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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：
7479375
负责人：
JARED C LEWIS
金额：
$ 4.68万
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 2010-07-22
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能够将紫穗槐二烯氧化为青蒿醇或青蒿醛，这两者都是青蒿素半合成的关键中间体，在E.杆菌该项目将从设计和验证筛选开始，以鉴定能够催化所需反应的P450，并分析Arnold组中保存的突变P450文库中的可行催化剂。诱变和纯化技术将用于基于从该筛选获得的命中物产生随后的突变体文库，并且该过程的重复应提供能够选择性催化所需反应的P450。最后，优化的酶将在E.大肠杆菌工程改造以过量生产紫穗槐二烯，以便从单糖提供高产率的青蒿醇或青蒿醛。一个简单的半合成路线可以用来将这种物质转化为青蒿素，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，并且由于对当前抗疟疾药物的耐药性增加而威胁主要位于热带环境中的大约3 - 5亿个体。青蒿素是从东亚灌木青蒿中分离出来的一种天然产品，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。该提议描述了一种新的细胞色素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，这两者都是青蒿素半合成的关键中间体，在E.杆菌