“Cold” Orthogonal Translation: A Psychrophilic Pyrrolysyl-tRNA Synthetase Boosts Genetic Code Expansion in E. coli

Using orthogonal translation systems (OTSs) is one of the most efficient strategies for producing unnatural proteins through the incorporation of 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 induced by necessary mutations. In contrast, we propose a radically different approach for the PylRS system: by starting with enzymes that evolved to cope with instability in order to adapt to cold conditions, potentially offering greater resilience to mutational changes. By finding and further engineering a psychrophilic (“cold”) OTS from Methanococcoides burtonii , we developed an alternative to the widely used mesophilic and thermophilic systems. This novel OTS demonstrated exceptional in vivo efficiency for a broad range of substrates, even at very low ncAA concentrations and low cultivation temperatures. The general versatility of the PylRS system across a wide range of applicable host organisms suggests that the Cold-OTS has the potential to also improve protein yields in these hosts and help to drive the transformation of the expanded genetic code from an academic pursuit into a high-value, chemistry-driven biotechnology.