基于稳定同位素示踪研究印楝素生物合成途径

The secondary metabolite azadirachtin is a tetranortriterpenoid obtained from the neem tree (Azadirachta indica). It is a natural insecticide, known to affect feeding, growth, reproduction and metamorphosis of the insect pests. Because of the broad-spectrum control of insects and the relatively low nontarget toxicity it has been widely used in agriculture. However, the supply of plant-derived azadirachtin is unstable, resulting in shortages and price fluctuations. A stable source of affordable azadirachtin is required. It is a highly oxygenated and complex molecule, which makes its chemical synthesis difficult as well as uneconomical. Commercial production of chemically complex molecules like azadirachtin can be made feasible via in vitro cultivations. But the biggest challenge is that the biosynthetic pathway of azadirachtin is poorly understood. The biosynthesis of higher-order azadirachtin, such as precursors, intermediate products, remains unclear, as well.. Supported by NSFC, the hairy root cultures will be established from the stem and leaf explants of neem following infection with Agrobacterium rhizogenes. Then, some stable-isotope labeled precursors and/or intermediate products will be administered to the neem hairy root, and the labeled metabolites will be tracked and measured with an accompanying high throughput analytical method based on HPLC-Q-TOF-MS. Furthermore, the specificity of inhibitors, lovastatin, fosmidomycin, and fluridon will be fed in the neem hairy root to investigate which pathway dominates in the biosynthesis of azadirachtin.. Based on these findings, we hope to clarify the biosynthetic origin and metabolic pathway of azadirachtin. It is essential to develop a process for the production of the azadirachtin by bioengineering techniques, and pave the way for an industrial process capable of supplementing the world supply of azadirachtin from a second source independent of the uncertainties associated with botanical production.

印楝素是从印楝中提取的一类四环三萜类化合物，具有拒食、忌避和干扰昆虫生长发育等多种活性，市场需求巨大。利用代谢工程手段调控印楝素生物合成，提高离体培养体系中印楝素产率在生产上具有重要意义。但是，目前印楝素的生物合成途径和机理研究基本处于空白，推测的合成途径特别是下游过程存在许多不确定点，缺乏实验依据，严重阻碍了印楝素生物合成及代谢调控研究的深入。申请项目拟以印楝毛状根为供试材料，采用稳定同位素示踪技术，结合HPLC-Q-TOF-MS检测，系统研究印楝素生物合成途径中存在的主要不确定问题(从2，3-环氧鲨烯到印楝素的合成步骤、中间产物和途径)，明确印楝素生物合成的主要中间产物，阐明印楝素生物合成的主导途径，推导印楝素在细胞中的合成过程。研究结果对于印楝素生物合成途径改造和代谢调控，构建印楝素高效生物反应器特别是利用微生物异源生产印楝素的高效生物反应器，均具有重要的科学意义和广阔的应用前景。

印楝素是从印楝中提取的一类四环三萜类化合物，具有拒食、忌避和干扰昆虫生长发育等多种生物活性，可杀灭蝗虫、小菜蛾等8目200余种农林、仓储和卫生害虫，已在中国、美国、加拿大等多个国家登记使用，被认为是完美的杀虫剂，市场需求巨大。印楝素主要存在于印楝种仁中，其主要活性成分为印楝素A和B，分别约占80%和20%。由于结构非常复杂，化学合成尚无法实现产业化生产，因此利用代谢工程手段调控印楝素生物合成，提高离体培养体系中印楝素产率在生产上具有重要意义。但是，目前印楝素的生物合成途径和机理研究基本处于空白，推测的合成途径特别是下游过程存在许多不确定点，缺乏实验依据，严重阻碍了印楝素生物合成及代谢调控研究的深入。. 本项目通过构建印楝素A无细胞合成体系，采用疑似前体物质添加、广泛靶向代谢组分析及全长转录组测序分析等技术，实验证实了印楝素A生物合成的6种前体/中间产物，包括2，3-环氧鲨烯、大戟二烯醇、丁酰鲸鱼醇、nimbin、脱乙酰茄碱苷和茄碱苷，否定了文献推测的1种合成前体(羊毛甾醇)，推导、绘制了印楝素A生物合成途径，为进一步阐明印楝素生物合成途径提供了依据。研究结果对于印楝素生物合成途径改造和代谢调控，构建印楝素高效生物反应器特别是微生物异源生产印楝素的高效生物反应器，均具有重要的科学意义和广阔的应用前景。