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Novel cytochrome P450 gene, expressed protein and application thereof

A gene, 1. CYP107Z13 technology, applied in the new cytochrome P450 gene, protein and its application field, can solve the problem of low transformation efficiency, achieve the effect of reducing production cost and improving biotransformation efficiency

Active Publication Date: 2012-03-28
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transformation efficiency of P450 heterologous expression products is improved to varying degrees, but the transformation efficiency is lower than 25% [I Molnar, et al.2006, the same as before]

Method used

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  • Novel cytochrome P450 gene, expressed protein and application thereof
  • Novel cytochrome P450 gene, expressed protein and application thereof
  • Novel cytochrome P450 gene, expressed protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Streptomyces biocatalytic transformation of abamectin to generate 4'-carbonyl abamectin activity detection

[0045] 1-1. Streptomyces strain culture and sample preparation

[0046] Streptomyces strains from different sources were isolated and cultured, and preserved in YEME medium. After the strains are activated, inoculate Streptomyces spores in ISP2 liquid medium (ISP2: yeast extract 4.0g; wort juice 10.0g; glucose 4.0g; agar 20.0g; distilled water 1000ml. pH 7.0), after 3 days of culture at 28°C and 120rpm , add 1g / L abamectin (concentration of stock solution 20g / LDS0:Tween-40=1:1, concentration of pure abamectin is 97%, wherein B1a content is 84%). Continue to cultivate for 24h. 30ml of the culture was extracted with 30ml of methyl tert-butyl ether (MTBE for short), and the ether phase was collected and vacuum-dried. The residue was redissolved in 1.2ml of acetonitrile, and the generation of abamectin derivatives was detected by HPLC.

[0047] 2. HPLC ...

Embodiment 2

[0049] Example 2. Cloning and sequence analysis of CYP107Z13 gene

[0050] 1. Extraction of Streptomyces sp.ZB01 Genomic DNA

[0051] YEME medium (yeast extract 3g; peptone 5g; malt extract 3g; glucose 10g; sucrose 340g; add distilled water to 1000ml, sterilize, add 5mM sterilized MgCl before use 2 ·6H 2 O) Cultivate Streptomyces ZB0148h on a liquid shaker at 30° C. and 200 rpm. The cells (50 mg) were collected by centrifugation, washed with STE buffer, centrifuged, and the supernatant was removed; the above steps were repeated. Suspend the bacteria with 500ul 2-3mg / ml lysozyme solution, and act in a water bath at 37°C for 60min. Add 1% SDS, 37 ° C water bath for 2h. Add an equal volume of neutral phenol / chloroform, mix carefully for about 10 minutes, and centrifuge to get the supernatant. Add 1 / 10 volume of 3M NaAc (pH4.8), add an equal volume of isopropanol, shake well, discard the supernatant, and suck out the precipitate. Add 70% ethanol to wash the precipitate. Dis...

Embodiment 3

[0082] Expression, purification and identification of embodiment 3 CYP107Z13 gene in Escherichia coli

[0083] 1. Construction of CYP107Z13 prokaryotic expression vector

[0084] According to the full-length coding sequence of CYP107Z13, design primers to amplify the coding reading frame (corresponding to more than 20 nucleotides at the 5' and 3' ends of the coding sequence, respectively), and introduce XbaI and XhoI restriction enzymes on the forward and reverse primers respectively site. The primer sequences are as follows:

[0085] Upstream: 5-TCTAGACACTGCACGCCACAGGAGA-3

[0086] Downstream: 5-CTCGAGGTTCAACCGCAGCGGCAG-3

[0087] Streptomyces ZB01 genomic DNA was used as a template to obtain the target fragment by PCR amplification. The PCR reaction conditions were: 94°C pre-denaturation for 3 minutes, 94°C pre-denaturation for 1 minute, 65°C for 1 minute, 72°C for 1 minute, 30 cycles, and 72°C extension for 10 minutes. The PCR product was recovered by gel cutting, ligat...

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Abstract

The invention relates to a novel cytochrome P450 gene, expressed protein and application thereof, which belong to the filed of biotechnology. The P450 gene encodes CYP107213 enzyme and has the nucleotide sequence shown as SEQ ID NO 1. The P450 gene has the function of oxidizing abamectin 4'-hydroxyl into 4'-carbonyl specifically in sites, provides a novel functional gene resource for biologicallyconverting abamectin, thereby laying the foundation of further reformation and recombination of the gene, improvement of biological conversion efficiency of abamectin and reduction of production costof methylamino abamectin benzoate. Wild Streptomyces sp.ZB01 containing the gene has the function of oxidizing abamectin 4'-hydroxyl into 4'-carbonyl specifically in sites with the conversion efficiency of 36.8% which is obviously greater than that of current wild Streptomyces and that of most ema heterologous expressed proteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel cytochrome P450 gene, protein and application thereof. Background technique [0002] Cytochrome P450 (cytochrome P450, CYP) is a heme- and thiol-containing protein that widely exists in organisms. . CYP enzymes can catalyze the biosynthesis of various endogenous substances in organisms, and also participate in the biological oxidation and degradation of many exogenous refractory organic substances, etc., and are considered to be one of the most catalytic biocatalysts in nature. Cytochrome P450 enzymes constitute a supergene family. Cytochrome P450 with more than 40% amino acid sequence identity belongs to the same family, while cytochrome P450 with more than 55% identity belongs to the same subfamily. Streptomyces contains a large number of CYPs of different families, which play an important role in the biosynthesis of Streptomyces secondary metabolites and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12P19/62C12R1/465C12R1/19
Inventor 李梅蒋细良刘卫德曾凡荣黄啟良李凤敏
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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