Functional analysis of tandem glycosyltransferases catalyzing the O-glycosylation of flavonoid 4′-O-glycosyl-6-C-glycosides biosynthesis from Isatis indigotica

Flavonoid O-glycosyl-C-glycosides with promising biological activities including antitumor, antidiabetes, and antibacterials are the primary active ingredients of the medicinal plant Isatis indigotica's leaves. However, O-glycosyltransferases catalyzing flavonoid O-glycosyl-C-glycosides biosynthesis in I. indigotica's leaves remain unclear. In this study, guided by genome-wide investigation and phylogenetic analyses, 89 glycosyltransferases were obtained and clustered into 18 families. In vitro enzyme assays demonstrated that eight novel glycosyltransferases could catalyze the 4′-O-glycosylation of flavonoid 6-C-glycosides, and exhibited different catalytic properties. Moreover, tandem UGT73Bs, IiUGT15 and IiUGT16, could catalyze not only 4′-O-glycosylate flavonoid 6-C-glycosides such as isovitexin, isoorientin, and isoscoparin but also 7-O-glycosylate flavonoids, and hydrolyze the glucose moiety of flavonoid O-glycosides. Homology modeling and site-directed mutagenesis uncovered that the amino acids interacting with acceptors were highly conserved between IiUGT15 and IiUGT16, with various degreed effects on 4′-O-glycosylation, potentially leading to differences in their catalytic activity. Isoorientin-4′-O-β-D-glucoside was synthesized in Escherichia coli without the exogenous supplementation of UDP-glucose. Overall, this study offered new biocatalysts to attain 4′-O-glycosylation of flavonoid C-glycosides for drug discovery and established a foundation for the biomanufacturing of critical flavonoid O-glycosyl-C-glycosides.