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. ? ? ?
| Lewis, Jared C; Bastian, Sabine; Bennett, Clay S et al. (2009) Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases. Proc Natl Acad Sci U S A 106:16550-5 |
