Rewiring hydrogenase-dependent redox circuits in cyanobacteria

Area of Science:

• Biotechnology
• Microbiology
• Bioenergetics

Background:

• Hydrogenases are crucial enzymes for reversible hydrogen production.
• Cyanobacteria are potential platforms for photobiological hydrogen generation.
• Optimizing hydrogenase activity is key to increasing yields.

Purpose of the Study:

• To enhance photobiological hydrogen production in cyanobacteria.
• To investigate the function of a heterologously expressed [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum.
• To assess the potential of engineered cyanobacteria for sustainable hydrogen fuel.

Main Methods:

• Gene expression of Clostridium acetobutylicum [FeFe] hydrogenase (HydA) in Synechococcus elongatus sp. 7942.
• In vitro and in vivo functional assays of hydrogenase activity.
• Measurement of light-dependent hydrogen evolution rates.
• Growth experiments under hydrogen and light conditions with Photosystem II inactivation.
• Modulation of redox flux using exogenous ferredoxins.

Main Results:

• Heterologously expressed HydA was functional in vitro and in vivo.
• HydA activity was linked to the light-dependent electron transport chain.
• Engineered cyanobacteria showed >500-fold increase in light-dependent hydrogen evolution compared to endogenous hydrogenase.
• HydA enabled limited growth using H(2) and light with Photosystem II inactivated.
• Exogenous ferredoxins enhanced hydrogen yields and facilitated dark fermentation into H(2).

Conclusions:

• Heterologous expression of [FeFe] hydrogenase (HydA) significantly enhances photobiological hydrogen production in cyanobacteria.
• Engineered cyanobacteria offer a viable platform for sustainable hydrogen fuel generation.
• Modulating redox flux presents a strategy for further optimizing hydrogen yields.