Genetic, structural, and functional characterization of allomelanin from black yeast Exophiala viscosa, a chassis for fungal melanin production

Melanized fungi are known for their remarkable resilience to environmental stress, largely attributed to the protective properties of melanin. In this study, we establish the black yeast Exophiala viscosa as a non-pathogenic, genetically tractable model for the scalable production and functional analysis of DHN-melanin (allomelanin). Cultivation in flasks and bioreactors yielded up to 8.6 g/L of melanin, with the majority tightly incorporated into the cell wall as "melanin ghosts". Chemical analyses including FTIR, XPS, ssNMR, and EPR confirmed the identity of the pigment as allomelanin and revealed a structural association with chitin. Gene deletions of Pks1, Arp2, and Abr2 validated the DHN-melanin biosynthetic pathway and enabled the generation of pigment-deficient mutants. Functional assays demonstrated that melanin contributes significantly to UV and cold tolerance, while offering limited protection against γ-radiation, suggesting that other pigments,such as carotenoids, may also play a protective role. The unique redox properties, structural integrity, and scalability of melanin production in E. viscosa highlight its potential for bio-derived materials used in radiation shielding, environmental remediation, and thermal regulation. This work establishes E. viscosa as a promising chassis for melanin biomanufacturing and a valuable model for studying fungal melanins in the context of materials science and environmental resilience. KEY POINTS: • Cultivation of E. viscosa in rich medium yielded up to 8.6 g/L of melanin. • Chemical and genetic analyses identified the pigment as allomelanin. • Melanin enhanced the tolerance of fungal cells to UV radiation and low temperatures.