Abstract
AbstractOrthogonal translation systems (OTSs) are the most expedient way to generate unnatural proteins by adding non-canonical amino acids (ncAAs) to the genetic code. Diversifying substrate specificity typically starts from a stable enzyme that is capable to withstand the structure-perturbing effects of the required mutations. We here take a radically different position, starting from an enzyme that has evolved to cope with instability and thus may better tolerate mutations. By engineering psychrophilic (“cold”) PylRS orthologs we develop “cold” OTS as an alternative to the commonly used mesophilic and thermophilic OTSs. In particular, the psychrophilic PylRS homolog fromMethanococcoides burtonii(MburPylRS) showed remarkable properties in terms of catalytic efficiency and promiscuity, even at very low ncAA concentrations. Outstanding multi-site incorporation efficiencies were achieved withNε-tert-Butoxycarbonyl-L-lysine (BocK) and especiallyS-allyl-L-cysteine in up to five sites. Given the broad range of host organisms (archaea, bacteria, and eukaryotes) in which the system can be used, we anticipate that Cold-OTS will greatly facilitate the transformation of the expanded genetic code from an academic discipline into a high-value chemistry-driven biotechnology.
Publisher
Cold Spring Harbor Laboratory
Cited by
2 articles.
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