AbstractThe ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N-and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a “drop-in” version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems—and protein translation apparatuses—to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such “noncanonical amino acids” (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.
Engineering Translation Components Improve Incorporation of Exotic Amino Acids
