H2-driven biocatalytic O-demethylation of lignin derived aromatics in a closed-loop flow system powered by water electrolysis

Abstract

Lignin is an abundant and renewable source of aromatic compounds, yet its utilization remains limited due to its recalcitrance and heterogeneity. Recent developments have enabled the catalytic fractionation of lignin into low molecular weight aromatics, which may be transformed into higher-value compounds. Here, we present a scalable biocatalytic platform for the selective O-demethylation of lignin-derived aromatic compounds, which integrates an O₂-tolerant, NAD⁺-reducing soluble hydrogenase from Cupriavidus necator for cofactor regeneration and NADH-dependent cytochromes P450 and Rieske-type monooxygenases. The process was implemented in a closed-loop flow system featuring dialysis membrane-entrapped multi-enzyme modules. H₂ and O₂ were precisely supplied via gas addition modules powered by water electrolysis. This configuration achieved >99% substrate conversion, high atom efficiency, and effective real-time management of the inhibitory byproduct formaldehyde. The hydrogenase-based cofactor regeneration system exhibits robust tolerance to formaldehyde and is adaptable to a broad range of gas-dependent biocatalytic processes, thereby advancing green, resource-efficient chemical production from renewable biomass.