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bioRxiv · 10.64898/2026.08.07.743378

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

Swartz, J.·Wang, W.·Liu, Q.
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Abstract

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 H2 can serve as a stoichiometric electron donor for cell-free enzymatic cofactor regeneration, coupling H2 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 M min-1 (27-fold enhancement) with superlinear dependence on H2 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 H2.

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Swartz, J., Wang, W., Liu, Q.. 2026-08-10. Hydrogen-Driven Cell-Free Cofactor Regeneration Enables Stoichiometric Bioconversion of Pyruvate to Lactate. https://doi.org/10.64898/2026.08.07.743378

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