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

Patrik Fladischer, Alexandra Weingartner, Johannes Blamauer, Barbara Darnhofer, Ruth Birner-Gruenberger, Tsvetan Kardashliev, Anna Joelle Ruff, Ulrich Schwaneberg, Birgit Wiltschi*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Article number1800125
JournalBiotechnology Journal
Issue number3
Early online date3 Jun 2018
Publication statusPublished - 1 Mar 2019
Externally publishedYes


  • amber suppression
  • biorthogonal conjugation
  • Desulfitobacterium hafniense
  • genetic code expansion
  • para-azido-phenylalanine
  • pyrrolysyl-tRNA synthetase

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine


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