LAUSR

Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non‐proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl–tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate‐based colorimetric assay, thereby combining high‐throughput capabilities with reliable and quantitative measurement of aaRS‐dependent ncAA incorporation efficiency. This two‐tier functional screening system was successfully applied to obtain a pyrrolysyl–tRNA synthetase (PylRS) variant (CrtK‐RS(4.1)) with significantly improved efficiency (+250–370 %) for mediating the incorporation of Nϵ‐crotonyl‐lysine and other lysine analogues of relevance for the study of protein post‐translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK‐RS(4.1) were found to localize within the noncatalytic N‐terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs.