School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
BiOrbic, Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland.
Biotechnol Lett. 2021 Jan;43(1):203-211. doi: 10.1007/s10529-020-02992-0. Epub 2020 Aug 26.
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.
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 k/K with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in k/K versus wild type YfkO in converting CB1954 to a genotoxic drug.
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.
利用定向进化提高 YfkO 介导的 5-硝基咪唑 PET 探针 SN33623 的还原活性，同时不损害抗癌前药 CB1954 的转化。
使用易错 PCR、纯化选择和在 DNA 损伤定量 GFP 报告菌株中的流式细胞分选进行了两轮迭代，鉴定出三种 YfkO 变体，能够使大肠杆菌宿主细胞对 SN33623 的敏感性提高至少 2.4 倍，低于天然酶。其中两种变体能够以功能性形式进行纯化，体外测定表明，与野生型 YfkO 相比，这些变体与 SN33623 的 k/K 值提高了两倍和四倍。偶然的是，与野生型 YfkO 相比，活性更高的变体在将 CB1954 转化为致基因突变药物方面的 k/K 值也提高了近四倍。
YfkO 的主导进化变体对 PET 成像探针 SN33623 和 CB1954 前药的增强激活，为提高酶-前药疗法提供了前景。
