Directed Evolution of a Cytochrome p450 for Synthesis of Artemisinic Alcohol

描述（由申请人提供）：该提案描述了一种新型细胞色素P450的定向进化，该细胞色素P450能够将amorphadiene氧化为青蒿醇或青蒿醛，这两种物质都是在大肠杆菌中半合成青蒿素的关键中间体。该项目将开始设计和验证筛选，以确定能够催化所需反应的P450，并分析Arnold组中维持的突变P450文库，以寻找可行的催化剂。突变和重组技术将用于基于从该筛选中获得的hit生成后续突变文库，并且该过程的迭代应该提供能够选择性催化所需反应的P450。最后，优化后的酶将在大肠杆菌菌株中过度表达，以过量生产amorphadiene，从而从单糖中获得高产量的青蒿醇或青蒿醛。一种简单的半合成途径可以将这种材料转化为青蒿素，这是一种非常重要的抗疟疾药物。疟疾每年夺去100多万人的生命，并威胁着大约3 -5亿人，这些人主要生活在热带环境中，原因是对现有抗疟疾药物的耐药性日益增强。青蒿素是一种从东亚灌木青蒿中分离出来的天然产物，被誉为治疗疟疾的一项突破，但供应短缺和成本高阻碍了其广泛使用。这一提议描述了一种新的细胞色素P450酶的定向进化，这种酶能够在大肠杆菌中将青蒿素氧化为青蒿醇或青蒿醛，这两种物质都是半合成青蒿素的关键中间体。

DESCRIPTION (provided by applicant): This proposal describes the directed evolution of a novel cytochrome P450 capable of oxidizing amorphadiene to artemisinic alcohol or artemisinic aldehyde, both of which are key intermediates in the semi-synthesis of artemesinin, in E. coli. The project will commence with the design and validation of a screen to identify P450s capable of catalyzing the desired reaction and analysis of mutant P450 libraries maintained in the Arnold group for viable catalysts. Mutagenesis and Recombination techniques will be used to generate subsequent mutant libraries based on the hits obtained from this screen, and iteration of this process should provide a P450 capable of selectively catalyzing the desired reaction. Finally, the optimized enzyme will be over-expressed in a strain of E. coli engineered to overproduce amorphadiene in order to provide high yields of artemisinic alcohol or artemisinic aldehyde from simple sugars. A simple semi-synthetic route can then be used to convert this material to artemesinin, a highly important anti- malarial. Malaria claims the lives of over one million people each year and threatens approximately 300-500 million individuals located predominately in tropical environments due to increasing resistance to current anti- malarial medicines. Artemisinin, a natural product isolated from the East Asian shrub Artemesia annua, has been heralded as a breakthrough in the treatment of malaria, but short supply and high cost have hindered its widespread use. This proposal describes the directed evolution of a novel cytochrome P450 enzyme capable of oxidizing amorphadiene to artemisinic alcohol or artemisinic aldehyde, both of which are key intermediates in the semi-synthesis of artemesinin, in E. coli.