Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Abstract

The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N-and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a “drop-in” version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems—and protein translation apparatuses—to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such “noncanonical amino acids” (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.

Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential

“…After induction with IPTG, 1.2 × 10 8 cells were sorted using fluorescence-activated cell sorting (FACS). FACS-based screens are increasingly being used over selection-based approaches to evolve orthogonal tRNA/aaRS pairs [ 17 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. Unsurprisingly, most of the cells screened in the first round of FACS did not aminoacylate tyrosine onto the M. barkeri tRNA and did not produce full-length, fluorescent GFP.…”

Chemical Diversification of Simple Synthetic Antibodies

“…NcAAs were incorporated using constitutively expressed orthogonal translation systems (OTSs; Figure 4A), which in this work are previously described aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. [62][63][64] Figure 4B depicts the ncAAs used in Differences in antigen binding following ncAA substitution indicate that these four clones exhibit a range of tolerances for ncAA substitutions in positions located outside of CDR-H3. To address one potential reason for these variable tolerances, we examined how ncAA incorporation efficiency affects antigen binding.…”

“…Three separate transformants per antibody clone + aaRS/tRNA combination were used to generate the data for RRE and MMF measurements. Data analysis was performed as described previously by Stieglitz et al 63 and Potts et al 64 Binding affinity (KD) data collection and analysis. 1.5 million freshly induced RJY100 cells displaying scFvs were aliquoted in 1.7 mL microcentrifuge tubes, pelleted, washed three times with 1× PBSA, and then resuspended in 1 mL PBSA.…”

“…To facilitate efficient synthetic antibody discovery and derivatization, we utilized the combination of yeast display and noncanonical amino acid (ncAA) incorporation. [62][63][64] We first developed an extremely simple, yeast-displayed synthetic single-chain variable fragment (scFv) library with amino acid and loop length diversity encoded solely in CDR-H3. 27,[65][66] Construction and screening of a billion-member yeast display library enabled the isolation of antibodies that exhibit specificity towards only one of three structurally similar antigens (donkey, bovine, or rabbit IgG).…”

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

“…[26][27][28][29] While these findings were applied to the residue-specific replacement of methionine, later findings evaluating OTSs for site-specific applications appear to be consistent with these earlier studies. [30][31][32][33] Eppinger, Rueping, and coworkers evolved Methanosarcina barkeri pyrrolysyl-tRNA synthetases (PylRSs) using a high-throughput FACS screen in E. coli, identifying a polyspecific variant capable of supporting stop codon readthrough with up to 31 aliphatic, cyclic, or fluorinated ncAAs. 33 Isaacs and coworkers used multiplex automated genome engineering (MAGE) to evolve chromosomally-encoded OTSs with diversity at 12 residues in the amino acid binding pocket and five residues in the tRNA anticodon recognition domain of the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS).…”