Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production.

l- threo - p -methylsulfonylphenylserine (compound 1b ) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa l-threonine transaldolase ( Bu LTTA) was rationally designed based on the sequence-structure-function relationship. A mutant M4 (Asn35Ser/Thr352Asn) could produce 35.5 mM 1b with 88.8% conversion and 93.8% diastereoselectivity, 314 and 129% of the values observed for wild-type Bu LTTA. Molecular dynamics simulations indicated that the shortened distance between key active site residues and the transition state (PLP- 1b ) and the improved hydrogen bond force enhanced the catalytic performance of the M4 variant. Then, the mutant M4 was combined with K. kurtzmanii alcohol dehydrogenase ( Kk ADH) to eliminate the Bu LTTA-inhibiting byproduct acetaldehyde, and a cosubstrate was added to regenerate the ADH cofactor NADH. Under optimized conditions, the yield of 1b reached 115.2 mM with a conversion of 96% and a diastereoselectivity of 95.5%. This work provides a new strategy for the efficient and sustainable production of 1b .