title: A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNAHispair.
abstract: 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 tRNAHisrecognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNAHispair 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.
  
  
  
  E. coli was genetically engineered to use a C. crescentus HisRS•tRNAHispair. 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.
  
  
  
  A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNAHispair 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•tRNAHispair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUAelevated its suppression efficiency by 2-fold.
  
  
  
  The C. crescentus HisRS•tRNAHispair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNAHisis orthogonal in MEOV1 cells. E. coli tRNAHisCUAis an efficient amber suppressor in MEOV1.
  
  
  
  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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Englert
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Vargas-Rodriguez
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Noah M
authors.initials: NM
authors.lastname: Reynolds
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Yane-Shih
authors.initials: YS
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: 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.
authors.forename: Takuya
authors.initials: T
authors.lastname: Umehara
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D006643:Histidine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D012354:RNA, Transfer, His / Q000235:genetics / Q000378:metabolism*
chemical_list: D012354:RNA, Transfer, His; D006643:Histidine-tRNA Ligase
keywords: Aminoacyl-tRNA synthetase; Genetic code expansion; Non-canonical amino acids; Orthogonal pair; Synthetic biology; tRNA