Molecular Cloning and Functional Analysis of Holin and Endolysin From Escherichia 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.