Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation

Genetic code expansion (GCE) is a critical approach to the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). However, evolving new aaRS/tRNA pairs traditionally relies on a labor-intensive process that often yields aaRSs with suboptimal ncAA incorporation efficiencies. Here, we present an OrthoRep-mediated strategy for aaRS evolution, which we demonstrate in 8 independent aaRS evolution campaigns, yielding multiple aaRSs that incorporate an overall range of 13 ncAAs tested. Some evolved systems enable ncAA-dependent translation at single amber codons with similar efficiency as natural translation at sense codons. Additionally, we discover an aaRS that regulated its own expression to enhance ncAA dependency. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms to advance the field of GCE.