Artemisinin Biosynthesis: Role of Reactive Oxygen

描述（由申请人提供）：青蒿素（AN），由植物青蒿（Artemisia annua L.）产生，是一种经证实的治疗疟疾的药物，也可用于治疗各种细菌、病毒和寄生虫病以及癌症。虽然AN作为治疗剂具有很高的价值，但其生产只有在从田间生长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，植物受到活性氧胁迫产生较高水平的AN及其前体。由于这种现象有可能连接在一个统一的模型中的各种既定的AN激发子，我们建议调查活性氧应激作为AN生物合成的关键调节器。我们的三个目标是：1。研究了植物受到不同来源的氧化胁迫（包括光、干旱和化学诱导的氧化）时早期（HMGR、FPS）和晚期专用（ADS、CYP71AV1、DBR2、ALDH1）基因的相对表达水平。这些数据将用于确定ROS诱导AN产生的转录基础。2.比较正常和氧胁迫生长条件下植物的代谢概况和氧化还原状态。测量关键的代谢中间体使我们能够形成初步的代谢平衡图，显示ROS诱导的扰动后的关键变化点，以帮助进一步阐明ROS在AN生物合成和代谢物平衡控制的推定非酶最后一步中的作用，而这一步通过转录本分析不容易识别。3.研究并证明在土壤生长的植物中，通过生物和非生物手段诱导氧化应激来增加AN产量的过程，这将允许优化AN的生产，同时允许最大的植物生长。这将进一步关联潜在的简单方法，可以立即用于诱导土壤生长植物中青蒿素过度生产的温和氧化应激。这些结果将进一步加深我们对青蒿素在A. annua，并将进一步提高我们的能力，以提高其生产，以满足不断增长的治疗需求。 公共卫生相关性：我们提案中提到的疟疾和被忽视的热带疾病造成5亿多人感染，200多万人死亡。青蒿素已被证明对所有这些疾病都有效，但即使是治疗疟疾，这种药物也极度短缺。我们对活性氧的研究将有助于更好地了解生物合成途径中的最后步骤，从而提高植物中药物产量的潜力。

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.