Abstract: 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.

Key words: C‐glycosyltransferase, Dracocephalum moldavica, flavonoids, synthetic biology, UDP‐glucuronosyl/ glucosyltransferase