Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (P_SH) fusion to gfp (green fluorescent protein)

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H₂). Amongst a number of promising candidates for application in the oxidation of H₂ is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise P_SH promoter activity using several gene cloning approaches. A P_SH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the P_SH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study P_SH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate P_SH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.