Enhancing Orthogonal Behavior and Amber Suppression Activity for Heterologous tRNA and tRNA Synthetase Variants

There have been considerable advancements in the translational incorporation of non-canonical or unnatural amino acids (UAA) into proteins over the last two decades.  As the most widely used method for site-specific UAA incorporation, amber stop codon suppression employs an orthogonal translation system (OTS) consisting of a heterologous tRNA synthetase:tRNA pair, which function to augment or expand an organism’s genetic code.  More recent advances such as “Amberless” E. coli as well as engineering UAA-dependence or “addiction” build on earlier work by expanding the utility of OTSs.  However, heterologous tRNA synthetase:tRNA pairs remain critical to establishing efficient OTSs.  To this end, we have conducted a complete overhaul of the heterologous tRNA from Methanocaldococcus janaschii (Mj) tyrosyl pair.  Using compartmentalized partnered replication (CPR) and liquid-based pheS negative selection, we evolved a new set of orthogonal Mj-tRNA variants with enhanced amber suppression activity for incorporation of tyrosine as well as three 3-halo-tyrosine UAAs (3-Cl-Y, 3-Br-Y, and 3-I-Y) in E. coli.  These tRNAs show up to three-fold reduction in promiscuous aminoacylation by endogenous tRNA synthetases while increasing amber suppression activity by almost 40%.  With respect to host translation machinery, these new Mj-tRNA variants also display enhanced orthogonality, thus eliminating organismal fitness costs associated with tRNA mischarging.  Our study identified a tRNA variant displaying tyrosine-dependent suppression activity, which demonstrates the capacity to engineer suppressor tRNA variants with amino acid specificity.  Additionally, we successfully employed our CPR selection to enhance the activity of the Methanocaldococcus janaschii (Mj) tyrosyl tRNA synthetase with some mutant variants demonstrating over fifteen-fold increase in amber suppression activity.