Evolution and enrichment of CYP5035 in Polyporales: functionality of an understudied P450 family

Bioprospecting for innovative basidiomycete cytochrome P450 enzymes (P450s) is highly desirable due to the fungi’s enormous enzymatic repertoire and outstanding ability to degrade lignin and detoxify various xenobiotics. While fungal metagenomics is progressing rapidly, the biocatalytic potential of the majority of these annotated P450 sequences usually remains concealed, although functional profiling identified several P450 families with versatile substrate scopes towards various natural products. Functional knowledge about the CYP5035 family, for example, is largely insufficient. In this study, the families of the putative P450 sequences of the four white-rot fungi Polyporus arcularius, Polyporus brumalis, Polyporus squamosus and Lentinus tigrinus were assigned, and the CYPomes revealed an unusual enrichment of CYP5035, CYP5136 and CYP5150. By computational analysis of the phylogeny of the former two P450 families, the evolution of their enrichment could be traced back to the Ganoderma macrofungus, indicating their evolutionary benefit. In order to address the knowledge gap on CYP5035 functionality, a representative subgroup of this P450 family of P. arcularius was expressed and screened against a test set of substrates. Thereby, the multifunctional enzyme CYP5035S7 converting several plant natural product classes was discovered. Aligning CYP5035S7 to 102,000 putative P450 sequences of 36 fungal species from Joint Genome Institute-provided genomes located hundreds of further CYP5035 family members, which subfamilies were classified if possible. Exemplified by these specific enzyme analyses, this study gives valuable hints for future bioprospecting of such xenobiotic-detoxifying P450s and for the identification of their biocatalytic potential. Graphical abstract Key points • The P450 families CYP5035 and CYP5136 are unusually enriched in P. arcularius. • Functional screening shows CYP5035 assisting in the fungal detoxification mechanism. • Some Polyporales encompass an unusually large repertoire of detoxification P450s.

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass

White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found the fungus possessed an unexpectedly large set of genes coding for enzymes related to lignin degradation, and that these were highly expressed and massively secreted under solid-state fermentation conditions. The examination of interrelated multi-omics patterns revealed the coordinated regulation of lignin-active peroxidases and H2O2-generating enzymes along with the activation of cellular mechanisms for detoxification, which combined to result in the efficient lignin breakdown by the fungus.ImportancePlant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e. agriculture and forestry by-products) are major obstacles for biomass conversions. From a biotechnological aspect, these compounds could be a potential source of aromatic platform molecules for bio-based polymers. Here we describe the extraordinary ability of Polyporus brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. We observed unusual expansions of gene families coding for; 1) Class II peroxidases involved in lignin degradation; and 2) GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity. Our findings suggested the fungus massively mobilizes this oxidative machinery during growth on wheat straw. Overall, we identified sets of co-regulated enzymes, which could potentially augment the efficiency of biotechnological plant biomass conversions.

De Novo Whole-Genome Sequencing of the Wood Rot Fungus Polyporus brumalis, Which Exhibits Potential Terpenoid Metabolism

ABSTRACT Polyporus brumalis is able to synthesize several sesquiterpenes during fungal growth. Using a single-molecule real-time sequencing platform, we present the 53-Mb draft genome of P. brumalis, which contains 6,231 protein-coding genes. Gene annotation and isolation support genetic information, which can increase the understanding of sesquiterpene metabolism in P. brumalis.