Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

Qing Liu,Jules Beekwilder,Francel W A Verstappen,Sander Van Der Krol,Miriam Goedbloed,Mohammad Majdi,Tatsiana Charnikhova,Katarina Cankar,Ric C H De Vos,Harro J Bouwmeester,Miguel A Blazquez

doi:10.1371/journal.pone.0023255

Abstract

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

Highlights

Sesquiterpene lactones (SLs) are a major class of plant secondary metabolites
The TpGAS cDNA - using its native targeting to the cytosol or equipped with a mitochondrial targeting signal - was cloned into a binary expression vector under control of the Rubisco promoter and introduced into Agrobacterium tumefaciens
Partial reconstruction of the sesquiterpene biosynthesis pathways in yeast has been demonstrated for the antimalarial drug artemisinin [30] and for germacra1(10),4,11(13)-trien-12-oic acid [25,26]

Summary

Introduction

Sesquiterpene lactones (SLs) are a major class of plant secondary metabolites. These bitter tasting, lipophilic molecules form the active constituents of a variety of medicinal plants used in traditional medicine [1,2]. While the detailed structure of those SLs varies, their backbones are constrained to a limited set of core skeletons, such as germacranolide, eudesmanolide and guaianolide [7,8,9]. For all these three types of sesquiterpene lactones costunolide is generally considered the common precursor [6]. Parthenolide has been reported to have anti-inflammatory and anti-cancer activity [16,17]

Methods

Results

Conclusion