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

A technology of hyaluronic acid and expression vector, which is applied to recombinant expression vectors pQHK and pHK of high-efficiency hyaluronic acid-producing engineering bacteria and their construction fields, can solve the problems of shortage of hyaluronic acid resources and low safety.

Active Publication Date: 2011-08-17
MICROBIAL FERMENTATION ENG RES CENT CO LTD OF YUNNAN PROVINCE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0011] 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

[0058] Example 1: (Construction of engineering E. coli recombinant expression vector pQHK for efficient production of hyaluronic acid)

[0059] Derived from Pasteurella multocida ( Pasteurella multocida subsp. Multocida ) Hyaluronic acid synthase gene pmHas Cloning and containing hyaluronic acid synthase gene pmHas The specific steps for constructing the expression vector pQEpmHas are as follows:

[0060] (1) Construct the expression vector pQEpmHas containing the hyaluronic acid synthase gene, and clone it from Pasteurella multocida ( Pasteurella multocida subsp. Multocida ) In the hyaluronic acid synthase gene ( P asteurella m ultocida h yaluronic a cid synthase PmHas) , The primers used for PCR amplification cloning are designed as follows:

[0061] The upstream primer pmHas P1 is 5'-TA GGATCC ATG AACACATTATCACAAGCAAT-3', (SEQ ID No: 3 in the Sequence Listing) contains Bam H I site GGATCC and start codon ATG;

[0062] The downstream primer is pmHas P2 is 5'-TA GAGCTC ...

Embodiment 2

[0087] Construction of the engineered E. coli recombinant expression vector (wide spectrum of gram-negative bacteria host) pHK for efficient production of hyaluronic acid:

[0088] To make pmHas with kfi D can be expressed in other Gram-negative bacteria including Escherichia coli. The primers are designed according to the DNA sequence (GenBank No. Y14439) of the Gram-negative bacteria host broad-spectrum plasmid pBBR122 from MoBiTec, Germany:

[0089] Upstream primer PBBR P1: 5'-TTTGGT GTCGAC CTTGCCAGCCCGTGGATATGTGG-3' (SEQ ID NO: 10 in the sequence listing); contains Sal I restriction site, GTCGAC

[0090] Downstream primer PBBR P1: 5'-TTAGGT GTCGAC TCTGTGATGGCTTCCATGTCGGCAG-3' (SEQ ID NO: 11 in the sequence listing), containing Sal I restriction site, GTCGAC);

[0091] The above-mentioned upper and lower primers were used to amplify the fragment of plasmid pBBR122 containing its replication site and kanamycin resistance gene. The PCR amplification parameters were: 95℃, 2 min, ...

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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

[0001] Technical field [0002] The invention relates to the technical field of genetic engineering of microorganisms, in particular to constructing recombinant expression vectors pQHK and pHK for efficient hyaluronic acid production engineering bacteria and construction methods thereof. Background technique [0003] The E. coli K12 strain has been widely used in the field of metabolic engineering due to its clear genetic background, numerous efficient genetic transformation systems and easy operability of the genome to produce a variety of high value-added products. At present, no natural E. coli strain has the ability to produce hyaluronic acid. [0004] Hyaluronic acid (Hyaluronic acid, HA) is widely present in human and animal tissues, and its molecular weight is 5×10 4 To 8×10 6 Between Daltons, there are glucuronic acid linked by β-1,4-glycosidic bonds and aza-acetylglucuronic acid linked by β-1,3-glycosidic bonds (β1-4 D-Glucuronic acid, GlcA, β1- 3 DN-acetylglucosamine, Glc...

Claims

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Application Information

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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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