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

ABSTRACT Orthogonal translation systems (OTSs) enable site‐specific incorporation of non‐canonical amino acids (ncAAs) and are central to genetic code expansion. Current engineering strategies typically rely on hyperstable aminoacyl tRNA synthetase (aaRS) scaffolds to tolerate destabilizing mutations required for substrate diversification. Here, we introduce an alternative design principle: exploiting the intrinsic conformational flexibility of a psychrophilic pyrrolysyl‐tRNA synthetase (PylRS) enzyme to enhance mutational tolerance and in vivo performance. We identified a cold‐adapted PylRS from Methanococcoides burtonii and established a psychrophilic OTS (“Cold‐OTS”) compatible with Escherichia coli . This system consistently outperformed established mesophilic and thermophilic PylRS variants in single‐ and multi‐site ncAA incorporation. Notably, Cold‐OTS maintained high suppression efficiency at low ncAA concentrations and exhibited enhanced absolute performance at reduced cultivation temperatures, even under globally diminished protein synthesis rates. In addition, engineered variants accommodated a broader set of substrates, consistent with elevated substrate promiscuity. These findings establish psychrophilic aaRS scaffolds as potentially powerful resources for genetic code expansion. Given the broad host compatibility of the PylRS platform, Cold‐OTS provides a scalable strategy for efficient production of ncAA‐modified proteins across diverse biological systems.