Understanding and Exploiting the Biosynthesis of Diterpenoids in Euphorbia peplus

萜烯是最大和最多样化的一类特殊代谢物，包括世界卫生组织（世卫组织）列出的基本药物，如紫杉醇和青蒿素。多氧戊烯醇酯是由植物大戟产生的一种临床上重要的二萜类化合物。2012年，它被美国食品和药物管理局（FDA）批准用于治疗光化性角化病（一种癌前皮肤状况）。目前，它正在进一步评估治疗基底细胞癌和激活人体内潜伏的HIV储存库。尽管ingenol mebutate的药用重要性日益增加，但这种有价值的代谢物的供应有限，因为它仅在植物中少量生产，目前还没有可行的全合成报道。在这项研究中，我们旨在应用“多组学”方法来阐明大戟中戊烯醇酸的生物合成途径。基因组和RNA测序数据将获得和分析，以确定参与生物合成壬烯醇酸酯和伴随产生的二萜的基因。候选基因将在农杆菌介导的烟草瞬态表达系统中进行活性测试，并在大戟毛状根组织培养中进行rnai实验。一旦确定了所有的酶，我们的目标是重建烟草中的整个生物合成途径，从而提供第一个在异源宿主中全生物合成戊烯醇酸酯。该项目将揭示具有重要药理意义的、尚未被充分研究的一类植物特化代谢的生物合成，并为未来组合和半合成具有微调生物活性的新天然二萜奠定基础。

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.