Hydrogen-Driven Cell-Free Cofactor Regeneration Enables Stoichiometric Bioconversion of Pyruvate to Lactate

The declining cost of green hydrogen—projected below 1.5 USD/kg by 2030—opens new avenues for its use beyond fuel cells and industrial heating. Here we demonstrate that H ₂ can serve as a stoichiometric electron donor for cell-free enzymatic cofactor regeneration, coupling H ₂ oxidation to NADPH production and driving the complete bioconversion of pyruvate to lactate. A partially purified enzyme ensemble from Escherichia coli overexpressing Clostridium pasteurianum ferredoxin, augmented with [FeFe]-hydrogenase CpII, delivers NADP ⁺ reduction rates of 103 μM min ⁻¹ (27-fold enhancement) with superlinear dependence on H ₂ partial pressure. Reconstitution from purified components (CpI or CpII, CpFd, AnFNR, LDH) uncovers a redox-potential-dependent lag phase: the NADPH/NADP ⁺ ratio must exceed 0.85 before pyruvate reduction becomes thermodynamically spontaneous, after which the rate accelerates exponentially. These results position hydrogen-driven cofactor regeneration as a scalable, byproduct-free platform for reductive biotransformations powered by renewable H ₂ .