Exploration of diverse glycosyltransferases in Dracocephalum moldavica and engineering the production of bioactive flavonoid glycosides

Dracocephalum moldavica L., an annual herb valued for its medicinal and ornamental properties, produces flavonoid glycosides like apigenin 7-O-glucuronide, scutellarein-7-O-glucuronide, and vitexin, which offer cardiovascular benefits. However, the UDP-glycosyltransferases (UGTs) involved in their biosynthesis have not been fully characterized. In the present investigation, we identified five UGTs, which comprise two bifunctional flavonoid UDP-glucuronosyl/glucosyltransferase genes, DmUGT1 and DmUGT2; two flavonoid UDP-glucosyltransferase genes, DmUGT3 and DmUGT4; and one type I di-C-glycosyltransferase gene, DmCGT1. The UDP-glucuronosyl/glucosyltransferase DmUGT1 showed effective glycosylation activity and exhibited a wide substrate promiscuity, facilitating the synthesis of the principal flavonoid glycosides in D. moldavica, including bioactive compounds such as scutellarein-7-O-glucuronide. Homology modeling and site-directed mutagenesis of the bifunctional DmUGT1 indicated that the amino acids Ser127 and Tyr373 are critical determinants of sugar donor specificity. DmCGT1 could catalyze phloretin to form phloretin-3'-C-glycoside and phloretin-3',5'-di-C-glycoside. Additionally, we engineered Escherichia coli strains that utilized DmUGT1 and DmCGT1, complemented with plasmids designed to enhance the intracellular supply of UDP-glucuronic acid and UDP-glucose in E. coli. These engineered strains successfully enabled the in vivo production of scutellarein-7-O-glucuronide and phloretin-3',5'-di-C-glycoside, achieving yields of 195 and 196 mg/L, respectively. This study provides a systematic elucidation of the glycosylation mechanisms of flavonoids in D. moldavica and offers candidate genes and methodologies for the biosynthesis of bioactive glycoside compounds through synthetic biology approaches.