Strategies for Quantifying and Enhancing Genetic Code Manipulation in Yeast

Noncanonical amino acids (ncAAs) facilitate a diverse range of applications in protein science and engineering ranging from dissecting key protein functions to engineering proteins with more “druglike” properties. Genetically encoding ncAAs in proteins produced within cells and in vitro provides the basis for preparing ncAA-containing proteins that would be difficult or impossible to produce via chemical synthesis. However, our understanding of the performance of these systems remains limited, especially from a quantitative perspective. Here, we present our efforts to (1) establish tools to measure ncAA incorporation in Saccharomyces cerevisiae in response to the amber stop codon; and (2) use these tools to enhance ncAA incorporation efficiency and fidelity in yeast. We have identified a series of dual reporter systems that support flow cytometry-based measurements of ncAA incorporation efficiency and fidelity. These reporters enable quantitative measurements for a diverse range of orthogonal translation systems (OTSs) and for a wide range of yeast strains. We surveyed a number of previously described OTSs in yeast and determined that these systems typically support ncAA incorporation at approximately 15-20% of wild-type protein translation events. Many of these OTSs also exhibit polyspecificity (incorporation of several ncAAs). Ongoing work in the laboratory seeks to use these reporter systems to identify OTSs with improved or altered ncAA incorporation. We have also initiated studies in which we use our reporters to screen a broad range of yeast strains for phenotypes of enhanced ncAA incorporation. Our early findings demonstrate the feasibility of this approach using single-gene knockout strains with known alterations in stop codon readthrough. The measurement tools we have developed form the basis for evolving yeast to better accommodate alternative genetic codes.