Discovery of anti-enterococcal phage lysins from environmental metagenomes using protein embedding-based classification

Phage lysins, enzymes encoded by bacteriophages that degrade bacterial cell walls, are emerging as a promising class of antimicrobial agents. This study aimed to discover novel lysins with activity against Enterococcus species using a sequence-based metagenomic discovery pipeline. Viral metagenomic DNA was extracted and sequenced from five environmental samples originating from pig feces or sewage. Putative lysins were first predicted with SUBLYME, a protein embedding-based classifier. Subsequently, a specific protein embedding-based classifier was developed to predict lysins with potential activity against Enterococcus . A total of 8 825 candidate lysins were predicted, including 129 with potential anti-enterococcal activity. Comparative analysis revealed differences in domain architectures and physicochemical properties between lysins derived from fecal and sewage samples, suggesting distinct phage host origins. A subset of the predicted lysins was expressed in Escherichia coli , partially purified and tested for muralytic activity against three enterococcal species ( Enterococcus faecium , Enterococcus faecalis , and Enterococcus hirae ). Among the 21 expressed lysins with variable expression yields, four exhibited lytic activity against all three Enterococcus species, two were active against Ent. faecalis and Ent. hirae , and seven showed activity exclusively against Ent. hirae . Six of these active proteins contained previously unreported domain architectures, indicating that this approach can uncover structurally novel functional lysins. While this pipeline was applied to Enterococcus , it is broadly adaptable for the discovery of lysins targeting other bacterial pathogens, offering a scalable approach to expand the antimicrobial arsenal.