High-Throughput Aminoacyl-tRNA Synthetase Engineering for Genetic Code Expansion in Yeast

Abstract

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in Escherichia coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting to screen libraries of aaRSs in high throughput for (1) the incorporation of ncAAs not previously encoded in yeast; (2) the improvement of the performance of an existing aaRS; (3) highly selective OTSs capable of discriminating between closely related ncAA analogues; and (4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific or polyspecific properties is invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE

“…Thus, our finding highlights the importance of applying selective pressure or phenotypic screening to identify cells with genomic rearrangements subjected to the SCRaMbLE system for a broader range of applications, as demonstrated in previous studies 32,33 . We also envision that the recombination frequency mediated by GCE-SCRaMbLE would be further enhanced by improving the suboptimal incorporation efficiency of OMeY into Cre enzyme via the engineering of LeuOmeRS/tRNA CUA pair 34 and eukaryotic release factor 1 (eRF1) that competes with unnatural amino acid insertion in response to stop codons 35 .…”

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

“…For the Ec LeuRS/tRNA Leu pair, we used the polyspecific aaRS variant LeuOmeRS with a T252A mutation in its editing domain and employed the ncAA O -methyl-L-tyrosine ( 1 ; OmeY) as a substrate (Wu et al, 2004). For the Ec TyrRS/tRNA Tyr pair, we employed a polyspecific aaRS variant, T2RS5, identified through our high-throughput screening and used it with the ncAA p -propargyloxyl-L-phenylalanine ( 2 ; OPG) as a substrate (Stieglitz and Van Deventer, 2022). Lastly, for the PylRS/tRNA Pyl pair, we used the M. alvus PylRS with the ncAA N ε - Boc-L-Lysine ( 3 ; BocK) as a substrate (Stieglitz et al, 2022) (Figure 1C).…”

“…Numerous engineering strategies are known to augment stop codon readthrough, including single-gene deletions, whole-genome synthesis, and engineering various components of the translation machinery (DeBenedictis et al, 2021; Dunkelmann et al, 2021; Gan et al, 2017; Sanders et al, 2022; Stieglitz and Van Deventer, 2022; de la Torre and Chin, 2021; Zackin et al, 2022). Prior work from our group has shown that the use of single-gene knockout strains of S. cerevisiae can enhance ncAA incorporation efficiencies in response to the TAG codon for a range of OTSs and ncAAs (Potts et al, 2020; Stieglitz et al, 2021; Zackin et al, 2022).…”

“…This Aga1p-Aga2p-based display system facilitates the use of induction procedures identical to those used for dual fluorescent reporters. For preparation of a “mutually orthogonal” Ec TyrRS/tRNA CUA Tyr + Ec LeuRS/tRNA UCA Leu combination, we used the TyrAcFRS variant as part of an Ec TyrRS/tRNA CUA Tyr OTS to suppress TAG, and a newly-identified variant termed LysAlkRS3 as part of the Ec LeuRS/tRNA UCA Leu OTS to suppress TGA (Stieglitz and Van Deventer, 2022). These OTSs were chosen based on their nonoverlapping substrate specificities, where the ncAA AzF ( 4 ) can be encoded with TyrAcFRS/tRNA CUA Tyr , and the ncAA LysAlk ( 5 ) can be encoded with LysAlkRS3/tRNA UCA Leu .…”

“…For instance, recent reports by He et al and Tan et al demonstrated the isolation of E. coli aaRS variants exhibiting unique aminoacylation properties by utilizing custom selection schemes in yeast; these studies led to the identification of variants that support translation with new ncAAs including sulfotyrosine (He et al, 2020; Tan et al, 2020). Parallel efforts from our lab have led to the discovery of a broad range of E. coli tyrosyl-tRNA synthetase ( Ec TyrRS) and E. coli Leucyl-tRNA synthetase ( Ec LeuRS) variants with a variety of ncAA incorporation properties, including new-to-yeast ncAA incorporation and improved selectivity and activity for known ncAAs (Stieglitz and Van Deventer, 2022). In addition, work by Chatterjee and coworkers in engineered E. coli strains has led to the identification of additional Ec aaRS variants derived from Ec TyRS and Ec TrpRS with unique substrate preferences for use in mammalian cells (Grasso et al, 2022; Osgood et al, 2022).…”