巨大戟醇是一种结构复杂的大环二萜类成分，微量分布于少数大戟科植物中，其不仅具有显著的抗癌抗艾滋等活性，还是新药Picato的原料，但化学合成或植物提取的产率极低，基于机理明确的生物合成是解决巨大戟醇低成本生产难题的最佳途径。本项目拟针对目前巨大戟醇生物合成途径仍存在较大争议的问题，在前期研究基础上，通过愈伤组织碳13前体饲喂、氧18氧气培养和整株植物碳13二氧化碳培养，分离得到经稳定同位素标记的巨大戟醇及其中间体，进行核磁共振及质谱结构解析，确定其标记位点、同位素丰度及达到稳定态速率，依次阐明巨大戟醇的碳骨架来源、氧原子来源和合成机理；运用激光显微切割-液质联用技术，按器官—组织—细胞次序，逐步定位续随子中巨大戟醇的合成位点；并运用无细胞酶促反应对上述结论进行验证，阐明巨大戟醇的生物合成途径和位点，为巨大戟醇的生物合成奠定理论基础，为其他复杂天然产物的生物合成研究提供参考。

Ingenol, a complex macrocyclic diterpenoid possessing important anticancer and anti-HIV activity, only distributes in several Euphorbiaceae plants with trace content. And it is also the current industrial source of Picato, a first-in-class drug for the treatment of actinic keratosis. However, the problem of current supply of ingenol is that either the isolation from its natural source or the semisynthesis is fraught with difficulties for the very low yield. Completing elucidation of the biosynthetic pathways and establishing a metabolic engineering and synthetic biology approaches for ingenol shall be a novel promising way to solve the problem of supply. To elucidate the biosynthetic pathway of ingenol, we have established callus tissue of Euphorbia lathyris for ingenol. On this basis, to clarify the source of carbon skeleton, origin of oxygen, and biosynthetic mechanism of ingenol gradually, 13C-labeled precursors and 18O2 will be administered to plants and a retrobiosynthetic approach using 13CO2 for labeling will be used. To pinpoint the biosynthetic sites of ingenol from organ and tissue to cell, a method of microchemical analysis using LMD-LCMS will be used to study their cell-specific localization in E. lathyris. Additionally, to prove the biosynthetic pathway and localization of ingenol, cell free enzyme incubations with different supposed precursors will be systemically investigated. The discovery of the biosynthetic pathway and localization of ingenol may pave the way for biotechnological production of ingenol, and provide valuable reference to elucidate the biosynthetic pathways of other complicated natural products.