Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A^{<a href="#fn4" rid="fn4" class=" fn">1</a>,}^{<a href="#fn5" rid="fn5" class=" fn">[OPEN]</a>}

Abstract

Diabetes and obesity are diseases that are approaching globally epidemic proportions with tremendous human health and economic consequences. Type 2 diabetes, which is characterized by high blood glucose (Glc) levels, afflicts ∼6% of the population of the western world, making it the third most prevalent disease (Health Canada, 2019; The World Health Organization, 2019).

A major goal in treating diabetes and obesity is reducing high levels of blood Glc, which is a particular problem in patients with diets that are rich in starch and other sugars. Digestion of starch begins with the activity of salivary α-amylase. The main enzyme of starch degradation is the human pancreatic α-amylase (HPA), which produces linear and branched malto-oligosaccharides. These oligosaccharides are degraded to Glc and passed into the bloodstream by gut wall α-glucosidases. The α-glucosidase inhibitors Acarbose and Miglitol are two pharmaceuticals currently used for controlling blood Glc levels. These drugs cause the passage of undigested oligosaccharides to the lower gut, which commonly results in gastrointestinal upsets due to adverse osmotic effects and bacterial fermentation. Selective inhibitors of HPA, which act upstream in the starch degradation cascade by reducing the breakdown of starch into oligosaccharides, are emerging as alternatives for the treatment of type 2 diabetes. In a large-scale screen of 30,000 natural products for HPA inhibitors, the complex acylated flavonol glycoside montbretin A [MbA; myricetin 3-O-(6′-O-caffeoyl)-β-d-glycosyl 1,2-β-d-glucosyl 1,2-α-l-rhamnoside 4′-O-α-l-rhamnosyl 1,4-β-d-xyloside] was discovered as a potent (inhibitory constant [Ki] = 8 nm) and specific amylase inhibitor (Fig. 1; Tarling et al., 2008; Williams et al., 2015). The simpler MbA precursor mini-MbA is active with a K_i = 90 nm. MbA does not significantly affect human gut α-glucosidases or gut bacterial amylases and effectively controls blood Glc levels in Zucker Diabetic Fatty rats (Yuen et al., 2016). The promising preclinical studies prompt methods for producing large quantities of MbA for further drug development.

Figure 1.

Structure of MbA and schematic structures of metabolites in MbA biosynthesis. A, The building blocks of MbA are labeled: myricetin, rhamnose (Rha), glucose (Glc), xylose (Xyl), and caffeic acid. The dotted line marks the mini-MbA structure. Red hydroxyls mark groups specific to myricetin compared with kaempferol, and the blue hydroxyl marks the group specific to the caffeoyl compared with a coumaroyl moiety. B, Schematic of myricetin 3-O-α-l-rhamnoside (MR), myricetin 3-O-β-d-glucosyl 1,2-α-l-rhamnoside (MRG), myricetin 3-O-(6′-O-caffeoyl)-β-d-glucosyl 1,2-α-l-rhamnoside (mini-MbA), and MbA. Identified MbA biosynthesis steps and enzymes are labeled. C, Caffeic acid (blue); G, Glc (green); M, myricetin (pink); R, Rha (yellow); X, Xyl (orange).

MbA occurs in the corms (belowground bulb-like organs) of the ornamental plant montbretia (Crocosmia × crocosmiiflora, family Iridaceae), which is the only known source for this compound (Asada et al., 1988; Offen et al., 2006; Irmisch et al., 2018). MbA is present in the corms at low levels (Irmisch et al., 2018) that allow isolation of sufficient amounts for animal studies. However, much larger amounts will be required than can be harvested from the natural source for treatment of patients with type 2 diabetes as well as those diagnosed as prediabetic. The low amounts of MbA in montbretia corms, lack of agricultural production systems for this plant, and the destructive harvest of the belowground corms make it unrealistic to support MbA production via montbretia cultivation and harvest. Alternative avenues for MbA production are chemical synthesis and synthetic biology-enabled metabolic engineering. The complexity of the MbA molecule mitigates strongly against production by classical synthesis. Production by synthetic biology appears a viable approach, but it requires knowledge of the genes and enzymes of the MbA biosynthetic system.

The general flavonoid biosynthetic system (Fig. 2A) is widely conserved across diverse plant species (Schijlen et al., 2004; Martens et al., 2010). The first committed step of flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Chalcone isomerase (CHI) converts chalcone into the 4′OH-flavanone naringenin, which is the precursor for all other groups of flavonoids, including flavones, flavonols, flavandiols, anthocyanins, and proanthocyanidins (Martens et al., 2010). In the biosynthesis of flavonols, which includes the myricetin core of MbA, naringenin is hydroxylated by flavanone 3-hydroxylase (F3H) to form the 4′OH-dihydroflavonol dihydrokaempferol (DHK). F3H also converts the 3′4′OH-flavanone eriodictyol into dihydroquercetin (DHQ) and the 3′4′5′-OH flavanone pentahydroxyflavanone (PHF) into dihydromyricetin (DHM). Flavonol synthase (FLS) catalyzes the desaturation of dihydroflavonols to flavonols. F3H and FLS are members of the large enzyme family of 2-oxoglutarate-dependent dioxygenases (2OGDs). The flavonols kaempferol, quercetin, and myricetin differ by their hydroxylation pattern on the B-ring with 4′-, 3′4′-, or 3′4′5′-hydroxylation, respectively. Hydroxylation of the B-ring is produced by the cytochromes P450 (P450s) flavonoid 3′-hydroxylases (F3′Hs) and flavonoid 3′5’-hydroxylases (F3′5H).

Figure 2.

General schematic of myricetin biosynthesis and transcript profiles of myricetin pathway genes in montbretia. A, Enzymes and metabolites of flavonol biosynthesis leading to the formation of the 3′4′5′-hydroxylated flavonol myricetin. B, Heat map showing relative transcript abundance of putative myricetin biosynthetic pathway genes in yC and oC of montbretia.

MbA is derived from the flavonol myricetin (Irmisch et al., 2018). The biosynthesis of MbA occurs in young developing corms and proceeds via the formation of MR, MRG, and mini-MbA (or MRG-Caff; Fig. 1; Irmisch et al., 2018). We recently discovered two UDP-dependent glycosyltransferases (UGTs), CcUGT1 (UGT77B2) and CcUGT2 (UGT709G2), and two BAHD-acyltransferases, CcAT1 and CcAT2, which catalyze the first three pathway-specific steps of MbA biosynthesis. CcUGT1 converts myricetin into MR, CcUGT2 converts MR into MRG, and CcAT1 and CcAT2 both convert MRG into MRG-Caff (mini-MbA; Irmisch et al., 2018). Transient coexpression of the two UGTs and CcAT1 in Nicotiana benthamiana resulted in the formation of small amounts of kaempferol 3-O-(6′-O-coumaroyl)-glucosyl rhamnoside, termed surrogate mini-MbA (Irmisch et al., 2018). In comparison with mini-MbA, the surrogate mini-MbA contained the 4′-hydroxylated flavonol kaempferol, instead of the 3′4′5′-hydroxylated flavonol myricetin, as the core flavonol structure. Instead of the caffeoyl moiety of mini-MbA, the surrogate mini-MbA had a coumaroyl moiety. These results demonstrated that N. benthamiana could be developed as a production system for MbA, but the availability of myricetin and caffeoyl-CoA may be limiting and require additional pathway engineering.

Myricetin is not a ubiquitous or abundant metabolite across the plant kingdom, due in part to the limited occurrence of F3′5′H, which is missing, for example, in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa; Tohge et al., 2013). Myricetin formation via DHM may be limited in cases where DHM is a poor substrate for FLS, such as in tomato (Solanum lycopersicum) or potato (Solanum tuberosum; Forkmann et al., 1986; Bovy et al., 2002; Tanaka et al., 2008). Research on flavonol biosynthesis has focused mostly on the more ubiquitous kaempferol and quercetin, while information about myricetin biosynthesis is missing. Here, we explore the genes and enzymes of myricetin biosynthesis in montbretia with the goal of engineering the availability of myricetin in N. benthamiana as a substrate for MbA production.

Acknowledgments

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