Biosynthesis of the anti‐diabetic metabolite montbretin A: glucosylation of the central intermediate mini‐MbA

Abstract

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic a-amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia 3 crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3-O-(6 0 -O-caffeoyl)-glucosyl rhamnoside (mini-MbA). Here, we describe the sequence of reactions from mini-MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini-MbA. The UDP-dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2-glucosylation of mini-MbA to produce myricetin 3-O-(glucosyl-6 0 -O-caffeoyl)-glucosyl rhamnoside. Co-expression of CcUGT3 with genes for myricetin and mini-MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.Keywords: plants and human health, plant secondary metabolism, plant specialized metabolism, UDP-dependent glycosyltransferase, flavonoid, type 2 diabetes, metabolic engineering, synthetic biology, bioproduct, Crocosmia 3 crocosmiiflora.

Complete Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin A

“…and spatial expression with highest transcript levels in young developing corms in the early summer and low expression in old corms of the previous year, matching the dynamics of MbA accumulation (Irmisch et al, 2018;Irmisch et al, 2019a;Irmisch et al, 2019b). These patterns of differential gene expression have enabled the gene discovery of the first four steps of MbA assembly as well as montbretia genes affecting myricetin biosynthesis.…”

“…Using CcUGT1, CcUGT2, CcUGT3 and CcAT1 or CcAT2, together with montbretia genes of myricetin biosynthesis, specifically a MYB-The formation of the disaccharide moiety at the 4'-hydroxy of myricetin defines the final two steps of MbA biosynthesis. Based on in vitro enzyme assays with corm protein extracts (Irmisch et al, 2019b), we proposed the final two reactions involve two UGT-mediated glycosylation reactions with UDP-Xyl and UDP-Rha, respectively ( Figure 1A). Glycosylation of specialized metabolites in plants is typically catalyzed by UGTs of the GT-1 family, which use UDP-activated sugar donors and contain a highly conserved Plant Secondary Product Glycosylation (PSPG) motif (Gachon et al, 2005;Caputi et al, 2012;Lombard et al, 2014).…”

“…The six individual biosynthetic reactions of the MbA assembly pathway, and their specific sequence as they occur in montbretia corms, have also recently been described (Irmisch et al, 2018;Irmisch et al 2019b). In addition, the genes and encoded enzymes for the first four of the six biosynthetic steps have been cloned and functionally characterized ( Figure 1A) (Irmisch et al, 2018;Irmisch et al, 2019b).…”

Imitation is the sincerest form of flattery: reconstructing the biosynthesis of montbretin A

“…But identifying the compound is just the beginning: often, it is necessary to uncover each step of the biochemical pathway that delivers the compound before recreating it in the laboratory. In this issue of The Plant Journal , Irmisch et al () report on the characterization of the biosynthesis pathway of a potential medicine for treating diabetes.…”

“…Steps 1–3 have been described previously (Irmisch et al , ). The sequence of glycosylation steps 4–6 and the enzyme CcUGT3 (bold) are described in this issue of TPJ (Irmisch et al , ). C, caffeic acid; G, glucose; M, myricetin; R, rhamnose; X, xylose.…”