Zhu, X.; Somerville, V.; Shi, R.; Rakotoharisoa, R. V.; Chica, R. A.; Moineau, S.; Oechslin, F.

Bacteriophages release progeny by producing endolysins that degrade the bacterial cell wall. While the frequent horizontal transfer of endolysins suggests substantial evo-lutionary plasticity, the mechanisms by which these enzymes adapt to new phage-host contexts remain poorly understood. Here, we investigated the evolutionary dy-namics and structural mechanisms governing endolysin adaptation following experi-mental evolution of a chimeric phage generated via heterologous endolysin exchange between phages infecting different hosts. Using replicate experimental evolution and time-resolved PacBio sequencing, we iden-tified a dominant, highly reproducible adaptive trajectory characterized by the stepwise fixation of three key mutations. This constrained mutational order correlated with in-cremental gains in enzymatic activity, reflecting a rugged yet predictable fitness land-scape. High-resolution structural analyses revealed that these substitutions lie exclu-sively outside the catalytic site; instead, they enhance substrate recognition through electrostatic tuning, optimized hydrophobic packing, and local conformational refine-ment, resulting in significantly higher binding affinity. While the adaptive trajectory was largely conserved, one replicate followed an alterna-tive path, highlighting the interplay between selection and historical contingency. Adap-tation was further shaped by a functional trade-off, whereby increased lytic activity on the novel host was accompanied by reduced activity on the ancestral host, consistent with antagonistic pleiotropy. Genome-wide sequencing additionally identified a com-pensatory mutation in a lytic transglycosylase, suggesting coordinated evolution of the broader lysis machinery. Together, these results demonstrate that endolysins evolve through reproducible adaptive walks constrained by structure, selection, and trade-offs, providing a mechanistic framework for understanding enzyme evolution and in-forming rational protein engineering.