An efficient C-glycoside production platform enabled by rationally tuning the chemoselectivity of glycosyltransferases

Despite the broad potential applications of C -glycosides, facile synthetic methods remain scarce. Transforming glycosyltransferases with promiscuous or natural O -specific chemoselectivity to C -glycosyltransferases is challenging. Here, we employ rational directed evolution of the glycosyltransferase MiCGT to generate MiCGT-QDP and MiCGT-ATD mutants which either enhance C- glycosylation or switch to O -glycosylation, respectively. Structural analysis and computational simulations reveal that substrate binding mode govern C -/ O -glycosylation selectivity. Notably, directed evolution and mechanism analysis pinpoint the crucial residues dictating the binding mode, enabling the rational design of four enzymes with superior non-inherent chemoselectivity, despite limited sequence homology. Moreover, our best mutants undergo testing with 34 substrates, demonstrating superb chemoselectivities, regioselectivities, and activities. Remarkably, three C -glycosides and an O -glycoside are produced on a gram scale, demonstrating practical utility. This work establishes a highly selective platform for diverse glycosides, and offers a practical strategy for creating various types of glycosylation platforms to access pharmaceutically and medicinally interesting products.