Construction of a Highly Efficient System for Fisetin-3-O-Glucoside Synthesis Using a Novel Glycosyltransferase AaUGT from <i>Arabis alpina</i>

Fisetin is a natural polyhydroxyl flavonoid with anti-inflammatory, antioxidant, and neuroprotective activities, but its application in foods and agriculture is limited by poor stability, low absorption, and low bioavailability. Glycosylation can effectively improve its physicochemical and biological properties. Enzymatic glycosylation is attractive owing to its green, efficient, and highly specific features. In this study, a novel glycosyltransferase AaUGT from Arabis alpina was cloned and expressed in Escherichia coli . It showed optimal activity at 45 °C and pH 7.5, strict 3-O regioselectivity, and high catalytic efficiency ( K m = 0.37 mM, k cat / K m = 1884.18 M –1 s –1 ). By strengthening uridine diphosphate glucose (UDPG) supply via metabolic engineering, an efficient whole-cell system was constructed. Under optimized conditions, the titer of fisetin-3-O-glucoside reached 3.51 g/L with a conversion rate of 89.6%. This work supports the development of fisetin glycosides and provides a reference for flavonoid glycoside biosynthesis.