Complete Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin A.

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 antidiabetes and antiobesity 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 × crocosmiiflora) corms during the early 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. Genes and enzymes for the first four steps of MbA biosynthesis, starting from the flavonol precursor myricetin, have recently been identified. 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 that converts 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.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with 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 coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg 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.

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

A Conifer UDP-Sugar Dependent Glycosyltransferase Contributes to Acetophenone Metabolism and Defense against Insects.

Acetophenones are phenolic compounds involved in the resistance of white spruce (Picea glauca) against spruce budworm (Choristoneura fumiferiana), a major forest pest in North America. The acetophenones pungenol and piceol commonly accumulate in spruce foliage in the form of the corresponding glycosides, pungenin and picein. These glycosides appear to be inactive against the insect but can be cleaved by a spruce ß-glucosidase, PgßGLU-1, which releases the active aglycons. The reverse glycosylation reaction was hypothesized to involve a family 1 UDP-sugar dependent glycosyltransferase (UGT) to facilitate acetophenone accumulation in the plant. Metabolite and transcriptome profiling over a developmental time course of white spruce bud burst and shoot growth revealed two UGTs, PgUGT5 and PgUGT5b, that glycosylate pungenol. Recombinant PgUGT5b enzyme produced mostly pungenin, while PgUGT5 produced mostly isopungenin. Both UGTs also were active in vitro on select flavonoids. However, the context of transcript and metabolite accumulation did not support a biological role in flavonoid metabolism but correlated with the formation of pungenin in growing shoots. Transcript levels of PgUGT5b were higher than those of PgUGT5 in needles across different genotypes of white spruce. These results support a role of PgUGT5b in the biosynthesis of the glycosylated acetophenone pungenin in white spruce.