Expanding the genetic code: phage-driven evolution of pyrrolysyl-synthetase for site-specific incorporation of synthetic phenylalanine and tyrosine derivatives.

Introduction: Pyrrolysyl-tRNA synthetase (PylRS) is a key enzyme for the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. However, its native form has a limited substrate scope. This study aimed to evolve PylRS from Methods: We used phage-assisted non-continuous evolution (PANCE) to generate a library of PylRS variants under selective pressure for the target ncAAs. Evolved variants were sequenced to identify mutations. Their aminoacylation efficiency and specificity were quantitatively assessed using fluorescence-based incorporation assays and mass spectrometry. Results: Sequencing revealed a polymorphic population of mutations, with a significant cluster located within the enzyme's amino acid binding pocket. Several evolved variants showed an orders-of-magnitude increase in the efficiency of incorporating their target ncAAs compared to the wild-type enzyme. Discussion/Conclusion: Our findings confirm that PANCE is a highly effective method for engineering PylRS variants with strong and specific activity towards desired non-canonical amino acids. The identified mutations, particularly those in the binding pocket, provide a basis for understanding and further optimising substrate specificity in synthetic biology applications.