Flavonol biosynthesis genes and their use in a synthetic biology approach to engineering the plant antidiabetic metabolite montbretin A.

The plant metabolite montbretin A (MbA) and its simpler precursor mini-MbA are being developed as new drugs against Type-2 diabetes. The only known source for these complex acylated flavonol glycosides is the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA occurs in small amounts in below-ground storage organs, called corms. Our long-term goal is to metabolically engineer Nicotiana benthamiana as a heterologous production system for mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside, the first two pathway-specific intermediates in MbA biosynthesis. Expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides, but not myricetin glycosides, suggesting myricetin is limiting. We investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana. We characterized two flavanone hydroxylases (F3H), a flavonol synthase (FLS), a flavonoid 3'-hydroxylase (F3'H) and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia FLS converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence-relatedness to F3'Hs in the CYP75B subfamily of cytochromes P450 than those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient co-expression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg x g-1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.