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A Semi‐Rationally Engineered Bacterial Pyrrolysyl‐tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry
Author(s) -
Fladischer Patrik,
Weingartner Alexandra,
Blamauer Johannes,
Darnhofer Barbara,
BirnerGruenberger Ruth,
Kardashliev Tsvetan,
Ruff Anna Joelle,
Schwaneberg Ulrich,
Wiltschi Birgit
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800125
Subject(s) - transfer rna , biochemistry , escherichia coli , amino acid , chemistry , phenylalanine , aminoacyl trna synthetase , bacteria , biology , gene , genetics , rna
The site‐specific incorporation of non‐canonical amino acids (ncAAs) at amber codons requires an aminoacyl‐tRNA synthetase and a cognate amber suppressor tRNA (tRNA CUA ). 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/tRNA CUA 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 ( Dh AzFRS) using a semi‐rational approach. Dh AzFRS 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 Dh AzFRS 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.