Diabetes and obesity are affecting human health worldwide. Their occurrence is increasing with lifestyle choices, globalization of food systems, and economic development. The specialized plant metabolite montbretin A (MbA) is being developed as an anti-diabetes and anti-obesity treatment due to its potent and specific inhibition of the human pancreatic α-amylase. MbA is a complex acylated flavonol glycoside formed in small amounts in montbretia (Crocosmia x crocosmiiflora) corms during the

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... summer. The spatial and temporal patterns of MbA accumulation limit its supply for drug development and application. We are exploring MbA biosynthesis to enable metabolic engineering of this rare and valuable compound. Recently, we reported genes and enzymes for the first four steps of MbA biosynthesis starting from the flavonol precursor myricetin. Here, we describe the gene discovery and functional characterization of the final two enzymes of MbA biosynthesis. The UDP glycosyltransferases, CcUGT4 and CcUGT5, catalyze consecutive reactions in the formation of the disaccharide moiety at the 4'-hydroxy position of the MbA flavonol core. CcUGT4 is a flavonol glycoside 4'-O-xylosyltransferase that acts on the second to last intermediate (MbA-XR2) in the pathway. CcUGT5 is a flavonol glycoside 1,4-rhamnosyltransferase converting the final intermediate (MbA-R2) to complete the MbA molecule. Both enzymes belong to the UGT family D-clade and are specific for flavonol glycosides and their respective sugar donors. This study concludes the discovery of the MbA biosynthetic pathway and provides the complete set of genes to engineer MbA biosynthesis. We demonstrate successful reconstruction of MbA biosynthesis in Nicotiana benthamiana.