Noncanonical amino acids (ncAAs) are powerful tools for precisely manipulating the structures and functions of proteins with applications in therapeutics and basic biological studies. A common strategy for genetically encoding ncAAs is to encode these amino acids in response to stop codons. However, this process is typically inefficient because cells did not evolve to accommodate such stop codon suppression events. Little is known about the range of genetic or genomic manipulations that may enhance ncAA incorporation efficiency. Here, we present a strategy for screening collections of yeast strains in search of clones that support enhanced noncanonical amino acid incorporation. This approach is capable of leveraging the extensive genetic resources available in yeast, and has the potential to provide new insights into the eukaryotic translation apparatus. In this work, we 1) transformed a barcoded yeast knockout collection comprising more than 5,000 gene knockouts with a series of ncAA incorporation reporter constructs; and 2) performed high throughput screening based on the reporter-enabled phenotype of enhanced stop codon readthrough in a flow cytometry format. These efforts yielded multiple populations exhibiting apparent increases in ncAA incorporation levels. Sequencing of isolated colonies revealed dozens of candidate knockout genes. Preliminary validations of two commonly identified candidates using strains acquired from independent sources confirmed that the candidate knockouts are capable of enhancing ncAA incorporation based on qualitative evaluation of flow cytometry experiments. Quantitative evaluations of ncAA incorporation efficiency and fidelity in these strains are ongoing. Interestingly, a number of knocked out genes, including the two lead candidate genes, have no known connections to protein translation or termination of translation. This suggests that unbiased genetic and genomic screens have great potential to enhance ncAA incorporation. Taken as a whole, our findings indicate that engineering and evolving yeast strains to enhance noncanonical amino acid incorporation is a promising strategy to improve the biosynthesis of proteins containing noncanonical amino acids. Such strategies are likely to identify previously unknown biological phenomena within the eukaryotic translation apparatus in addition to practical applications involving the efficient biosynthesis of proteins containing an expanded range of chemical functionalities.

Screening a yeast knockout collection to enhance noncanonical amino acid incorporation

ACS Spring 2022

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