Olive triterpenoids: identification and functional characterization of key biosynthetic genes
Alagna F, Thimmappa R, Reed J, Cultrera NGM, Calderini O, Baldoni L, Osbourn A
In: “SIGA LXIII Convegno Annuale "Science and innovation for sustainable agriculture intensification: the contribution of plant genetics and breeding"”. Napoli Italy, 10-13 Settembre 2019. (2019)
Olive (Olea europaea L.) produces large amounts of highly valuable specialized metabolites. Among them, triterpenoids have numerous pharmaceutical, agricultural, and industrial biotechnology applications ranging from flavors and fragrances, defense compounds against herbivores or pathogens, to bioactive components of drugs. Olive is among the species accumulating the highest amount of the triterpenoids oleanolic and maslinic acids, however the genes underpinning their biosynthesis have not been yet identified in this species. Based on the homology to other plant systems, we searched for terpene synthases and cythocromes P450 involved in the biosynthesis of oleanolic and maslinic acids. Candidate genes have been cloned, sequenced and expressed in yeast (Saccharomyces cerevisiae) and/or plant (Nicotiana benthamiana) host systems, then functionally characterized by in vivo enzymatic assays. Three ?-amyrin synthases, a C-28 oxidase able to oxidase ?-amyrin for the synthesis of erythrodiol and oleanolic acid and a C-2 oxidase able to catalyze the following oxidation for the synthesis of maslinic acid have been identified. Moreover, the cythocromes P450 have been shown to catalyze also the oxidation of ?-amyrin and derivated oxidized compounds for the synthesis of ursane-type triterpenoids. With these results we identified and characterized the main players involved in the biosynthesis of olive triterpenoids. In addition, we have transiently expressed the entire pathway for the synthesis of olive oleanane- or ursane-type triterpenoids. At this aim, we used a previously developed synthetic biology platform based on N. benthamiana that enables the production of gram-scale quantities of target compounds 1. Our results will be exploited in various ways for commercial applications. These include the development of molecular markers for triterpenoid-determined traits, the development of novel improved olive varieties, and the production of a high amount of olive triterpenoids by metabolic engineering for industrial applications.
