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

Abstract

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.

Keywords: Exophiala; Allomelanin; Biomanufacturing; DHN-melanin; Melanized fungus; Radiation resistance.

Fig. 1

Melanin production from E. viscosa in bioreactor and flask. Melanin yields (g/L) were generated from four biological replicates under each growth condition

Fig. 2

FE-STEM images of fungal melanin granules (A) and melanin ghosts (B)

Fig. 3

Chemical characterization of fungal melanin. (A) ¹³C multi-CPMAS ssNMR spectra of fungal melanin (top) and 1,8-DHN monomer (bottom); (B) FTIR spectra of fungal melanin after 1 st and 2nd round of extraction compared to synthetic p-1,8-DHN allomelanin; (C) XPS spectra of fungal melanin after 1 st and 2nd round of extraction compared to synthetic p-1,8-DHN allomelanin

Fig. 4

Characterization of redox properties of E. viscosa melanin. (A) EPR characterization of fungal melanin granule, fungal melanin ghost, and bacterial melanin (eumelanin). (B, C) Cyclic voltammetry analysis of fungal melanin and eumelanin. A chitosan film without melanin was used as a null control. Three replicate samples of each type of melanin were measured. The p-values for the comparison of the N (oxidation) values and N (reductive) values between fungal melanin and eumelanin by a two-tailed T-test are 0.0009 and 0.0844, respectively

Fig. 5

Colony phenotypes of E. viscosa wild-type and three genetic mutants (EvPks1Δ, EvArp2Δ, and EvAbr2Δ) in the DHN-melanin biosynthesis pathway

Fig. 6

Cellular phenotypes of E. viscosa wild-type and Pks1Δ mutant. (A) Microscope images at visible Light and 385 nm wavelength; (B) FESEM images

Fig. 7

Survival of E. viscosa WT and Pks1Δ cells following exposure to varying total doses of (A) UV-C radiation with a dose rate of 0.273 mJ/cm² s⁻¹, (B) γ-radiation with a dose rate of 22.7 Gy/min, and (C) varying temperatures (the representative of three biological replicates)