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Biocatalytic conversion of avermectin to 4''-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution.

Authors :
Trefzer A
Jungmann V
Molnár I
Botejue A
Buckel D
Frey G
Hill DS
Jörg M
Ligon JM
Mason D
Moore D
Pachlatko JP
Richardson TH
Spangenberg P
Wall MA
Zirkle R
Stege JT
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.

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