetic engineering of microorganisms, in particular to the construction of recombinant expression vectors pQHK and pHK for high-efficiency hyaluronic acid-producing engineering bacteria and their construction methods. Background technique [0002] Escherichia coli K12 strain is widely used in the field of metabolic engineering to produce a variety of high value-added products due to its clear genetic background, numerous efficient genetic transformation systems and the ease of genome manipulation. No natural strain of E. coli has been found to have the ability to produce hyaluronic acid. [0003] Hyaluronic acid (Hyaluronic acid, HA) widely exists in human and animal tissues, and its molecular weight is 5×10 4 to 8×10 6 Between Daltons, it is glucuronic acid linked by β-1,4-glycosidic bonds and nitrogen acetylglucosamine linked by β-1,3-glycosidic bonds (β1-4 D-Glucuronic acid, GlcA, β1- 3 D-N-acetylg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66
Inventor 毛自朝杨发祥尚海丽程倩
Owner MICROBIAL FERMENTATION ENG RES CENT CO LTD OF YUNNAN PROVINCE
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