(64b) Engineering Bacteria for the Production of Human-Like Metabolites of Diclofenac

Drug metabolism describes the process in which a drug is degraded or modified for elimination from the body. These degradation products, or metabolites, can be characterized by varying toxicity profiles compared to the original drug. As such, metabolites must also be studied in an effort to accurately depict the full effects of a specific drug. Microbial models for human drug metabolism have been an effective method to produce metabolites in quantities suitable for testing and are more attractive than other methods such as chemical synthesis. In both microbes and mammals, cytochrome P450 enzyme activity is responsible for the breakdown of drugs into metabolites. The bacterium, Actinoplanes sp. has been found to have similar drug metabolism profile for several drugs as humans. However, further engineering towards scaling up metabolite production has proven difficult. To address these concerns, we are working to identify the cytochrome P450s responsible for the biotransformation of these drugs for production of specific metabolites in order to optimize expression through metabolic engineering for increased production of drug metabolites.

To date we have identified at least 14 unique P450 genes in Actinoplanes, five of which we have the entire ORF. We have successfully cloned, expressed, and purified three of the P450s in the active form. In order to characterize the ability of each P450 to metabolize a range of drugs we are using a combination of heterologous expression as well as gene disruption. The two methods are useful when combined to work around the lack of native redox partners associated with the P450s in E. coli as well as to characterize genes not expressed well. Later optimization of redox partners will be possible once we have identified the specific P450s we are interested in working with to develop a system for in vivo drug metabolism in E. coli.

We have identified two of the P450s as possible targets for the observed drug metabolism in Actinoplanes. Substrate binding assays show one of the P450s binds diclofenac and lovastatin, while another P450 binds rapamycin. Studies are currently underway to identify the metabolite profile of each P450 using LC-MS. In summary, we have identified P450 genes in Actinoplanes that are responsible for the observed drug metabolism of diclofenac, and are working towards engineering a system in E. coli for the production of drug metabolites.