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. • Genome-wide analysis obtained 89 UGTs in the GT1 family from I. indigotica. • Eight novel glycosyltransferases exhibited different catalytic properties in the 4′- O- glycosylation of flavonoid 6- C- glycosides. • Tandem UGT73Bs, IiUGT15 and IiUGT16, likely serve as the most efficient catalysts in the biosynthesis of 4′- O -glycosyl- C -glycosylflavonoids in the cell factory, which might play pivotal roles in modulating flavonoid glycosides' accumulation in I. indigotica.