Biocatalytic conversion of avermectin to 4''-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution.
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.
Trefzer
Axel
A
Diversa Corporation, San Diego, CA 92121, USA.
Jungmann
Volker
V
Molnár
István
I
Botejue
Ajit
A
Buckel
Dagmar
D
Frey
Gerhard
G
Hill
D Steven
DS
Jörg
Mario
M
Ligon
James M
JM
Mason
Dylan
D
Moore
David
D
Pachlatko
J Paul
JP
Richardson
Toby H
TH
Spangenberg
Petra
P
Wall
Mark A
MA
Zirkle
Ross
R
Stege
Justin T
JT
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
D004187:Disaccharides; D007306:Insecticides; D007559:Ivermectin; C019264:avermectin; C443359:emamectin; D003577:Cytochrome P-450 Enzyme System