Directed Evolution of a Cytochrome p450 for Synthesis of Artemisinic Alcohol

细胞色素 p450 的定向进化用于合成青蒿醇

基本信息

批准号：
7658151
负责人：
JARED C LEWIS
金额：
$ 2.5万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-23 至 2010-01-22
项目状态：
已结题

项目摘要

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.

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能够在大肠杆菌中将紫穗槐二烯氧化成青蒿醇或青蒿醛，这两者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选开始，以确定能够催化所需反应的 P450，并对 Arnold 小组维护的突变 P450 文库进行分析，寻找可行的催化剂。诱变和重组技术将用于根据从该筛选获得的命中生成后续突变体文库，并且该过程的迭代应提供能够选择性催化所需反应的P450。最后，优化的酶将在大肠杆菌菌株中过度表达，该菌株被改造为过量生产紫穗槐二烯，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，一种非常重要的抗疟药。由于人们对当前抗疟疾药物的耐药性日益增强，疟疾每年夺走超过一百万人的生命，并威胁着主要生活在热带环境中的大约 300-5 亿人。青蒿素是从东亚灌木青蒿中分离出来的天然产物，被誉为治疗疟疾的突破，但供应短缺和成本高昂阻碍了其广泛使用。该提案描述了一种新型细胞色素 P450 酶的定向进化，该酶能够将紫穗槐二烯氧化为青蒿醇或青蒿醛，这两者都是大肠杆菌中青蒿素半合成的关键中间体。