Format

Send to

Choose Destination
Biochim Biophys Acta Gen Subj. 2017 Nov;1861(11 Pt B):3009-3015. doi: 10.1016/j.bbagen.2017.03.003. Epub 2017 Mar 10.

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

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
2
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Chemistry, Yale University, New Haven, CT 06520, USA. Electronic address: dieter.soll@yale.edu.
3
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan. Electronic address: tumehara@rs.noda.tus.ac.jp.

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. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

KEYWORDS:

Aminoacyl-tRNA synthetase; Genetic code expansion; Non-canonical amino acids; Orthogonal pair; Synthetic biology; tRNA

PMID:
28288813
PMCID:
PMC5592127
DOI:
10.1016/j.bbagen.2017.03.003
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center