A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNA His pair

Abstract

Background Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNAHis recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNAHis pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus. Methods E. coli was genetically engineered to use a C. crescentus HisRS•tRNAHis pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo. Results A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNAHis pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNAHis pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUA elevated its suppression efficiency by 2-fold. Conclusions The C. crescentus HisRS•tRNAHis pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNAHis is orthogonal in MEOV1 cells. E. coli tRNAHisCUA is an efficient amber suppressor in MEOV1. General significance We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli.

Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes

“…However, functionally replacing an endogenous aaRS/tRNA pair of E. coli with a eukaryotic/archaeal counterpart can enable its reintroduction in the resulting “altered translational machinery” (ATM) strain as an orthogonal nonsense suppressor (Figure 1B). (Englert et al, 2017; Iraha et al, 2010; Italia et al, 2017a) Recently, we demonstrated that such an ATM strain can serve as the selection host for altering the substrate specificity of the “liberated” bacterial tryptophanyl-tRNA synthetase/tRNA pair. (Italia et al, 2017a) The resulting engineered variants of this pair then can be used for Uaa mutagenesis both in eukaryotes and in the engineered E. coli strain.…”

Rational Design of Aptamer-Tagged tRNAs

“…It is well known that a large subgroup of α-Proteobacteria has a non-canonical tRNA His lacking the G-1:C73 pair and has a non-canonical HisRS species that recognizes the non-canonical G1:U72 pair and A73 in addition to the GUG anticodon [ 17 , 57 , 58 , 59 , 60 ]. The two α-proteobacterial species are Afifella pfennigii DSM 17,143 ( Ap ) [ 17 ] and Consotaella salsifontis USBA 369 ( Cs ) [ 61 ], because the well-studied α-proteobacterial Caulobacter crescentus HisRS was not active enough in E. coli [ 60 ]. Two kinds of allo-tRNA chassis, S072 and S005, were engineered to have a typical α-proteobacterial tRNA His anticodon arm as their V-arms, to develop H072-αψHis-3 and αH005-αψHis-3/4/5 variants ( Figure 7 A).…”

“…For example, the pairs of Ca. M. alvus TyrRS and Y072/Y005M and the pairs of Cs HisRS variants and αH005-αψHis-4 variants may be orthogonal in E. coli and might be useful for incorporating non-canonical amino acids [ 17 , 58 , 60 ]. Alternatively, some of the endogenous tRNAs or tRNA-aaRS pairs might be replaceable with allo-tRNAs or orthogonal pairs composed of allo-tRNAs [ 60 , 63 , 64 , 65 ].…”

“…M. alvus TyrRS and Y072/Y005M and the pairs of Cs HisRS variants and αH005-αψHis-4 variants may be orthogonal in E. coli and might be useful for incorporating non-canonical amino acids [ 17 , 58 , 60 ]. Alternatively, some of the endogenous tRNAs or tRNA-aaRS pairs might be replaceable with allo-tRNAs or orthogonal pairs composed of allo-tRNAs [ 60 , 63 , 64 , 65 ]. For reprogramming the genetic code, the allo-tRNA sequestration system may be useful for use in vivo and in vitro [ 66 ] and for the purification and elimination of allo-tRNA molecules from tRNA mixtures [ 66 , 67 , 68 , 69 ].…”

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

“…High-throughput sfGFP stop codon read-through assays were carried out in 96-well plates as previously described with minor modifications [ 20 ]. Briefly, E. coli strains were either transformed with pET-sfGFP-sepT or co-transformed with pET-sfGFP-sepT and pCDF2-SepRS.…”