Identifying Yeast Gene Deletions That Enhance Noncanonical Amino Acid Incorporation

Efficient incorporation of noncanonical amino acids (ncAAs) in proteins is a prerequisite for leveraging their chemical diversity in the engineering of proteins with expanded capabilities. Potential applications for ncAAs span from engineering biological therapeutics with chemistries beyond those found in canonical amino acids to expanding our fundamental understanding of protein biosynthesis. Efforts to improve ncAA incorporation have focused on engineering orthogonal translation systems (OTSs): the translation machinery introduced into cells that allows for the encoding of ncAAs in proteins. However, the efficacy of these efforts is limited by complex cellular factors that inhibit genetic encoding of ncAAs at repurposed stop codons. Identification of these cellular factors is necessary to engineer cells that better accommodate expanded genetic codes. In this work, we performed a genome-wide screen of Saccharomyces cerevisiae using a pooled yeast knockout (YKO) collection to identify single-gene deletions that enhance ncAA incorporation efficiency and fidelity. We utilized dual-fluorescent reporter systems that facilitated fluorescence-activated cell sorting (FACS) of the YKO collection to isolate deletion strains with increased ncAA incorporation efficiency. Our early screens identified several unique deletions that appear to exhibit improved stop codon suppression and ncAA incorporation. Identified genes are involved in diverse cellular activities, and genes with no previously characterized function have also been identified. Current efforts focus on further validating these deletions, including verifying that these single-gene deletions enhance ncAA incorporation efficiency in other strains, and quantitatively evaluating ncAA incorporation efficiency and fidelity. Future work will interrogate these single-gene deletion strains to elucidate how they affect ncAA incorporation for applications such as click chemistry. To our knowledge, this work is the first genome-wide investigation of factors that influence ncAA incorporation. Understanding the cellular processes that dictate stop codon suppression and ncAA incorporation are imperative to augmenting genetic code manipulation strategies for a multitude of applications.