A novel family of fungal protein biosurfactants: Discovery and sustainable production.

Abstract

Protein-based biosurfactants remain underexplored compared to glycolipids and lipopeptides, despite their unique interfacial properties and self-assembly behavior. PAC3, a surface-active protein produced by the marine fungus Acremonium sclerotigenum, exhibits dual behavior as both a biosurfactant and bioemulsifier. For this reason, it can be seen as a high molecular weight proteinaceous compound, able to efficiently reduce surface tension. Here, we identify PAC3 as the first member of a previously unrecognized family of fungal protein biosurfactants. The complete amino acid sequence of PAC3 was determined through a combined de novo transcriptomic and mass spectrometry approach, revealing an 83-residue protein that lacks the canonical eight-cysteine motif typical of hydrophobins, the most surface-active proteins known. Sequence, phylogenetic, and structural analyses revealed a distinct fold and amphipathic architecture, with a negatively charged surface and a hydrophobic planar region, providing a molecular basis for its strong interfacial activity. The identification of homologous sequences across fungi supports the existence of a novel protein family. Notably, we show through spectroscopy and confocal microscopy that PAC3 fibrils exhibit deep-blue intrinsic fluorescence, a property recently associated with amyloid architecture. To support industrial application, we developed a simplified downstream process based on methanol/chloroform extraction, reducing costs while preserving functionality. In parallel, the use of waste frying oil enhanced fungal biomass production and supported efficient PAC3 synthesis, demonstrating a sustainable production strategy. Overall, this study introduces a new class of fungal biosurfactant proteins and provides a foundation for their biotechnological exploitation.