High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation

Noncanonical amino acids (ncAAs) enable diversification of protein functions, but the efficiency of genetic code expansion (GCE) in eukaryotes is hindered by competition between suppressor tRNAs and release factors. Prior work has identified eukaryotic release factor 1 (eRF1) mutants that improve ncAA incorporation, suggesting that screens for improved variants may lead to further enhancements. Here, we developed a high-throughput system to screen eRF1 mutants in Saccharomyces cerevisiae where eRF1 mutants are coexpressed on a plasmid alongside genomically encoded, wild-type eRF1. This strategy enabled recovery of live cells expressing eRF1 variants that enhance ncAA incorporation, even with mutants known to severely affect cell viability in the absence of WT eRF1 expression. We prepared and screened a million-member library of randomly mutated eRF1 variants for clones exhibiting improved ncAA integration phenotypes. Deep sequencing revealed a diverse set of enriched mutations across all three major domains of eRF1. Interestingly, several enriched mutations identified here are also found in naturally occurring eRF1 homologs from species that recode canonical stop codons. When eRF1 variants were combined with yeast knockout strains also known to enhance ncAA incorporation, this resulted in further improvements to efficiency, highlighting the complementarity of release factor engineering to other GCE enhancement strategies. This work demonstrates that high-throughput engineering of the eukaryotic translational apparatus is a powerful approach to identify previously unknown solutions for enhancing ncAA incorporation, with implications for elucidating and precisely manipulating the molecular functions of essential translational machinery.