A facile platform to engineer E. coli tyrosyl-tRNA synthetase adds new chemistries to the eukaryotic genetic code, including a phosphotyrosine mimic

Abstract

The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells

“…This is in contrast with the paradigm in aaRS engineering, where enhanced mutants developed in E. coli are routinely imported into mammalian cells with retention of function. 28,38,39…”

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

“…While a portion of this challenge can be mitigated by using high ncAA concentrations during induction of protein synthesis, the discovery of additional high-performing aaRS variants that discriminate against the canonical amino acids will be important going forward. Dual ncAA incorporation in yeast will certainly benefit from ongoing efforts to expand the availability and performance of OTSs to access a broader palette of genetically encodable ncAAs with useful chemistries (Dunkelmann et al, 2020; Grasso et al, 2022; He et al, 2020; Italia et al, 2017; Osgood et al, 2022; Stieglitz and Van Deventer, 2021; Tan et al, 2020).…”

“…These machineries were identified from an extensive repository of OTSs available in these organisms that facilitate specific and efficient decoding of multiple orthogonal codons with structurally and functionally diverse ncAAs. (Beránek et al, 2018; Biddle et al, 2022; Cervettini et al, 2020; Gan et al, 2017; Grasso et al, 2022, 2022; Italia et al, 2017; Kelemen et al, 2022; Lang et al, 2012; Wang et al, 2014; Zheng et al, 2018a).…”

“…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). While not yet evaluated in yeast, such Ec aaRS variants have the potential to further broaden the range of OTSs available for use in this organism.…”