Engineering Efficient Biosynthesis of Baicalein-7- <i>O</i> -xyloside in <i>Escherichia coli</i> via Glycosyltransferase Evolution and Metabolic Pathway Optimization

Glycosylation expands natural product diversity. Baicalein, a bioactive compound from <i>Scutellaria baicalensis</i>, suffers from poor solubility and bioavailability. Hence, we engineered a novel baicalein xyloside using <i>Arabidopsis thaliana</i> 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 <i>ushA</i>, <i>pgi</i>, and <i>zwf</i> did not increase yield, but introducing a cellobiose phosphorolysis pathway (CPP) into knockout strains significantly boosted output. The engineered strain <i>B73uc</i> 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.