Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering.

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.