Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof

A technology of Escherichia coli and construction methods, applied in the field of microbial genetic engineering, to achieve the effect of high-efficiency accumulation

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some foreign companies have carried out relevant research, but the conditions for industrialization need to be further optimized

Method used

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  • Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof
  • Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof
  • Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, construction of mutant strain CICIM B0013 SA4

[0037] 1.1 Mutant strain CICIM B0013·SA1 ( △aroL ) build

[0038] used to aroL Primers designed upstream and downstream of the gene aL up, aL dn amplifies the chromosomal DNA of Escherichia coli aroL gene, the fragment size is 575 bp. The PCR amplification system is 50 μL (Taq enzyme 1 μL, Taq enzyme buffer 5 μL, dNTPs 4 μL, upstream and downstream primers 1 μL, template 1 μL, ddH 2 O 17μL), the conditions are: 94°C, 5min; 94°C, 30s; 52°C, 1min; 72°C, 1min, 30 cycles. The PCR product was cloned into the pMD18-T-simple vector to obtain the recombinant plasmid pMD- aroL . Using the recombinant plasmid as a template, primers L verup, L verdn for inverse PCR amplification. The PCR amplification system was 50 μL, and the conditions were: 94°C, 5min; 94°C, 30s; 58°C, 1min; 72°C, 3.5min, 30 cycles. PCR product with difGm The fragments were ligated to obtain the recombinant plasmid pMD- aroL ':: ...

Embodiment 2

[0070] Embodiment 2, construction of recombinant plasmid pTHGAA

[0071] 2.1 Cloning of recombinant plasmid pTHG

[0072] Using the genome of Escherichia coli CICIM B0013 as a template, primers aoGm-up, aoGm-dn, aoG-up, aoG-dn were used to perform overlapping PCR to amplify the site-directed mutation aroG* gene. The primer sequences are as follows:

[0073] aoGm-up: 5′-gtgagtttctcaatatgatcaccc-3′

[0074] aoGm-dn: 5′-tggggtgatcatattgagaaact-3′

[0075] aoG-up: 5′-ccggaattcaggaggccatccatgaattatcagaacgac-3′

[0076] aoG-dn: 5′-cggggtaccttacccgcgacgcgcttttact-3′

[0077] Fragment P1 was obtained by PCR amplification with primers aoG-up and aoGm-dn, and fragment P2 was obtained by second-round PCR amplification with primers aoGm-up and aoG-dn. The PCR amplification system is 50 μL (Taq enzyme 1 μL, Taq enzyme buffer 5 μL, dNTPs 4 μL, upstream and downstream primers 1 μL, template 1 μL, ddH 2 O 17 μL), the PCR amplification conditions were: 94°C, 5min; 94°C, 30s; 56°C, 1min; ...

Embodiment 3

[0091] Embodiment 3, the acquisition of shikimic acid expression strain

[0092] The mutant strain CICIM B0013 SA4 ( △aroL , △aroK , △ptsG , △ydiB ) as a host bacterium, the recombinant expression plasmid pTHGAA obtained in Example 2 was transformed into it by electric shock transformation method, and the Escherichia coli host bacterium SA4 / pTHGAA expressing shikimic acid was obtained.

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Abstract

An Escherichia coli recombinant strain producing shikimic acid, and a construction method and application thereof belong to the technical field of microbial gene engineering. The invention firstly utilizes a molecular biology technique to delete shikimic acid kinase I gene (aroK) and shikimic acid kinase II gene (aroL) of Escherichia coli CICIMB0013, and a gene (ptsG) of a key protein EIIBC<Glc> and a quinin acid/shikimic acid dehydrogenase gene (ydiB) of a glucose phosphotransferase system to obtain an Escherichia coli mutant strain CICIMB0013.SA4 (delta aroK, delta aroL, delta ptsG, delta ydiB). The invention also constructs a recombinant expression plasmid pTHGAA containing key genes comprising aroG*,ppsA and tktA in a metabolic pathway of shikimic acid; and the recombinant expression plasmid pTHGAA is transferred into the recombinant strain CICIMB0013.SA4 to obtain a recombinant Escherichia coli B0013 (SA4/pTHGAA) capable of producing shikimic acid efficiently. The Escherichia coli recombinant strain provided by the invention can realize efficient accumulation of shikimic acid in a fermentation process.

Description

technical field [0001] The invention relates to a shikimic acid-producing Escherichia coli recombinant bacterium and its construction method and application. By studying and improving the influence of related genes and nodes in the shikimic acid production pathway, excessive synthesis and accumulation of shikimic acid by E. coli cells is realized. The invention belongs to the technical field of microbial genetic engineering. Background technique [0002] Shikimic acid is an aromatic compound containing three hydroxyl groups, one carboxyl group and one double bond, and has a chiral isomer structure. It is not only the raw material of chiral drugs (such as antiviral drugs), but also the synthetic raw material of many alkaloids, aromatic amino acids and indole derivatives. More importantly, it is the key raw material of the neuraminidase inhibitor GS4104 (Tamiflu), which can be used to synthesize drugs for the prevention and treatment of avian influenza and influenza A. Its p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
Inventor 陈献忠李明明王正祥
Owner JIANGNAN UNIV
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