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The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA EcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli -based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNA CUA EcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNA CUA EcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine., Competing Interests: The authors declare the following competing financial interest(s): A patent application on the EcTyrRS mutants and ncAAs reported here has been submitted. A.C. is a cofounder and senior advisor of BrickBio, Inc., which focuses on applications of the ncAA mutagenesis technology., (© 2022 The Authors. Published by American Chemical Society.)

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii -derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli -derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

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 N ϵ -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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)