Cell-free Engineering of LPST153 Bacteriophage Receptor-Binding Proteins Enables Tunable Host-Range Modulation

Abstract Bacteriophages are increasingly explored and applied to combat antibiotic-resistant bacteria, yet rapid resistance development and narrow host range can limit their broader application. Engineering phage receptor-binding proteins (RBPs) to expand phage host ranges offers a promising strategy to overcome some of these constraints. However, current approaches remain labor-intensive and difficult to control. Here, we use a cell-free phage engineering workflow to rapidly modify and characterize the RBP of the Salmonella phage LPST153. Using the PHEIGES platform and engineering targeted RBP mutations, we identified residues 389 and 402 within the tail fiber as determinants of host specificity. The G389R substitution significantly increased phage adsorption (%), reaching 65% on E. coli BW25113 and 51% on the LPS-truncated E. coli BW25113 ΔwaaC, compared with 27% and 5.7% for the wild-type LPST153, respectively. Targeted engineering of tail fiber residue 389 generated phage variants with distinct host-range phenotypes, with a G389T variant that exhibited an alternative infection profile relative to G389R. Together, these findings demonstrate that minimal, site-specific modifications of RBPs can substantially modulate LPST153 host specificity. This work establishes a framework for rapid engineering of phages with tailored host ranges, advancing phage-based antimicrobial strategies.