Enzymatic depolymerization of hyaluronic acid into well-defined oligosaccharides offers a sustainable alternative to chemical methods, providing precise control over product size and functionality. Yet, fungal enzymes capable of this transformation remain largely unexplored, despite fungi being prolific producers of carbohydrate-active enzymes through their complex secretomes. Here, we report the extracellular degradation of high molecular weight hyaluronic acid by the filamentous fungus Purpureocillium lilacinum. Hyaluronan depolymerization was monitored directly in culture supernatants, revealing the progressive accumulation of hyaluronan fragments and oligosaccharides with varying degrees of polymerization. Chromatographic time-resolved analyses showed the sequential appearance of long and short fragments, while mass spectrometric analysis demonstrated a complex product profile composed of even-numbered saturated and both even- and rare odd-numbered unsaturated species. This pattern differs from the uniform profiles typically generated by canonical hyaluronidases, suggesting a non-canonical degradation mechanism. Comparative cultivation of different Purpureocillium species showed that this activity is restricted to P. lilacinum among the species tested. To explore the genetic basis of this phenotype, a de novo transcriptome was assembled and functionally annotated. Although numerous carbohydrate-active enzymes were identified, none of the secreted candidates could be confidently assigned to known canonical hyaluronidase families. In particular, polysaccharide lyases associated with β-elimination mechanisms were not predicted to be secreted, and only a small fraction of glycoside hydrolases from families previously linked to hyaluronan degradation were extracellular. Together, our results establish P. lilacinum as a novel fungal system capable of extracellular hyaluronic acid degradation and support the existence of a non-canonical, potentially multienzymatic pathway. This work expands current knowledge of fungal glycosaminoglycan metabolism and highlights filamentous fungi as an underexplored source of hyaluronan-processing activities with biotechnological potential. KEY POINTS: • Purpureocillium lilacinum is a new hyaluronic acid degrader • Degradation yields diverse saturated and unsaturated hyaluronan oligosaccharides • Transcriptomics suggests a non-canonical, multienzymatic degradation system.
Minguet-Lobato et al. (Sun,) studied this question.