Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria

Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient Listeria monocytogenes L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, Listeria L-form cells not only support rebooting of native and synthetic Listeria phage genomes but also enable cross-genus reactivation of Bacillus and Staphylococcus phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.