Unlocking catalytic capacity of (S)-selective ω-transaminases toward aryl methyl ketones via pocket engineering and droplet screening.

ω-Transaminases (ω-TAs) exhibit remarkable efficiency and stereoselectivity in catalyzing the asymmetric synthesis of chiral amines, rendering them prime biocatalysts for pharmaceutical chiral intermediate production. Nevertheless, developing a generalizable strategy to significantly enhance the acceptance of bulky substrates across different ω-TAs remains a major challenge, as most enzyme engineering efforts are case-specific and lack of high-efficiency screening approaches. Herein, we presented an efficient ω-TAs engineering strategy, established via the direct conversion of 1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one (1a) to the high-value chiral intermediate ((S)-1b) of the anticancer drug pralsetinib. Based on a thermophilic enzyme scaffold, a combination of pocket engineering and fluorescence-activated droplet sorting (FADS) yielded the robust variant W62L/I263G, showing dramatically improved activity toward 1a through pocket expansion. This engineering approach overcomes the catalytic limitations of wild-type ω-TA, achieving 95% conversion with >99.9% enantiomeric excess (ee) within one hour. Notably, the I263G mutation unlocks the ability of diverse (S)-selective ω-TAs to accept a range of aryl methyl ketones, boosting their catalytic efficiency from negligible to high levels. Our broadly applicable strategy, intergratig pocket engineering with high-throughput screening, provides a new paradigm for ω-TAs engineering and offers general potential for the biocatalytic synthesis of bulky chiral amines.