Genetically encoding noncanonical amino acids (ncAAs) advances applications ranging from proteomics to bioconjugation. A major factor limiting the use of ncAAs in numerous organisms is the lack of engineered aminoacyl-tRNA synthetase/tRNA pairs supporting efficient genetic code expansion at stop codons. In S. cerevisiae, versatile ncAA incorporation strategies would synergize well with genetic and engineering tools unique to yeast. Here, we describe how a yeast display-based reporter enables careful tuning of aminoacyl-tRNA synthetase (aaRS) activity, specificity, and polyspecificity. We used our reporter to perform flow cytometry-based sorting of libraries of orthogonal translation systems (OTSs) based on the E. coli tyrosyl-tRNA synthetase/tRNACUA and E. coli leucyl-tRNA synthetase/tRNACUA pairs. Positive and negative screens yielded OTSs that support protein translation with ncAAs that have never been genetically encoded in yeast, including L-DOPA, and several variants exhibiting improved ncAA incorporation efficiency compared to prior mutants. To enhance specificity, we induced libraries of OTSs in the presence of several “off-target” ncAAs during negative screens to enrich for clones capable of discriminating between ncAAs with closely related structures. This strategy was used to isolate OTSs that support protein translation with O-propargyl-L-tyrosine (OPG) while discriminating against five closely related ncAAs, suggesting that genetic code expansion with structurally related ncAAs can be achieved without causing loss of fidelity. To enhance polyspecificity, two distinct strategies were successful: (1) induction of libraries in the presence of several ncAAs simultaneously, followed by positive screens; and (2) induction of libraries in the presence of individual, distinct ncAAs in successive screening rounds. Unexpectedly, following enrichments with only aromatic ncAAs, some clones supported high levels of incorporation with aliphatic derivatives of lysine; even conservative polyspecificity screens may lead to broad ncAA recognition. Access to polyspecific OTSs may enable “protein medicinal chemistry” strategies to improve properties of therapeutically relevant proteins. Our findings have important implications regarding the evolvability of aaRSs. Further, access to OTSs with diverse activities, specificities, and polyspecificities may prove invaluable for applications within chemical and synthetic biology.

Expanding the toolkit for noncanonical amino acid incorporation in yeast: High-throughput screens for aminoacyl-tRNA synthetase activity, specificity, and polyspecificity

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.