**1. Introduction**

The term melanin refers to a diverse set of dark polymeric pigments found in all kingdoms of life. In fungi, melanin plays a panoply of protective roles against stress (Table 1; Figure 1) [1]. For example, fungal melanin can protect against ionizing radiation, including ultraviolet, X-ray, gamma-ray, and particulate radiation [2–4]. In addition, melanin can play a role in thermoregulation and protection against both heat and cold shock [5,6]. For example, melanized *Cryptococcus neoformans* cells were more likely to survive both heat shock and cold shock compared to non-melanized species [6]. Melanized endophytes in a mutualistic relationship with plants will often help their symbionts thermoregulate by dissipating heat and absorbing ROS [7]. Darkly pigmented yeasts and mushrooms tend to be more common at higher absolute latitudes and colder climates, suggesting that the pigment's ability to capture radiation energy and dissipate it as heat provides an advantage in generating thermal energy [5,8]. Part of melanin's role in thermotolerance may be attributable to its ability to react with and neutralize Reactive Oxygen Species (ROS), helping fungal organisms withstand the oxidative stress that often accompanies higher temperatures [9].

In addition, melanin can help fungi withstand chemical stressors. In halotolerant black yeast, melanin synthesis inhibitors diminished the yeast's ability to grow in hypersaline environments [10]. Researchers posited that this could be due to the stabilizing effect that melanin has on the fungal cell wall, which would have permitted a more effective response to osmotic stress [10]. In addition, melanization has also been shown to protect fungal cells from heavy metal stress and hydrolytic enzymes [11,12]. In areas with low water content, melanin is associated with stress response to dry conditions. For example, heavy melanization was associated with survival in microcolonial rock fungi exposed to long periods of desiccation [13]. Some fungal species in spalted woods will melanize in response to periods of low water content, creating black zones in the wood [14]. These numerous protective functions enable melanized fungi to reside in some of civilization's most extreme environments, from deep-sea vents to the International Space Station [15,16].

**Figure 1.** Panel of four images of different melanized fungi. *Xylaria polymorpha* (top left), a mushroom also referred to as 'Dead Man's Fingers.' The stromata of *X. polymorpha* have an average total length, including rooting bases, of 5 to 8 cm by an average 2 cm diameter [27]. "Black fungi—*Xylaria polymorpha* (dead man's fingers)" by ohi007 is licensed under CC BY-NC-SA 2.0; *Cladophialosphora bantiana* (top right), a pathogenic mold. *C. bantiana* conidia are approximately 5 to 10 μm in length [28]. "File:Cladophialophora bantiana UAMH10767.jpg" by Medmyco is licensed under CC BY-SA 4.0; *Inonotus obliquus* (bottom left), also known as the Chaga mushroom. Chaga appears as a sclerotia ranging from 5 to 40 cm in diameter [29]. "Chaga mushroom (*Inonotus obliquus*)" by Distant Hill Gardens is licensed under CC BY-NC-SA 2.0; *Cryptococcus neoformans* (bottom right), a pathogenic yeast. Cryptococcal cells range from 5 to 10 μm in diameter [30]. "Cryptococcosis—GMS stain" by Pulmonary Pathology is licensed under CC BY-SA 2.0. Images presented are not to scale.

**Table 1.** Examples of the functions of fungal melanin in living fungal organisms.


Given the numerous functional groups present in melanotic pigments, melanin can bind and interact with many different organic and inorganic molecules [1,17]. One no-

table example is melanin's affinity for chelating metal ions, which can be toxic to fungal cells [17–19]. Fungal melanin also plays a role in protecting the fungus against the human immune response and is often associated with pathogenicity in *Aspergillus fumigatus*, *Cryptococcus neoformans*, and *Talaromyces marneffei*, among others [20].

Lastly, melanin is correlated with fungal virulence and has been implicated as a possible antifungal target [21]. While fungal melanin's role in virulence will be limited in this review, please see [20] for a more thorough overview of this topic.

Some of the functions that melanin holds in biology can be utilized for societal benefit. These biological functions of radioprotection, stress response, and substrate binding, among others, often stem from melanin's unique physical and chemical properties. When properly understood, scientists may be able to use melanin to carry out parallel purposes in the fields of industry, healthcare, and bioremediation.

## **2. Fungal Melanins**

Investigators wishing to work with melanin have several different options for sourcing the material, whether from animals such as cuttlefish, bacteria, fungi, or even synthetic reservoirs. Each of these options has different chemical properties and extraction protocols, conferring various advantages and disadvantages depending on the desired application [31]. For example, there are significant differences between fungal eumelanin and synthetic eumelanin, likely due to the fact that in vivo melanogenesis is vesicle-associated and enzymatically catalyzed, while synthetic melanogenesis often relies on spontaneous autopolymerization in solution [32]. In addition, synthetic melanins can be costly to produce [33]. Melanin from animal and plant sources can be less expensive to produce than the synthetic route, but it can be difficult to purify, as melanin is often associated with other biomolecules. Thus, microbial melanin is often touted as a low-cost and high-yield alternative to synthetic, animal, or plant melanin [33]. More specifically, fungal melanin is attractive due to the well-known protective functions in fungal organisms adapted to survive extreme environments [1].

Fungal organisms can produce different kinds of melanins (Figure 2). Most fungal melanins are allomelanins, which do not contain nitrogen [34]. DHN melanin is a common type of allomelanin derived from the polymerization of 1,8-dihydroxynaphthalene (DHN) [35]. The polyketide pathway that produces allomelanins begins with an endogenously produced molecule of acetyl coA or malonyl coA, which undergoes several reductions and dehydrations to produce 1,8-DHN [36]. The complete literature for scientific studies of fungal melanins dates back to the 1960s [37].

Some basidiomycetous fungi, such as *Cryptococcus neoformans*, produce eumelanin [38]. Eumelanins are derived from the amino acid tyrosine, and unlike allomelanins, they contain nitrogen [39]. Fungi synthesize eumelanins via the L-3,4-dihydroxyphenylalanine (L-dopa) pathway, which begins by using laccase to oxidize L-dopa into dopaquinone [40]. Eventually, the pathway produces dihydroxyindoles that can polymerize into eumelanins [40]. *Cryptococcus neoformans* cannot carry out this pathway without an exogenous substrate [40]. Pyomelanins are also derived from tyrosine, but they are produced by fungi such as *Aspergillus fumigatus* via the tyrosine degradation pathway, which involves the oxidative phosphorylation of homogentisate (HGA) [41].

Although not heavily discussed in recent reviews, some mushrooms and basidiomycetes have been shown to produce melanin from the precursor glutaminyl-hydroxybenzene (GHB) [42]. The presence of GHB on mushroom caps of *Agaricus biosporus* has been correlated with their susceptibility to browning, and the transformed product is commonly dubbed GHB-melanin [43]. Although a synthesis pathway has not been fully outlined, it is thought to occur as GHB is transformed by a polyphenol oxidase (typically tyrosinase) into 2-hydroxy-*p*-iminobenzoquinone (2-HpIBQ) and polymerized [42,44]. The diversity of pathways that organisms use to synthesize melanin explains in part why these polymers are not as well understood as other biopolymers such as proteins or nucleic acids [45].

**Figure 2.** A simplified diagram showing the precursors for the three different kinds of fungal melanin. GHB-melanins (far left, green) are synthesized through a series of reactions with tyrosinase. Eumelanins (center left, pink) are synthesized via the L-Dopa pathway, which uses tyrosine as a precursor. Pyomelanins (center right, red) also use tyrosine as a precursor but are ultimately derived from HGA. Allomelanins (far right, blue) are derived from 1,8-DHN.

When extracting fungal melanins for human applications, eumelanins are often preferred as allomelanins are attached to the inner side of the fungal cell walls [46]. In contrast, eumelanins are used by fungi to neutralize toxic environmental compounds, making these melanins easier to extract extracellularly [46]. Currently, there are several different methods used to culture melanotic fungi, but most methods use a combination of tyrosine and metal ions [46]. Recently, fungal strains were genetically modified to become more melanotic for extraction purposes, principally by overexpressing genes for tyrosinases [33].

Prior to melanin extraction, isolating melanotic yeast can be a challenge depending on the yeast used. Some melanized yeasts are polyextremophiles, which can be difficult to isolate due to their slow growth rates and low competitive ability [47]. However, researchers have proposed the method of enriching black extremophile yeast of the order Chaetothyriales on aromatic hydrocarbon, which can inhibit the growth of other microbial species while allowing black fungi to be cultured [47].

Another challenge associated with the industrial use of fungal melanin is the potential pathogenicity of fungal species. This requires scientists to identify non-pathogenic species of melanized fungi, attenuate pathogenic melanotic fungi, or to introduce melanization to a non-pathogenic species [48]. Alternatively, when using pathogenic species for melanin extraction, operations must be carried out in laboratories certified for a given biosafety level.

While the challenges of fungal melanin extraction have made industrial-scale implementation difficult in the past, newer extraction techniques are simpler and produce higher yields of the compound [49]. For example, a 2015 paper recorded a 10% yield of fungal melanin from *Auricularia auricula* after several steps, including treatment with lytic enzymes, guanidinium thiocyanate, chloroform, and HCl [50]. In contrast, a 2019 paper recorded that an optimized strain of *Armillaria cepistipes* was able to produce a 99% yield of melanin following a simpler extraction procedure [49].

This review provides an overview of the various developments attempting to use specifically fungi-derived melanin for human application in fields including healthcare, bioremediation, and industry. The outlined projects are in varying stages of development, mostly due to the complicated nature of melanin extraction.
