Antibiotic resistance represents a great challenge for health care worldwide, and novel strategies to control infection by drug-resistant bacteria are urgently needed. While the natural enemies of bacteria, bacteriophages, offer great host specificity and killing activity, their therapeutic potential is naturally limited by narrow host-ranges, insufficient antimicrobial activity, lysogeny, and rapid emergence of resistance. However, synthetic biology and genetic engineering of phage genomes can overcome these limitations and offers new possibilities for the design of smart and effective phage-based antimicrobials.

We use a synthetic phage engineering approach, based on in-vitro DNA assembly and subsequent reactivation (rebooting) of synthetic phage genomes within suitable host cells. To enable efficient rebooting of phage genomes in Gram-positive bacteria, we developed a bacterial L-form based platform, providing less stringent surrogate hosts for phage amplification. To enhance recombination-based engineering of very large phage genomes unsuitable for synthetic fragment assembly, CRISPR-Cas based counterselection systems were established in various phage hosts. Using these platforms, we (i) converted temperate phages to virulent ones, (ii) produced phages carrying a broad variety of additional payload genes for expression in the infected host, (iii) created phages showing extended killing of completely unrelated bacteria by phage-encoded cross-acting antimicrobials, (iv) designed transducing but non-replicating killer phage, (v) broadened phage host ranges by structure-guided design of receptor binding proteins, and (vi) provided a corresponding arsenal of reporter phages for companion diagnostics prior to administration in clinical trials.

Besides using bacteriophages, another very successful approach is to harness the bacteriolytic endolysins. Here, we have made significant progress by not only optimizing enzyme activity and in vivo half-life by domain shuffling and fusion to non-phage sequence, but also targeted modification of the enzymes for fine-tuned application in serum and blood, tissue, and intracellular environments.