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

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 tRNA ^His recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNA ^His 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•tRNA ^His 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•tRNA ^His 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•tRNA ^His pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNA ^His _CUA elevated its suppression efficiency by 2-fold.
Conclusions: The C. crescentus HisRS•tRNA ^His pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNA ^His is orthogonal in MEOV1 cells. E. coli tRNA ^His _CUA 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.
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