Biocatalytic conversion of avermectin into 4″-oxo-avermectin:Discovery, characterization, heterologous expression and specificity improvement of the cytochrome P450 enzyme

4″-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strainswith the ability to oxidize avermectin to 4″-oxoavermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP+ reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in coexpression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly™, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening.