GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE300nnn/GSE300708/primaryOK200Transcriptomics Gelatoporia subvermisporaTrametes versicolorExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE300708GEOGSEfalseCatabolism of lignin-related methoxylated compounds in white-rot fungi utilizes a non-canonical extradiol dioxygenaseWhite-rot fungi (WRF) are the most effective lignin-degrading organisms in nature, making them essential to Earth’s carbon cycle. Lignin is a highly methoxylated, heterogeneous biopolymer derived from plants. However, the pathways WRF use to metabolize methoxylated aromatic compounds as carbon sources remain unidentified. Here, we employ a systems biology approach to elucidate the intracellular catabolism of vanillate – a monomethoxylated aromatic compound – in two white-rot fungi (WRF), Gelatoporia subvermispora and Trametes versicolor. We identified and biochemically validated a four-enzyme pathway that converts vanillate into β-ketoadipate – a metabolite that enters central carbon metabolism. This pathway deviates from typical bacterial pathways, where vanillate is initially demethylated and ring-cleaved by intradiol dioxygenases; instead, oxidative decarboxylation occurs prior to ring cleavage by extradiol dioxygenases. Thus, we conducted an in-depth investigation of ring cleavage and further downstream catabolism by the identified fungal enzymes using biochemical and structural approaches. This revealed non-canonical enzymes, including a highly substrate-specific extradiol dioxygenase and a metal-free, promiscuous reductase, the latter capable of acting on catabolic intermediates derived from both methoxylated and non-methoxylated aromatic compounds. This work emphasizes the potential of WRF and their enzymes to advance lignin valorization and enhance our understanding of their role during wood decay.2026/04/01GSE300708GSM9066989GSM9066999GSM9066988GSM9067009GSM9066998GSM9066987GSM9067000GSM9067010GSM9067008GSM9066997GSM9067007GSM9066996GSM9067006GSM9066995GSM9067005GSM9066994GSM9066993GSM9067004GSM9067003GSM9066992GSM9067002GSM9066991GSM9067001GSM90669902861928618300708 Gelatoporia subvermisporaTrametes versicolor