Deep Antimicrobial Activity and Stability Analysis Inform Lysin Sequence-Function Mapping

Antibiotic-resistant infectious disease is a critical challenge to human health. Antimicrobial proteins offer a compelling solution if engineered for potency, selectivity, and physiological stability. Lysins, which lyse cells via degradation of cell wall peptidoglycans, have significant potential to fill this role. Yet, the functional complexity of antimicrobial activity has hindered high-throughput characterization for discovery and design. To dramatically expand knowledge of the sequence-function landscape of lysins, we developed a depletion-based assay for library-scale measurement of lysin inhibitory activity. We coupled this platform with a high-throughput proteolytic stability assay to assess the activity and stability of ∼5 × 10⁴ lysin catalytic domain variants, resulting in the discovery of a variant with increased activity (70 ± 20%) and stability (7.2 ± 0.4 °C increased midpoint of thermal denaturation). Ridge regression of the resulting data set demonstrated that libraries with a higher average Hamming distance better informed pairwise models and that coupling activity and stability assays enabled better prediction of catalytically active lysins. The best models achieved Pearson’s correlation coefficients of 0.87 ± 0.01 and 0.61 ± 0.04 for predicting catalytic domain stability and activity, respectively. Our work provides an efficient strategy for constructing protein sequence-function landscapes, drastically increases screening throughput for engineering lysins, and yields promising lysins for further development.