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.Emamectin benzoate is a potent semisynthetic insecticide used to control many agriculturally important pests. However, wider application of emamectin has been hindered by the high cost of its chemical synthesis, which involves regioselective oxidation of the 4Љ alcohol of the natural product avermectin, followed by the reductive amination of the resulting key intermediate 4Љ-oxo-avermectin (see Fig. 1). Achievement of high regioselectivity in the oxidation reaction requires the costly protection and deprotection of the very reactive allylic hydroxyl function at C-5 of the avermectin molecule.As an alternative to chemical synthesis, microbial conversion has been used successfully in other regioselective oxidation reactions to bypass complex protection and deprotection strategies. Many microbial oxidative bioconversion reactions are carried out by dehydrogenases (4) or oxidases (3, 11) while some use cytochrome P450 monooxygenases (CYPs) (12a, 12b, 15-17). The cytochrome P450 family is a large class of enzymes that catalyze hydroxylation, epoxidation, and demethylation reactions. Class II CYPs from eukaryotes accept electrons from NADPH via a NADPH-cytochrome P450 reductase, whereas class I CYPs in prokaryotes use distinct ferredoxins and ferredoxin reductases for electron transfer from NADH. Biotransformations using CYPs have to regenerate the cofactor, which can be achieved cost-effectively on an industrial scale by using metabolically active whole cells as biocatalysts. Examples of CYP-catalyzed biotransformations include the 11␤-hydroxylation of Reichstein S to hydrocortisone (2) and the microbial hydroxylation of zofimarin, a sordarin-related antibiotic (32).Recently, we have reported the isolation and characterization of 17 CYP enzymes, collectively named the Ema CYPs, capable of regioselectively oxidizing avermectin to 4Љ-oxo-avermectin (13,19,20). The Ema CYPs were isolated from closely related Streptomyces spec...

Reactions that were once the exclusive province of synthetic catalysts can increasingly be addressed using biocatalysis. Through discovery of unnatural enzyme reactions, biochemists have significantly expanded the reach of enzymatic catalysis to include carbene transfer chemistries including olefin cyclopropanation. Here we describe hemoprotein cyclopropanation catalysts derived from thermophilic bacterial globins that react with diazoacetone and an unactivated olefin substrate to furnish a cyclopropyl ketone, a previously unreported reaction for enzyme catalysts. We further demonstrate that the resulting cyclopropyl ketone can be converted to a key cyclopropanol intermediate that occurs en route to the anti‐hepatitis C drug grazoprevir.