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 phages 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.