Abstact

White-rot fungi (WRF) are the most effective lignin-degrading organisms in nature. Lignin is a highly methoxylated plant biopolymer, yet the pathways WRF use to metabolize methoxylated aromatic monomeric compounds as carbon sources remain unidentified. Using systems biology, we elucidate the intracellular catabolism of vanillate-a monomethoxylated aromatic compound-by Gelatoporia subvermispora and Trametes versicolor. We identify and biochemically validate a four-enzyme pathway that converts vanillate into beta-ketoadipate, which enters central carbon metabolism. Unlike bacteria, which demethylate vanillate before ring-cleavage by intradiol dioxygenases, WRF employ oxidative decarboxylation followed by extradiol dioxygenase-mediated cleavage. A previously uncharacterized hydrolase is also shown to catalyze the terminal step of this pathway. Biochemical and structural approaches reveal non-canonical enzymes, including a highly substrate-specific extradiol dioxygenase and a metal-free, promiscuous reductase that acts on both methoxylated and non-methoxylated intermediates. These findings highlight distinct fungal strategies for aromatic degradation, offering insights into lignin valorization and wood decay mechanisms.