Modular plug-and-play engineering of Klebsiella phages with dual receptor-binding proteins for efficient host range design

BACKGROUND: The high capsular diversity restricting the host range of many Klebsiella phages has driven the evolution of branched receptor-binding protein (RBP) systems as a strategy for host range expansion. These dual RBP systems offer a unique opportunity for modular engineering. However, most previous approaches lacked a standardized and systematic framework to exploit an engineering platform that enables efficient and modular reprogramming of Klebsiella podophages with dual RBP systems. RESULTS: The workflow integrates (I) the VersaTile technique for rapid assembly of chimeric RBP gene clusters, (II) Gibson assembly for in vitro genome construction, and (III) electroporation-based rebooting. By retaining one native RBP, the system ensures that a suitable host is always available for rebooting, reducing technical failure, and allowing a clear distinction between biological incompatibility and assembly issues. Using this approach, we systematically swapped full-length RBPs and receptor-binding domains (RBDs) at both positions of the dual RBP system in podophages K11 and KP32, confirming the modularity and interchangeability of structural and enzymatic domains. Advanced designs, including cross-swapping and position swapping, were successfully implemented, while attempts to graft phylogenetically distant RBPs revealed structural constraints that inform future design strategies. CONCLUSION: This work introduces a standardized, scalable, and plug-and-play framework for phage engineering that leverages the modularity of dual RBP systems. By ensuring rebooting through an unmodified RBP, the platform provides a robust foundation for systematic host range reprogramming and functional studies of RBP architecture, paving the way for rational design of therapeutic phages.