A kind of 3-dehydroquinic acid synthase mutant and its gene and application

A technology for synthesizing enzymes and quinic acid, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of insufficient activity and low yield of shikimic acid, and achieve the effects of improving enzyme activity, increasing activity, and increasing yield

Inactive Publication Date: 2016-04-20
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for synthesizing 3-dehydroquinic acid in order to overcome the defect that the activity of 3-dehydroquinic acid synthetase is not high enough in the existing biosynthesis method, resulting in low yield of shikimic acid. Enzyme mutants and their genes and applications

Method used

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  • A kind of 3-dehydroquinic acid synthase mutant and its gene and application
  • A kind of 3-dehydroquinic acid synthase mutant and its gene and application
  • A kind of 3-dehydroquinic acid synthase mutant and its gene and application

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Embodiment 13

[0030] The construction of the mutant library of embodiment 13-dehydroquinic acid synthase gene

[0031](1) Design primers for error-prone PCR amplification of 3-dehydroquinate synthase gene. The sequence of the upstream primer B1 (as shown in SEQ ID No: 2) is 5'-AAAACCATGGAGAGGATTGTCGTTACTCTCGG-3', and the downstream primer B2 ( As shown in SEQ ID No: 3), the sequence is 5'-GACAAAGCTTACGCTGATTGACAATCGGCAAT-3', which respectively contain NcoI and HindIII restriction sites.

[0032] (2) Using the genome of Escherichia coli W3110 (purchased from E.coliGeneticStockCenter, YaleUniversity, Yale University E.coli Collection Center) as a template, use primers B1 and B2 to carry out PCR amplification under the catalysis of TaqDNA polymerase to obtain 3-de Hydroquinic acid synthase gene fragment, the gene fragment obtained by the first round of error-prone PCR was recovered from agarose gel, and the second round of error-prone PCR was performed using it as a template. The PCR reaction...

Embodiment 23

[0034] Example 23 - Screening of a library of mutants of dehydroquinic acid synthase gene

[0035] (1) Construction of 3-dehydroquinic acid synthase gene-deficient strain

[0036] The plasmid pKD46 expressing Red recombinase (purchased from Yale University Escherichia coli Collection Center, the plasmid information is disclosed in the literature BabaT, AraT, HasegawaM, et al.ConstructionofEscherichiacoliK-12in-frame, single-geneknockoutmutants:theKeiocollectionMolSystBiol2006:2-8) Introduce E.coliBL21(DE3), and select transformants on LB solid plates containing 100 mg / mL ampicillin (cultivation temperature is 30°C). Inoculate the transformant into LB liquid medium, shake culture at 200r / min at 30°C, when the cell OD 600 When it reaches 0.2, add 1mML-arabinose to induce the expression of Red recombinase, the induction time is not less than 1h, OD 600 Harvest the cells at about 0.6 to prepare competent cells. The fragments for homologous recombination were introduced into com...

Embodiment 33

[0039] Example 33 - Expression of dehydroquinic acid synthase gene mutants

[0040] Inoculate the mutant strains grown on the M9 solid medium in Example 2 into a 250ml Erlenmeyer flask containing 20ml of seed medium, and after overnight shaking at 37°C, inoculate into fresh LB medium according to 1% inoculum size, 37 Cultivate to OD 600 At about 0.6, add IPTG to a final concentration of 1 mM, continue to induce culture for 4 hours, centrifuge the bacterial solution at 12,000 r / min for 5 minutes, discard the supernatant, and use the bacterial cells for SDS-PAGE detection. The result is as figure 2 As shown, compared with the No. 7 host strain, No. 1-6 strains expressed the target protein of 38 kDa, and the expression level was higher.

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Abstract

The invention discloses a 3-dehydroquinic acid synthase mutant as well a gene and an application thereof. The 3-dehydroquinic acid synthase mutant is a protein having the activity of 3-dehydroquinic acid synthase which is derived from the 3-dehydroquinic acid synthase and obtained by substituting amino acids at positions 153, 175, 177 and 336 of the protein having an amino acid sequence shown in a sequence table SEQ ID No: 1 into an amino acid. According to the recombinant 3-dehydroquinic acid synthase disclosed by the invention, the activity of the parental enzyme is greatly improved and the yield of shikimic acid in the shikimic acid pathway can be greatly enhanced.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and specifically relates to a 3-dehydroquinic acid synthetase mutant and its gene and application. Background technique [0002] Shikimic acid (Shikimicacid, SA), the chemical name is 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid. It is the precursor for the synthesis of aromatic amino acids, alkaloids, indole derivatives and chiral drugs, and it has analgesic, anti-inflammatory and anti-thrombotic effects. [0003] The preparation methods of shikimic acid mainly include: plant extraction method, chemical synthesis method and biosynthesis method. The plant extraction method is mainly to obtain shikimic acid from the fruit of star anise. Although the content of shikimic acid in star anise is as high as more than 10%, star anise belongs to the small oriental tree species of Magnoliaceae, which is only distributed in very few areas of the world. Its growth is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12P7/42C12R1/19
CPCC12N9/88C12P7/42C12Y402/03004
Inventor 朱宝泉刘宇林军胡海峰周斌
Owner SHANGHAI INST OF PHARMA IND CO LTD
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