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：
7220822
负责人：
JARED C LEWIS
金额：
$ 4.48万
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的定向演化，能够将二苯乙烯氧化为artimisisic醇或氨基甲性醛，这两者都是Artemesinin的半合成中的关键中间体，在大肠杆菌中。该项目将从屏幕的设计和验证开始，以识别能够催化Arnold组中的突变P450文库的所需反应和分析的P450，用于可行的催化剂。诱变和重组技术将根据从此屏幕获得的命中来生成随后的突变库，并且该过程的迭代应提供能够选择性地催化所需反应的P450。最后，优化的酶将以高出生产的大肠杆菌菌株的过度表达，以便从简单糖中提供高产量的artemisicanic醇或artemisisicalic醛。然后，可以使用简单的半合成途径将这种材料转换为阿耳震宁，这是一种非常重要的抗臭虫。疟疾每年宣称一百万人口的生命，并威胁着大约300亿人，主要是在热带环境中，由于对当前抗毒害药物的抵抗力增加。阿尔雷木辛（Artemisinin）是一种从东亚灌木洋努亚（Artemesia Annua）隔离的天然产品，已被认为是疟疾治疗的突破，但短暂的供应和高成本阻碍了其广泛使用。该提议描述了一种新型细胞色素P450酶的定向演变，该酶能够将非甲二烯氧化为artemisicic饮酒或artemisicinic醛，这两种都是甲r小毛蛋白的半合成中的关键中间体。