Soumillion, PatriceGhislain, MichelLernoux, FrançoisFrançoisLernoux2025-05-142025-05-142025-05-142023https://hdl.handle.net/2078.2/33809Although our knowledge of enzymes is vast, our understanding of the relationship between enzyme structure and function is still very limited, and attempts at engineering them rationally are often doomed to failure. In contrast, the directed evolution approach consists of allowing natural selection to take place among a large number of mutants, allowing those that are functional to emerge thanks to the competitive advantage they have acquired. In recent times, Gol Mohammad Dorrazehi undertook an informal directed evolution experiment involving libraries of PBP-A variants expressed within Escherichia coli. Throughout this experiment, these libraries were exposed to sublethal concentrations of ampicillin for a duration of slightly less than 20 days. Remarkably, a mutant emerged from the PBP-A L158E variant library displaying an estimated minimum inhibitory concentration of approximately 1500 µg/mL. Subsequent analysis indicated that by the conclusion of the evolutionary trajectory, PBP-A was either no longer responsible for conferring resistance, or was significantly altered in its role. Instead, substantial chromosomal mutations were identified, principally within genetic loci such as marR, ftsi, and most notably, ampC, serving as explanatory factors for the observed resistance phenomenon. In the course of this study, this experiment was repeated, augmented and improved, to continue to learn both about the emergence of ampicillin resistance in E. coli, but also about the possibility of PBP-A conversion from DD-peptidase activity to beta-lactamase activity, a conversion that has failed in all previous attempts. The DNA sequences of the pbpA copies possessed by the mutants that have emerged from the evolutionary process reveal mutations in the Ω-loop, a crucial distinction between PBP-A and class A β-lactamases, otherwise close phylogenetically. Chromosome sequencing of the mutants revealed mutations in the marR (typically observed in many antibiotic resistant bacteria), acrR (an efflux pump repressor) and yfhM (unknown function) loci, as well as deletion of the main protagonists of the elongasome and RhsA (unknown function). These intriguing mutations suggest that further investigations are desirable in the future. Our observations enabled us to put forward a hypothesis of importance concerning the effect of freezing PBP-A libraries on the diversity they contain. Experiments need to be carried out to confirm this hypothesis, which, if it proves to be true, will enable this experiment to be greatly improved in the future.Escherichia coliDirected evolutionAmpicilline resistanceAntibiotics resistanceBeta-lactamasePenicillin-binding-proteintext::thesis::master thesisthesis:43447