C‐Glycosyltransferases catalyzing the formation of di‐C‐glucosyl flavonoids in citrus plants

Summary Citrus plants accumulate many kinds of flavonoids, including di‐C‐glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di‐C‐glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C‐glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di‐C‐glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2‐hydroxyflavanones, dihydrochalcone, and their mono‐C‐glucosides as sugar acceptors and produced corresponding di‐C‐glucosides. The Km and kcat values of FcCGT toward phloretin were