TY - JOUR
T1 - A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry
AU - Fladischer, Patrik
AU - Weingartner, Alexandra
AU - Blamauer, Johannes
AU - Darnhofer, Barbara
AU - Birner-Gruenberger, Ruth
AU - Kardashliev, Tsvetan
AU - Ruff, Anna Joelle
AU - Schwaneberg, Ulrich
AU - Wiltschi, Birgit
PY - 2019/3/1
Y1 - 2019/3/1
N2 - 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.
AB - 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.
KW - amber suppression
KW - biorthogonal conjugation
KW - Desulfitobacterium hafniense
KW - genetic code expansion
KW - para-azido-phenylalanine
KW - pyrrolysyl-tRNA synthetase
UR - http://www.scopus.com/inward/record.url?scp=85056609210&partnerID=8YFLogxK
U2 - 10.1002/biot.201800125
DO - 10.1002/biot.201800125
M3 - Article
C2 - 29862654
AN - SCOPUS:85056609210
SN - 1860-6768
VL - 14
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 3
M1 - 1800125
ER -