Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop antibacterial agents with novel modes of action. A promising alternative to antibiotics are the naturally occurring endolysin enzymes from bacteriophage. Endolysins cause bacterial lysis by degrading the bacterial peptidoglycan cell wall. Exogenous application of endolysins results in rapid and specific elimination of Gram-positive bacteria making them an excellent alternative and/or adjunct to traditional antibiotics. The streptococcal C1 phage lysin, PlyC, is the most potent endolysin described to date and can rapidly lyse Group A, C and E Streptococci . We intend to engineer the specificity of PlyC using a directed evolution approach, to retarget its bacteriolytic activity to other groups of Streptococci. We have previously determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of two catalytic domains bound to an octameric cell-wall docking assembly . In this assembly, five residues, previously shown to be important for cell–wall binding, were targeted for site-saturation mutagenesis. This generated five libraries of several hundred clones, which are currently being screened for lytic activity against different bacterial strains. These results will provide several new PlyC mutants that display lytic activity against previously untargeted bacterial strains. In addition, these results will provide an exhaustive mutational analysis of the known cell-wall binding site in PlyC, delivering valuable insight into the interaction between PlyC and its target bacterial cell wall.