Integrated Gelidium amansii biorefinery for selective HMF and green enzymatic FDCA production via ketohexose bias and engineered oxidase

Gelidium amansii , a marine red alga rich in cellulose and agar, is a promising blue-biorefinery feedstock for the sustainable production of furan-based platform chemicals. This study introduces, an integrated, fully aqueous, and metal-free biorefinery approach that combines a ketohexose-biased dehydration pathway with an engineered 5-hydroxymethylfurfural (HMF) oxidase (8BxHMFO) to enable efficient HMF formation and subsequent production of 2,5-furandicarboxylic acid (FDCA). Thermo-acid hydrolysis was first applied to fractionate agarose into a galactose-rich liquid while retaining cellulose in the solid residue, achieving 25.8% solids recovery, and 97.5% glucan recovery. The recovered cellulose was subsequently hydrolyzed enzymatically to glucose. Inhibitory ions were then removed through electrodialysis, followed by selective adsorption of HMF on activated carbon. Pre-isomerization and epimerization (D-galactose → D-tagatose via gtAI; D-glucose → D-fructose/D-psicose via smXI ± pcTE) were critical for enhancing HMF formation. Response surface analysis revealed that HMF yields increased by more than 50-fold, rising from < 1% for aldoses to as high as 80% for ketohexoses. Under identical acid dehydration conditions, HMF titers increased from 0.20 to 1.15 mg mL -1 following galactose-to-tagatose conversion and from 0.38 to 3.80 mg mL -1 after glucose-to-fructose or psicose conversion. The resulting HMF fractions, ~2 mM in concentration, were oxidized to FDCA by 8BxHMFO under mild aqueous conditions, yielding 1.72 mM FDCA with 84.9% conversion and negligible accumulation of intermediates. Overall, this integrated process enables efficient HMF generation and sustainable FDCA production from G. amansii , establishing a scalable marine-biomass route to bio-based PEF monomers within a fully aqueous and metal-free framework. • Gelidium amansii was valorized as a marine feedstock for furanic chemicals • A fully aqueous, metal-free route coupled ketohexose-biased dehydration and oxidation • Pre-isomerization markedly increased HMF yields from GA-derived hexose streams • Engineered 8BxHMFO oxidized HMF to FDCA with 84.9% conversion under mild conditions • The workflow enables a scalable marine-biomass route for sustainable PEF monomers