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

Authors

Qin Fan, Stevanie Stevanie, Stefan Frielingsdorf, Peter Neubauer, Oliver Lenz, Matthias Gimpel

Published in

Microbial cell factories. Jul 06, 2026. Epub Jul 06, 2026.

Abstract

The functional diversification of O₂-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.

PMID:
42410413
Bibliographic data and abstract were imported from PubMed on 07 Jul 2026.

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