Understanding and Exploiting the Biosynthesis of Diterpenoids in Euphorbia peplus

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Terpenes represent one of the largest and most diverse class of specialized metabolites, including world health organisation (WHO)-listed essential drugs such as Taxol and Artemisinin. The polyoxygenated ingenol mebutate is a clinically important diterpenoid produced by the plant Euphorbia peplus. In 2012 it was approved by the Food and Drug Administration (FDA) for the treatment of actinic keratosis, a precancerous skin condition. At the moment it is under further evaluation for the treatment of basal cell carcinoma and the activation of latent HIV reservoirs in the human body. Despite the growing pharmaceutical importance of ingenol mebutate current supplies of this valuable metabolite are limited, as it is only produced in minute quanitites in planta and currently no feasible total synthesis has been reported. In this study we aim to apply a `multi-omics´ approach to elucidate the biosynthetic pathway towards ingenol mebutate in Euphorbia peplus. Genome and RNA sequencing data will be obtained and analysed to identify genes involved in the biosynthesis of ingenol mebutate and concomitantly produced diterpenoids. Candidate genes will be tested for activity in a robust Agrobacterium-mediated transient expression system in tobacco and via RNAi-experiments in hairy root tissue cultures of Euphorbia peplus. Once all enzymes are identified we aim to reconstitute the entire biosynthetic pathway in tobacco, thus providing the first total biosynthesis of ingenol mebutate in a heterologous host. The proposed project will shed light on the biosynthesis of a pharmaceutically important, understudied class of plant specialized metabolism and lay the foundation for future combinatorial and semi-synthetic synthesis of new-to-nature diterpenoids with fine-tuned biological activities.