title: Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.
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.
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.forename: Mohammad
authors.initials: M
authors.lastname: Majdi
authors.forename: Katarina
authors.initials: K
authors.lastname: Cankar
authors.forename: Miriam
authors.initials: M
authors.lastname: Goedbloed
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Charnikhova
authors.forename: Francel W A
authors.initials: FW
authors.lastname: Verstappen
authors.forename: Ric C H
authors.initials: RC
authors.lastname: de Vos
authors.forename: Jules
authors.initials: J
authors.lastname: Beekwilder
authors.forename: Sander
authors.initials: S
authors.lastname: van der Krol
authors.forename: Harro J
authors.initials: HJ
authors.lastname: Bouwmeester
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D018651:Cichorium intybus / Q000201:enzymology / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry / Q000378:metabolism; D020903:Tanacetum parthenium / Q000201:enzymology / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*; D007783:Lactones
chemical_list: D019883:Alkyl and Aryl Transferases; D003545:Cysteine; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D010088:Oxidoreductases; D010940:Plant Proteins; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C431525:germacra-1(10),4,11(13)-trien-12-oic acid; C471077:germacrene A; C002602:costunolide; D007783:Lactones; C462022:germacrene A synthase, Cichorium intybus