Rapid and high-throughput evaluation of diverse configura-tions of engineered lysins using the VersaTile technique

The emerging and spreading antibiotic resistance in bacteria is a severe and global problem and poses a worrying threat to our health care. Hence, the demand for alternative antibiotics is increasingly urgent. One of the most promising novel, alternative classes of antibiotics are lysins or bacteriophage-encoded peptidoglycan hydrolases, also called enzybiotics. A first lysin has recently successfully passed a superiority clinical phase II trial and got a Breakthrough Therapy designation by the FDA. Here, I present a high-throughput engineering and selection experiment for enzybiotics targeting Gram-negative Klebsiella species. These pathogens are a frequent cause of nosocomial infections that are often associated with a fatal outcome. The most prevalent species is Klebsiella pneumoniae, which belongs to group of carbapenem-resistant Enterobacteriaceae (CRE). With VersaTile technology, a quick and efficient method for combinatorial shuffling, we constructed five libraries of shuffled lysins, totaling to more than 400,000 different modular variants. Each library had a different configuration, comprising an outer membrane permeabilizing peptide, cell wall binding domain, enzymatically active domain and a possible linker, each time with a different order and composition. We analysed 940 variants from these libraries with a microtiter plate based growth inhibition assay targeting Klebsiella pneumoniae and Klebsiella oxytoca under different conditions. We found different outcomes depending on the lysin configuration and composition. The large number of constructed variants, facilitated by VersaTile technology, allows to explore the practically infinite sequence space of shuffled variants, to an unprecedented extent.