Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H production. Direct H generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H /L h , the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H production biosystems and artificial photosynthesis.

To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

Green hydrogen production through water splitting at low temperatures is highly desired for hydrogen economy. Herein, we demonstrate an in vitro non‐natural enzymatic pathway to utilize the chemical energy stored in xylooligosaccharides from biomass to split water to produce a nearly theoretical yield of H2 (i.e., ≈9.5 H2 per xylose plus water). This pathway was constructed on the basis of the novel activities of phosphopentomutase catalyzing the conversion of d‐xylose 1‐phosphate into d‐xylose 5‐phosphate and of ribose 5‐phosphate isomerase catalyzing the conversions of d‐xylose 5‐phosphate and d‐xylulose 5‐phosphate. This study suggests that the discovery of novel promiscuous enzyme activities is important to implement complicated biotransformations catalyzed by synthetic enzymatic pathways.