<p><i>Methylorubrum extorquens</i> formate dehydrogenase I (Me-FDH1) is a tungsten-dependent heterodimeric enzyme with high activity for CO₂/formate interconversion, making it an attractive biocatalyst for carbon capture, formate production, and bioelectrocatalysis. Its broader application, however, is limited by the lack of a heterologous production host for this complex metalloenzyme. Here, we systematically evaluated <i>Escherichia coli</i> as a host for Me-FDH1 production and compared its performance with that of the native host and a previous heterologous host. Across multiple <i>E. coli</i> strains, active Me-FDH1 was obtained only when tungstate uptake was supported by a functional <i>Mod</i>ABC system or by heterologous expression of the <i>Tup</i>BCA transporter, demonstrating that <i>E. coli</i> can synthesize and incorporate the W-bis-MGD cofactor. Nevertheless, expression remained low (&lt; 1% of total cellular protein), and the purified enzyme displayed only 4–14 U mg^− 1 specific activity, far below the 80–100 U.mg^− 1 observed for the enzyme produced in <i>M. extorquens</i>. Operon redesign, altered gene order, stronger ribosome-binding sites, SUMO fusion, chaperone co-expression, and codon harmonization did not improve α-subunit production. In both <i>E. coli</i> and <i>M. extorquens</i>, the α-subunit was poorly produced in the absence of the β-subunit, indicating that the β-subunit contributes to α-subunit stabilization and/or maturation. Cell-free translation produced both subunits efficiently, showing that the principal barrier in <i>E. coli</i> is not transcription or translation, but post-translational instability and likely proteolytic loss of the α-subunit. These findings define the key bottleneck for Me-FDH1 production in <i>E. coli</i> and provide a roadmap for engineering hosts for tungsten-containing enzymes.</p> Graphical abstract <p></p>