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

Hikaru Seki,Tomoyoshi Akashi,Ery Odette Fukushima,Satoru Sawai,Toshiya Muranaka,Kiyoshi Ohyama,Toshio Aoki,Masaharu Mizutani,Toshiyuki Ohnishi,Hiroshi Sudo,Kazuki Saito

doi:10.1105/tpc.110.082685

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 β-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 β-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-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-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 β-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.

Highlights

Triterpenoid saponins consist of a triterpenoid aglycone and one or more sugar moieties and belong to a class of natural plant products that includes various bioactive compounds found in medicinal plants (Waller and Yamasaki, 1996)
The early stages of triterpenoid saponin biosynthesis involve the dimerization of two farnesyl diphosphate molecules catalyzed by Squalene epoxidase–mediated oxidation produces 2,3oxidosqualene, a common substrate of oxidosqualene cyclases (OSCs), as a precursor of both triterpenes and sterols (Abe et al, 1993) (Figure 1)
The mass spectrum of the major product was an excellent match with that of authentic 30-hydroxy-11-oxo-bamyrin (4a) (Figure 2C). Both minor products have ion fragmentation patterns similar to that of peak 4a and were identified as isomers of 30-hydroxy-11-oxo-b-amyrin (4a); they are likely to differ in the position of the hydroxyl group introduced by CYP72A154

Summary

Introduction

Triterpenoid saponins consist of a triterpenoid aglycone and one or more sugar moieties and belong to a class of natural plant products that includes various bioactive compounds found in medicinal plants (Waller and Yamasaki, 1996). The early stages of triterpenoid saponin biosynthesis involve the dimerization of two farnesyl diphosphate (an intermediate product of the mevalonate pathway) molecules catalyzed by Squalene epoxidase–mediated oxidation produces 2,3oxidosqualene, a common substrate of oxidosqualene cyclases (OSCs), as a precursor of both triterpenes and sterols (Abe et al, 1993) (Figure 1). Solid black arrows indicate a dimerization reaction of two farnesyl diphosphate (FPP) molecules catalyzed by squalene synthase (SQS) originating squalene, oxidation by squalene epoxidase (SQE) to 2,3-oxidosqualene, or cyclization catalyzed by bAS. A dashed arrow between mevalonic acid and farnesyl diphosphate indicates multiple enzyme reactions.

Methods

Results

Conclusion