"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 incorporating non-canonical amino acids (ncAAs) into the genetic code. Traditionally, efforts to expand substrate specificity start with a (hyper-)stable enzyme capable of withstanding the structural changes resulting from necessary mutations. However, we adopt a radically different approach by starting with an enzyme that evolved to cope with instability, potentially offering 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 exhibited remarkable in vivo efficiency and promiscuity, even at very low ncAA concentrations. Given the broad range of applicable host organisms, we anticipate that Cold-OTS will significantly advance the transformation of the expanded genetic code from an academic discipline into a high-value chemistry-driven biotechnology.