Personalized Production, Non-Genomic Engineering, and Time-Resolved Proteomics of Therapeutic Phages for Biohazardous and Multidrug-Resistant Bacteria

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is currently hampered by a lack of rapid phage production and efficient bioengineering and characterization capabilities. Thus, we have developed a comprehensive cell-free platform for time-efficient and personalized manufacturing, transient bioengineering, and proteomic characterization of a broad spectrum of phages. We validated 40  hypothetical proteins, including non-structural proteins, from T7 and CLB-P3 phages using high-resolution mass spectrometry and identified a new late-expressing gene cluster. Just a few microliters of the one-pot reaction produce effective doses of phages against enteroaggregative E. coli (EAEC) and potential biological warfare agents such as Yersinia pestis. By coexpressing suitable host factors, we could extend the range of cell-free phage production to include selective phages targeting gram-positive bacteria. We also introduce a transient, non-genomic phage engineering method, which adds functionalities for only one replication cycle. Finally, we showcase a pipeline for personalized phage therapy of a multidrug-resistant ESKAPE pathogen using a newly isolated and in-vitro produced phage against Klebsiella pneumoniae. We expect our cell-free methodology to enable accelerated reverse and forward phage engineering and the safe and customized production of clinical-grade therapeutic bacteriophages.