Genetically encoding noncanonical amino acids (ncAAs) in proteins is a promising strategy for enhancing the properties of biological therapeutics. There are now tantalizing hints of the power of this approach in the literature, including antibody-drug conjugates and cyclic peptides with properties that would be difficult to access using other approaches. However, not all protein engineering platforms are yet compatible with ncAAs. This is especially true in the yeast <i>S. cerevisiae</i>, where the underdeveloped ncAA incorporation toolkit limits integration with powerful yeast display technology. One major barrier to effective integration is a lack of quantitative understanding of ncAA incorporation events. Here, we describe the evaluation of a number of reporters of ncAA incorporation efficiency and fidelity in response to the amber (TAG) codon in yeast that may help shed light on this question: single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters. Across these varying reporter architectures, we found that flow cytometry-based evaluations with dual reporters yielded consistent quantitative trends across numerous incorporation conditions. In addition, using dual-fluorescent protein reporters, we found that ncAA incorporation efficiency and fidelity is influenced by both stop codon location and N- and C-terminal fluorescent protein identity. Finally, we investigated the feasibility of using several reporters to quantify the effects of single-gene knockouts on ncAA incorporation events. Both dual-fluorescent protein reporters and a modified “drop-in” version of yeast display revealed that several gene knockouts enhance ncAA incorporation efficiency without compromising fidelity. These findings highlight the important roles that quantitative reporter systems play in identifying high efficiency, high fidelity ncAA incorporation events, and suggest opportunities for improving ncAA insertion using genome engineering. Using these reporters to facilitate the encoding of a diverse set of ncAAs in yeast is expected to set the stage for engineering more “druglike” proteins that leverage the broad set of chemistries present in noncanonical amino acid side chains.

Quantifying noncanonical amino acid incorporation events in yeast: Laying the groundwork for engineering more druglike proteins

ACS Fall 2020 Virtual Meeting

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