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Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby

A technology of shikimic acid and recombinant engineering bacteria, applied in the field of genetic engineering, can solve problems such as low purity, low shikimic acid synthesis rate and yield, achieve simple operation process, increase the synthesis of gluconeogenesis and glycogen, and reduce glycolysis solution effect

Inactive Publication Date: 2008-03-05
SHANGHAI HAITAI PHARMA
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Problems solved by technology

[0009] In order to improve the defects of low shikimic acid synthesis rate, yield and low purity in the current method for synthesizing shikimic acid using genetic engineering technology, the present invention discloses a method for preparing shikimic acid using biosynthesis technology

Method used

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  • Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby
  • Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby

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Embodiment 1

[0036] The construction of embodiment one shikimic acid expression bacterial strain

[0037] (1) PCR amplification of target genes aroF, aroB, aroE, Ptac, shiA, kan; using E.coli31884 full DNA as a template, using corresponding oligonucleotide primers to amplify target genes; artificially synthesizing the full sequence of Ptac, and Using this as a template, the corresponding primers were used for amplification.

[0038] The strong Ptac I promoter is efficient in controlling the expression of foreign genes in E. coli. It is reported that its initiation efficiency is 11 times higher than that of 1acUV5 promoter. On the one hand, the kan resistance gene in the gene cassette is to facilitate the screening of strains after gene knockout and gene replacement; . Alternatively, kan gene completion can be eliminated using the FLP recombinase system that recognizes FRT sites.

[0039] (2) Construction of recombinant plasmids pUC-aroA, pUC-shiA, pET22b-aroB, pET22b-aroC, pET22b-kan r...

Embodiment 2

[0044] Example Dishikimic acid expression and purification

[0045] (1) Expression of shikimic acid: shake flask fermentation, purification of shikimic acid and identification of conversion rate;

[0046] Shake flask fermentation conditions: temperature: 37°C; pH: 6.5-7.5; time 12h;

[0047] Inoculated on the fermentation medium;

[0048] (2) Purification of shikimic acid: the expressed shikimic acid was separated by ion exchange chromatography, and then shikimic acid was further purified by recrystallization.

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Abstract

The present invention discloses biosynthesis process of preparing shikimic acid and the recombinant colibacillus therefor. The biosynthesis process of preparing shikimic acid includes constituting vector including the polygene kit of shikimic acid metabolizing pathway rate-limiting enzyme, replacing the polygene kit for shiA gene, knocking out shikimate kinase isozyme with Red recombinant system, fermenting recombinant engineering bacteria to obtain shikimic acid, purifying shikimic acid and other steps. The process has high shikimic acid expressing yield and lowered shikimic acid producing cost, and can provide material for preparing Oseltamivir phosphate as influenza preventing and treating medicine.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing shikimic acid through biosynthesis technology, and also relates to engineering bacteria used in the method. Background technique [0002] In recent years, bird flu has been proved to be able to directly infect humans, with ferocious onset and high fatality rate. According to statistics, bird flu has infected more than 100 people, and more than half of them died. As of October 2005, more than ten countries and regions, including Cambodia and China, have experienced the H5N1 virus epidemic, and it is still spreading. At present, governments of various countries are actively responding to the possible large-scale outbreak of bird flu in humans. Influenza virus experts believe that a new influenza pandemic is inevitable and may not be far away. At a seminar hosted by the World Health Organization on July 4, 2005, scientists pointed out that bird flu is sti...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C12N15/52C12P7/42
Inventor 汪莉王瑾峰
Owner SHANGHAI HAITAI PHARMA
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