Improved selections and assessments for engineering orthogonal translation systems

Over twenty-five years have passed since researchers first demonstrated that introduction of a heterologous tRNA and tRNA synthetase pair could be used as an effective in vivo amber suppression system. This hallmark achievement began an era of site-specific unnatural or non-canonical amino acid incorporation through amber stop codon suppression. Of course, this all follows years of work using both semi-synthetic and global replacement methods. As with the preceding approaches and technologies, there have been considerable advances in the field of site-specific non-canonical amino acid incorporation. New technologies are now driving the development of expanded genetic codes, including codon capture, genetically recoded organisms, creation of an orthogonal tethered ribosome in E. coli, as well as expansion of the genetic alphabet. Despite these advances, amber suppression continues to be the principle technique for site-specific incorporation of non-canonical amino acids and will serve as a foundation for developing the strategies used in more advanced orthogonal translation systems (OTS). Herein we describe advances to selection schemes used to evolve orthogonal translation system apparatus as well as improved techniques for assessing and understanding the performance of evolved translation machinery. Our research focused upon the well-characterized Methanocaldococcus jannaschii (Mj) tyrosyl tRNA synthetase and tRNA [superscript Tyr] pair. We improved upon previous work by evolving a suite or lineage of new heterologous amber suppressing MjY-tRNA variants that have orthogonal behavior in E. coli equivalent to background read-through while identifying and eliminating fitness effects caused by heterologous tRNA expression. In addition, we have selected tRNA variants for greatly increased incorporation of non-canonical amino acids (ncAAs) while improving selection methods for increasing the activity of paired heterologous tRNA synthetases. We have also advanced the study of tRNA modifications as it relates to amber suppressor tRNA performance. Our work in studying tRNA modification will both impact selection strategies for heterologous tRNAs and may provide insight to understanding endogenous tRNA structure and function. We have also improved selection strategies and techniques for dramatically increasing the performance of heterologous tRNA synthetases through both positive and negative CPR-based selection. Finally, we developed a very effective dual CPR-pheS selection strategy that greatly enhanced the performance of an L-Dopa responsive transcriptional regulator. The insight gained from our dual CPR-pheS selection can be applied to other directed evolution efforts such as further enhancing ncAA-incorporating OTSs.