Exploring PhaLP, an open source portal for Phage Lytic Proteins, to engineer modular enzybiotics against vancomycin-resistant Enterococcus faecalis

Enzybiotics are considered as one of the most promising alternatives to conventional antibiotics. They are derived from enzymes used by bacteriophages to degrade the bacterial cell wall, also called phage lytic proteins. We have created PhaLP (https://www.phalp.org/), an elaborated and comprehensive database of phage lytic proteins with 17,356 entries (and growing). PhaLP aims to serve as an open portal for the phage community, particularly those researchers interested in phage lytic proteins. The database is easily searchable through two user-friendly web interfaces and integrates nine data types originating from multiple source databases.1 PhaLP can be used as a starting point to explore the broad diversity of phage lytic proteins, for example to study their host-specific evolution. In addition, PhaLP can also serve as a resource to perform protein engineering with phage lytic proteins. Indeed, due to their modular architecture, they can be engineered by recombining protein domains of native phage lytic proteins to create new modular variants with improved properties. Here, we describe a dataset that describes a complete design space of 780 modular variants. Every possible variant was constructed in a rational way using the VersaTile technique, starting from a small number of diverse building blocks (six enzymatically active domains and three cell wall binding domains) selected from PhaLP. The muralytic activity of each variant against vancomycin-resistant Enterococcus faecalis was determined in synthetic urine as a surrogate condition for a urinary tract infection, yielding hits that by far exceed the activity of the bulk of the variants