Directed Evolution of a Cytochrome p450 for Synthesis of Artemisinic Alcohol

描述(申请人提供)：本提案描述了一种新型细胞色素P450在大肠杆菌中的定向进化，该细胞色素P450能够将氨基二烯氧化为青蒿醇或青蒿醛，这两种物质都是青蒿素半合成的关键中间体。该项目将从设计和验证筛查开始，以识别能够催化所需反应的P450，并分析Arnold集团保存的突变P450文库以寻找可行的催化剂。突变和重组技术将被用来根据从这个筛选获得的点击来产生后续的突变体文库，并且这个过程的迭代应该提供能够选择性地催化所需反应的P450。最后，优化后的酶将在一株过量生产甲二烯的大肠杆菌中过度表达，以便从单糖中提供高产率的青蒿醇或青蒿醛。然后，可以使用简单的半合成路线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。疟疾每年夺走100多万人的生命，由于对当前抗疟疾药物的抗药性日益增强，威胁着主要位于热带环境中的大约3-5亿人。青蒿素是从东亚灌木青蒿素中分离出来的天然产物，曾被誉为治疗疟疾的突破性进展，但供应短缺和高昂的成本阻碍了其广泛使用。这项建议描述了一种新型的细胞色素P450酶在大肠杆菌中的定向进化，该酶能够将氨基二烯氧化为青蒿醇或青蒿醛，这两种物质都是青蒿素半合成的关键中间体。

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.