Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

Abstract

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to a lack of the available selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support the separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS both led to the enrichment of a strain known to support a higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well behaved in yeast and provide a convenient alternative to DPSs. SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

Genome-wide screen for enhanced noncanonical amino acid incorporation in yeast

“…The two reporters have different stop codon positions that have previously been shown to exhibit different readthrough efficiencies ( 27 ). The reporter architecture enables high-throughput evaluations of ncAA incorporation efficiency at the level of both individual clones and whole populations; the reporters are also compatible with fluorescence-activated cell sorting (FACS) ( 29 ). The OTS is the machinery that enables ncAA incorporation at amber stop codons and consist of an aminoacyl-tRNA synthetase (aaRS) and a suppressor tRNA CUA .…”

“…Transformed knockout strains were quantitatively evaluated using two metrics: relative readthrough efficiency (RRE) and maximum misincorporation activity (MMF). These metrics were originally described by Barrick and coworkers in E. coli and extended to S. cerevisiae by our group ( 27–29, 35, 36 ). RRE measures ncAA incorporation efficiency, where a value of 1 indicates wildtype incorporation efficiency at the amber stop codon and a value of 0 indicates total translation truncation at the amber stop codon (SI Equation 1).…”

“…MMF measures the extent of misincorporation of a canonical amino acid at the amber stop codon in the absences of ncAA. An MMF value of 0 indicates no detectable misincorporation, while values approaching 1 indicate high levels of canonical amino acid misincorporation (SI Equation 2) ( 27–29, 35, 36 ).…”

“…ppq1Δ was used because its enhanced ncAA incorporation efficiency has been previously demonstrated; in the absence of OTSs, deletion of the gene leads to a known phenotype of increased stop codon readthrough ( 27, 37 ). Using a two-plasmid reporter system that we have previously determined to be functionally equivalent to the single-plasmid systems used during screening ( 29 ), we cotransformed a plasmid encoding a BXG-altTAG reporter along with a plasmid encoding the TyrOmeRS/tRNA CUA Tyr OTS into these six strains and evaluated their incorporation efficiency and fidelity with flow cytometry. Both RRE values and the flow cytometry dot plots suggest that enhanced ncAA incorporation due to deleting PPQ1 are only observed in the MAT a (BY4741) and heterozygous diploid (BY4743) genetic backgrounds (SI Figure 3).…”

“…YKO collections have also facilitated genome-wide functional profiling in various growth stress conditions and studies for identifying genes and protein involved within cellular pathways, such as genes that regulate the yeast mating pathway (24)(25)(26). In addition, there exist multiple ways to analyze ncAA incorporation quantitatively and qualitatively in yeast using intracellular fluorescent protein reporters in high throughput (27)(28)(29).…”