Abstract
Lignin-derived aromatic aldehydes are key intermediates in lignocellulosic biomass degradation but can exert cytotoxic effects on microorganisms. Ceriporiopsis subvermispora exhibited enhanced glucose consumption and mycelial growth when cultured in the presence of vanillin, suggesting its ability to metabolize lignin-derived aldehydes. We identified 16 putative aldehyde dehydrogenase genes (Cs-aldhA-P) from C. subvermispora, based on sequence homology to known aromatic aldehyde dehydrogenases (Pc-ALDH1 and Pc-ALDH2) from Phanerochaete chrysosporium. These Cs-Aldhs shared 65.7-89.7% amino acid identity with Pc-ALDHs and contained conserved functional motifs. They were grouped into six clades based on phylogenesis and AlphaFold2-based structural analyses, with pronounced divergence in the architecture of their substrate-binding pockets. Fourteen of these genes were successfully expressed in Rhodococcus erythropolis, and their enzymatic activities were evaluated using fluorescence-based NADH assays. Cs-AldhJ showed broad specificity and high activity (1.86 ± 0.34 to 5.22 ± 0.05 U/mg), whereas Cs-AldhA and Cs-AldhK exhibited lower activities, ranging from 0.02 ± 0.00 to 0.08 ± 0.01 U/mg and 0.05 ± 0.01 to 0.38 ± 0.01 U/mg, respectively. The corresponding oxidation products were confirmed by GC-MS, and kinetic analyses revealed mechanistic diversity among the enzymes. These findings highlight the functional diversification of the Cs-Aldh family and provide molecular insights into the cellular responses of C. subvermispora to lignin-derived aromatic compounds.