Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck
TL;DR
The α-subunit was the major bottleneck in Me-FDH1 production and was defined as the key bottleneck for Me-FDH1 production in E. coli and provides a roadmap for engineering hosts for tungsten-containing enzymes.
Abstract
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. E. coli produced active Me-FDH1 when tungstate uptake was enabled. ModABC or TupBCA supported W-bis-MGD-dependent Me-FDH1 activity. The α-subunit was the major bottleneck in Me-FDH1 production. The β-subunit promoted α-subunit accumulation and/or maturation. Cell-free translation revealed post-translational loss of the α-subunit.