We have developed a scalable platform that employs electrolysis for an in vitro synthetic enzymatic cascade in a continuous flow reactor. Both H₂ and O₂ were produced by electrolysis and transferred through a gas‐permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H₂ and O₂ served as electron mediators for the biocatalysts. We demonstrate the production of methylated N‐heterocycles from diamines with up to 99 % product formation as well as excellent regioselective labeling with stable isotopes. Our platform can be applied for a broad panel of oxidoreductases to exploit electrical energy for the synthesis of fine chemicals.

Molecular hydrogen (H₂) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H₂ production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H₂ syntheses rely mainly on light, water, and CO₂ and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H₂-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O₂) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O₂-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O₂-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O₂ sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O₂ tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH⁺ to utilize H₂ as sole electron source to fix CO₂. Syn_ReSH⁺ also was able to produce H₂ under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H₂ production.

We designed a novel H₂-driven cascade for the biosynthesis ofN-heterocycles.

A bacterial whole-cell system was designed for hydroxylation of n -octane to 1-octanol at the expense of molecular hydrogen and oxygen.

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H₂ conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by ⁵⁷Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD⁺-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective ¹³C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

We show that an O₂-tolerant hydrogenase is capable to regenerate flavins and to power flavin- and H₂O₂-dependent biocatalysts with H₂.