Biotechnology Letters, Vol.43, No.1, 203-211, 2021
Directed evolution of theB. subtilisnitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug
Objectives To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. Results Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitizeE. colihost cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved ink(cat)/K(M)with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved ink(cat)/K(M)versus wild type YfkO in converting CB1954 to a genotoxic drug. Conclusions The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.