Engineering and Directed Evolution of Cellular Protein Translation

Rapid methods to engineer evolve and cellular translation machinery would enable applications beyond canonical protein synthesis. Unsurprisingly, manipulations to cellular translation often yield pleiotropic results, confounding experimental interpretation as researcher-dictated activities cannot be easily uncoupled from cellular fitness. Here, we develop a series of systematic advances to engineer and evolve novel translational capabilities in living cells. We apply orthogonal translation to define general requirements for efficient heterologous rRNA processing. We discover that supplementation with a small subset of cognate r-proteins enhances heterologous ribosome activity for rRNAs derived from organisms with as little as 76.1% 16S rRNA sequence identity to E. coli. We leverage these advances to develop oRibo-PACE, a novel method for continuous evolution of orthogonal rRNAs. Using oRibo-PACE, we evolved orthogonal ribosomes from E. coli, P. aeruginosa and V. cholerae for >260 generations under increasing selection regimes. Evolved o-ribosomes exhibited improved protein translation activities, up to 400% of corresponding wild-type ribosomes, at similar or reduced cellular burdens. Finally, we develop an engineering approach that affords quadruplet-decoding tRNAs using representative scaffolds for all 20 canonical amino acids, and employ novel PACE methodologies to substantially improve their translation efficiency without compromising amino acid selectivity.Collectively, our results simplify translational engineering and directed evolution pipelines, and provide extensible strategies for affecting ribosome function in vivo.