An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes

In Escherichia coli , replacement of the endogenous tryptophanyl–tRNA synthetase–tRNA pair with its counterpart from Saccharomyces cerevisiae liberates the bacterial counterpart for directed evolution to incorporate unnatural amino acids in both E. coli and eukaryotes. In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl–tRNA synthetase and tRNA (TrpRS–tRNA Trp ) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli –optimized counterpart from Saccharomyces cerevisiae , and then reintroducing the liberated E. coli TrpRS–tRNA Trp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS–tRNA Trp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl–tRNA synthetase (aaRS)–tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS–tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.