Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.
Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
Iraha
Fumie
F
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Oki
Kenji
K
Kobayashi
Takatsugu
T
Ohno
Satoshi
S
Yokogawa
Takashi
T
Nishikawa
Kazuya
K
Yokoyama
Shigeyuki
S
Sakamoto
Kensaku
K
D001386:Azides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D017353:Gene Deletion; D005815:Genetic Code; D005816:Genetic Complementation Test; D017015:Methanococcales / Q000201:enzymology / Q000235:genetics; D007470:Monoiodotyrosine / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012365:RNA, Transfer, Tyr / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013489:Suppression, Genetic; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C523964:3-iodotyrosine; D001386:Azides; D012365:RNA, Transfer, Tyr; C065394:3-azidotyrosine; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase; D007470:Monoiodotyrosine