title: Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae.
abstract: The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies.
authors.affiliation: University of Calgary, Department of Biological Sciences, Calgary, T2N 1N4, Canada.
authors.forename: Trinh-Don
authors.initials: TD
authors.lastname: Nguyen
authors.affiliation: John Innes Centre, Department of Metabolic Biology, Norwich, NR4 7UH, United Kingdom.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.affiliation: John Innes Centre, Department of Metabolic Biology, Norwich, NR4 7UH, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Vardakou
authors.affiliation: John Innes Centre, Department of Metabolic Biology, Norwich, NR4 7UH, United Kingdom.
authors.forename: Melissa
authors.initials: M
authors.lastname: Salmon
authors.affiliation: John Innes Centre, Department of Metabolic Biology, Norwich, NR4 7UH, United Kingdom; Institute of Food Research, Food and Health Programme, Norwich, NR4 7UA, United Kingdom. Electronic address: paul.omaille@sri.com.
authors.forename: Paul E
authors.initials: PE
authors.lastname: O'Maille
authors.affiliation: University of Calgary, Department of Biological Sciences, Calgary, T2N 1N4, Canada. Electronic address: daekyun.ro@ucalgary.ca.
authors.forename: Dae-Kyun
authors.initials: DK
authors.lastname: Ro
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019659:Asteraceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D044822:Biodiversity; D003001:Cloning, Molecular; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D007783:Lactones; D010940:Plant Proteins; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; D019883:Alkyl and Aryl Transferases
keywords: Asteraceae; Barnadesia spinosa; Germacrene A synthase; Sesquiterpene lactone; Terpene synthase