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Gene Cloning, Expression and Functional Verification of Avermectin 4"-Carbinol Group Oxidase CYP107Z13

Author: LiuWeiDe
Tutor: LiMei
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Avermectin Oxidase CYP107Z13 Function Enzyme kinetics
CLC: S482.3
Type: Master's thesis
Year: 2011
Downloads: 41
Quote: 0
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Abstract

Avermectin is an important bioinsecticide. Emamectin benzoate, an avermectin derivative, is more efficient and has a wider insecticidal spectrum than avermectin. It is chemically synthesized from the natural product mixture of avermectin via the key intermediate 4″-oxo-avermectin. Direct regiospecific chemical oxidation of 4″-OH of avermectin to 4″-oxo-avermectin is precluded by the high reactivity of 5-OH in the molecule, necessitating a protection-deprotection strategy. Avoiding these additional steps would greatly reduce the complexity of the production process along with the final cost of emamectin benzoate. Some streptomyces strains were demonstrated to be able to carry out the regioselective oxidation of avermectin at the 4"-position. We screened a strain named S.ahygroscopicus ZB01 （CGMCC NO. 2804）, which can oxidize avermectin to 4″-oxo-avermectin with high regioselectivity and efficiency. Resting S. ahygroscopicus ZB01 cells can convert 36% of the avermectin substrate to 4″-oxo-avermectin in 72 h at avermectin concentrations of 1 g·l^-1 measured by HPLC, which was twice more than the highest convert rate of functional strain reported, and shows great potential in synthesis of emamectin benzoate. In this study, a series of studies on the avermectin 4"-carbinol group oxidase gene in S. ahygroscopicus ZB01 were conducted as follows:1. A cyp107z homologous gene was cloned from S. ahygroscopicus ZB01 genome using homology cloning and genome walking, which was 1290 bp in length encoding 429 amino acid residues. It was the thirteenth new member of cyp107z subfamily gene and was named as cyp107z13 by cytochrome P450 Nomenclature Committee according to the Nomenclature of CYP. The sequence of cyp107z13 was analyzed with bioinformatics softwares, and the structure model of CYP107Z13 protein was predicted.2. cyp107z13 gene was cloned into the multiple copies E. coli-Streptomyces shuttle vector pKC1139, and the recombinant plasmid was transformed into the S. lividians TK54 protoplast. Under the control of Streptomyces constitutive ermE*P promoter, cyp107z13 gene was expressed in Streptomyces lividians TK54 demonstrated by CO difference spectrum analysis. The expressed CYP107Z13 protein can accept electron from S. lividians TK54 electron transport system and oxidize the 4"-carbinol group of avermectin in a regioselective way.3. The whole-cell reaction conditions of the S. lividians TK54 recombinant containing cyp107z13 were optimized. ISP-2 at pH 6 was more conducive for the expression of CYP107Z13 than PYG1, and the active cells of the recombinant strain have higher biocatalytic activity than resting cells.4. cyp107z13 was cloned into the expression vector pET28a, and introduced into Escherichia coli BL21（DE3）. The expression of CYP107Z13 recombination protein with His6 tag was detected by ELISA and CO difference spectrum analysis, and the active recombinant protein was purified by Ni-NTA affinity chromatography.5. An in vitro catalysis system was constructed of purified recombinant CYP107Z13 protein together with ferredoxin and ferredoxin reductase, and the kinetic parameters of the recombinant protein were determined by monitoring the consumption of NADPH in the system, The K_m of purified recombinant protein of CYP107Z13 expressed in E.coli was 1.4μmol/L, the V_max was 0.041μmol/min·mg and the k_cat was 0.033 s^-1 with avermectin as substrate.

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