Sequence-guided stereo-enhancing and -inverting of (R)-styrene monooxygenases for highly enantioselective epoxidation

To improve the enantioselectivity of newly identified (R)-selective styrene monooxygenases, a concise sequence-guided approach was exploited to efficiently identify candidate mutations in SeStyA (from Streptomyces exfoliates). Positions 86 and 219 of SeStyA were experimentally confirmed to be critical for stereo-control, and subsequent saturation mutagenesis revealed multiple beneficial mutations that could enhance or invert the enantioselectivity. In the epoxidation of a panel of styrene derivatives, mutant W86A yielded (R)-oxides with 95–99% ee, a significant increase from the wild-type (83–96% ee); and mutant A219H showed drastic inversion of stereoselectivity, yielding (S)-oxides with 92–99% ee. Such mutational effects could be extended to other (R)-SMOs. Corresponding mutants of NaStyA, AaStyA, and StStyA contributed to an increase from 91–94% ee to 98–99% ee for (R)-styrene oxide, as well as to the formation of (S)-styrene oxide with up to 92% ee.