Shikimic acid prepared bacterial strain and constructing method

A technology for producing strains and constructing methods, applied in the biological field

Inactive Publication Date: 2008-03-12
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some foreign companies have carried out relevant research, but further optimization of conditions is needed to realize industrialization

Method used

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  • Shikimic acid prepared bacterial strain and constructing method
  • Shikimic acid prepared bacterial strain and constructing method
  • Shikimic acid prepared bacterial strain and constructing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 , Construction of double knockout strain W3110 (ΔaroLΔaroK)

[0028] 1.1 Knockout of aroL and aroK genes in BW25113 (E.coli Genetic Stock Center)

[0029] With reference to the literature of Gust.B. et al. (PCR-targeting system in Streptomyces coelicolor, Gust.B., KieserT.et al, 2002), the PCR-Targeting method was used to knock out the aroL and aroK genes in the Escherichia coli genome, as follows :

[0030] 1.1.1. Obtainment of single knockout strain BW25113 (ΔaroL)

[0031] Using the plasmid pIJ773 (E.coli Genetic Stock Center) as a template, the aac (Apra) gene in the plasmid pIJ773 was amplified by PCR using primers aroK-up and aroK-down. The primer sequences are as follows:

[0032] aroK-up: 5'-tacgctaatcttacccggtgattatcgccagagcggtgattccggggatccgtcgacc-3';

[0033] aroK-down: 5'-tactgtacccgcagacgagtgtatataaagccagaattatgtaggctggagctgcttc-3';

[0034] The amplification conditions are: 94°C, 2min, 94°C, 45sec, 55°C, 45sec, 72°C, 90sec, 30 cycles.

[00...

Embodiment 2

[0053] Example 2 1. Construction of recombinant plasmid pSUFEBPT

[0054] 2.1. Cloning of recombinant plasmid pSU-F

[0055] Using the genome of E.coli DH5α (Amersham Company) as a template, use primers P148L(+), P148L(-), aroFSac I(+) and aroFSac I(-) to perform overlapping PCR to amplify the aroF gene of E.coli DH5α , the primer sequences are as follows:

[0056] P148L(+): 5'-ttagatctgaatagcccgcaatacctgggc-3';

[0057] P148L(-): 5'-gctattcagatctaacgcttccgtcgccagtgg-3';

[0058] aroFSac I(+): 5'-aacgagctcaccggaaagtcctcgggcataag-3';

[0059] aroFSac I(-): 5'-aacgagctccgacttcatcaatttgatcgcgtaa-3';

[0060] First, a fragment was amplified with the primer pair aroFSac I(+) and P148L(+), the amplification conditions were: 94°C, 2min, 94°C, 45sec, 56°C, 45sec, 72°C, 1min, 30 cycles; The primer pair aroFSac I(-) and P148L(-) amplified another fragment. The amplification conditions were: 94°C, 2min, 94°C, 45sec, 56°C, 1min, 72°C, 1min, 30 cycles; gel recovery as above Two PCR...

Embodiment 3

[0096] Example 3 , Obtaining of shikimic acid expression strain

[0097] The Escherichia coli host bacterium W3110 (ΔaroLΔaroK) obtained in Example 1 that knocked out the aroL and aroK genes was made into competent cells, and then the recombinant expression plasmid pSUFEBPT obtained in Example 2 was transferred into competent cells by chemical transformation (specific method Referring to molecular cloning III), Escherichia coli host strain W3110 (ΔaroLΔaroK) expressing shikimic acid was obtained.

[0098] The shikimic acid expression strain W3110 (ΔaroLΔaroK) obtained above was submitted to the China Center for Type Culture Collection (CCTCC) located in Wuhan on September 4, 2006 for deposit, and the deposit number is CCTCC M206083.

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Abstract

The invention discloses a shikimic-acid-producing bacterial strain and a constructing method for the bacterial strain. First, bacterial strain W3110 (delta aroL delta aroK) with gene aroL and aroK knocked out is constructed, and a recombination expression plasmid pSUFEBPT containing key gene aroF, aroE, aroB, ppsa and tktA in the metabolism of shikimic acid is constructured, then the recombination expression plasmid is transferred into the bacterial strain W3110 (delta aroL delta aroK) with gene aroL and aroK knocked out, so as to get the shikimic-acid-producing bacterial strain. The shikimic-acid-producing bacterial strain in the invention can achieve the effective accumulation of shikimic acid in fermentation, and lay a foundation for the industrialization of shikimic-acid production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a shikimic acid producing strain and a construction method thereof. Background technique [0002] Shikimic acid is an intermediate in the synthesis of metabolic compounds in organisms, and it is also a raw material for the synthesis of many alkaloids, aromatic amino acids and indole derivatives, and chiral drugs (such as antiviral drugs). There are three hydroxyl groups, one carboxyl group, and one double bond in the molecular structure of shikimic acid. It has chiral isomers, and it can form esters or salts. The chemical name of shikimic acid is 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid, and its structural formula is as follows: [0003] [0004] Tamiflu is currently considered to be the most effective drug against H5N1 highly pathogenic avian influenza virus, and shikimic acid is the upstream key raw material for the production of Tamiflu. [0005] At presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12P7/42C12R1/19
Inventor 杨晟郑华宝胡世元杨俊杰孙周通范文超黄鹤詹剑芦银华姜世民王金刚李宇伟陈磊关春鸿邵丽君刁刘洋陈军杨蕴刘姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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