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

Tungsten-dependent formate dehydrogenases catalyze CO₂ reduction, yet their heterologous production remains limited by the absence of tungsten-specific cofactor maturation in standard laboratory hosts. Although molybdenum-dependent formate dehydrogenases have been implemented in Escherichia coli , analogous maturation of tungsten-dependent enzymes has proven unsuccessful because native metal homeostasis does not support selective tungstate uptake or W-bis-MGD assembly. Here we reconstitute tungsten-specific metal metabolism in E. coli by reconstructing tungsten cofactor biosynthesis, installing a high-affinity tungstate uptake system, and reinforcing Fe–S cluster biogenesis. This enables aerobic production of catalytically competent tungsten-dependent formate dehydrogenase from Methylorubrum extorquens AM1, achieving native-level specific activity and a tungsten occupancy of 0.92 mol per mol enzyme—nearly double that of the native host. The engineered strain delivers a volumetric activity exceeding 3,500 U L⁻¹. In a coupled whole-cell CO-to-formate biotransformation, the strain generated 44 mM formate within 4 hours, outperforming the native host system by more than threefold. The platform also supports activation of W-bis-MGD-dependent enzymes from Cupriavidus necator H16 and Lutibaculum baratangense AMV1. This work establishes a general design framework for W-bis-MGD maturation, expanding access to tungsten-dependent biocatalysts for CO₂-to-formate and related reductive biotransformations.