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Recombinant pichia pastoris, construction method and application of recombinant pichia pastoris

A Pichia pastoris, gs115-ppic9-novq technology, applied in the field of recombinant Pichia pastoris, can solve the problems of low expression amount, complicated purification process, etc., and achieves high catalytic activity, low substrate specificity, and simplification. The effect of the production process

Inactive Publication Date: 2016-10-26
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can produce a low-specificity aromatic prenyltransferase that does not depend on living cells and has high application value. However, due to the limitation of the E. coli expression system, the expression level is not high, and the enzyme protein is concentrated after expression. Inside the cell, the subsequent separation, extraction and purification process is complicated

Method used

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  • Recombinant pichia pastoris, construction method and application of recombinant pichia pastoris
  • Recombinant pichia pastoris, construction method and application of recombinant pichia pastoris
  • Recombinant pichia pastoris, construction method and application of recombinant pichia pastoris

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Effect test

Embodiment 1

[0034] A recombinant strain of Pichia pastoris (GS115-ppic9-novQ), which was preserved in the China Center for Type Culture Collection (CCTCC) on March 11, 2016, address: 299 Bayi Road, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province No., China Center for Type Culture Collection), the deposit number is CCTCC M 2016105.

[0035] Constructed by the following steps:

[0036] Step 1: Construction of recombinant Pichia pastoris expression vector ppic9-novQ:

[0037] (1) Prenyl transferase novQ gene acquisition

[0038] Use Gao's No. 1 medium, 50mL / 250mL liquid volume, 28°C, 180rpm shake flask to culture Streptomyces snow white (purchased from China Pharmaceutical Microorganism Culture Collection Management Center) for 72h, filter to obtain mycelia, and use the CTAB method to extract bacteria Total silk DNA. Using the total DNA as a template, PCR amplification was performed using the NovQ gene forward primer SEQ ID No.1 and reverse primer SEQ ID No.2. The amplificatio...

Embodiment 2

[0049] A recombinant Pichia pastoris GS115-ppic9-novQ is used to produce aromatic prenyl transferase NovQ, comprising the following steps:

[0050] (1) Pick a single colony of recombinant Pichia pastoris GS115-ppic9-novQ, inoculate it in a 250mL Erlenmeyer flask filled with 25mL of MGY medium, and cultivate it at 28°C with shaking at 250rpm for 36h to obtain activated recombinant Pichia pastoris GS115 - ppic9-novQ bacteria solution.

[0051] (2) The activated recombinant Pichia pastoris GS115-ppic9-novQ bacterial solution was inserted into a 250 mL Erlenmeyer flask filled with 25 mL of BMGY medium at an inoculum volume of 10% by volume, and cultured at 28°C and 250 rpm for 36 hours with shaking. The fermentation broth was centrifuged at 4000 g for 15 min to remove the culture medium to obtain recombinant Pichia pastoris GS115-ppic9-novQ cells.

[0052] (3) Resuspend the recombinant Pichia pastoris GS115-ppic9-novQ bacteria in the same volume of BMMY medium as before centrifug...

Embodiment 3

[0055] A recombinant Pichia pastoris GS115-ppic9-novQ is used to produce aromatic prenyl transferase NovQ, comprising the following steps:

[0056] (1) Pick a single colony of recombinant Pichia pastoris GS115-ppic9-novQ, inoculate it in a 250mL Erlenmeyer flask filled with 12.5mL of BMGY medium, culture at 20°C, 180rpm shaking for 24 hours, and obtain activated recombinant Pichia pastoris GS115-ppic9-novQ bacterial fluid.

[0057] (2) The activated recombinant Pichia pastoris GS115-ppic9-novQ bacterial solution was inserted into a 250mL Erlenmeyer flask containing 12.5mL MGY medium at an inoculum volume of 5% by volume, and cultured at 20°C and 180rpm for 24h with shaking. The fermentation broth was centrifuged at 2000 g for 10 min to remove the culture medium to obtain recombinant Pichia pastoris GS115-ppic9-novQ cells.

[0058] (3) Resuspend the recombinant Pichia pastoris GS115-ppic9-novQ in the same volume of BMMY medium as before centrifugation, 12.5mL / 250mL liquid volu...

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Abstract

The invention discloses recombinant pichia pastoris (GS115-ppic9-novQ) preserved in CCTCC (China center for type culture collection) on March 11, 2016 with the preservation number of CCTCC M 2016105. The invention further discloses a construction method of the recombinant pichia pastoris and an application in production of aromatic prenyltransferase. By means of induction culture of the engineering bacterium, soluble recombinant aromatic prenyltransferase NovQ with biological activity can be directly obtained in supernatant of a fermentation liquid, and processes such as cell collection, cell disruption and the like are omitted, accordingly, the production process is simplified. Compared with enzymes from other sources, the aromatic prenyltransferase has the advantages that the activity does not depend on structures such as a living cell or a biological membrane, the substrate specificity is low, prenylation of multiple substrates can be catalyzed, the catalytic activity is high and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering of aromatic prenyl transferase, and in particular relates to a strain of recombinant Pichia pastoris, its construction method and its application. Background technique [0002] Aromatic prenyltransferase (aromatic prenyltransferase) is a large class of transferases that can catalyze the prenylation of aromatic compounds to generate prenylated aromatic compounds and their derivatives. Prenylation is a key step in the biosynthesis of a variety of aromatic active molecules of important biological origin, such as coenzyme Q, vitamin K2, shikonin, and naphthalenediol. Aromatic prenyl transferase plays an important role and significance in the fields of pharmaceutical industry, food and health product processing, and chemical synthesis. [0003] There have been many reports on the classification, function and application in the synthesis of biologically active substances of aromatic prenyltransferase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/66C12R1/84
CPCC12N9/1085C12N15/66C12N15/815C12N2800/102
Inventor 郑之明李哲敏赵根海刘会王鹏王丽方雪孙小雯吴锡华吴荷芳尉鸿飞
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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