17 janvier 2022
Seminar room of the LIBST, Place Croix du Sud 4-5 - will also take place in video conference
The catalytic activity of a DD-peptidase impairs its evolutionary conversion into a beta-lactamase by Gol Mohammad DORRAZEHI
Pour l’obtention du grade de Docteur en sciences
Despite the wealth of knowledge on enzymes, their evolutionary emergence is still poorly understood. The classical view that enzymes are highly precise molecular machines has been replaced with the fact that enzymes, as ensemble of conformers, are able to carry secondary activities. Although these unwanted activities are less intense compared to what the enzymes have been specialized for, they may interfere with the metabolism and impose a cost to the cell. Therefore, in in-vivo directed evolution of enzymes, restraining effects of fitness costs on the host organism would become beneficial for success.
Through successes and failures on directed evolution of a D-alanyl-D-alanine-peptidase from Thermosynechococcus elongatus, named PBP-A, into a beta-lactamase, we have accumulated evidences that this conversion follows a counter-intuitive evolutionary trajectory that is hindered by fitness costs on the host bacteria. In this study, we envisioned to further explore the molecular origins of this fitness cost, to evaluate molecular mechanisms that may contribute to fitness improvement and then to subject different variants of PBP-A to directed evolution under the fitness-improving parameters.
One of the activities that was found to be effective on peptidoglycan of the host Escherichia coli cells, was DD-endopeptidase activity. Analysis of the peptidoglycan of the E. coli cells under expression of PBP-A showed a less-crosslinked peptidoglycan as the main origin of the fitness cost. We found that reducing the expression level of PBP-A, lowering its isoelectric point and avoiding acidic pH conditions, significantly improves the fitness.
Furthermore, introduction of a disulfide bridge that is conserved among class A serine β-lactamases, into PBP-A, showed significant fitness improvement and characterization of the enzymes revealed a severe impact of the disulfide bridge on the DD-peptidase activity, without compromising the reactivity against penicillin. Suggesting that disulfide bonds may play an intragenic epistatic role along evolutionary trajectories to secure the enzymes against deleterious effects of gaining the hydrolytic power.
To benefit from the single expression in directed evolution, we developed a novel scarless method for building chromosomal libraries in genome of E. coli, based on a combination of CRISPR-Cas9 cleavage and λ-Red recombination systems. This method allows generation of chromosomal libraries up to million independent clones.
The results of this thesis clearly demonstrates that fitness cost issues are multi-factorial and that an initial careful attention to every biological parameter may be crucial for the success of a directed evolution campaign.
Jury members :
- Prof. Patrice Soumillion (UCLouvain), supervisor
- Prof. François Chaumont (UCLouvain), chairperson
- Prof. Pascal Hols (UCLouvain), secretary
- Prof. Waldemar Vollmer (Newcastle University, UK)
- Prof. Florian Hollfelder (University of Cambridge, UK)
Pay attention :
The public defense of Gol Mohammad Dorrazehi scheduled for Monday 17 January at 2:00 p.m will take place in the form of a video conference