Directed Evolution of a Plant Glycosyltransferase for Chemo- and Regioselective Glycosylation of Pharmaceutically Significant Flavonoids

Glycosyltransferases have attracted increasing interest for the ability to construct glycosylated molecules in a facile way. However, promiscuous chemoselectivity and poor regioselectivity hinder their widespread application in the synthetic field, especially in the pharmaceutical area. Here, a plant glycosyltransferase, MiCGT, was engineered by directed evolution to catalyze the glycosylation of flavonoids, which opens the door to pharmaceutical applications. Combining an alanine scan and iterative saturation mutagenesis, mutants with enhanced chemo- and regioselectivity and significantly improved activities toward seven different flavonoids were evolved, and two glycosylated products were prepared on a large scale. The best quadruple mutant VFAH enables strict 3-O glycosylation selectivity and a 120-fold activity enhancement toward the model substrate quercetin relative to the wild type (WT). Moreover, the crystal structures of the WT and mutant VFAH were obtained, a breakthrough of its kind in plant glycosyltransferase research. The origin of substrate specificity and regioselectivity was elucidated by combining the experimental data with the unique structure information. We anticipate that this work will aid future protein engineering of this type of enzyme.