A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry

Biotechnol J. 2019 Mar;14(3):e1800125. doi: 10.1002/biot.201800125. Epub 2018 Jun 11.


The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

Keywords: Desulfitobacterium hafniense; amber suppression; biorthogonal conjugation; genetic code expansion; para-azido-phenylalanine; pyrrolysyl-tRNA synthetase.

MeSH terms

  • Amino Acids / genetics*
  • Amino Acyl-tRNA Synthetases / genetics*
  • Azides / chemistry*
  • Azides / metabolism*
  • Desulfitobacterium / genetics
  • Escherichia coli / genetics
  • Methanosarcina / genetics
  • Substrate Specificity / genetics


  • Amino Acids
  • Azides
  • Amino Acyl-tRNA Synthetases