Engineering Efficient Biosynthesis of Baicalein-7-O-xyloside in Escherichia coli via Glycosyltransferase Evolution...

Glycosylation expands natural product diversity. Baicalein, a bioactive compound from Scutellaria baicalensis, suffers from poor solubility and bioavailability. Hence, we engineered a novel baicalein xyloside using Arabidopsis thaliana glycosyltransferase AtUGT73B1, although the enzyme also produced oroxin A. Structural analysis indicated that Gly157's carbonyl interacts with UDP-glucose. Mutating Gly157 to serine (G157S) reduced UDP-glucose activity while slightly enhancing UDP-xylose activity. Simulations confirmed weakened UDP-glucose binding but strengthened UDP-xylose interactions in G157S. To improve production, we regulated intracellular sugar donors via two approaches: single or combined knockout of ushA, pgi, and zwf did not increase yield, but introducing a cellobiose phosphorolysis pathway (CPP) into knockout strains significantly boosted output. The engineered strain B73uc achieved efficient biotransformation. With metabolic and fermentation optimization, the titer reached 198.4 ± 3.9 mg/L. This study provides an efficient, selective route to baicalein xyloside by combining enzyme and metabolic engineering, enabling scalable production of bioactive glycosides.