Directed Evolution of Glucosyltransferase ZmDo and Its Application in the Synthesis of Luteolin-5-O-Xyloside.

Luteolin is a plant-derived flavone widely distributed in edible plants and medicinal herbs, attracting attention due to its antioxidant and anti-inflammatory properties. However, efficient biosynthesis of specific luteolin glycosides, particularly luteolin-5-O-xyloside, remains challenging due to low donor specificity, limited catalytic efficiency of natural glycosyltransferases, and the high cost of UDP-xylose (UDP-Xyl). In this study, a novel 5-O-xylosyltransferase and an engineered Escherichia coli whole-cell system were developed for luteolin-5-O-xyloside production. Structure-guided engineering identified Thr142 as a key residue for UDP-sugar recognition. The mutant ZmDo-T142 V showed enhanced catalytic efficiency toward UDP-Xyl (kcat/Km = 1302.8 s-1 M-1), 2.3-fold higher than the wild type. A de novo UDP-Xyl biosynthetic pathway was constructed, enabling intracellular cofactor regeneration. Under optimized conditions, a titer of 4451 mg/L with an 87% conversion rate was achieved.