A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic

Abstract

The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA^EcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNA_CUA ^EcTyr 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 tRNA_CUA ^EcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the 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 incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

Figure 1

Structures of ncAAs used in this study.

Figure 2

Development of an orthogonal tRNA_CUA^EcTyr variant through directed evolution. (A) The sequence of the original tRNA_CUA^EcTyr, where the highlighted segment in the acceptor stem was randomized to all possible combinations. (B) Selecting this library of mutants led to the identification of tRNA_CUA^EcTyr-h1, which showed dramatically attenuated cross-reactivity in ATMY. The mutations relative to its precursor are shown in red. (C) These assays were performed in an ATMY5 strain that did not encode any tRNA_CUA^EcTyr, which was instead expressed from the pRepTrip2.3 plasmid that also harbored the CAT-TAG reporter. pBPARS was used as the cognate EcTyrRS, and the activity was measured in the presence and absence of pBPA. These assays were performed without overexpressing tRNA^Gln, which was previously necessary to reduce the cross-reactivity of the original tRNA_CUA^EcTyr. Consequently, tRNA_CUA^EcTyr shows significantly higher background activity (survival at concentrations up to 60 μg/mL chloramphenicol) in the absence of pBPA that cannot be differentiated from the activity of pBPARS in the presence of pBPA (top panel). In contrast, tRNA_CUA^EcTyr-h1 shows no activity in the absence of pBPA and survival at concentrations up to 30 μg/mL chloramphenicol in the presence of pBPA, showcasing its high orthogonality and the ability to adequately discern the weak activity of pBPARS from the background.

Figure 3

Directed evolution of highly active pBPA-selective EcTyrRS mutants. (A) The active site of EcTyrRS. The bound substrate is shown in magenta, and the key active site residues that were subjected to randomization are highlighted. Mutations in the isolated pBPARS clones are also shown in the table below. (B) Activity of the pBPARS mutants in the ATMY E. coli strain. The activity was measured through the expression of the sfGFP-151-TAG reporter. (C) The scheme for the WiDR antibiotic selection to identify pBPARS mutants with higher activities from a random mutagenesis library. (D) Mutations associated with enhanced pBPARS mutants (shown in red). (E) Activity of the new pBPARS mutants in the ATMY E. coli strain. The activity was measured through the expression of the sfGFP-151-TAG reporter. (F) Activity of the pBPARS mutants in the HEK293T cells. The activity was measured through the expression of the EGFP-39-TAG reporter. (G) Representative fluorescence images of the HEK293T cells from panel F expressing the EGFP-39-TAG reporter.

Figure 4

A highly polyspecific EcTyrRS mutant capable of charging ncAAs 2–10. (A) Mutations associated with the EcTyrRS variants identified from the selection. (B) Activity of pAAFRS-9 in the ATMY E. coli strain. The activity measured using sfGFP-151-TAG as the reporter. (C) Incorporation of ncAAs 2–10 in HEK293T cells. EGFP-39-TAG was used as the reporter. Isolated yields for the reporter proteins and the observed molecular weight of each are shown. (D) Representative fluorescence images of the HEK293T cells from panel expressing the EGFP-39-TAG reporter.

Figure 5

Genetically encoding pCMF in eukaryotes. (A) pCMF mimics a phosphorylated tyrosine residue. (B) Mutations associated with pCMFRS, which were identified through the selection of a EcTyrRS library. (C) In ATMY E. coli, pCMFRS facilitates the selective expression of the sfGFP-151-TAG reporter in the presence of pCMF. (D) Activity of pCMFRS in HEK293T cells, demonstrating selective reporter expression in the presence of pCMF (fluoresence in the cell-free extract). Activities were measure using the EGFP-39-TAG reporter. (E) Representative fluorescence microscopy images of HEK293T cells expressing the EGFP-39-TAG reporter. Images were taken using pCMFRS in both the presence and absence of pCMF. (F) SDS-PAGE and ESI-MS analysis of the EGFP-39-pCMF reporter isolated from HEK293T cells.

Figure 6

Phosphotyrosine mimicry by pCMF activates STAT3 in mammalian cells. (A) The transfection of STAT3–705-TAG in HEK293T cells in the presence of the pCMF-selective EcTyrRS/tRNA_CUA^EcTyr pair results in the pCMF-dependent expression of STAT3. (B) A STAT3-dependent luciferase reporter was used to evaluate STAT3 activity in HEK293T cells, showing the significantly higher activity of STAT3-705-pCMF relative inactivated STAT3 (Y705F) and WT STAT3. Activities were normalized relative to the inactive STAT3 (Y705F) control. (C) The expression of each STAT3 construct was optimized to achieve similar expression levels.