| Summary: | Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting <i>Klebsiella pneumoniae</i>. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of <i>Klebsiella pneumoniae</i> could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP–linker–CBD–EAD) and the inverse configuration (CBD–EAD–linker–OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.
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