Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Abstract

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Figure 1.

The tyrS complementation tests with pMjYS, pScYS, pMjYSYR and pScYSYR (A). The mutant cells transformed with these plasmids were inoculated on the LB plates containing 1% d-glucose and 34 µg/ml Cm. The three sectors of each half plate represent dilutions of the cells. An illustration of the genetic modifications and plasmid systems in FT3 and FB3 cells (B). The absence of the E. coli TyrRS activity in the strain FT1 (C). TOP10 and FT1 cells were each transformed with pACamKsupF and were then inoculated on the LB plate with 10 µg/ml Cm.

Figure 2.

E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS-ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo-l-tyrosine (0.5 mg/ml) (A) and 3-azido-l-tyrosine (0.3 mg/ml) (B). FT3 cells expressing iodoTyrRS-ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo-l-tyrosine (C). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

Figure 3.

(A) Site-specific incorporation of 3-iodo-l-tyrosine (IY) and 3-azido-l-tyrosine (AzY) into GST in FT3 cells (lanes 1–4) and FB3 cells (lanes 5–10). The full-length GST was detected by western blotting. The growth media were supplemented with IY (lanes 2 and 6) and AzY (lane 8). The wild-type GST (WT) was expressed from pRGexGST (lane 3) or pRSFGST (lane 10). IPTG(–) means no induction of the gst expression (lanes 4 and 9). (B) The full-length GST product, indicated by the arrow, was detected by western blotting in extracts from the FB3 and BL21(DE3) cells. The applied quantity of total protein from the extract is indicated at the bottom of each lane. (C) Fluorescent labeling of rat calmodulin containing 3-azido-l-tyrosine. Extracts from the cells grown in the absence and presence of AzY were mixed with the fluorescein-triarylphosphine conjugate and then applied to SDS–PAGE. The band of calmodulin was indicated by the arrow.

Figure 4.

(A) The amino-acid binding pocket in the crystal structure of iodoTyrRS-ec bound with 3-azido-l-tyrosine. The mutated residues are denoted in red. The carbon atoms of the ligand are shown in green. The nitrogen, oxygen and sulfur atoms are in blue, red and yellow, respectively. The hydrogen bonds are shown as pink broken lines. (B) The surface structures of the binding pockets of iodoTyrRS-ec and iodoTyrRS-mj accommodating 3-aido-l-tyrosine or 3-iodo-l-tyrosine, as indicated below each panel. The enzyme surface and section are shown in light blue. The iodine atom is in purple.