期刊
NATURE CHEMICAL BIOLOGY
卷 13, 期 12, 页码 1253-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEMBIO.2474
关键词
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资金
- US National Institutes of Health (NIH) [R01 EB022376, R35 GM118062, R21 AI119813, R01 GM022854, R35 GM122560]
- Department of Energy [FG02-98ER2031]
- Defense Advanced Research Projects Agency [N66001-12-C-4207]
- Howard Hughes Medical Institute
- Ruth L. Kirschstein National Research Service Award [F32 GM106621]
Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (K-cat/K-M(tRNA)) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
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