A superfamily of bioactive proteins from fungi - are these secondary metabolites?

Since decades, fungi are leveraged in biotechnology to produce high-value compounds used in multiple economic sectors. Strain and process optimisation is based on a comprehensive understanding of the production organism on the cellular and molecular level. Among three antifungal protein families in fungi, consisting of small cysteine-stabilised proteins, it has been shown that, for some family members, bioactivities are also associated with additional functions in their hosts, e.g., carbon metabolism, autophagy, or asexual development. These proteins are interesting as alternative source of novel antifungal drugs. However, their potential impact on biotechnological production is not yet elucidated.

In this study, we introduce the antifungal bubble protein "AgBP", from Aspergillus giganteus and further elucidate the reservoir of bioactive proteins in fungi. We used NCBI PSI-BLAST and subsequent phylogenetic and structural analyses of the Antifungal Protein (AFP), Bubble Protein (BP), and Neosartorya fischeri antifungal protein 2 (NFAP2) family members. We could identify further putative members: 165 AFP-, 102 BP-, and 219 NFAP2-like proteins. Six of the AFP and all 219 NFAP2 family members are not yet assigned on InterPro. All proteins were exclusively identified in fungi. To our best knowledge, this is the first study to report this group of bioactive proteins is shared among the two divisions of Ascomycetes and Basidiomycetes. Phylogenetic tree analyses demonstrate restricted taxonomic distribution within single genera. Furthermore, the comparison of the tertiary structures of all members of the three AFP families clearly separates them from each other and from non-fungal small cysteine-stabilised antifungal proteins.

We hypothesise that the three protein families represent a distinct superfamily of evolutionary related proteins. We further hypothesise that these proteins could be categorised as secondary metabolite like molecules of ribosomal origin. For brevity, we named this superfamily BPF (bioactive proteins from fungi). The distribution of BPF members is presumably driven by horizontal gene transfer. Furthermore, we hypothesise that BPF members likely serve rather different biological roles than merely acting as antimicrobials. Their hypothetical classification as potential secondary metabolite like proteins in combination with their occurrence among several biotechnologically relevant fungal genera, e.g., Aspergillus, Penicillium, Trichoderma, Schizophyllum, etc., emphasises their potential relevance for genetic and metabolic engineering.