Engineering Endolysins for Enhanced Bacteriolytic Activity Using Genetically Encoded Azido-Containing Noncanonical Amino Acids

The emergence of multidrug resistance in Gram-negative bacteria poses a significant challenge to global health, necessitating the development of effective antibiotics and therapeutic approaches. Endolysins derived from phages exhibit specificity toward bacteria, making them desirable candidates for the treatment of infectious bacteria. Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) with unique side chains, endowing proteins with new functions and properties. In this study, we applied genetic code expansion to introduce ncAAs into endolysin LysPA26, which targeted a broad spectrum of Gram-negative bacteria. Incorporating p-azido-l-phenylalanine (pAzF) at position R16 in LysPA26 increased its broad-spectrum bacteriolytic activity without an apparent change in its secondary structure. Furthermore, the engineered LysPA26-R16pAzF variant exhibited higher bacteriolytic activity than the wild type after exposure to varying temperatures (4 to 70 °C) or freezing (-25 °C). Additionally, the incorporation of another azido-containing ncAA, azidonor-leucine (AnzL), at position R16 of LysPA26 also improved bacteriolytic activity. Using a similar strategy, the insertion of pAzF into LysDLP1, a lysin from Acinetobacter phage vB AbaM DLP1, yielded a variant with enhanced bacteriolytic activity. These results collectively indicate that ncAAs can be effectively incorporated to engineer endolysins with enhanced antimicrobial performance, offering significant implications for lysin engineering and the development of antibiotics.