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Recombinant strain for producing shikimic acid, and preparation method and application thereof

A kind of technology of recombinant Escherichia coli, bacterial strain, applied in the recombinant bacterial strain of producing shikimic acid and preparation field thereof, can solve the problems such as unfavorable industrial production, output is not ideal enough

Active Publication Date: 2017-01-04
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide an improved recombinant shikimic acid production process in order to overcome the problems that antibiotics and inducers need to be added in the production process of the existing microbial synthesis of shikimic acid, and the yield is not ideal and unfavorable for industrial production. Bacterial strain and its preparation method

Method used

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  • Recombinant strain for producing shikimic acid, and preparation method and application thereof
  • Recombinant strain for producing shikimic acid, and preparation method and application thereof
  • Recombinant strain for producing shikimic acid, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Construction of targeting plasmid pTKIP-GBAE

[0054] (1) All required genes were amplified using the chromosome of strain BW25113 as a template. The gene aroG was amplified with primers G1 (SEQ ID NO: 1) and G2 (SEQ ID NO: 2), and the PCR amplification conditions were as follows: PCR system (20 μL): template 10 ng, 10 μM primers 1 μL each, 2 × pfu enzyme PCR Reaction mixture 10μL, add ddH 2 0 to 20 μL. Amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 68°C for 30 s (minus 0.5°C for each cycle), extension at 72°C for 1 min / kb, a total of 35 cycles; extension at 72°C for 10 min.

[0055] The amplified gene aroG and plasmid pETDuet-1 were digested with restriction endonuclease BamH I and ligated to obtain plasmid pETDuet-G; amplified with primers B1 (SEQ ID NO: 3) and B2 (SEQ ID NO: 4) To obtain the gene aroB, the gene aroB and the plasmid pETDuet-1 were digested with restriction endonucleases BamH I and Hind III and...

Embodiment 2

[0059] Construction of targeting plasmid pTKIP-glk-galP

[0060] (1) Using the bacterial strain BW25113 as a template, use primers glk1 (SEQ ID NO: 15) and glk2 (SEQ ID NO: 16) to amplify the gene glk fragment, the amplification conditions are the same as above, and use restriction The plasmid pETDuet-glk was obtained after digestion with endonucleases EcoR I and Pst I; the gene galP fragment was amplified with primers galP1 (SEQ ID NO: 17) and galP2 (SEQ ID NO: 18), and the amplification conditions were the same as above , digest it with the plasmid pETDuet-glk with restriction endonucleases Bgl II and Xho I and connect it to obtain the plasmid pETDuet-glk-galP;

[0061] (2) Gene glk and galP on the plasmid pETDuet-glk-galP are amplified with primers Duet-F (SEQ ID NO: 13) and Duet-R (SEQ ID NO: 14) together with their respective rbs sequences and the T7 promoter terminator Amplification is obtained, the amplification conditions are the same as above, digested with restricti...

Embodiment 3

[0063] Construction of targeting plasmid pTKIP-ppsA

[0064] (1) Using the bacterial strain BW25113 as a template, the gene ppsA fragment was amplified with primers ppsA1 (SEQ ID NO: 19) and ppsA2 (SEQ ID NO: 20). The plasmid pETDuet-ppsA was obtained after digestion with endonucleases EcoR I and Xho I;

[0065] (2) The gene ppsA on the plasmid pETDuet-ppsA together with its rbs sequence and T7 promoter terminator were amplified with primers Duet-F (SEQ ID NO: 13) and Duet-R (SEQ ID NO: 14). The multiplication conditions are the same as above, and the restriction endonucleases Apa I and Nhe I are digested and connected to the plasmid pTKIP-cat to obtain the plasmid pTKIP-ppsA, whose structure is as follows: image 3 shown.

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Abstract

The invention provides a recombinant strain for producing shikimic acid, and a preparation method and application thereof. The recombinant strain does not comprise the following genes: aroK, aroL, tyrR and ptsHIcrr. The ptsHIcrr site and tyrR site comprise the following genes: aroG, aroB, tktA, aroE, glk, galP and ppsA. The transcription expression of the genes aroG, aroB, tktA, aroE, glk, galP and ppsA is activated by an active promoter in Escherichia coli and terminated by an active terminator in the Escherichia coli. The application is as follows: the recombinant Escherichia coli strain is fermented and cultured to obtain the shikimic acid, wherein the glucose / glycerol mass ratio in the fermentation culture medium is 1:5-5:1. The recombinant strain has high shikimic acid fermentation yield, and has the advantages of low production cost and simple technique since neither antibiotics nor inducers are needed in the shikimic acid production process.

Description

technical field [0001] The invention relates to the field of genetically engineered recombinant bacteria, in particular to a recombinant bacterial strain producing shikimic acid, a preparation method and application thereof. Background technique [0002] Shikimic acid, the chemical name is [3R-(3α, 4α, 5β)]-3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid. With the successive outbreaks of H5N1, H1N1 and H7N9 subtype influenza viruses, Roche's patented drug "Tamiflu" (Oseltamivir Phosphate, Oseltamivir) has become the preferred anti-influenza virus drug that countries around the world are scrambling to reserve. As the key chiral starting material for the synthesis of "Tamiflu", shikimic acid has also attracted widespread attention. [0003] There are three main ways to obtain shikimic acid: plant extraction, chemical synthesis and microbial synthesis. The source of plant extraction is mainly Magnoliaceae star anise, but using star anise to extract shikimic acid, the product...

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 朱宝泉刘向磊林军胡海峰周斌
Owner SHANGHAI INST OF PHARMA IND
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