Coupling corncob fractionation with covalent enzyme assembly for resveratrol biosynthesis from lignin-derived p-coumaric acid.

Abstract

Lignin is an abundant renewable source of aromatic carbon, yet its biological valorization remains limited by poor coordination between biomass fractionation and microbial pathway design. We combined urea-KOH (UK) fractionation with pathway-entry optimization and SpyTag/SpyCatcher-mediated enzyme assembly to match the recovery of lignin-derived precursors with microbial resveratrol biosynthesis. UK fractionation released 191.97 ± 7.42 mg p-coumaric acid (p-CA) per g Klason lignin and retained cellulose-rich solids suitable for saccharification. Purified lignin-derived p-CA and glucose-rich hydrolysate from the same corncob were then used as the aromatic precursor and principal carbon source, respectively. In E. coli, replacing plant 4-coumarate: CoA ligase with a lignin-catabolic feruloyl-CoA synthetase improved p-CA activation. Tests using matched vector backgrounds showed that reactive SpyTag/SpyCatcher pairs formed complex-sized species without increasing the soluble abundance of either enzyme. In reconstituted cascades, this assembly increased resveratrol formation and reduced p-coumaroyl-CoA exposure, even though upstream-enzyme activity remained unchanged. These results are consistent with tighter coupling between the cascade enzymes. Following optimization, 5-L fed-batch fermentation produced 803.85 ± 10.06 mg L-1 resveratrol. Exploratory structural modelling and molecular dynamics simulations suggested closer enzyme-pair arrangements and smaller ligand displacements in several assembled models, offering a possible explanation for the improved cascade coupling. The resulting process combines complementary corncob fractions for resveratrol production through coordinated fractionation and pathway engineering.

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