Artemisinin Biosynthesis: Role of Reactive Oxygen

说明(申请人提供)：青蒿素(AN)，由植物青蒿素(Artemisia annua L.)生产，已被证明是一种治疗疟疾的药物，也可用于治疗多种细菌、病毒和寄生虫病以及癌症。虽然AN具有很高的治疗价值，但它的生产只有在从田间种植的植物中收获时才具有成本效益。了解这种重要的萜类药物的生物合成控制对于满足日益增长的全球需求至关重要。最近我们发现，处于活性氧胁迫下的植物会产生更高水平的AN及其前体。由于这一现象有可能将各种已建立的激发子连接在一个统一的模型中，我们建议将活性氧胁迫作为生物合成的关键调节因素进行研究。我们的三个目标是：1.研究植物在不同来源的氧化胁迫下的早期(HMGR，FPS)和晚期专用(ADS，CYP71AV1，DBR2，ALDH1)基因的相对表达水平，包括光、干旱和化学诱导的氧化。这些数据将有助于确定ROS诱导产生的转录基础。2.比较植物在正常和氧气胁迫条件下的代谢特征和氧化还原状态。通过测量关键的代谢中间体，我们可以形成一个初步的代谢平衡图，显示ROS诱导的扰动的关键点，以帮助进一步阐明ROS在生物合成和代谢物平衡控制的假定的非酶最后步骤中的作用，而通过转录分析很难识别这一点。3.在土壤种植的植物中研究和演示一种通过生物和非生物手段诱导氧化胁迫来增加产量的过程，这将允许优化AN的生产，同时允许植物最大限度地生长。这将进一步关联潜在的简单方法，这些方法可以立即用于诱导土壤植物中过量产生青蒿素的轻微氧化应激。这些结果将加深我们对青蒿素在青蒿素中生物合成的基本了解，并将进一步提高我们提高青蒿素产量以满足日益增长的治疗需求的能力。 与公共卫生相关：我们的提案中涉及的疟疾和被忽视的热带病造成5亿人感染和200多万人死亡。青蒿素已被证明对所有这些疾病都有效，但这种药物严重短缺，甚至无法治疗疟疾。我们对活性氧的研究将有助于更好地了解生物合成途径的最后几步，从而增强在植物中增加药物产量的潜力。

DESCRIPTION (provided by applicant): Artemisinin (AN), produced by the plant Artemisia annua L., is a proven therapeutic for treating malaria and also for treatment of a large range of bacterial, viral and parasitic diseases, and cancers. While AN has high value as a therapeutic, its production is only cost effective when harvested from field grown plants. Understanding biosynthetic control of this important terpenoid drug is crucial to meeting the growing worldwide demand. Recently we showed that plants subjected to reactive oxygen stress produce higher levels of AN and its precursors. Since this phenomenon has the potential to link a variety of established AN elicitors in a unified model, we propose investigating reactive oxygen stress as a key regulator of AN biosynthesis. Our 3 objectives are: 1. Investigation of the relative expression levels of early (HMGR, FPS) and late dedicated (ADS, CYP71AV1, DBR2, ALDH1) genes as plants are subjected to diverse sources of oxidative stress, including photo, drought, and chemically induced oxidation. These data would serve to identify a transcriptional basis for ROS induction of AN production. 2. Compare metabolic profiles and the redox state of plants under normal and oxygen stressed growing conditions. Measuring key metabolic intermediates allows us to form a preliminary metabolic balance map showing key points of change upon ROS-induced perturbations to help further elucidate the ROS role in the putative non-enzymatic last step of AN biosynthesis and metabolite balance control not readily identifiable through transcript analysis. 3. Investigate and demonstrate in soil-grown plants a process for increasing AN production through the induction of oxidative stress by both biotic and abiotic means that will allow for optimizing production of AN while simultaneously allowing for maximum plant growth. This will further correlate potentially simple methods that could be employed immediately to induce mild oxidative stress for over production of artemisinin in soil-grown plants. These results will further our fundamental understanding of the biosynthesis of artemisinin in A. annua and will further our ability to enhance its production to meet the ever growing therapeutic demand. PUBLIC HEALTH RELEVANCE: Malaria and the neglected tropical diseases addressed in our proposal account for >500 million infections and upwards of 2 million deaths. Artemisinin has been shown to be effective against all of these diseases, but the drug is in desperately short supply to treat even malaria. Our studies on reactive oxygen will facilitate a better understanding of the last steps in the biosynthetic pathway thereby enhancing the potential to increase the drug produced in the plant.