(61e) Engineering Dual Noncanonical Amino Acid Incorporation in Yeast

The biosynthesis of proteins containing noncanonical amino acids (ncAAs) provides opportunities for dissecting basic biological processes and engineering proteins with “druglike” features using chemistries not found in the 20 canonical amino acids. The production of proteins with more than one ncAA provides opportunities for simultaneously introducing multiple new features into a protein, such as new molecular recognition and crosslinkability. Here we describe our progress in introducing multiple, distinct ncAAs into proteins prepared in the yeast S. cerevisiae. To complement the use of the UAG (amber) stop codon in yeast, we evaluated suppression of the UGA (opal) stop codon using three distinct orthogonal translation systems (based on E. coli tyrosyl-tRNA synthetase/tRNA, E. coli leucyl-tRNA synthetase/tRNA, and M. alvus pyrrolysyl-tRNA synthetase/tRNA pairs). Following mutation of the tRNAs of the orthogonal translation systems and complementary conversion of amber codons to opal codons in ncAA incorporation reporter systems, we observed stop codon readthrough for all three UGA suppression systems without detectable crossreactivity from native translation components. In general, stop codon readthrough efficiencies at UGA codons decreased by a factor of two or more when compared with readthrough efficiencies at corresponding UAG codons. We also observed that readthrough efficiencies at UGA exhibited position-dependent changes in efficiencies similar to those observed at UAG codons. Furthermore, previously identified yeast single-gene knockout strains known to increase readthrough efficiencies at UAG codons supported similar enhancements in readthrough efficiencies at UGA codons (by approximately 2-fold). We then used combinations of two orthogonal translation systems to investigate the feasibility of dual ncAA incorporation at UAG and UGA codons within the same mRNA transcript. Two different system combinations supported the production of full-length proteins based on fluorescent protein reporters with estimated overall efficiencies in the range of 1 to 5 percent of wild-type protein production levels (E. coli tyrosyl-tRNA synthetase/tRNA and E. coli leucyl-tRNA synthetase/tRNA pairs or E. coli tyrosyl-tRNA synthetase/tRNA and M. alvus pyrrolysyl-tRNA synthetase/tRNA pairs). We also investigated the application of the dual incorporation system in yeast display format by preparing a synthetic antibody containing two ncAAs. The full-length antibody containing two distinct ncAAs was detected and retained its binding function (though with a decrease in target binding affinity of approximately one order of magnitude). Ongoing work focuses on applications of the dual ncAA incorporation system as well as improvements in the efficiency of ncAA incorporation. Routine access to proteins containing multiple chemical features, especially in yeast display format, will further expand opportunities for the discovery and application of “druglike” proteins.