Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug
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Updated: Dec 10, 2025
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Michelle H Rich1,2, Abigail V Sharrock1,3, Amir Ashoorzadeh4,5
1School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
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.
