Journal
BIOTECHNOLOGY JOURNAL
Volume 14, Issue 3, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201800125
Keywords
amber suppression; biorthogonal conjugation; Desulfitobacterium hafniense; genetic code expansion; para-azido-phenylalanine; pyrrolysyl-tRNA synthetase
Funding
- Austrian Science Fund (FWF) [W901]
- Federal Ministry of Science, Research and Economy (BMWFW)
- Federal Ministry of Traffic, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol
- Government of Lower Austria
- ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program [282482]
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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 (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.
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