Engineered clostridial [FeFe]-hydrogenase shows improved O₂ tolerance in Chlamydomonas reinhardtii

Hydrogenase intolerance to oxygen remains a critical hurdle on the road to photosynthetic hydrogen production for sustainable energy demands. Although the engineering of the intrinsic oxygen tolerance mechanism of hydrogenase using mutagenesis is an ambitious approach, recent in-vitro studies reported a novel and improved synthetic [FeFe]-Hydrogenase variants. To corroborate these findings in-vivo, we expressed either an engineered variant or its cognate wild type enzyme in the chloroplast genome of Chlamydomonas reinhardtii. We characterized their activity using a customized photosynthetic hydrogen production in-vivo assay to test whether the improved variant could maintain a greater fraction of its activity following oxygen exposure. We found that the mutated variant exhibited a superior oxygen tolerance while persevering its photosynthetic performance in terms of hydrogen production yield. Importantly, we show for the first time that this approach can potentially address the inherent O₂ sensitivity of [FeFe]-Hydrogenases for photosynthetic hydrogen production.