The direct objective of this grant is to establish conditions in which Escherichia coli cells can be used co-expressing multi-gram quantities of cytochrome P450 metabolite(s). The technology is based on recombinant strains of E. Coli coexpressing individual cytochrome P450s and NADPH: cytochrome P450 reductase. Fermentation conditions will be optimized such that maximal CYP450 catalytic activity is achieved. These conditions will then be incorporated into a partitioning bioreactor that is capable of simultaneous CYP450-mediated biotransformation and recovery of the metabolites. The N-demethlylation of the prodrug imipramine to the antidepressant desipramine catalyzed by the human liver CYP2Cl9-producing E. Coli strain will be used as the model reaction during the optimization phase of the proposal. This novel technology could be used to economically produce sufficient pharmaceutical metabolites for animal toxicity studies and clinical trials, and could be scaled up to produce commercial quantities of therapeutic drugs. Similarly, the catalytic power of the bioreactor could be used to perform difficult industrial chemical reactions on complex organic molecules in a much more economical and environmentally friendly fashion. Given the potential scale of the bioreactor, these efforts could also form the basis of using CYP450 catalysis in a bioremediation strategy.
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