Development of a Universal Platform for the Heterologous Expression of Bidirectional [Ni–Fe]-Hydrogenases in <i>E. coli</i>

Bidirectional [Ni-Fe]-hydrogenases are useful tools for integrating hydrogen into existing chemical processes by utilizing H₂ to regenerate expensive cofactors such as NAD(P)H. One enzyme broadly applied to this purpose is the soluble [Ni-Fe]-hydrogenase from <i>Cupriavidus necator</i> (<i>Cn</i>SH). However, the homologous production of <i>Cn</i>SH suffers from slow growth rates and complex growth medium requirements of the native host. In the present study, we developed a simple approach for the production of <i>Cn</i>SH in <i>Escherichia coli</i> based on the coexpression of the maturation factors from <i>C. necator</i>. By optimizing the artificial operons coding for the hydrogenase proteins as well as the maturation factors, we were able to produce <i>Cn</i>SH with similar yields and activities compared to the native host. Additionally, we used our system to express three functional novel soluble [Ni-Fe]-hydrogenases, demonstrating its applicability for future enzyme screening and discovery.