Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck

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🔬研究者:Provides a clear roadmap for engineering E. coli to produce active tungsten-containing formate dehydrogenases, highlighting the critical role of α-subunit stabilization.

🏢実務担当者:May inform biotech companies targeting enzyme-based carbon capture or formate production, but immediate applicability is limited due to low yields.

<title>Abstract</title> <p>Methylorubrum extorquens 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 Escherichia coli as a host for Me-FDH1 production and compared its performance with that of the native host and a previous heterologous host. Across multiple E. coli strains, active Me-FDH1 was obtained only when tungstate uptake was supported by a functional ModABC system or by heterologous expression of the TupBCA transporter, demonstrating that E. coli can synthesize and incorporate the W-bis-MGD cofactor. Nevertheless, expression remained low (<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 M. extorquens. 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 E. coli and M. extorquens, 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 E. coli 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 E. coli and provide a roadmap for engineering hosts for tungsten-containing enzymes.</p>

• Research Square https://doi.org/10.21203/rs.3.rs-9133619/v1first seen 2026-05-14 21:23:32