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

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 Cupriavidus necator (CnSH). However, the homologous production of CnSH 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 CnSH in Escherichia coli based on the coexpression of the maturation factors from C. necator. By optimizing the artificial operons coding for the hydrogenase proteins as well as the maturation factors, we were able to produce CnSH 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.