System analysis and improved [FeFe] hydrogenase O2 tolerance suggest feasibility for photosynthetic H2 production.

Abstract Photosynthetic H 2 production has been a compelling but elusive objective. Here we describe how coordinated bioreactor, metabolic pathway, and protein engineering now suggest feasibility for the sustainable, solar-powered production of a storable fuel to complement our expanding photovoltaic and wind based capacities. The need to contain and harvest the gaseous products provides decisive solar bioreactor design advantages by limiting O 2 exposure to prolific, but O 2 -sensitive H 2 producing enzymes—[FeFe] hydrogenases. CO 2 supply and cell growth can also be limited so that most of the photosynthetic reduction capacity is directed toward H 2 production. Yet, natural [FeFe] hydrogenases are still too O 2 sensitive for technology implementation. We report the discovery of new variants and a new O 2 tolerance mechanism that significantly reduce the sensitivity to O 2 exposure without lowering H 2 production rates or losing electrons to O 2 reduction. Testing the improved hydrogenases with a biologically derived, light-dependent electron source provides evidence that this game changing technology has the potential for sustainable large-scale fuel production. Highlights • A single Cys replacement improved O 2 tolerance with only a minor activity loss. • Saturation mutagenesis found synergistic, non-Cys mutations for improving O 2 tolerance. • Higher O 2 tolerance enabled more than 300% improvement in light-driven H 2 production. [ABSTRACT FROM AUTHOR]