Gol Mohammad Dorrazehi, UCL - SST / LIBST - Louvain Institute of Biomolecular Science and Technology, UCL - Faculty of Sciences, Soumillion, Patrice, Chaumont, François, Hols, Pascal, Vollmer, Waldemar, and Hollfelder, Florian
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 (DD-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 and to evaluate molecular mechanisms that may contribute to fitness improvement. For that, we initially characterized the PBP-A enzyme by in vitro assays and found that it carries several activities, beside the previously reported DD-carboxypeptidase (DD-CPase) activity. One of the activities that was found to be effective on peptidoglycan of the host Escherichia coli cells, was DD-endopeptidase (DD-EPase) 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. To overcome this cost, we examined several parameters including expression level, pH dependence and potential electrostatic interaction between PBP-A and the peptidoglycan. We found that reducing the expression level of PBP-A, lowering its isoelectric point and avoiding acidic pH conditions, significantly improves the fitness cost. We further investigated the introduction of a disulfide bridge, which is conserved in many class A serine β-lactamases, in PBP-A. Fitness improvement of disulfide-bonded PBP-A was significant and analysis of peptidoglycan indicated that the interfering activity on peptidoglycan was significantly reduced. Further in vitro characterization of the enzymes shows a severe impact of the disulfide bridge on the DD-peptidase activity, without compromising the reactivity against penicillin. Based on these results we propose that disulfide bonds played an intragenic (positive) epistatic role, along evolutionary trajectories from PBPs to β-lactamase, to secure the enzyme against deleterious effects of gaining the hydrolytic power, highlighting an overlooked role of disulfide bonds to impact the structure-function relationship of proteins by constraining the conformational dynamics. To benefit from the single-copy chromosomal expression in the directed evolution approach, 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 system. This method allows generation of chromosomal libraries up to million independent clones. We then subjected different variants of PBP-A to directed evolution under the fitness-improving parameters. Although all the attempts to convert PBP-A to a beta-lactamase were not successful, a slightly higher resistance was achievable from the libraries of disulfide-bonded PBP-A, that was strangely not stable upon re-growing of the resistant population. The results of this thesis clearly demonstrate 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, especially when dealing with potentially dangerous enzymatic activity such as a hydrolase. (SC - Sciences) -- UCL, 2022