Directed evolution of a sulfoxidation biocatalyst

The chirality of sulfoxides has been exploited in many organic syntheses. The persistent use of non-racemic sulfoxides directly or as chiral relay agents in organic synthesis has spurred the development of improved routes to each sulfoxide enantiomer. The long term goal of this project is to develop a biocatalytic system for quick and selective production of both stereoisomers of a chiral sulfoxide (methyl p-tolyl sulfoxide). The directed evolution of the enzyme flavin-containing monooxygenase was used to develop this biocatalytic system.; FMO enzymes from rabbit (FMO1) and rhesus macaque (FMO2) have opposite stereochemical preference for the sulfoxidation of methyl p-tolyl sulfide. They have been cloned into an Escherichia coli based expression and biocatalytic system. Libraries of mutants of these FMO's have been developed for screening in order to identify an enzyme that produces high yields of the (S) sulfoxide. Genes with single amino acid mutations were generated from rabbit FMO1, which was also shuffled with the monkey enzyme to generate a library of chimeric genes.; These libraries were screened for enantioselectivity in the sulfoxidation of methyl p-tolyl sulfide. A screening method developed using a flow cell optical rotation and a UV detector proved slow (<100 clones/day) and had low sensitivity for identifying differences in enantiomeric compositions of reactions. Instead, using chiral HPLC the activities of 6000 clones were tested (6min/clone) for the sulfoxidation of methyl p-tolyl sulfide. From both the mutant libraries one clone (176E70) displayed an increase in selectivity for the (S) sulfoxide of 30% ee . and there were 4 clones which displayed up to 35% increased activity. Of the two directed evolution strategies the single mutation library of FMO1 yielded interesting clones while none were observed from the chimeric enzymes.