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Biocatalytic conversion of avermectin to 4''-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 2007 Jul; Vol. 73 (13), pp. 4317-25. Date of Electronic Publication: 2007 May 04. - Publication Year :
- 2007
-
Abstract
- Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.
- Subjects :
- Biotransformation
Disaccharides metabolism
Gene Library
Genes, Bacterial
Ivermectin metabolism
Molecular Sequence Data
Mutation
Oxidation-Reduction
Streptomyces enzymology
Streptomyces genetics
Substrate Specificity
Cytochrome P-450 Enzyme System genetics
Cytochrome P-450 Enzyme System metabolism
Directed Molecular Evolution
Insecticides metabolism
Ivermectin analogs & derivatives
Subjects
Details
- Language :
- English
- ISSN :
- 0099-2240
- Volume :
- 73
- Issue :
- 13
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 17483257
- Full Text :
- https://doi.org/10.1128/AEM.02676-06