sst |
In vivo versus in vitro directed evolution of PBP-A by Julia Wessel -
Vendredi 18 septembre 2026 à 16h00 - Salle Jean-Baptiste Carnoy (B.059) - Bâtiment Carnoy - Croix du Sud - 1348 Louvain-la-Neuve -
This thesis focuses on the directed evolution of PBP-A, a penicillin-binding protein from cyanobacteria, towards a beta-lactamase by acquiring ampicillin-degrading activity. Two approaches were compared: in vivo transformation of E. coli with a PBP-A mutant library and selection on ampicillin, and in vitro creation of covalently linked units of PBP-A mutant genes with their mutant proteins, followed by screening in artificial monodisperse compartments for hydrolysis of fluorocillin (fluorogenic cephalosporin). In vivo directed evolution produced an E. coli phenotype resistant to 250 µg/mL ampicillin after six rounds. However, it is hypothesized that the selected protein, which has 13 amino acid substitutions including 5 near the active site, operates mechanistically differently from a beta-lactamase. Its activity is likely that of a fragmentase, showing only a ~10-fold increase in ampicillin turnover, far below the >10,000-fold faster hydrolysis of ampicillin by beta-lactamases. In contrast, the in vitro approach, after two rounds of screening, yielded a mutant with two amino acid substitutions that conferred E. coli resistance to 16 µg/mL ampicillin. Purified mutant protein displayed kinetics consistent with low hydrolysis activity. While during the first round of in vivo directed evolution, the selected mutant primarily showed reduced toxicity towards the host, with higher cell densities and reduced activity of the purified protein on mimics of natural metabolites and on ampicillin, by contrast, the initial round of in vitro evolution identified a mutant protein with eventually improved ampicillin hydrolysis. The comparison indicates that although in vitro evolution was more time- and cost-intensive, screening based solely on substrate hydrolysis allows the desired function to emerge without selection for alternative catalysis mechanisms that could be connected to the diminishing of harmful promiscuous activities.
Jury members :
Prof. Patrice Soumillion (UCLouvain) (Supervisor)
Prof. Bernard Hallet (UCLouvain) (Chairperson)
Prof. Corentin Claeys Bouuaert (UCLouvain) (Secretary)
Prof. Florian Hollfelder (Cambridge University)
Prof. Anastassia Vorobieva (VIB-VUB Belgium)
Pay attention : the public defense of Julia Wessel will also take place on the form of a videoconference
