(608d) Strategies for Expanding Genetically Encoded Chemical Diversity in Yeast

The genetic code normally encodes 20 canonical amino acids, but nature also uses posttranslational modifications and cofactors to enhance the range of chemistries in naturally occurring proteins. In the laboratory, genetic code expansion methods provide the means to precisely add chemistries to proteins beyond naturally occurring modifications, with applications ranging from proteomics to bioconjugation and single-molecule studies. However, a major barrier to expanding the genetic code is engineering the protein translation apparatus. Despite the importance of the yeast Saccharomyces cerevisiae in engineering and synthetic biology, very little work has been done to efficiently add amino acids to the genetic code of this organism. To understand the current state of the field, we established a quantitative, yeast display-based reporter platform for rapid evaluation of noncanonical amino acid incorporation in response to the UAG stop codon (Stieglitz, J.T.; et al. (2018) ACS Synthetic Biology 7:2256-2269). The reporter system exhibits improved precision over a fluorescent protein-based reporter evaluated on a spectrophotometric plate reader and allows for direct comparison of aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs and other components of orthogonal translation systems. We used the reporter to conduct fluorescence activated cell sorting with libraries of Escherichia coli tyrosyl- and leucyl-tRNA synthetase variants for two purposes: (1) to expand the range of chemical diversity that can be site-specifically encoded in proteins; and (2) to evolve aaRSs that are either highly specific against similarly structured ncAAs or highly promiscuous for incorporation of several ncAAs. Library screening yielded several aaRSs capable of encoding ncAAs that have not previously been encoded in yeast. Furthermore, initial screens for specificity or promiscuity have yielded populations of aaRSs that exhibit increased discrimination against undesired ncAAs or enhanced promiscuity, respectively; such “specificity tuning” would be extremely challenging with alternative aaRS engineering strategies. Our successful screening suggests that our reporter may facilitate evolution of the translation apparatus in order to generate organisms that better accommodate expanded genetic codes. In addition, the ability to tune the specificity or promiscuity of translation machinery could prove vital to the many synthetic and chemical biological applications that utilize site specific ncAA incorporation. In summary, our reporter platform is allowing us to further expand the repertoire of ncAAs that can be encoded in eukaryotes and to tune the specificity and promiscuity of aaRSs in ways that have not previously been possible via alternative methods.