Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface─(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.