Introduction: Lignin, a key component of lignocellulosic biomass, is an abundant and renewable resource that remains vastly underexploited. Selective depolymerization of lignin yields valuable lignin-derived aromatic compounds (LDACs) like alkylguaiacols and vanillate, which can serve as precursors for high-value platform chemicals. Enzymatic valorization of these LDACs encounters critical bottlenecks. Key enzymes, such as cytochrome P450s and Rieske oxygenases, enable efficient O-demethylation but are inherently unstable and depend on costly, stoichiometric amounts of cofactors, limiting both scalability and economic feasibility. Using H₂-driven cofactor regeneration via hydrogenases (SH) offers an effective solution.
Goals: (1) Develop high-performance, safe H₂-driven O-demethylation by coupling SH with cytochrome P450s and Rieske oxygenases; (2) Establish a scalable platform for H₂-driven reactions, linking LDACs valorization to renewable energy through a continuous flow chemistry system with in-situ H2 and O2 generation.
Methods: AgcA/AgcB, PbdA/HaPuX/HaPuR (P450s), and VanA/VanB (Rieske-type) have been expressed in E. coli, with SH produced homologously in C. necator; all were purified. Systems were tested in gas-tight batch reactions with varied NAD⁺ levels and H2 held below 4%. A flow chemistry setup with PEM electrolysis enabled in situ H₂ production and scaled reaction volumes. Product yield and purity were assessed via real-time gas monitoring, HPLC, NMR.
Results: Batch reactions achieved high turnover numbers (TTNs) and >99% yield, effectively balancing cofactor cost and efficiency with minimized NAD⁺ levels. Gas studies confirmed that maintaining hydrogen concentrations below 4% ensured safe operation without compromising performance. The flow system sustained high conversions (>99%) at an efficient Faradaic rate of 27.1%, with a final product yield of 82.3% after extraction. Together, these metrics underscore the system's scalability and efficiency, highlighting its potential as a viable option for sustainable, industrial-scale LDAC valorization.
Summary: This study presents a scalable platform for LDACs valorization, integrating H2- and electro-driven cofactor regeneration in continuous flow. With efficient enzymes, safe gas handling, and renewable PEM technology, our approach offers a sustainable model for industrial biocatalysis and expands the potential for NAD(P)H-dependent oxidoreductase applications beyond LDACs valorization.