Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations.

Background: O-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks.

Results: In this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity.

Conclusions: This work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies.