Cascade reactions with two non‐physiological partners for NAD(P)H regeneration via renewable hydrogen

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides.Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28 +/- 2 nmol NADPH regenerated s-1 mg of hydrogenase-1 (i.e., 1.68 +/- 0.12 U mg-1, TOF: 126 +/- 9 min-1) and 0.46 +/- 0.04 nmol NADH regenerated s-1 mg of hydrogenase-1 (i.e., 0.028 +/- 0.002 U mg-1, TOF: 2.1 +/- 0.2 min-1), meaning up to 74 mg of NADPH and 1.23 mg of NADH produced per hour by a system involving 1 mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.This work demonstrated a novel concept of an artificial and stable two-component protein system able to sustain a good NADPH regeneration rate from hydrogen. The system proposed here is the first of its kind, based on a highly active and oxygen-resistant [FeFe]-hydrogenase and a reductase (BMR) as non-physiological partners. As a proof-of-concept, a cascade reaction involving the [FeFe]-hydrogenase, BMR and a mutant Baeyer-Villiger monooxygenase is presented, showing how the cascade can be exploited for indigo production in multiple reaction cycles.