title: Biocatalytic conversion of avermectin to 4''-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution.
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.
authors.affiliation: Diversa Corporation, San Diego, CA 92121, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Trefzer
authors.forename: Volker
authors.initials: V
authors.lastname: Jungmann
authors.forename: István
authors.initials: I
authors.lastname: Molnár
authors.forename: Ajit
authors.initials: A
authors.lastname: Botejue
authors.forename: Dagmar
authors.initials: D
authors.lastname: Buckel
authors.forename: Gerhard
authors.initials: G
authors.lastname: Frey
authors.forename: D Steven
authors.initials: DS
authors.lastname: Hill
authors.forename: Mario
authors.initials: M
authors.lastname: Jörg
authors.forename: James M
authors.initials: JM
authors.lastname: Ligon
authors.forename: Dylan
authors.initials: D
authors.lastname: Mason
authors.forename: David
authors.initials: D
authors.lastname: Moore
authors.forename: J Paul
authors.initials: JP
authors.lastname: Pachlatko
authors.forename: Toby H
authors.initials: TH
authors.lastname: Richardson
authors.forename: Petra
authors.initials: P
authors.lastname: Spangenberg
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wall
authors.forename: Ross
authors.initials: R
authors.lastname: Zirkle
authors.forename: Justin T
authors.initials: JT
authors.lastname: Stege
mesh_terms: D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004187:Disaccharides / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial; D007306:Insecticides / Q000378:metabolism*; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004187:Disaccharides; D007306:Insecticides; D007559:Ivermectin; C019264:avermectin; C443359:emamectin; D003577:Cytochrome P-450 Enzyme System