Genomically integrated orthogonal translation system in Escherichia coli enables production of functional modified [NiFe]-hydrogenases

The functional diversification of O 2 -tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator . Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.