| Current studies on drug metabolism are often hindered by the ability to obtain the drug metabolites in a pure form in suitable quantities for detailed study. Traditional methods for producing metabolites involve reconstituted enzyme systems that have limited half lives and result in low yields, or by chemical synthesis which is time consuming and expensive. Many microbes are able to produce the same drug metabolites as humans, but the strains are often uncharacterized and have poor growth characteristics. Our approach to improving drug metabolism studies is to identify the cytochrome P450 enzymes responsible for the observed drug metabolism ability of Actinoplanes sp. ATCC 53771 and, through metabolic engineering, create a system for drug metabolism in an industrially relevant host organism such as Escherichia coli. We have identified and characterized the drug metabolism abilities of two P450s from Actinoplanes, CYP105AA8 and CYP105E4. Both enzymes have been expressed and purified from E. coli and found to bind the drugs rapamycin and diclofenac. The metabolism of diclofenac by CYP107E4 has been confirmed in a reconstituted system using electron transport enzymes from spinach. Work is underway to identify a bacterial electron transport system that can support CYP107E4 activity. Additionally, an improvement to active P450 expression through the coexpression of the hemA gene from R. capsulatus has been studied. This work has advanced the understanding of Actinoplanes P450s responsible for drug metabolism, and is the first step towards creating a system for high-level production of drug metabolites. |
