Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

Abstract

SummaryGlycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins.

A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis

“…Next, we engineered a glucoglycyrrhizin-producing platform yeast strain (GLU) expressing β-amyrin synthase, CYP88D6, CYP72A63 and UGT73P12v. UGT73P12 catalyses the second glucuronosylation in glycyrrhizin biosynthesis, and its natural variant UGT73P12v, obtained from glucoglycyrrhizin-producing G. uralensis 83-555 strain, has lost its specificity for UDP-glucuronic acid 14 . Instead, UGT73P12v catalyses transfer of glucose, yielding glucoglycyrrhizin.…”

“…Glycyrrhizin and soyasaponins are oleanane-type triterpenoid saponins, which are derived from β-amyrin, and most of their biosynthetic enzymes have been identified (Fig. 1) [12][13][14][15][16][17] . However, the enzyme that catalyses the transfer of the conserved glucuronosyl moiety at the C-3 position of the aglycones has not yet been determined.…”

“…However, the massive number of UGTs found per plant genome and the low degree of correlation between substrate selectivity and the primary UGT sequence have made identification of the target UGT difficult 21 . Although GuUGAT 22 (UGT73B27) reportedly catalyses continuous two-step glucuronosylation to yield glycyrrhizin directly from its aglycone, glycyrrhetinic acid, our recent study on UGT73P12 14 , which catalyses only the second glucuronosylation, strongly suggests that a separate enzyme is responsible for the first glucuronosylation. Here, we demonstrate that a cellulose-synthase superfamily-derived glycosyltransferase (CSyGT) catalyses the transfer of glucuronic acid from UDP-glucuronic acid to the C-3 position of oleanane-type triterpenoid aglycones.…”

Glycoside‐specific glycosyltransferases catalyze regio‐selective sequential glucosylations for a sesame lignan, sesaminol triglucoside

“…(OG2), and licorice UGT73P12(OG1) are involved in sugarsugar O-glycosylation for the specialized triterpenoid soyasaponin (b1?3), diterpenoid sweetener (b1?3), and triterpenoid sweetener (b1?2), respectively (Richman et al, 2005;Sayama et al, 2012;Nomura et al, 2019;Lee et al, 2019). Moreover, saffron UGT707B1 catalyzes the sugarsugar b1?2-O-glucosylation of flavonol glucoside, resulting in the formation of flavonol sophoroside, whereas rice UGT707A3 has flavonol 3-O-glucosylating activity (Ko et al, 2008;Trapero et al, 2012).…”

Saponins in Insect Pest Control

“…Various cultivated crops are supplemented with multiple saponins, which are responsible for plant health [34,55]. Likewise, triterpenoid saponins are the main part of the defense system of several agricultural crops, e.g., Spinacia oleracea, Beta vulgaris, Glycyrrhiza glabra, Helianthus annuus, Aesculus hippocastanum, Quillaja saponaria, Smilax ornata, and Camellia sinensis [20,34,[56][57][58]. On the other hand, various steroidal glycosides are also a prominent part of many plants, which are helpful in the plant immune system, e.g., Allium spp., Asparagus officinalis, Avena sativa, Capsicum spp., Dioscorea spp., Panax ginseng, Solanum melongena, S. lycopersicum, as well as Trigonella foenumgraecum [59][60][61][62][63].…”