Engineering stereoselectivity of phenylserine dehydratase and in situ byproduct upcycling for efficient production of enantiopure phenylserine and d-phenyllactic acid.

Efficient production of enantiopure phenylserine (PhSer) is hampered by poor economy and challenging byproduct separation in conventional resolution methods. Phenylserine dehydratase (PSDH) offers an alternative by catalyzing irreversible dehydration, yet its strict stereospecificity toward the l-threo isomer limits its synthetic scope. Herein, structure-guided site-saturation mutagenesis of l-PSDH from Paraburkholderia xenovorans yielded the G93C mutant, which acquired activity against the previously inaccessible d-erythro isomer. Both wild-type and G93C enabled preparative-scale kinetic resolution, affording enantiopure l-threo- and d-erythro-PhSer with improved isolated yields of 43.6% and 43.0%, respectively. To upgrade the byproduct, a multi-enzyme cascade combining PSDHs, d-lactate dehydrogenase, and formate dehydrogenase was constructed, which simultaneously resolved racemic PhSer and converted the phenylpyruvate byproduct in situ into high-value d-phenyllactic acid. This work expands the synthetic utility of PSDHs through stereoselectivity engineering and cascade design, establishing an efficient biocatalytic platform for co-producing enantiopure PhSer and d-phenyllactic acid.