Molecular Cloning and Functional Analysis of Holin and Endolysin From <i>Escherichia</i> Phage UE‐M6 as Potential Antibacterial Agents

The rise of antibiotic-resistant bacteria has intensified the search for alternative antibacterial strategies. Bacteriophage (phage) therapy is gaining attention as a promising approach, utilizing phage-derived proteins such as holins and endolysins to combat bacterial infections. In this study, the endolysin (UE-lysin) and holin (UE-holin) genes from Escherichia phage UE-M6 were characterized, and their antimicrobial activity was evaluated. In silico analysis revealed that UE-lysin has a modular architecture, with the N-terminal enzymatic activity domain that contains an N-acetylmuramidase of the glycoside hydrolase family GH108, and the C-terminal cell wall-binding domain that contains the peptidoglycan binding family PG_binding_3 domain. UE-holin was predicted to belong to class II holins, featuring two transmembrane helices. Furthermore, the genes encoding the UE-lysin and UE-holin were cloned and their expression optimized in Escherichia coli BL21 (DE3). The purified recombinant UE-lysin (27 kDa) and UE-holin (15 kDa) exhibited antibacterial activity against the E. coli host strain PSU-5266 (UE-17). The addition of the outer membrane permeabilizer ethylenediaminetetraacetic acid further enhanced their activity. Notably, the combined application of UE-holin and UE-lysin demonstrated greater antibacterial efficacy than either enzyme alone, highlighting a synergistic effect. Furthermore, UE-lysin and UE-holin exhibited high lytic activity against E. coli, Bacillus, and Staphylococcus aureus strains, underscoring their potential as candidates for treating both Gram-negative and Gram-positive bacterial infections.