Rational design of a bifunctional glycosyltransferase for enhanced substrate promiscuity and thermostability

Glycosylation of natural products significantly enhances their physicochemical properties, with glycosyltransferases (GTs) serving as the catalytic core of this biotransformation. Rational design of tailored GTs aligned with green chemistry principles is pivotal for the targeted synthesis of high-value glycosides, yet precise functional engineering remains challenging. This study employed a multi-scale computational strategy (molecular docking, multiple sequence alignment, molecular dynamics simulations) to systematically re-engineer the substrate recognition and stability modules of the Bacterial GT BsGT-1. By reverse-engineering the active pocket of a plant-derived hyperpromiscuous GT (FiGT-2), we orthogonally mapped and identified six functional hotspots in BsGT-1. Site-directed mutagenesis and screening yielded the double variant S128T/T229S, achieving dual optimization: improved substrate scope (121-140 % increase in conversion with UDP-Gal/UDP-Rha compared to wild-type) and enhanced thermostability (>70 % residual activity after 4 h at 50℃). Structural dynamics analyses revealed that mutation-induced global conformational rigidity and localized hydrogen-bond network optimization primarily drove thermostability improvement and substrate affinity enhancement, respectively. This work establishes a closed-loop engineering paradigm of "computational prediction → rational mutagenesis → mechanistic decoding", providing a scalable framework for precision engineering of GTs and glycoside biomanufacturing.

Epothilone B; Glycosyltransferase; Molecular docking; Rational design; Substrate scope improvement.