Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation

Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage.  The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec.  In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound 4-nitroimidazole was found to be capable of detecting nitroreductase expression at the level of a single colony. While no novel nitroreductase enzymes were discovered in the scope of this work, the preliminary results are encouraging that a screening strategy centred on 4-nitroimidazole in particular could successfully do so in the near future.