Hydrogen is widely believed as the best future clean energy carrier to replace oil-derived liquid fuels due to increased energy utilization efficiencies and decreased reliance of fossil fuels. Microbial anaerobic fermentation for biological hydrogen production suffers from a low theoretical hydrogen yield (i.e., 4 H<sub>2</sub> per glucose). Previously we designed in vitro synthetic enzymatic pathways to implement high-yield hydrogen production from biomass sugars (e.g., glucose and xylose) and water (Rollin et al. 2015). However, the complicated structure of starch results in great challenge of the complete utilization of starch. Different from enzymatic hydrolysis of starch to glucose, we design a novel starch phosphorolysis pathway including isoamylase, alpha-glucan phosphorylase, 4-glucanotransferase, and polyphosphate glucokinase for ATP-free generation of glucose 1-phosphate from starch. Inspired by high-speed hydrogen production for dark fermentation, we applied an abiotic mediator (benzyl viologen) instead of ferredoxin between NADPH, generated from the pentose phosphate pathway, and [NiFe]-hydrogenase to decrease the activation energy for H<sub>2</sub> production (Kim et al. 2016). A total of 15 enzymes were used to build an in vitro enzymatic pathway for the complete utilization of starch, generating 12 moles of hydrogen from one mole of glucose and six moles of water, for the first time. Also, enhanced hydrogen generation rates via the artificial electron transport chain are fast enough for stationary hydrogen production.

High-yield & high-speed biohydrogen production from starch and water via in vitro synthetic enzymatic pathways

253rd National Meeting (2017)

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