Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

ABSTRACT: 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 ambe

SUBMITTER: Furuhata Y 

PROVIDER: S-EPMC12103617 | biostudies-literature | 2025 May

REPOSITORIES: biostudies-literature