Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (PyoFun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The PyoFun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of PyoFun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (PyoSyn). The results confirmed successful production of PyoFun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of PyoFun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, PyoFun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by PyoSyn. The findings of this study suggest that PyoFun has significant potential as a biological shield against harmful radiation. Notably, PyoFun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes PyoFun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation.

Applied and Molecular Microbiology Institute of Biotechnology Technische Universität Berlin Berlin Germany

Department of Microbiology Faculty of Medicine Masaryk University and St Anne's Faculty Hospital Brno Czechia

Institute of Scientific Instruments of the Czech Academy of Sciences Brno Czechia

Radiation Biology Department Aerospace Microbiology Research Group German Aerospace Center Institute of Aerospace Medicine Cologne Germany

Zobrazit více v PubMed

Aunsbjerg S. D., Andersen K. R., Knøchel S. (2015). Real-time monitoring of fungal inhibition and morphological changes. J. Microbiol. Methods. 119, 196–202. 10.1016/j.mimet.2015.10.024 PubMed DOI

Blin K., Medema M. H., Kazempour D., Fischbach M. A., Breitling R., Takano E., et al. . (2013). antiSMASH 2.0—a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204–W212. 10.1093/nar/gkt449 PubMed DOI PMC

Bos C. J., Debets A. J. M., Swart K., Huybers A., Kobus G. (1988). Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr. Gen. 14, 437–443. 10.1007/BF00521266 PubMed DOI

Cairns T. C., Nai C., Meyer V. (2018). How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol. Biotechnol. 5, 13. 10.1186/s40694-018-0054-5 PubMed DOI PMC

Cao W., Zhou X., McCallum N. C., Hu Z., Ni Q. Z., Kapoor U., et al. . (2021). Unraveling the structure and function of melanin through synthesis. J. Am. Chem. Soc. 143, 2622–2637. 10.1021/jacs.0c12322 PubMed DOI

Castellano-Pellicena I., Morrison C. G., Bell M., O'Connor C., Tobin D. J. (2021). Melanin distribution in human skin: Influence of cytoskeletal, polarity, and centrosome-related machinery of stratum basale keratinocytes. Int. J. Mol. Sci. 22, 3143. 10.3390/ijms22063143 PubMed DOI PMC

Chen Z., Bertin R., Froldi G. (2013). EC50 estimation of antioxidant activity in DPPH· assay using several statistical programs. Food Chem. 138, 414–420. 10.1016/j.foodchem.2012.11.001 PubMed DOI

Cordero R. J. B., Casadevall A. (2017). Functions of fungal melanin beyond virulence. Fungal Biol. Rev. 31, 99–112. 10.1016/j.fbr.2016.12.003 PubMed DOI PMC

Cortesão M., de Haas A., Unterbusch R., Fujimori A., Schütze T., Meyer V., et al. . (2020a). Aspergillus niger spores are highly resistant to space radiation. Front. Microbiol. 11, 560. 10.3389/fmicb.2020.00560 PubMed DOI PMC

Cortesão M., Holland G., Schütze T., Laue M., Moeller R., Meyer V., et al. . (2022). Colony growth and biofilm formation of Aspergillus niger under simulated microgravity. Front. Microbiol. 13, 975763. 10.3389/fmicb.2022.975763 PubMed DOI PMC

Cortesão M., Schütze T., Marx R., Moeller R., Meyer V. (2020b). “Fungal biotechnology in space: Why and how?” in Grand Challenges in Biology and Biotechnology, eds. H., Nevalainen (Cham: Springer). 10.1007/978-3-030-29541-7_18 DOI

Dadachova E., Bryan R. A., Howell R. C., Schweitzer A. D., Aisen P., Nosanchuk J. D., et al. . (2008). The radioprotective properties of fungal melanin are a function of its chemical composition, stable radical presence and spatial arrangement. Pigment Cell Melanoma Res. 21, 192–199. 10.1111/j.1755-148X.2007.00430.x PubMed DOI

De Maesschalck R., Jouan-Rimbaud D., Massart D. L. (2000). The Mahalanobis distance. Chemom. Intell. Lab. Syst. 50, 1–18. 10.1016/S0169-7439(99)00047-7 DOI

Esbelin J., Mallea S., Ram A. F., Carlin F. (2013). Role of pigmentation in protecting Aspergillus niger conidiospores against pulsed light radiation. Photochem Photobiol. 89, 942–951. 10.1111/php.12037 PubMed DOI

Fiedler M. R. M., Gensheimer T., Kubisch C., Meyer V. (2017). HisB as a novel selection marker for gene targeting approaches in Aspergillus niger. BMC Microbiol. 17, 41. 10.1186/s12866-017-0960-3 PubMed DOI PMC

Gao J., Wenderoth M., Doppler M., Schuhmacher R., Marko D. (2022). Fungal melanin biosynthesis pathway as source for fungal toxins. MBio 13, e00219–e00222. 10.1128/mbio.00219-22 PubMed DOI PMC

Green M. R., Sambrook J. (2012). “Molecular cloning: A Laboratory Manual,” in The Quarterly Review of Biology.

Heinekamp T., Thywißen A., Macheleidt J., Keller S., Valiante V., Brakhage A. A., et al. . (2013). Aspergillus fumigatus melanins: interference with the host endocytosis pathway and impact on virulence. Front. Microbiol. 3, 440 10.3389/fmicb.2012.00440 PubMed DOI PMC

Jørgensen T. R., Park J., Arentshorst M., van Welzen A. M., Lamers G., Vankuyk P. A., et al. . (2011). The molecular and genetic basis of conidial pigmentation in Aspergillus niger. Fungal Genet. Biol. 48, 544–553. 10.1016/j.fgb.2011.01.005 PubMed DOI

Larroude M., Nicaud J-M., Rossignol T. (2021). Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway. AMI Microbial. Biotechnol. 14, 2420–2434. 10.1111/1751-7915.13745 PubMed DOI PMC

Lorquin F., Piccerelle P., Orneto C., Robin M., Lorquin J. (2022). New insights and advances on pyomelanin production: from microbial synthesis to applications. JIMB J. Ind. Microbiol. Biotechnol. 49, 13. 10.1093/jimb/kuac013 PubMed DOI PMC

Lorquin F., Ziarelli F., Amouric A., Di Giorgio C., Robin M., Piccerelle P., et al. . (2021). Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments. Scient. Rep. 11, 1–16. 10.1038/s41598-021-87328-2 PubMed DOI PMC

Mlynáriková K., Samek O., Bernatová S., RuŽička F., JeŽek J., Hároniková A., et al. . (2015). Influence of culture media on microbial fingerprints using raman spectroscopy. Sensors. 15, 29635–29647. 10.3390/s151129635 PubMed DOI PMC

Mózsik L., Pohl C., Meyer V., Bovenberg R. A. L., Nygård Y., Driessen A. J. M., et al. . (2021). Modular synthetic biology toolkit for filamentous fungi. ACS Synthet. Biol. 10, 2850–2861. 10.1021/acssynbio.1c00260 PubMed DOI PMC

Nikodinovic-Runic J., Martin L. B., Babu R., Blau W., O'Connor K. E. (2009). Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6. FEMS Microbiol. Lett. 298, 174–183. 10.1111/j.1574-6968.2009.01716.x PubMed DOI

Norbury J. W., Slaba T. C., Aghara S., Badavi F. F., Blattnig S. R., Clowdsley M. S., et al. . (2019). Advances in space radiation physics and transport at NASA. Life Sci. Space Res. 22, 98–124. 10.1016/j.lssr.2019.07.003 PubMed DOI

Perez-Cuesta U., Aparicio-Fernandez L., Guruceaga X., Martin-Souto L., Abad-Diaz-de-Cerio A, Antoran A, et al. . (2020). Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction. Int. Microbiol. 23, 55–63. 10.1007/s10123-019-00078-0 PubMed DOI

Peters B. A., Wu J., Hayes R. B., Ahn J. (2017). The oral fungal mycobiome: Characteristics and relation to periodontitis in a pilot study. BMC Microbiol. 17, 1–11. 10.1186/s12866-017-1064-9 PubMed DOI PMC

Pohl C., Kiel J. A., Driessen A. J., Bovenberg R. A. (2016). CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synthet. Biol. 5, 754–764. 10.1021/acssynbio.6b00082 PubMed DOI

Polli F., Meijrink B., Bovenberg R. A. L., Driessen A. J. M. (2016). New promoters for strain engineering of Penicillium chrysogenum. Fungal Genet. Biol. 89, 62–71. 10.1016/j.fgb.2015.12.003 PubMed DOI

Pralea I. E., Moldovan R. C., Petrache A. M., Ilie? M., Heghe? S. C., Ielciu I., et al. . (2019). From extraction to advanced analytical methods: The challenges of melanin analysis. Int. J. Molec. Sci. 20, 3943. 10.3390/ijms20163943 PubMed DOI PMC

Rebrošová K., Šiler M., Samek O., RuŽička F., Bernatová S., JeŽek J., et al. . (2017). Differentiation between Staphylococcus aureus and Staphylococcus epidermidis strains using Raman spectroscopy. Future Microbiol. 12, 881–890. 10.2217/fmb-2016-0224 PubMed DOI

Richter L., Wanka F., Boecker S., Storm D., Kurt T., Vural Ö., et al. . (2014). Engineering of Aspergillus niger for the production of secondary metabolites. Fungal Biol. Biotechnol. 1, 1–13. 10.1186/s40694-014-0004-9 PubMed DOI PMC

Romsdahl J., Blachowicz A., Chiang A. J., Singh N., Stajich J. E., Kalkum M., et al. . (2018). Characterization of Aspergillus niger isolated from the International Space Station. mSystems. 3, 10–1128. 10.1128/mSystems.00112-18 PubMed DOI PMC

Schmaler-Ripcke J., Sugareva V., Gebhardt P., Winkler R., Kniemeyer O., Heinekamp T., et al. . (2009). Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus. Appl. Environ. Microbiol. 75, 493–503. 10.1128/AEM.02077-08 PubMed DOI PMC

Singh S., Nimse S. B., Mathew D. E., Dhimmar A., Sahastrabudhe H., Gajjar A., et al. . (2021). Microbial melanin: Recent advances in biosynthesis, extraction, characterization, and applications. Biotechnol. Adv. 53, 107773. 10.1016/j.biotechadv.2021.107773 PubMed DOI

Stemmer M., Thumberger T., del Sol Keyer M., Wittbrodt J., Mateo J. L. (2015). CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS ONE 10, e0124633. 10.1371/journal.pone.0124633 PubMed DOI PMC

Tahar I. B., Kus-Liśkiewicz M., Lara Y., Javaux E., Fickers P. (2020). Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica. Biotechnol. Progress. 36, e2912. 10.1002/btpr.2912 PubMed DOI

Tokuhara Y., Shukuya K., Tanaka M., et al. . (2018). Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate. Sci. Rep 8, 11364. 10.1038/s41598-018-29769-w PubMed DOI PMC

Vasileiou T., Summerer L. (2020). A biomimetic approach to shielding from ionizing radiation: The case of melanized fungi. PLoS ONE. 15, e0229921. 10.1371/journal.pone.0229921 PubMed DOI PMC

Vasileiou T., Summerer L. (2021). Correction: A biomimetic approach to shielding from ionizing radiation: The case of melanized fungi. PLoS ONE. 16, e0257068. 10.1371/journal.pone.0257068 PubMed DOI PMC

Winters M., Aru V., Howell K., Arneborg N. (2022). Reliable budding pattern classification of yeast cells with time-resolved measurement of metabolite production. Biotechniques. 72, 100–103. 10.2144/btn-2021-0120 PubMed DOI