Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast

Abstract

Numerous applications of noncanonical amino acids (ncAAs) in basic biology and therapeutic development require efficient protein biosynthesis using an expanded genetic code. However, achieving such incorporation at repurposed stop codons in cells is generally inefficient and limited by complex cellular processes that preserve the fidelity of protein synthesis. A more comprehensive understanding of the processes that contribute to ncAA incorporation would aid in the development of genomic engineering strategies for augmenting genetic code manipulation. In this work, we used a series of fluorescent reporters to screen a pooled Saccharomyces cerevisiae molecular barcoded yeast knockout (YKO) collection. Fluorescence-activated cell sorting enabled isolation of strains encoding single-gene deletions exhibiting improved ncAA incorporation efficiency in response to the amber (TAG) stop codon; 55 unique candidate deletions were identified. The deleted genes encoded for proteins that participate in diverse cellular processes, including many genes that have no known connection with protein translation. We then verified that two knockouts, yil014c-aΔ and alo1Δ, exhibited improved ncAA incorporation efficiency starting from independently acquired strains possessing the knockouts. Using additional orthogonal translation systems and ncAAs, we determined that yil014c-aΔ and alo1Δ enhance ncAA incorporation efficiency without loss of fidelity over a wide range of conditions. Our findings highlight opportunities for further modulating gene expression with genetic, genomic, and synthetic biology approaches to improve ncAA incorporation efficiency. In addition, these discoveries have the potential to enhance our fundamental understanding of protein translation. Ultimately, cells that efficiently biosynthesize ncAA-containing proteins will streamline the realization of applications utilizing expanded genetic codes ranging from basic biology to drug discovery.

Keywords: amber suppression; aminoacyl-tRNA synthetases; fluorescence-activated cell sorting; noncanonical amino acids; yeast knockout collection.

Figure 1.

Overview of screening campaign. (A) Diagram of the general architecture of the four unique plasmid systems transformed into the YKO collection containing the reporter and orthogonal translation system (OTS). The reporters consisted of a BFP linked to GFP by a peptide linker containing an amber (TAG) stop codon. Two reporters, BXG and BXG Alt-TAG, that differ by the location of the stop codon within the peptide linker were used. The OTS contained an aaRS and suppressor tRNA_CUA. Two aaRSs were used: TyrOmeRS and LeuOmeRS. (B) Structure of ncAA, O-methyl-L-tyrosine (OmeY), used for screens. (C) General schematic of the screening process. A pooled YKO collection transformed with the reporter/OTS was induced in the presence of 0, 0.1, or 1 mM OmeY. FACS was used to isolate cells with high GFP expression, which indicated successful OmeY incorporation in the reporter (or, in some cases, canonical amino acid incorporation). Following multiple rounds of screening, the barcodes of isolated colonies from enriched populations were sequenced to identify their respective gene deletions. (D) Representative flow cytometry overlay dot plots comparing readthrough of naïve (pre-sort) and enriched (post-sort) populations of three sorting paths. Populations were induced with the same concentration of OmeY used for screening. Refer to Table S4 for information on sort path nomenclature.

Figure 2.

Quantitative evaluation of ncAA incorporation efficiency and fidelity with yil014c-aΔ and alo1Δ with OTSs used during screening. (A) RRE and MMF metrics based on a flow cytometry ncAA incorporation assay following the induction of cells in the presence of 1 mM OmeY. (B) RRE and MMF metrics based on flow cytometry ncAA incorporation assay following the induction of cells in the presence of 1 mM AzF (see Figure 3A for chemical structure). All samples in this figure were evaluated in biological triplicate. Error bars represent the standard deviation based on the propagated error. All experiments utilized BXG Alt-TAG single plasmid reporter constructs. Median fluorescence intensity (MFI) data and statistical tests are available in Figure S5.

Figure 3.

Quantitative evaluation of ncAA incorporation efficiency of BY4741ΔALO1 and BY4741ΔYIL014C-A with additional OTSs and ncAAs. (A) Structures of additional ncAAs used in these studies. (B) RRE metric based on a flow cytometry ncAA incorporation assay following the induction of cells expressing SpecOPGRS-3 in the absence of ncAA and in the presence of 1 mM OPG and five additional ncAAs. (C) RRE and MMF metric based on a flow cytometry ncAA incorporation assay following the induction of cells expressing a pyrrolysyl-tRNA synthetase/tRNA OTS in the presence of 10 mM BocK. Experiments shown in (B) and (C) utilize the BXG Alt-TAG fluorescent reporter cotransformed with the appropriate OTS on two separate plasmids. All samples were evaluated in biological triplicate. Error bars represent the standard deviation based on the propagated error. Median fluorescence intensity (MFI) data and statistical tests are available in Figure S6.