Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution

Axel Trefzer, Volker Jungmann, Istvan Molnar, Ajit Botejue, Dagmar Buckel, Gerhard Frey, D. Steven Hill, Mario Jörg, James M. Ligon, Dylan Mason, David Moore, J. Paul Pachlatko, Toby H. Richardson, Petra Spangenberg, Mark A. Wall, Ross Zirkle, Justin T. Stege

Research output: Contribution to journalArticle

22 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)4317-4325
Number of pages9
JournalApplied and Environmental Microbiology
Volume73
Issue number13
DOIs
Publication statusPublished - Jul 2007

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ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

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