Metal cofactor-level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia...

Tungsten-dependent formate dehydrogenases are among the most active biological catalysts for carbon dioxide reduction, but their production is limited by complex metal cofactor requirements and poor compatibility with standard expression hosts. Here, we address this limitation by engineering an Escherichia coli chassis through reconstruction of tungsten cofactor biosynthesis, installation of a high-affinity tungstate transporter, and reinforcement of iron-sulfur cluster biogenesis. The resulting chassis produces the formate dehydrogenase from Methylorubrum extorquens with native-level specific activity, near-stoichiometric tungsten incorporation, and high volumetric activity, while substantially enhancing whole-cell CO-driven CO₂-to-formate conversion compared with the native-host system. The engineered host demonstrates broad applicability by supporting maturation of tungsten-dependent formate dehydrogenases from Cupriavidus necator, Lutibaculum baratangense, Thermococcus onnurineus, and Thermoanaerobacter kivui. In this work, we show that engineering host metal cofactor metabolism provides a strategy for aerobic production of active tungsten-dependent formate dehydrogenases, expanding access to this enzyme family for biological and biotechnological applications.