Toward a High-throughput Screening Platform for Directed Evolution of Enzymes That Activate Genotoxic Prodrugs

Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90,000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.