"Cold" Orthogonal Translation by Psychrophilic Pyrrolysyl-tRNA Synthetase Boosts Genetic Code Expansion

Abstract Using orthogonal translation systems (OTSs) is the most efficient way to produce unnatural proteins by adding non-canonical amino acids (ncAAs) to the genetic code. In the quest to expand substrate specificity the conventional approach begins with a (hyper-)stable enzyme capable of withstanding the structural changes resulting from necessary mutations. However, we here take a radically different position by starting with an enzyme that has evolved to cope with instability, and thus may exhibit greater resilience to mutations. By engineering a psychrophilic ("cold") OTS from Methanococcoides burtonii we developed an alternative to the commonly used mesophilic and thermophilic systems. This OTS showed remarkable properties in terms of catalytic efficiency and promiscuity, even at very low ncAA concentrations. Given the broad range of applicable host organisms, 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.