Deciphering Key Enzymes Involved in Colorant Biosynthesis and Lignocellulose Degradation in White-Rot Basidiomycota

Nature has evolved an extraordinary chemical diversity of natural products and an equally diverse enzymatic machinery over millions of years, yet only a fraction of this potential has been harnessed for biotechnological and industrial applications. The present work aims to utilize this biodiversity focusing on the enzymatic degradation of lignocellulosic biomass and the biosynthesis of colored secondary metabolites by white-rot fungi of the phylum Basidiomycota.
In the first part of this work, the species Cerrena zonata was investigated with respect to enzymes involved in the effective utilization of lignocellulosic biomass, a capability exclusive to white-rot Basidiomycota. In a broader context, this work aims to lay the foundation for the usage of these enzymes in industrial processes, which currently rely on costly physical or chemical pretreatments. Employing a multi-omics approach integrating genomics, transcriptomics, and proteomics, the Carbohydrate-Active enZyme (CAZyme) repertoire of C. zonata was systematically characterized. Of the 20,816 predicted protein-coding genes in the genome, 487 were annotated as CAZymes. Among the 147 proteins detected in the secretome, 36 were CAZymes. Notably, laccase, manganese peroxidase, and versatile peroxidase activities were confirmed in the culture supernatants and detailed structural and sequence analyses allowed the unambiguous assignment of specific activities to certain auxiliary activities family 2 (AA2) peroxidases embedded in C. zonatas genome.
The second part of the work focused on the investigation of coloring secondary metabolites in white-rot Basidiomycota. In submerged cultures of the veiled oyster mushroom Pleurotus dryinus, supplementation with L-phenylalanine triggered the formation of a deep burgundy-red coloration, a phenomenon not previously described for this ordinarily colorless edible mushroom. Isolation and comprehensive structural elucidation via high-resolution-tandem mass spectrometry (HR-MS/MS), UV/vis and multidimensional nuclear magnetic resonance (NMR) spectroscopy revealed two novel meroterpenoid colorants, named dryinones, featuring an aminoquinone moiety as the central chromophore. The absolute configuration of one colorant, dryinone A, was confirmed through nuclear Overhauser effect spectroscopy (NOESY) correlations, electronic circular dichroism (ECD) experiments, and density functional theory (DFT) computations.
The last species investigated in this work was Phlebia centrifuga, isolated from the Black Forest National Park and investigated for its dark red coloration in submerged culture, when cultivated in potato dextrose medium. The respective colorant was identified as the bibenzoquinone oosporein, a promising biocontrol agent against insect pests. Furthermore, a scalable submerged fermentation system was developed, achieving yields of up to 1.60 g L⁻¹ in the culture supernatant upon supplementation with orsellinic acid as a key biosynthetic intermediate, a compound that causes a strong up-regulation of genes for oosporein biosynthesis. Using an integrated multi-omics and in silico approach, encompassing genomics, transcriptomics, molecular docking and molecular dynamics simulations, the complete biosynthetic pathway was predicted, implicating a type I polyketide synthase, two monooxygenases, and a heme peroxidase as the core enzymes.
The organisms investigated in this work highlight the largely untapped potential of Basidiomycota. Both enzymes relevant for the pretreatment of lignocellulosic biomass were identified and novel insights into the structure and biosynthesis of fungal colorants were obtained. Overall, this work contributes to a more comprehensive understanding of the metabolic and enzymatic capacities encoded within Basidiomycota and emphasizes their broader potential as a source of industrially and economically valuable biocatalysts and secondary metabolites.