Multifaceted Cross-Rational Modification of the α2,3-Sialyltransferase MsST for High-Efficiency Production of 3'-Sialyl-3-fucosyllactose via One-Pot Cascade Reaction.

3'-Sialyl-3-fucosyllactose (3'S3FL) is a representative fucose-containing sialylated human milk oligosaccharide with distinctive biological functions, but its in vitro synthesis is limited by the poor tolerance of α2,3-sialyltransferases toward fucosylated acceptors. Here, we report an engineered α2,3-sialyltransferase variant, R60V, with enhanced catalytic performance toward the 3-fucosyllactose (3-FL) substrate. R60V exhibited a 3.6-fold enhancement in transglycosylation activity and a nearly 5-fold lower apparent Km for 3-FL. Its half-life at 40 °C extended from 42.55 to 89.82 h. Mechanistic analyses revealed that R60V alleviated steric hindrance for 3-FL binding, promoted productive interactions with the catalytic residue, and balanced local flexibility with global stability. Furthermore, a one-pot cascade system comprising R60V and a CMP-sialic acid synthetase was established, enabling in situ sialyl donor generation and affording 9.31 mM 3'S3FL in 93.13% yield within 3 h under optimized conditions. This study provides a practical biocatalytic cascade platform for efficient 3'S3FL production.