Triterpene Functional Genomics in Licorice for Identification of CYP72A154 Involved in the Biosynthesis of Glycyrrhizin    

Abstract

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton b-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding b-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-b-amyrin (a possible biosynthetic intermediate between b-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-b-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of b-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

“…Our experiments demonstrate that the CPMV-HT expression system provides a rapid and effective means of expressing enzymes for the synthesis of low molecular weight specialized compounds alone and in combination. Previous work on triterpene biosynthetic enzymes by other groups has focused on yeast as a heterologous expression system (24)(25)(26)(27)(28). Here, we have used the CPMV-HT system for the synthesis and modification of the simple triterpene β-amyrin, thereby reconstructing the first two committed steps in the avenacin biosynthetic pathway.…”

“…Several triterpene-modifying P450s from eudicots have been characterized recently by heterologous expression in yeast. These include CYP93E1 from soybean, which hydroxylates β-amyrin and sophoradiol at position C24 (24); two P450s from liquorice, one (CYP88D6) that converts β-amyrin to 11-oxo-β-amyrin and a second (CYP72A154) that converts 11-oxo-β-amyrin to glycyrrhizin acid (25,26); and CYP716A12 from Medicago truncatula, a β-amyrin 28-oxidase that converts β-amyrin to oleanolic acid (27,28). These enzymes belong to different P450 families, indicating that the ability to oxygenate β-amyrin has arisen multiple times during evolution.…”

A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

“…In Catharanthus roseus, the CYP72A1 gene encodes a secologanin synthase that converts loganin into secologanin (Irmler et al, 2000). CYP72A154 and CYP72A63 are involved in the glycyrrhizin biosynthesis pathway in licorice (Glycyrrhiza glabra) and Medicago truncatula, respectively (Seki et al, 2011). Such findings reveal that CYP72A proteins work as ring-opening and triterpene-oxidizing enzymes.…”

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

“…P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11)(12)(13)(14)(15)(16)(17)(18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19)(20)(21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified.…”