Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug

Biotechnol Lett. 2021 Jan;43(1):203-211. doi: 10.1007/s10529-020-02992-0. Epub 2020 Aug 26.


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 sensitize E. coli host 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 in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM 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.

Keywords: Cancer gene therapy; Error-prone PCR; GDEPT; Niclosamide; Nitroimidazole; Nitroreductase.

MeSH terms

  • Antineoplastic Agents / metabolism
  • Aziridines / metabolism
  • Bacillus subtilis* / enzymology
  • Bacillus subtilis* / genetics
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Directed Molecular Evolution / methods*
  • Enzyme Therapy
  • Nitroimidazoles / metabolism*
  • Nitroreductases / genetics*
  • Nitroreductases / metabolism


  • Antineoplastic Agents
  • Aziridines
  • Bacterial Proteins
  • Nitroimidazoles
  • SN33623
  • tretazicar
  • Nitroreductases