**Sphinganine-Analog Mycotoxins (SAMs): Chemical Structures, Bioactivities, and Genetic Controls**

**Jia Chen <sup>1</sup> , Zhimin Li <sup>1</sup> , Yi Cheng <sup>1</sup> , Chunsheng Gao <sup>1</sup> , Litao Guo <sup>1</sup> , Tuhong Wang <sup>1</sup> and Jianping Xu 1,2,\***


Received: 31 October 2020; Accepted: 22 November 2020; Published: 24 November 2020 -

**Abstract:** Sphinganine-analog mycotoxins (SAMs) including fumonisins and *A. alternata* f. sp. *Lycopersici* (AAL) toxins are a group of related mycotoxins produced by plant pathogenic fungi in the *Fusarium* genus and in *Alternaria alternata* f. sp. Lycopersici, respectively. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. This review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death. We also reviewed the detoxification methods against SAMs and how plants develop resistance to SAMs. Genetic and evolutionary analyses revealed that the *FUM* (fumonisins biosynthetic) gene cluster was responsible for fumonisin biosynthesis in *Fusarium* spp. Sequence comparisons among species within the genus *Fusarium* suggested that mutations and multiple horizontal gene transfers involving the *FUM* gene cluster were responsible for the interspecific difference in fumonisin synthesis. We finish by describing methods for monitoring and quantifying SAMs in food and agricultural products.

**Keywords:** sphinganine-analog mycotoxins; fumonisins; AAL-toxin; chemical structure; toxicity; genetics and evolution; biosynthesis

### **1. Introduction**

Mycotoxins are secondary metabolites produced by various fungi. These metabolites have important ecological functions on living systems in their natural habitats. As secondary metabolites, mycotoxins are regarded as not essential for fungal growth or reproduction. However, their toxic effects to plants, animals, as well as humans are attracting increasing attention from chemists, biologists, food scientists, and healthcare professionals. Many fungi are capable of synthesizing mycotoxins, including certain saprophytic molds, poisonous mushrooms, human fungal pathogens, and plant fungal pathogens. Mycotoxins produced by plant pathogenic fungi can be divided into two groups: (i) host-selective (or host-specific) toxins (HSTs) and (ii) non-host-specific toxins (nHSTs), depending on whether they are specifically toxic to host plant (HSTs) or to a wide range of species (nHSTs). The known mycotoxins are typically low molecular-weight chemicals but with diverse structures and modes of actions. One group of mycotoxins are structurally analogous to sphingosine, the backbone precursor of sphingolipids that play essential structural and cellular roles in eukaryotic cells. These toxins are called

sphinganine-analog mycotoxins (SAMs), with fumonisins and the *Alternaria alternata* f. sp. *Lycopersici* (AAL) toxins as the two most widely studied groups of SAMs. SAMs are toxic to plants and animals. They act by inhibiting the ceramide synthase (CerS), thereby influencing the sphingolipid metabolism and initiating apoptosis in animals and programmed cell death (PCD) in plants [1–3]. The objective of this paper is to provide an updated review on the structural diversity, syntheses, modes of action, and health impacts of SAMs.

The discovery of fumonisin was first reported in 1988 and the organism producing it was *Fusarium verticillioides* (syn. *Gibberella fujikuroi* mating population A, syn. *G. moniliformis* Wineland, syn. *F. moniliforme* Sheldon) [4]. Fumonisins have since been found to be produced by at least 18 species of the *Fusarium* genus, with *F. verticillioides* and *F. proliferatum* being the most prominent, and by three unrelated fungal genera, *Aspergillus* section *Nigri* (such as *Asp. niger*, *Asp. Welwitschiae* (syn. *Asp. awamori*) and so on, known as black aspergilli), *Tolypocladium* (*T. inflatum*, *T. cylindrosporum*, and *T. geodes*), and *Alternaria* (the tomato pathotype of *A. alternata,* formerly known as *A. arborescens*) [5–10]. Species of the *Fusarium* genus can be found as saprophytes in soil and as endophytes and pathogens of many plants worldwide. A common group of diseases caused by *Fusarium* pathogens is rotting that can happen to all tissues during all stages of plant development [11,12]. In addition, the *Fusarium* species can infect crops at the post-harvest period during storage [13]. The fungal propagules surviving in the soil can also infect new crop plants and can be carried to new fields by wind or by anthropogenic activities, such as when seedlings are transplanted [14]. *Fusarium* strains can synthesize fumonisins during all stages of their growth, including the saprophytic stage in the soil, during their pathogenesis, and as endophytes in different parts of plants, as well as during crop storage after harvest [15].

Fumonisins, as a nHST, are major contaminants of cereals and grains, including corn, rice, wheat, barley, rye, oat, millet, and products made based on these crops [16]. The consumption of food contaminated by fumonisins significantly increases health problems for humans, leading to a variety of cancers such as esophageal cancer and neurological defects [17,18]. For example, the International Agency for Research on Cancer (IARC) characterized fumonisin FB<sup>1</sup> as a group 2B carcinogens for humans [16]. Fumonisins can also cause diseases and adverse effects in other species, especially in livestock when the feeds are contaminated [19]. Well-known diseases in livestock caused by fumonisins include leukoencephalomalacia in horses and pulmonary edema syndrome in pigs [20,21].

Similar to fuminisins, the AAL-toxins include a family of structurally analogous metabolites. AAL-toxins are a group of HST produced by the ascomycete fungal pathogen *A. alternata* f. sp. *Lycopersici*, the causal agent of tomato stem canker disease [22]. It should be noted that several other pathotypes of *A. alternata* could also produce other HSTs responsible for fungal pathogenesis on their specific host plants, respectively [23]. Unlike other HSTs produced by *A. alternata*, besides the susceptible tomato host, AAL-toxins can also affect many other weeds and crops of dicotyledonous species and at least 25 species of *Solanaceae* [24,25]. Furthermore, the tomato pathotype of *A. alternata* was also reported to produce fumonisins B (FBs) [8,26]. AAL-toxin and FBs were not only detected in the necrosis plant tissues and culture media inoculated by *A. alternata* but also in spores and mycelia of this pathogen [27]. However, AAL-toxin remains the only toxin as a pathogenicity factor for stem canker disease of sensitive tomato varieties, while fumonisins are toxigenic virulence factors [28].

Because of the adverse impacts of SAMs on animal and human health, these toxins are also attracting increasing attention from food inspectors and public health agencies. Over the last three decades, significant progress has been made in our understanding of SAMs. Our objectives of this review are to capture these developments on SAMs with regard to their chemical structural diversity, the relationship between structure and activity, PCD induction, detoxification, genetics and evolution of SAMs biosynthesis, and laboratory detections.

#### **2. Chemical and Structural Properties**

#### *2.1. Chemical and Structural Properties of Sphingolipids*

SAMs have a distinct structural similarity to sphinganine (Figure 1). Sphinganine (dihydrosphingosine, DHS) is the simplest class of sphingolipids and has a backbone that consists of a linear aliphatic group with 18-carbon, an amino at C-2, and two hydroxyls (-OH) at C-1 and C-3, respectively. Phytosphingosine is obtained if a hydroxyl is introduced at C-4. Sphingosine consists of the sphinganine backbone but with a double bond at C-4. Ceramides are synthesized by linking an amide fatty acid at C-2 of sphingosine. Ceramides is a waxy lipid molecule, which is found in high concentrations in the membrane of eukaryotic cells. More complex sphingolipids can be formed by linking different chemical groups to hydroxyl (C1) of ceramides. Sphingolipids are one type of lipids widely found in their membranes in eukaryotes and a few prokaryotes, and they form complex and diverse interactions with other molecules [29]. Sphingolipids play important structural and functional roles, they are involved in a variety of signal transductions and crucial cellular processes [30,31]. For example, in humans, ceramides help form the skin's barrier and regulate immune response, protecting the skin against environmental irritants, pollutants, and water loss. Without the proper ratio of ceramides on our epidermal cells, the barrier of the skin will be damaged, resulting in dryness, itching, and irritation [32]. . For example, in humans, ceramides help form the skin's barrier and regulate immune

**Figure 1.** Chemical structure of sphingolipids. The table shows the different substituents in the chemical scaffold of the most essential sphingolipid.

#### *2.2. Chemical and Structural Properties of Fumonisins*

– – SAMs consist of two main types of toxins, fuminisins and AAL-toxins. Fumonisins can be divided into seven groups (FA, FB, FC, FD, FP, FPy, and FLa). These groups differ in the nitrogen functional group and the length of the carbon backbone [5]. Most fumonisins contain a 19–20 (FD contain 17 or 18 carbon) linear backbone similar to sphinganine with one nitrogen functional group (except for FPys and FLas), two to four hydroxyl, two methyl, and two propane-1,2,3-tricarboxylic acid (PTCA) side chains esterified to the backbones [26,33]. The structural features of the seven groups of fumonisins are shown in Figure 2. Among them, the B group is the dominant one. For example, FB<sup>1</sup> accounts for 70–80% of the total fumonisins produced by *F. verticillioides* and is the predominant toxic form [5]. FB<sup>2</sup> and FB<sup>3</sup> are isomers of each other but with one less hydroxyl group than FB1. The B series of fumonisins (FBs) are also the main food contaminants. Group A fumonisins (FA) are acetylated derivates of group B toxins, with lower toxicity and bioactivity than their FB counterparts [34]. Group C fumonisins (FC) have the same nitrogen functional group as FB<sup>1</sup> but lack the terminal methyl group at C-1 [35]. Three forms of acetylated FC<sup>1</sup> have been discovered in *F. oxysporum* [36]. Group P fumonisins (FP) have a nitrogen functional group of 3-hydroxypyridinium instead of the amino group in FB at the R<sup>2</sup> position [37]. The FC and FP groups have similar phytotoxic and cytotoxic effects to those caused by FB<sup>1</sup> or AAL-toxin [38]. Aside from these four main groups, there are several other lesser-known fumonisin analogs, with one or two PTCA replaced by a hydroxyl or carbonyl or other carboxylic acids group at C-13 and/or C-14 of the backbone (for example, HFB1, as show in

Figure 2). Rheeder et al. summarized the 28 fumonisin analogs that have been characterized between 1988 and 2002 [5]. By reversed-phase high-performance liquid chromatography/electrospray ionization ion trap multistage mass spectrometry (RP-HPLC/ESI-IT-MS n ), Bartok et al. detected 58 fumonisins (including FD) or fumonisin-like compounds from *F. verticillioides* in rice cultures, and 28 isomers of FB<sup>1</sup> [33,39]. Indeed, the recent application of a semi-targeted method revealed over 100 structurally related compounds from SAMs-producing fungi, including a hydroxyl-FB1, and two new classes of non-aminated fumonisins (FPys and FLas) [26].



**Figure 2.** Chemical structure of sphinganine-analog mycotoxins (SAMs). Panel A shows the AAL-toxins, Panel B shows fumonisin. In the table of each panel, the different substituents present in the chemical scaffolds of individual compounds are shown.

#### *2.3. Chemical and Structural Properties of AAL-Toxin*

The AAL-toxins have a structural similarity to fumonisins (Figure 2). The main difference between fumonisins and AAL-toxins is that AAL-toxins have one fewer PTCA side chain than fumonisins. The AAL-toxins have been divided into five pairs based on their side chain structures: A, B, C, D, and E pairs (TA, TB, TC, TD and TE). These pairs differ in their nitrogen functional group and hydroxylation at C-4 or C-5 positions of the backbone [40–42]. Each pair of AAL-toxins is composed of two regioisomers

–

with PTCA esterified to C-13 or C-14 of the backbone, respectively. The TA pair is the major pair of toxins, with the TB and TC pairs formed by removing hydroxyl groups one by one from C-5 and C-4 of the TA pair. The TD and TE pairs were acetylated derivatives of TB and TC respectively, while the acetylated form of TA and keto derivatives of AAL-toxins (2-keto or 14-keto analogues predicted) were also found in 2015 [26]. These four regioisomeric pairs (TB, TC, TD, and TE) of AAL-toxins can all induce genotype-specific necrosis characteristics in tomato leaflets in the same pattern as that of the TA pair, but they differ as much as 1000-fold in their relative toxicity [42].

#### *2.4. Chemical and Structural Properties of Analogs of SAMs*

In addition to fumonisins and AAL-toxins, several fungal secondary metabolites have also been identified as structural analogs of sphinganine and CerS inhibitors (summarized in Figure 3 and Table 1). These metabolites include myriocins, sphingofungins, viridiofungins, 2-amino-14,16-dimethyl-octadecan-3-ol (2-AOD-3-ol), and a new C17-SAM identified from mussels contaminated by marine fungi including *Aspergillus*, *Fusarium,* and *Trichoderma.* Australifungin, a structurally unrelated mycotoxin produced by *Sporormiella australis,* was also shown to inhibit sphingolipid synthesis in plants, similar to those of SAMs.

**Figure 3.** Chemical structures of other sphinganine-analog metabolites.

Myriocins, sphingofungins, and viridiofungins inhibit serine palmitoyltransferase (SPT), while fumonisins, AAL-toxin, and australifungin inhibit sphinganine-N acyltransferase. Serine palmitoyl transferase, one of the key enzymes in the synthesis of sphingolipids, was also reported to play a positive role in PCD regulation. The increase of SPT activity promoted PCD in plants. In contrast, by inhibiting SPT activity, the excessive accumulation of sphingosine can be alleviated, leading to reduced PCD [43]. Therefore, myriocin are usually used as a SPT inhibitor to pretreat *Arabidopsis thaliana* and tomato plants to induce their resistance to FB<sup>1</sup> and AAL-toxin, respectively [44,45].


#### **Table 1.** Analogs of sphinganine-analog mycotoxins (SAMs), their fungal producer(s), and their activities.

#### **3. Relationships between SAMs' Structure and Biological Activities**

The biological effects of SAMs, such as their toxicity, are similar among different SMAs. Many SAMs have a similar spectrum of susceptible plant species [34]. Tomato tissues and cells are similarly sensitive to AAL-toxins and to FB<sup>1</sup> and FB<sup>2</sup> toxins. In some other plants, AAL-toxins can cause necrotic cell death, similar to that of fumonisins [66]. For animal tissue cultures, the TA toxins can induce cytotoxicity in both rat liver and dog kidney cells as FB<sup>1</sup> toxin [67,68]. Besides, AAL-toxin and *F. verticillioides* could also inhibit larval growth and reduced pupal weights of tobacco budworrn *Heliothis virescens* [69]. Such similarities have been attributed to the structural similarities between the SAMs and sphinganine. However, there are differences among SAMs in their biological effects and those differences are related to their structural differences. Below, we summarize the main findings in this area.

The amino functional group of SAMs is essential for their toxic activity. The peracetylated derivatives of AAL-toxins and FB<sup>1</sup> are biologically inactive or have significantly reduced toxicity in both the plant bioassay and the animal tissue culture systems [66,70,71]. These results were consistent with initial reports on these toxins showing that blocking the free primary amines of AAL-toxins by specific reagents could abolish the biological activities of these toxins in plants [72]. In an in vitro test of rat primary hepatocytes, it was noted that the N-acetyl analogue of FB1, FA1, also showed CerS inhibition [68]. Later, FA was found to spontaneously undergo isomerization, rearranging its O-acetylation group to form different analogs. The impact of these rearrangement products on inhibition of CerS in rat liver slices also supported the important role of a primary amino for both CerS inhibition and toxicity [73]. Derivatization of the amino group with fluorogenic reagents also makes the FBs' detection possible by the high-performance liquid chromatography (HPLC) assay [74]. FBs can bind covalently to proteins by reacting with amino groups in abiogenic conversions, which may increase the toxicity of those conversion products [75]. Similarly, the terminal amino group of FB<sup>1</sup> can conjugate to bovine serum albumin (BSA) and work as an immunogen to produce monoclonal antibodies for enzyme-linked immunosorbent assay (ELISA) detection [76]. Amino group of fumonisins can also work as an electron donor and react with the electrophilic carbon within the isothiocyanate (ITC) group. Consequently, FBs can be degraded by fumigation treatment with ITC-containing compounds [77].

The hydrolysis product of FB<sup>1</sup> (HFB1) was shown as less toxic than both FB<sup>1</sup> and TA to plants [78]. Neither HFB<sup>1</sup> nor the yeast sphingolipids (completely acetylated) contain PTCA. While both had adverse effects on duckweed growth, they showed lower phytotoxicity than TA and FB<sup>1</sup> that contained one and two PTCA, respectively [79]. In contrast, the hydrolysis products of AAL-toxins largely maintain the toxicities of their parental compounds to the susceptible tomato lines [66]. These results indicate that PTCA is important to phytotoxicity of FBs and there is specificity of interaction between AAL-toxins and tomatoes.

Different from those in plants, an in vitro test using primary hepatocytes of rat showed that the HFBs had greater cytotoxicity than FBs. However, the HFBs could not initiate cancer development due to the lack of PTCA moiety, which was proposed to play an active role in the fumonisins absorption from the gut [70]. In the pregnant LM/Bc mouse model, HFB<sup>1</sup> did not cause neural tube defects. In contrast, 10 mg of FB1/kg body weight of mice disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the female mice, increased fetus mortality, and reduced fetus weight [80]. In the SAMs-sensitive pig model, HFB<sup>1</sup> was shown to have limited intestinal or hepatic toxicity but only slightly disrupted sphingolipids metabolism [81]. The toxic effects of FB<sup>1</sup> and HFB<sup>1</sup> exposure on intestinal barrier function and immunity in a pig intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells co-culture model was also investigated. FB<sup>1</sup> aggravated lipopolysaccharide (LPS)/deoxynivalenol (DON)-induced intestinal inflammation, while HFB<sup>1</sup> showed less toxicity to the immune system [82]. In addition, when HFB<sup>1</sup> and HFB<sup>2</sup> were acylated by CerS, the N-acyl-metabolites were toxic in vitro to the human colonic cell line and in vivo to the intraperitoneal rat tissues [83].

Fumonisins are capable of binding to polysaccharides and proteins via their two PTCA side chains in thermal-treated food and form fumonisin artifacts [84]. The activities of SAMs vary depending on where hydroxylation occurs along the carbon backbone. For example, FB<sup>2</sup> had a greater cytotoxic effect than FB<sup>3</sup> and FB<sup>1</sup> in primary rat hepatocytes [70]. However, different from most other side groups, the C-1 terminal methyl group, which differed between FC and AAL-toxin from other fumonisins, seemed not required for the biological activity in SAMs.

Similar symptoms but less phytotoxicities of SAMs were observed when long-chain sphingoid bases or simple sphingolipids were applied to duckweed, which indicated that the phytotoxicity of SAMs might be resulted from the accumulation of phytotoxic sphingolipid intermediates [71,85]. This result was consistent with the induction of PCD through ceramide-based signaling pathways (described below).

Although AAL-toxins and fumonisins are structurally related chemicals with similar phytotoxicity, the latter are 10 times less efficient. AAL-toxins have been considered to serve as an herbicide at a very low dosage against a wide variety of broadleaf weeds (e.g., jimsonweed, prickly sida, and black nightshade). However, monocotyledonous crops (e.g., maize, wheat, and resistant varieties of tomato) are tolerant to AAL-toxins [24,86,87]. Until 2013, the mode of action through CerS inhibition was not among the 21 molecular target sites of the commonly used herbicides. Using AAL-toxin as a lead compound has the potential to develop novel and safe bioherbicide, which has phytotoxicity but reduced or no mammalian toxicity [88,89].

#### **4. Detoxification of SAMs**

Using agricultural and manufacturing practices for preventing the spread and growth of toxin-producing fungi and limiting mycotoxin production is the preferred method to eliminate food contamination by fungal toxins at the pre-harvest period [90]. However, it is extremely difficult to completely prevent fungal pathogen growth and mycotoxin contamination in agricultural practices and in food storage and processing. Since the toxicity of SAMs is structurally dependent, our knowledge on the relationships between SAMs' structure and biological activity provides clues for developing effective management strategies to minimize the impact of SAMs in food and feed products. Indeed, structural modifications such as hydrolysis have been demonstrated as effective at reducing the toxicity of SAMs [91].

Over the last three decades, chemical, biological, and physical strategies have been developed to degrade mycotoxins in food and feed products [92]. For example, nixtamalization was applied to reduce FBs by cooking fumonisin-contaminated maize with lime, as well as by using atmospheric ammoniation treatment [93–95]. Chlorine dioxide also showed the ability to degrade FB<sup>1</sup> [96]. Two common cooking methods include extrusion and nixtamalization were shown to reduce the toxicity of FB1-contaminated corn [97]. Cold atmospheric pressure plasma was used as a physical treatment to successfully degrade pure FB<sup>1</sup> and AAL-toxins within 60 s, while the presence of the matrix slowed down the degradation [98,99]. Ozone was applied to disrupt fungal cells of *Fusarium* and *Aspergillus* by oxidizing sulfhydryl and amino acid groups of enzymes or attacking the polyunsaturated fatty acids of the cell wall [100]. However, not all SAMs are susceptible to physical and chemical treatments. In addition, some of these treatments may also result in derivatives with unknown toxicity and be detrimental for the treated commodities, as shown in some cases [101,102].

Another method to reduce SAM toxicity is through microbial actions. Microorganisms can carry out biotransformation reactions to detoxify SAMs [91]. Such methods include deamination, acetylation, hydrolysis, glucosylation, and decarboxylation. For example, Benedetti et al. isolated a Gram-negative rod bacterial strain from soil capable of degrading fumonisin to four metabolites when fumonisin was supplied as the sole carbon source [103]. The bacterium *Sphingopyxis* sp. could detoxify fumonisin B1 by at least two enzymatic steps, including an initial de-esterification reaction followed by de-amination of hydrolyzed product [104]. Chlebicz and Sli ˙zewska found that 12 strains of ´ *Lactobacillus* sp. bacteria and 6 strains of *Saccharomyces cerevisiae* yeast could reduce the concentration of FB<sup>1</sup> and FB<sup>2</sup> by 40% [105]. Similarly, Burgess demonstrated that fumonisin-producing *Asp. welwitschiae* have the ability to produce enzymes to synthesize non-aminated fumonisins that are less toxic than FB, and that those enzymes could be used for fumonisin detoxification [106]. Indeed, using enzymes to detoxify by

modification of chemical structures has become a promising method for mycotoxins control after grains harvest [107,108]. For example, fumD (carboxylesterase) from *Sphingopyxis* catalyze detoxification of FB<sup>1</sup> to the hydrolyzed form by hydrolysis of both PTCA side chains. Then, the aminotransferase FumI could degrade FB<sup>1</sup> by catalyzing the deamination of HFB1. FumD has also been tested for interference of fumonisins adsorption in turkey, swine, and pig [109–111]. Finally, several other enzymes such as manganese peroxidase from lignocellulose-degrading fungi and laccase from *Pleurotus eryngii* were all capable of degrading fumonisins [112,113].

Another potential method to reduce SAMs from food and feed products is to use adsorbent materials to soak up and remove the toxins. Many materials have shown the capacity to adsorb mycotoxins in vitro, thus the use of adsorbents in livestock diet as feed additives can potentially decrease the bioavailability of mycotoxins to humans and animals. As feed additives, cholestyramine, nanosilicate clay platelets, and refined calcium montmorillonite clay all reduced FB<sup>1</sup> toxicosis [114–116]. Moreover, natural products such as the phenolic compound chlorophorin, honey, and cinnamon oil have all shown promise as fumonisin-reducing agents [117–119].

#### **5. Programmed Cell Death and Sphingolipids**

Almost all cells die eventually. There are four main types of cell death: necroptosis, pyroptosis, ferroptosis, and apoptosis, classified based on their distinct molecular and cellular processes and different outcomes. Apoptosis or programmed cell death (PCD) is a kind of cell suicide that strictly regulates cells that are no longer needed or are a threat to the wellbeing of multicellular organisms. Both plants and animals have PCD and they are functionally analogous to each other [120–123]. PCD plays essential roles to maintain normal physiological activities in multicellular organisms such as plants and animals and is an active self-regulating process to selectively eliminate redundant, aged, and damaged cells. PCD can be predicted for specific cells at defined developmental stages. However, PCD can also be induced by membrane-bound and cytosolic proteins stimulated by stress-induced signals. Such signals can trigger cell death via intricate cascades of transcriptional changes and post-translational protein modifications [122,124]. The characteristics of PCD include reduced cell volume, chromatin marginalization and condensation, nuclear lamina disassembly, DNA fragmentation, and apoptotic body formation, etc. [1].

PCD triggers and propagation involve many factors, including the expressions of certain cell surface receptors, transmembrane domains of several membrane proteins, intracellular proteins related to the propagation of death signals, secondary messengers including inositol triphosphate and ceramides, calcium (Ca2+) fluxes, reactive oxygen species (ROS), regulatory factors of cell cycle, and other suppressors or activators proteins. Many of these subcellular components, genes, and signal transduction pathways involved in PCD are functionally conserved across all domains of cellular organisms, from bacteria to fungi to plants and animals. However, there are differences in the actual mechanisms among organisms, as summarized in References [125,126].

Sphingolipids have been implicated to play an important role in cell growth, development, response to external environment, and PCD. As the main component of the cell membrane system, sphingolipids help to maintain the structural stability and transport of molecules across cell membranes [127,128]. In mammals, sphingolipids are especially abundant in the nervous system cells, with important functions in cell contact, growth, differentiation, communication, response to stress signals, and apoptosis [30,129]. In plants, sphingolipids are involved in response to both biotic and abiotic stresses, such as to pathogen infection, drought, and low temperature [31,130,131]. Indeed, the linkage of ceramide signaling to apoptosis has been widely reported in both plants and animals. Consequently, actions by SAMs to disrupt the functions of sphingolipids could have significant negative consequences. However, our knowledge about the roles of sphingolipids on apoptosis have also led to increasing interests on potential novel therapies using sphingolipids as treatment targets against degenerative and proliferative diseases in humans and animals, such as cancer and Parkinson's disease [132,133].

A large number of studies have shown that sphingolipids could serve as critical secondary messengers in signal transduction to regulate PCD [134,135]. For example, in neutrophils, sphingolipids have been linked to increased superoxide formation and Ca2<sup>+</sup> influx, which are universal signaling molecules involved in many cellular functions [136]. The induction of PCD in *Arabidopsis* by ceramides was also verified to be partly dependent on ROS in mitochondria or regulated by the release of Ca2<sup>+</sup> [137,138]. In addition, increase of sphingosine level can activate the mitogen-activated protein kinase (MAPK) pathway in which MPK6 participates, promote the accumulation of sialic acid (SA), and then induce PCD [43]. Lachaud et al. found that the activated calcium-dependent kinase (CPK3) can regulate the process of sphingosine-induced PCD by dissociating CPK3 from the 14-3-3 protein-complex under increased calcium concentration induced by sphingosine. The activated CPK3 is then degraded followed by PCD induction [139].

Mutational studies of *Arabidopsis* and in vitro experiments have shown that ceramides, and free sphingoid bases such as sphingosine, sphinganine, and phytosphingosine, can all induce PCD. In contrast, phosphorylated products of these compounds can inhibit or alleviate PCD in plants [44,138,140–142]. However, phosphorylated sphingosine can inhibit the growth of yeast cells, which suggests that there are different mechanisms of action between plants and yeasts [143]. It is worth mentioning that not all ceramides can induce PCD. Nagano et al. found that when the C2 position of fatty acid in the side chain of ceramides was hydroxylated, PCD was inhibited rather than induced [144]. These results indicate that the occurrence of PCD in plants depends not only on the absolute content of sphingolipids, but also the relative ratios of various modified forms. In the next section, we will describe how SAMs are involved in PCD.

#### **6. SAMs Trigger PCD through Ceramide-Based Signaling Pathways**

In plants, pathogen invasions can lead to disruptions in host cellular homeostasis, and trigger cell death in susceptible varieties or even in resistant varieties with a hypersensitive response (HR). Because of its many similarities with PCD, HR is often considered as a form of PCD in plants. AAL-toxins, as the pathogenic factor of tomato stem canker disease, can induce PCD in sensitive tomato varieties resulting in fragmentation of chromosomal DNA and formation of apoptotic bodies in cells [145]. Similarly, when treated by FB<sup>1</sup> produced by pathogenic *Fusarium*, *Arabidopsis* protoplasts showed symptoms similar to PCD in animal cells [146]. At the tissue and organ levels, *Arabidopsis* leaves treated with the FB<sup>1</sup> toxin showed characteristic disease symptoms. Cells of the diseased leaves had overall phenotypes similar to HR, including callose accumulation, ROS production, and pathogenesis-related (PR) gene induction [147]. The damages caused by SAMs on host plants can further increase pathogen infection and colonization. Similar to those found in plants, SAMs can induce neuro-/renal-responses, heptatoxicosis, and neoplasms, as well as cell death in animals. The relationship between apoptosis and ceramide signaling has been established in both plants and animals in their response to SAMs [120]. For example, the induction of cell death in both tomato and African green monkey kidney (CV-1) cells occurred under similar toxin concentrations and time frames. For both types of cells, morphological markers characteristic of apoptosis were observed, including cells with positive terminal deoxynucleotidyl transferase end labeling (TUNEL), DNA fragmentations, and the formation of apoptotic-like bodies [145,148].

SAMs are structurally analogous to sphinganine and are thus effective inducers of PCD. The emerging mechanism of their actions is that SAMs can competitively bind to CerS in cells. Such binding leads to the accumulation of free sphingoid bases, the substrates of CerS, while ceramides as products of CerS were consumed and reduced, activating PCD in plant and animal cell lines [3,85]. For example, it was reported that FB<sup>1</sup> is a potent competitive inhibitor of CerS from liver and brain microsomes in several mammalian cell lines [149,150]. An increase in sphinganine was observed in an in vivo test of CerS inhibition, as well as in FB-fed animals treated at high concentrations [2]. In addition, TA and FB<sup>1</sup> can inhibit CerS in rat hepatocytes and green tomato fruits [150,151]. Furthermore, it was found that FB<sup>1</sup> not only induced apoptosis in animal cells, but also altered cell morphology, cell–cell

interactions, cell surface proteins behavior, protein kinase activity, and cell growth and viability in non-apoptotic cells [148,152]. In plant cells, after exposure to SAMs, sphingosine concentration increased significantly within a short time, followed by the accumulation of ROS in the cytoplasm and then apoptosis. These results suggested that the accumulation of sphingosine in cells was the upstream signal of ROS for cell death [43,44,85]. The induction of PCD by FB<sup>1</sup> is also related to the accumulation of ceramides. In *A. thaliana,* there are two types of CerSs that use different substrates. Class I CerSs use sphinganine and C-16 fatty acyl-CoA as substrates, while class II use phytosphingosine and very long-chain fatty acyl-CoA as substrates. FB<sup>1</sup> mainly inhibits the activity of class II CerSs. When treated with FB1, phytosphingosine in cells increases significantly. At the same time, as the product of a previous step, sphinganine also increases, which provides more substrates for class I CerS. Consequently, the products of class I CerS in cells increase, leading to induced PCD [153,154].

#### **7. Plant Resistance to SAMs**

Phytohormones are also involved in the defense reaction induced by SAMs. Changes in ethylene (ET) were first discovered in AAL-induced necrosis of tomato [155]. Alteration in ethylene perception in "never ripe" mutants of tomatoes can markedly alleviate the tissue damage caused by SAMs, which indicated an ethylene-associated signal transduction during plant cell death [156]. Later, a transcription analysis of AAL-toxin-induced cell death was carried out in *Arabidopsis*. Genes responsive to ROS and ET were among the earliest upregulated genes [157]. Mase used VIGS (virus-induced gene silencing) analyses and verified that the ET signaling pathway and MAPK cascades were required for AAL-toxin-induced PCD in tobacco [158]. By SA-mediated ET suppression, glutathione (GSH) may be involved in resistance primarily against AAL-toxin-induced stress in Arabidopsis [159].

Unlike ethylene in host basal defense responses against the tomato pathotype of *A. alternata*, the jasmonate (JA)-dependent signaling pathway is not involved in host defense against the toxigenic *A. alternata* pathogen. JA affects pathogen acceptability via a toxin-independent mannerin in the interactions between plants and toxigenic necrotrophic fungal pathogens. It may act upstream of ethylene biosynthesis in AAL-toxin-triggered tomato cell death [160,161]. Later, a comparative proteomics analysis revealed that the COI1 (coronatine insensitive 1, JA receptor)-dependent JA pathway enhances AAL-toxin-induced PCD of tomato through regulating the redox status of the leaves, other phytohormone pathways, and/or important PCD components [162].

The sensitivity of tomato plants to the fungal pathogen *A. alternata* f. sp. lycopersici is controlled by the *Alternaria* stem canker resistance locus (*Asc*-locus) on chromosome 3 [163]. Mutations of tomato *Asc* locus gives resistance to the pathogen, while overexpression of the tomato *Asc-1* gene mediates high insensitivity to SAMs in tomato and confers resistance to pathogen infection in sensitive *Nicotiana* plants [164,165]. *Asc-1* is a homolog of the yeast longevity assurance gene *LAG1*, which encodes components of sphinganine N-acyltransferase. This resistance gene could prevent the disruption of sphingolipid metabolism during AAL-toxin-induced PCD. Both *Nicotiana* and *Lycopersicon* genera belong to Solanaceae. In tomato, insensitivity to SAMs and susceptibility to the pathogen is determined by *Asc-1* [166]. In contrast, the SAM-sensitive species in the *Nicotiana* (except for *N. umbratica*) still have *Asc-1* homologs and are resistant to *A. alternata* f. sp. lycopersici infection with HR, which indicates an additional (non-host) resistance mechanism between *Nicotiana* and this pathogen [167]. The multilayered defense systems also exist in *Arabidopsis* non-host resistance to *A. alternata* [168]. Similarly, although many *Fusarium* species produce fumonisin, they cannot infect AAL-sensitive tomato. This non-host resistance includes a multi-layer defense system involving both pre- and post-invasion, and help plants defend against various pathogens [169]. In addition, Zélicourt demonstrated that two of three Lag1 homologs in the *Orobanche cumana* genome were responsible for an enhanced sensitivity to AAL-toxin [170].

Aside from the above-mentioned genes, several other genes were identified from Arabidopsis and found to be involved in the AAL-induced PCD pathway, including Zinc *A. thaliana* 11 (a zinc finger protein ZAT11), fbr41 (FB<sup>1</sup> Resistant41), and baculovirus p35 gene (inhibitor of a class of cysteine proteases). All of them showed protective effects on AAL-toxin-induced cell death and pathogen infection in plants [171–173]. Discovery of resistant genes has provided a potential strategy for SAMs' control in crop production by plant transgenic modification.

Because of the high toxicity of fuminisins, especially FB1, a large number of studies have focused on them. So far, the mechanism of FB<sup>1</sup> toxicity has been centered around its structural resemblance with sphinganine and consequent competitive inhibition of CerS and the disruption of lipidomic profiles. However, there is emerging evidence suggesting that FB<sup>1</sup> can disrupt mitochondrial function and generate excessive toxic ROS.

Table 2 shows a list of reviews summarizing the latest advances related to fumonisins, in their assessment, biosynthesis, detection, crop breeding of resistant varieties, and toxicity.


**Table 2.** Topical reviews on fumonisins over the last five years.

#### **8. Genes Responsible for SAMs Production**

In fungi, genes directly involved in the biosynthesis pathway of the same secondary metabolite are usually located at adjacent positions in gene clusters in the genome [191]. These genes are often co-expressed and co-regulated. The genes involved in fumonisin biosynthesis fit this general pattern. Specifically, *Fusarium* species capable of producing fumonisins typically contain one gene cluster involved in their synthesis, called the *FUM* gene cluster. At present, a total of 21 genes have been identified in *FUM* gene clusters of various species and verified to be involved in fumonisin biosynthesis and regulation or self-protection using a variety of approaches, such as gene knockouts, domain swapping, and heterologous expression (Table 4) [192–197]. In *F. verticillioides*, the *FUM* cluster responsible for fumonisin B biosynthesis includes 17 genes [193,197,198].

As described previously, SAMs are polyketide-derived compounds with structural similarity to sphinganine. Polyketides are synthesized by polyketide synthases (PKSs), which are large multifunctional enzymes. *FUM1* is a PKS gene previously designated as *FUM5* [199]. PKS encoded by *FUM1* catalyzes the synthesis of an octadecanoic acid precursor as the initial step for FB biosynthesis in *Fusarium* spp. [200,201]. The proposed biosynthetic pathway of FB is described in Figure 4 [197]. The precursor mentioned above undergoes condensation with L-alanine to synthesize the polyketide backbone, this reaction was catalyzed by the aminotransferase Fum8 [192,201]. In *F. oxysporum* strain O-1890, the orthologue of *FUM8* determines that *Fusarium* produces predominantly FCs [195]. The *fum8* deletion in some stains of *A. welwitschiae* is also considered to be associated with the loss of FB<sup>2</sup> production [202].

**Figure 4.** The *FUM* gene cluster, the proposed pathway of fumonisin biosynthesis, and the proposed mechanism for self-protection against fumonisins toxicity by the toxin-producing fungi (FBs and FCs). SAM, S-adenosyl methionine; VE, vesicles; MT, mitochondrion; ER, endoplasmic reticulum; NU, nucleus; PM, plasma membrane.

A likely mitochondrial carrier protein encoded by *FUM11* transport the substrate tricarboxylate for Fum7 (dehydrogenase) and Fum10 (acyl-CoA synthase) to produce CoA-activated tricarballylic acid, which are attached to the polyketide backbone by Fum14 (condensation-domain protein) [192,194]. The ensuring steps of fumonisin biosynthesis involving various modifications of the backbone (including primarily hydroxylation) were catalyzed by several enzymes, such as Fum6/Fum12/Fum15 (cytochrome P450 monooxygenase), Fum2 (hydroxylase of C10), Fum13 (short-chain dehydrogenase/3-ketoreduction), and finally, Fum3 (hydroxylase of C5, dioxygenase), catalyzed by *FUM9*-encoded protein (alleles of *FUM3*) [192,193,203–206].

In addition, *FUM21* encoding a GAL4-like Zn(II)2Cys6 transcription factor was verified to be involved in the regulation of fumonisin synthesis. However, it seemed that the deletion of *FUM16* had no apparent effect on fumonisin production in *F. verticillioides* [194,198,207–209]. Recently, *FUM17*–*FUM19* in *F. verticillioides* were found to help the fungus to avoid its own toxicity during fumonisin production. Fum19 is an ATP-binding cassette transporter (ABC transporter) and acts as a repressor of the *FUM* gene cluster. *FUM17* and *FUM18* are CerS homologs. *FUM18* could fully complement the yeast CerS null mutant *LAG1*/*LAC1*, while co-expression of *FUM17* and CER3 partially complemented. Both the Fum17 and Fum18 proteins enable *F. verticillioides* to increase its resistance of fumonisin by providing *FUM* cluster-encoded CerS activity as a first level of self-protection [197].

Aside from the *FUM* genes, other genes like *FST1* (transporter), *FUG1* (transcription or signal transduction factors), *CPP1* (protein phosphatase type 2A catalytic subunit), and *FvVEl* (regulator) in *F. verticillioides*, *PKS3* and *PKS11* in *F. proliferatum*, and GATA-type transcription factors *AreA* and *AreB* (known as the global nitrogen regulators) in *F. fujikuroi* have also been demonstrated to have an important role in fumonisin biosynthesis and regulation [209–214]. In addition, a degenerated, over-represented motif which is potentially involved in the cis-regulation of *FUM* genes and fumonisin biosynthesis was also identified from both *F. verticillioides* and *Asp. niger*, while it was not found in fumonisins non-producing fungi containing various *FUM* homologues [215].

Several abiotic and biotic factors have been found to affect the expression of *FUM* genes and regulate biosynthesis of fumonisin. These factors include water activity, temperature, carbon sources and other nutrients, host plant species and varieties or their extracts, and plant age [216–220]. Mature plants and extracts from those plants are often associated with higher concentrations of SAMs. It has been suggested that harvesting the crop at earlier stages other than full maturity could be one of the strategies to control fumonisin contamination [221,222].

The genome sequencing and analysis of *Asp. niger* revealed that its genome contained a gene cluster (*fum* cluster) homologous to the *FUM* cluster in *Fusarium* species (shown in Table 4). Specifically, 12 homologues of the fumonisin synthesis genes were found, including *fum1, fum3, fum6, fum7, fum8, fum10, fum13* to *fum16, fum19,* and *fum21* genes [208,223,224]. This gene cluster is also found in fumonisin-producing isolates of *Asp. welwitschiae* but is absent from the genomes of other sequenced *Aspergilli* that do not produce fumonisin, such as *Asp. fumigatus*, *Asp. oryzae*, and *Asp. nidulans* [7,208]. In addition, homologs of multiple *fum* genes have been found in several other *Aspergilli* spp. but where no fumonisin production has been detected (summarized in Table 3). Some of the *Aspergullus* spp. contain genes that are unique to them. For example, a dehydrogenase gene (*sdr1*) of a short-chain length was found in the *fum* cluster of *Asp. niger* but is absent in the *FUM* gene cluster of *Fusarium* spp. In contrast, the *Fusarium FUM2* gene with a function of hydroxylation at the C-10 backbone position of fumonisin is absent in the *Asp. niger fum* cluster [207,208]. This result is consistent with the study that shows that *Asp. niger* only produces fumonisins FB2, FB4, and FB6, which lack a hydroxyl at C-10 [7,225,226]. However, isolates of several black aspergilli (including *A. niger*, *Asp. foetidus,* and *A. tubingensis*) isolated from peanuts and maize also produced FB<sup>1</sup> and FB3, consistent with a complex biosynthesis pattern of the fumonisins in *Aspergilli* spp. [227].




#### **Table 4.** Homologous genes and their functional roles in the biosynthesis of SAMs.

AAL-toxins are produced by *A. alternata* f. sp. *Lycopersici,* a specific pathotype of a common plant fungal pathogen in a genus different from *Aspergillus* and *Fusarium.* This pathotype can produce polyketide-derived compounds similar in structure to fumonisins produced by *Fusarium* species. The *ALT* (AAL-toxins synthesis) genes are also located as a cluster on a conditional disposable (CD) chromosome of ~1.0 Mb in all strains of the tomato pathotype of *A. alternata* from different countries [237]. Such CD chromosomes carrying a toxin biosynthesis gene cluster were also found in other pathotypes of *A. alternata* [169]. They control other HSTs production and pathogenicity to their host. They could maintain stably in a new genetic background to an expanded range of pathogenicity, which was verified by a protoplast fusion test [238]. The AAL-toxin gene cluster includes at least 13 genes in a 120 kb region, some of which showed significant similarity to the *FUM* gene cluster consisting of 17 genes in a 45.5 kb region. However, the arrangement of genes in the *ALT* and *FUM* clusters differs between these two groups of fungi. In addition, in one strain, As-27 of *A. alternata*, there were two sets of the AAL-toxin biosynthetic gene cluster on the CD chromosome [169]. The synthesis of AAL-toxins was found to be initiated by *ALT1*-encoded PKSs to produce the aminopentol backbone, which was then modified by other enzymes [239]. The functional similarity between *ALT1* and *FUM1* was confirmed when fumonisin biosynthesis in *FUM1*-disrupted *F. verticillioides* was restored when complemented by the *ALT1* from *A. alternata* [240]. Similarly, expression of *ALT1* and production of AAL-toxins were also found to be regulated by the global regulator *LaeA* [241]. AAL-toxin accumulation also benefits from high water activity (0.995 aw) and high temperature (above 30 ◦C) during the incubation period of the pathogen [242].

Interestingly, a PKS gene similar to *FUM1* and orthologs of the *FUM* gene cluster were found in the genome of *Cochliobolus* spp. by phylogenetic analysis of fungal polyketide. These fungi were also speculated to produce a fumonisin or other SAMs [243,244]. To predict the potential distribution of SAM production in fungi, Kim et al. proposed a hypothesis on SAM biosynthetic gene clusters based on fumonisin biosynthesis model. This putative gene cluster should include a PKS, an aminotransferase, and a dehydrogenase gene. Their model showed that sixty-nine species of the *Fusarium* genus and species of twenty-four other fungal genera were predicted to have at least one SAM cluster [245].

#### **9. Evolution of SAMs Production**

Horizontal gene transfer (HGT) has been proposed as a major mechanism responsible for the acquisition and evolution of fumonisins and AAL-toxins biosynthetic gene clusters among divergent fungi [7,9,169,224]. In *Fusarium*, genome sequence analyses revealed that the fumonisin biosynthetic genes (*FUM*) are clustered and show a consistent gene organization among most species. For example, the *FUM* clusters in *F. oxysporum*, *F. proliferatum,* and *F. verticillioides* exhibit relatively little variability, with the order and orientation of genes within the clusters all being the same as each other. In addition, their sequence variability among the orthologues of coding regions from *F. oxysporum* and *F. verticillioides* is relatively low [193,195]. The two different species of *Aspergillus* that produce fumonisins, *Asp. niger* and *Asp. welwitschiae*, are also similar to each other in their gene order but different from that of the *FUM* cluster in *Fusarium* [224]. At present, the tomato pathotype of *A. alternata* was the only species of genus *Alternaria* capable of producing SAMs and this pathotype has clustered genes (involved in AAL-toxin biosynthesis) similar to the *FUM* cluster in *Fusarium* [169,237]. Together, the gene structure and sequence analyses suggested that the SAMs biosynthetic gene cluster likely originated in *Fusarium* and transferred to *Asp. niger* and *A. alternata* by HGT. The similarities in chemical structure and cytotoxicity on plants and animals between fumonisins and AAL-toxins are also supportive of this hypothesis. However, the differences between *FUM* clusters and the AAL-toxins biosynthetic gene cluster also suggested that there has been significant divergence between them.

Analyses of the *FUM* gene cluster among *Fusarium* species also revealed evidence for gene gain, loss, and mutations of different genes. For example, not all *Fusarium* species can produce fumonisins. Even for species that can synthesize fumonisins, some strains produce more than others under the same experimental conditions, while other strains do not produce the toxins at all [9,196]. Indeed, for certain

strains, while the *FUM* genes were detected, there was no detectable fumonisin. This was likely due to the accumulation of mutations leading to the *FUM* genes being nonfunctional. For example, several mutations have been found in *FUM7* and *FUM21* in *F. fujikuroi* [246]. Furthermore, several fumonisin-non-producing *Fusarium* species lack the fumonisin biosynthetic genes but retain homologs of several genes that flank the Fum cluster in *F. verticillioides* [247]. Interestingly, the flanking regions of the *FUM* cluster often differ between species, consistent with the independent origins of the *FUM* cluster, including independent acquisition and/or loss of the gene cluster by fumonisin-producing species [195]. For example, fumonisin-non-producing strains of *F. verticillioides* isolated from banana did not contain the functional fumonisin biosynthetic gene (*FUM*) cluster but did contain portions of *FUM21* and *FUM19* flanking the cluster, both of which are the terminal genes at each end of the *FUM* cluster. However, the banana strains are still pathogenic to banana, but they do not show the same pathology as the fumonisin-producing strains do on maize. When a banana strain was co-transformed with two overlapping cosmids containing the entire *FUM* gene cluster, fumonisin production and pathogenicity on maize seedlings were recovered [248]. Similar to *Fusarium*, FB-non-producing isolates of *Asp. niger* or other *Aspergillus* species also had an intact *fum* cluster or multiple patterns of *fum* gene deletion, respectively (shown in Table 3). Similarly, the AAL-toxin-production gene cluster in *A. alternata* was likely derived from *Fusarium* species by an HGT event. Evidence from the *ALT* gene cluster distributed in isolates of *A. alternata* also supports this hypothesis of HGTs within AAL-toxin-producing pathogens [237]. Together, these results suggest that there have been multiple HGTs of the cluster between species, as well as duplication and loss of the whole or part of the cluster after acquisition [9].

The hypothesis that multiple HGTs were involved in generating the current distribution of *FUM* gene clusters is further supported by phylogenetic studies. Specifically, phylogenetic trees based on genes from the *FUM* cluster often do not parallel that of the *Fusarium* species tree based on other genes [9]. In *Fusarium*, the translation elongation factor (tef-1α) gene is the most commonly used marker gene for taxonomic studies. However, sequences of the tef-1α are often insufficient for distinguishing fumonisin-producing isolates from different countries and/or host plants. In contrast, DNA sequence polymorphisms based on *FUM1* often provide better resolutions among pathotypes [196,249–253]. For example, phylogenetic analysis of 38 *F. proliferatum* isolates originating from different hosts showed that sequence variation among strains in the *FUM1* gene was correlated with that of the host plants. Specifically, phylogenetic analysis based on the partial *FUM1* sequences differentiated the host-related groups more clearly than that based on tef-1α sequences. The best distinguished group consists of garlic-derived isolates and formed a separate branch on a *FUM1*-based dendrogram [196,254]. Similarly, *FUM1* sequence divergence analysis on *F. proliferatum* and *F. verticillioides* strains isolated from pea also formed a distinct group when compared to strains derived from different host species [253].

Aside from gene differences among isolates from different host plants, variations of both toxigenic potential and growth patterns may also differ between isolates derived from the same host plants. While no difference was observed in FB levels measured among pea seeds, the FB productions differed between selected strains of *F. proliferatum* in rice cultures [253]. Among all these test isolates of *F. proliferatum*, the most varied group of isolates found so far were those isolated from maize [196,254]. Both the inter- and intra-specific variation in FBs synthesis level can at least partly be explained by the sequence differences inside the FUM cluster.

In summary, the analyses so far suggested that the *FUM* gene cluster was responsible for fumonisin biosynthesis. Mutation and deletion of some or all of the genes in the cluster could result in limited or no production of fumonisin, leading to a weaker disease development of the pathogen on the host plant. Sequence analyses showed that *A. alternata* has likely gained the ability for AAL-toxin production due to HGT of the SAMs gene cluster from fumonisin-producing *Fusarium* species followed by independent evolution in pathogen–host interaction. The divergent patterns of toxin biosynthesis gene sequence divergence may explain the differences between fumonisins and AAL-toxins in both their productions and their impacts on host–pathogen interactions.

#### **10. Detection Method of SAMs**

A variety of methods have been developed to detect SAMs, including HPLC with fluorescence/evaporative light scattering detection or mass spectrometry (MS), thin-layer chromatography (TLC), ELISA, Fourier transform near infrared (FT-NIR) spectroscopy, and so on [255–263]. While these traditional analytical methods were designed to detect and quantify known compounds for which standards are available, there is clear evidence that many unknown derivatives may exist in food and food products and some of these could be toxic to animals, including humans [39]. In 2015, a semi-targeted method combining product ion filtering and rapid polarity switching was designed for fast detection of all known fumonisins and AAL-toxins. Some new structurally related emerging toxins were also discovered by this method [26].

Aside from method development that targets potentially novel SAMs not reported before, there are also developments for efficient methods that target the detection of known SAMs. Indeed, several new methods based on immunoassay were developed for simple, rapid, and ultrasensitive on-site quantification of SAMs. For example, one method used chemiluminescent biosensors integrating a competitive lateral flow immunoassay and a charge-coupled device camera to detect FBs [264,265]. Another method uses gold nanoparticles or quantum dots nanobeads based on monoclonal antibodies against fumonisin and allows rapid detection of this mycotoxin in one step [266–268]. Furthermore, a loop-mediated isothermal amplification (LAMP) assay, based on the detection of *fum10*, could specifically detect the genes involved in FB<sup>2</sup> biosynthesis in *Aspergillus* species that could be evaluated using the naked eye in a short time [269]. This method was also applied to detect FB<sup>1</sup> targeting the *FUM1* gene in *Fusarium* [270]. The direct detection of genes involved in the biosynthesis of SAMs in agriculture production systems allows broad evaluations of the potential fumonisin-producing strains in food and feed products. To this end, multiplex PCR has shown great promise for detection of multiple fumonisin-producing *Fusarium* and *Aspergillus* species [271–273].

While the current focuses are on the known SAMs with known toxicities, there is increasing evidence that some of these toxins are masked and not easily detected or quantified. To ensure food safety, both the free forms and the masked forms of mycotoxins should be detected and quantified. The masked mycotoxins are usually modified forms of the mycotoxins by plant enzymes during infection and are not typically detectable during routine analysis. For example, the masked mycotoxins may conjugate with polar substances, store in the vacuole in the soluble form, or bind to macromolecules, and thus change their physiological properties. While the masked mycotoxins are often less toxic than the unmasked forms, they could be easily converted to the unmasked toxin forms, including during food digestion [274,275]. The most representative masked mycotoxins are the modified forms of Zearalenone (ZEN), DON, and fumonisins [276–279]. The so-called hidden fumonisins could form non-covalent bonds with food macro-constituents such as those in starch-based products. In certain situations, the masked fumonisins may be present in food at quantities much higher than the free forms. Many factors could influence the relative portions of the SAMs in masked forms, including crop growth conditions and food storage and processing techniques [279–282]. These hidden dangers require that novel method(s) be developed to allow the detection and quantification of the masked forms of SAMs as well as other mycotoxins.

#### **11. Concluding Remarks**

SAMs are highly toxic fungal compounds that have attracted significant attention from broad communities. They have toxicities to both plants and animals. These SAMs-producing pathogens are widely distributed in nature and closely related to agricultural production. Since its discovery in the mid-1980s, fumonisin has been among the mycotoxins with the greatest concern. As of now, Scopus database citations of fumonisin are above 5000, including 500+ reviews. SAMs are a series of compounds with structural similarity to sphingosine. As detection methods improve, additional new analogs have been continuously discovered. Research so far has shown that the toxicity and activity of SAMs are dependent on their structures. In this review, we summarized the detoxification method

based on their structural properties using chemical, biological, and physical strategies. The toxicity of SAMs is mostly due to their inhibitory effects on CerS, disruption on sphingolipid metabolism, and initiation on PCD. Except for the adverse effect of SAMs on animals and humans, its phytotoxicity (e.g., AAL-toxin) could potentially be used for herbicide development and a model for studying the molecular mechanism of PCD in plants. Horizontal gene transfers on the SAMs biosynthesis gene cluster seemed widespread in these toxin-producing fungi, especially among *Fusarium* spp. Such phylogenetic distribution patterns suggest that there are potentially other fungi capable of producing SAMs, including their various modified forms. These and other issues require continued efforts from the scientific community on SAMs.

**Author Contributions:** J.X. conceived the review and finalized the manuscript, J.C. conducted the literature search and drafted the manuscript, Z.L. drafted all the figures and tables, Y.C. coordinated the format and submitted the manuscript to the journal, C.G., L.G., and T.W. helped to revise the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Key R&D Program of China (N0: 2018YFD0201106), Natural Science Foundation of Hunan (2020JJ5637) and Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (Grant CAAS-ASTIP-2015-IBFC).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Fungal Pigments and Their Roles Associated with Human Health**

**Lan Lin 1,2 and Jianping Xu 2,\***


Received: 15 October 2020; Accepted: 9 November 2020; Published: 12 November 2020

**Abstract:** Fungi can produce myriad secondary metabolites, including pigments. Some of these pigments play a positive role in human welfare while others are detrimental. This paper reviews the types and biosynthesis of fungal pigments, their relevance to human health, including their interactions with host immunity, and recent progresses in their structure–activity relationships. Fungal pigments are grouped into carotenoids, melanin, polyketides, and azaphilones, etc. These pigments are phylogenetically broadly distributed. While the biosynthetic pathways for some fungal pigments are known, the majority remain to be elucidated. Understanding the genes and metabolic pathways involved in fungal pigment synthesis is essential to genetically manipulate the production of both the types and quantities of specific pigments. A variety of fungal pigments have shown wide-spectrum biological activities, including promising pharmacophores/lead molecules to be developed into health-promoting drugs to treat cancers, cardiovascular disorders, infectious diseases, Alzheimer's diseases, and so on. In addition, the mechanistic elucidation of the interaction of fungal pigments with the host immune system provides valuable clues for fighting fungal infections. The great potential of fungal pigments have opened the avenues for academia and industries ranging from fundamental biology to pharmaceutical development, shedding light on our endeavors for disease prevention and treatment.

**Keywords:** fungal pigments; melanin; carotenoids; polyketides; azaphilones; antitumor; medical roles

#### **1. Introduction**

Since prehistoric times, fungi have played critical roles in human daily activities, such as baking, brewing (wines and beers), and processing dairy products. With the advent of new technologies, especially biotechnology, over the past 60 years, the use of fungi has increased significantly for the production of commercially important products, such as edible mushrooms, drinks, alcohols, organic acids, enzymes, and colorants. After the breakthrough discovery of penicillin in the 1930s, fungi have become a very significant source of pharmaceutical products which give rise to life-saving medicines, including antibiotics, anticancer drugs, and cholesterol-lowering agents [1]. Apart from the benefits that fungi provide, some fungi can also cause significant diseases to a variety of crops, livestock, pet animals, and humans. Accumulating evidence has revealed that the incidence of human fungal diseases has been increasing rapidly since the 1980s, and is associated with excessive morbidity and mortality, particularly among immunosuppressed and immunocompromised patients [2]. Among the 625 fungal species known to be pathogenic to humans, those in the genera *Aspergillus*, *Candida*, *Cryptococcus*, and *Trichophyton* cause diseases in over 300 million people globally [2]. These diseases can be superficial, subcutaneous, or systemic. Some are acute infections, but others can be long-lasting chronic infections (e.g., athlete's foot). Accordingly, the detailed biological processes for generating

useful products from beneficial fungi and for virulence in pathogenic fungi are very active areas of research.

To the general public, when we talk about pigments in living organisms, we often think of the colorful flowers, insects, and birds. Indeed, pigments in these groups of organisms are the subjects of curiosity for people from diverse walks of life, including painters, photographers, writers, gardeners, and biologists. The biological roles for many of those pigments have been extensively studied. In contrast, unless we are in the woods and see colorful mushrooms, we often do not associate pigments with fungi, with the possible exception of "black" molds in spoiled foods. However, fungi are prolific producers of a myriad of pigments of different chemical structures and a diverse range of colors. The majority of well-studied fungal pigments are from fungi of four genera: *Aspergillus*, *Penicillium*, *Paecilomyces,* and *Monascus* [3,4]. When chemically categorized, fungal pigments are grouped into carotenoids, melanins, polyketides, azaphilones (polyketide derivatives), etc. [5,6].

Carotenoids constitute an abundant group of pigments in nature. They are found in all plants where they play an essential role in photosynthesis. In fungi, over 200 fungal species have been documented as capable of producing carotenes [7]. Carotenoids play very important roles in human health. They not only serve as the precursors of vitamin A in humans, but can also alleviate and/or prevent human age-related diseases, such as cataracts and macular degeneration. Furthermore, they can reduce the incidence of coronary heart disease and carcinomas, including lung, breast, prostate, and colorectal cancers [8]. The health-promoting effects of carotenoids are believed to be associated with their bioactivity as antioxidants and free radical scavengers.

Another broadly distributed group of fungal pigments is melanin. Melanin contributes significantly to the fungal capacity to survive and thrive in unfavorable habitats. Fungal melanin molecules are polyphenolic and/or polyindolic compounds with high molecular masses and negative charges. Due to its physiochemical properties, melanin can mediate an array of cellular functions, allowing fungal adaptation to diverse environmental factors, including ultraviolet (UV) light, heat, ionizing radiation, and oxidative stressors [9]. These properties make melanin a potential artificial stress-protecting agent (e.g., radioprotective) in bioinspired applications. However, the relevance of melanin to human health is probably best exemplified in human fungal pathogens as a virulence factor. For example, in the opportunistic human pathogens of the *Cryptococcus* genus, such as *Cryptococcus neoformans* and *C. gattii*, strains unable to produce melanin are avirulent or have significantly reduced virulence [10,11]. Similarly, in *Aspergillus fumigatus* as well as *Paracoccidioides brasiliensis*, the melanized cells exhibited elevated resistance to phagocytosis [12].

In this paper, we provide a comprehensive and updated review on fungal pigments that have demonstrated relevance to human health either directly or indirectly. We first describe the categories and chemical structures of these fungal pigments, including representative fungi that produce them. This is followed by descriptions of their biosynthesis pathways, how they interact with the human immune system, and the relationships between their structure and activities. We finish by providing a perspective on future developments.

#### **2. Types of Fungal Pigments and Their Relevance to Human Health**

The majority of organisms living on this planet synthesize some pigments. These biological pigments confer on our world a wide array of colors via the absorption and refraction of specific wavelengths of light. In nature there are many types of biological pigments, ranging from monomeric (such as carotenoids, flavonoids, luciferin, and heme/porphyrin-based pigments) to polymeric (i.e., melanin and humic compounds). Some pigments possess conjugated moieties (i.e., aromatics rings) that facilitate electronic resonances and mediate energy transfers within and between cells. The energy captured and/or reflected by pigments has been shown to play multiple biological roles ranging from the maintenance of life, including the utilization of solar energy for metabolic functions and protection against radiation damage, to camouflage, as well as mate and pollinator attraction. Below, we describe the main types of fungal pigments relevant to human health.

#### *2.1. Carotenoids*

Aside from β

ng β

In addition to being produced by photosynthetic organisms ranging from cyanobacteria to flowering plants, carotenoids are also synthesized in a variety of heterotrophic microbes including fungi [13]. Furthermore, carotenoids are present in animals that are unable to synthesize them de novo, but instead acquire them from their diet.

Carotenoids contain an aliphatic polyene chain usually composed of eight isoprene units that include light-absorbing conjugated double bonds, giving rise to characteristic colors such as yellow, orange, or red [14,15]. Representative structures of carotenoids are shown in Figure 1. Some carotenoids are precursors of vitamin A, specifically those with β-ring end groups, such as β-carotene, zeaxanthin, and β-cryotoxanthin [8]. From the standpoint of human health, carotenoids are among the bioactive products with significant medical value. For example, carotenoids have been found to lower the risks of diseases, including cancer, cardiovascular diseases, and age-related eye disorders, such as macular degeneration and cataracts [16].

**Figure 1.** Structure of representative carotenoids.

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Carotenoid-producing fungi are very diverse, including *Rhodotorula mucilaginosa* [17], *Blakeslea trispora, Phycomyces blakesleeanus, Mucor circinelloides, Fusarium sporotrichioides* [18], *Rhodosporium paludigenum*, and *Rhodotorula glutinis* [19]. Carotenoids protect fungi from oxidative stress and non-ionizing irradiation such as UV light. In addition, carotenoids are intermediates essential for synthesizing other biological molecules including bioactive apocarotenoids and related constituents. For instance, in many filamentous fungi, retinol, the precursor of vitamin A as well as an essential component of rhodopsins (membrane-bound photoreceptors), is produced via the oxidative cleavage of β-carotene [20]. In Mucorales, β-carotene is an intermediate during the synthesis of trisporoids, apocarotenoid derivatives that include the sexual hormones the trisporic acids. In addition, trisporoids have been proposed as substrates in the synthesis of sporopollenin, one of the most chemically inert biological polymers primarily found in the tough outer walls of plant spores and pollen grains [15]. In some fungi, such as the filamentous species *Blakeslea trispora* [21] and the yeast *Rhodotorula* spp. [22], the β-carotene biosynthetic pathway also produces lycopene, a carotenoid intermediate. Lycopene is a nutrient with antioxidant properties. It was originally identified in plants such as tomato and watermelon, giving their fruits the characteristic red and pink colours. Lycopene has been linked to several human health benefits ranging from improved cardiovascular health to protection against sunburns and certain types of cancers.

Aside from β-carotene and lycopene, another fungal carotenoid, astaxanthin, has attracted significant attention for its promise in the pharmaceutical, food and cosmetics industries. For example, astaxanthin has shown promise for treating Alzheimer's disease, Parkinson's disease, stroke, high cholesterol, age-related macular degeneration (age-related vision loss), and preventing cancer. *Xanthophyllomyces dendrorhous* (synonym *Pha*ffi*a rhodozyma*), a red- or orange-pigmented basidiomycetous yeast initially isolated from Japanese and Alaskan tree-exudates decades ago, is capable of producing astaxanthin [23,24]. Astaxanthin is synthesized via the mevalonate pathway starting with a condensation of two molecules of geranylgeranyl pyrophosphate, a four-step desaturation, cyclization, and a final 4-ketolation plus 3-hydroxylation [25,26]. In addition to astaxanthin, *X. dendrorhous* can also utilize the mevalonate pathway to produce several other types of carotenoids including β-carotene, canthaxanthin, and zeaxanthin. Canthaxanthin is used to reduce sensitivity to sunlight (photosensitivity) experienced by people who have a rare genetic disease called erythropoietic protoporphyria (EPP) [27]. In these people, sunlight can cause skin reactions such as rash, itch, and eczema. Similarly, zeaxanthin also protects human cells from the harmful effects of certain light sources such as the Sun. However, unlike canthaxanthin, that is preferentially deposited in the skin, zeaxanthin is an eye pigment that, once inside the body, accumulates in the eyes and helps to build a yellow-colored pigment shield to protect the eye cells. Indeed, the ability of *X. dendrorhous* to produce such a diversity of bioactive carotenoids makes it a potential model host to be engineered as a cell factory for the production of medically and industrially important carotenoids [28].

#### *2.2. Melanin*

Broadly speaking, melanins are heterogeneous polymers derived by the oxidation of phenols and subsequent polymerization of intermediate phenols and their resulting quinones. Fungal melanins are a subset of natural melanins and are polyphenolic and/or polyindolic compounds with high molecular masses and negative charges. Fungal melanins can appear dark green, brown, or black. In fungal cells, melanin serves as an antioxidant and a scavenger of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Commonly found in pathogenic fungi, melanin pigments are thought to provide those pathogens protection from UV and other radiation and aid in evasion from the host's immune system attacks [29]. Fungal melanins belong to three major types: 1,8-dihydroxynaphthalene (DHN) melanin, 3,4-dihydroxyphenylalanine (DOPA)-melanin (also called eumelanin), and pyomelanin [30,31]. DHN melanin is produced from acetyl-coenzyme A via the polyketide synthase pathway. The fungus *A. fumigatus* is able to synthesize DHN melanin, which contributes to the gray-green appearance of its conidia. The deletion of its polyketide synthase PksP in *A. fumigatus* gave rise to colorless spores

with weakened virulence [32]. Eumelanin is converted from *o*-diphenolic or *p*-diphenolic substrates by a polyphenol oxidase (laccase). In *Candida albicans*, eumelanin particles are observed in vitro and in infected mammalian tissues, such as murine kidney and human skin [33]. In this ascomycete fungus, melanin is externalized in the form of electron-dense melanosomes that are extracellularly secreted or loosely attached to the cell wall surface through binding with chitins [34]. Although *C. albicans* has laccase activity [33], the association of melanin with pathogenicity remains to be clarified for this fungus. Pyomelanin is an extracellular water-soluble pigment, in contrast to both DHN- and DOPA-melanins that are cell wall-immobilized and insoluble in water [31]. Pyomelanin is produced by the polymerization of homogentisic acid degraded from L-tyrosine/L-phenylalanine. In *A. fumigatus*, pyomelanin plays an important role in the germination of conidia and in the defensive responses toward external oxidative stresses [31].

Apart from the above-stated three major types of fungal melanins, there is another pathway of melanin biosynthesis in mushrooms such as the common cultivated mushroom *Agaricus bisporus* and others [35]. In these mushrooms, melanin is formed from a benzoquinone. Benzoquinone is converted from the precursor γ-glutaminyl-4-hydroxybenzene (GHB) by tyrosinase. GHB is the main phenolic compound in *A. bisporus* fruiting bodies and spores [36].

In addition to its involvement as a virulence factor in fungal pathogens, melanin enables fungi to survive and thrive in adverse and even extreme environments, providing protection against desiccation, non-ionizing (i.e., UV light) and ionizing radiation, as well as oxidative and nitrosative stresses [37,38]. Over the past decade, melanin pigments and their subunits have kindled great interest as soft biocompatible functional materials with antioxidant characteristics for engineering high-performance, low-impact biocompatible optoelectronic devices, such as light emitting diodes, memory devices, etc. [39]. Intriguingly, halophilic fungal isolates of two species, *Trimmatostroma salinum* and *Phaeotheca triangularis,* from the eastern coast of the Adriatic Sea, have been found to produce melanin in sodium chloride solutions at saturated concentrations [40]. Similarly, high-dose radiation resistant melanin was found in an Antarctic desert-dwelling (or desert-inhabiting) fungus, *Cryomyces antarcticus*. These fungal extremophiles could serve as promising sources of melanin with excellent physicochemical properties for a variety of industrial and medical applications.

#### *2.3. Polyketides*

Many fungal pigments are polyketide-based compounds. This group of fungal pigments is abundantly produced by most ascomycetous fungi, including the filamentous ascomycete genera *Neurospora* and *Monascus* [41].

Fungal polyketide pigments are made of tetraketides and octoketides which have eight C<sup>2</sup> units giving rise to the polyketide chain. Anthraquinones (including hydroxyanthraquinones), and naphthoquinones are representative classes which display an arsenal of various colors [42,43]. The basic structure of the anthraquinone class of pigments is a polycyclic aromatic hydrocarbon which is derived from anthracene or phthalic anhydride, i.e., the merger of three benzene rings. Anthraquinone itself is highly insoluble and is generally used in the manufacture of dyes for the textile and pulp industries [44,45]. However, other members of the anthraquinone class have been used in the pharmaceuticals industry.

Approximately 700 representatives of anthraquinones have been found in fungi, plants, and lichens, conferring a yellow, orange, or brown color to the mycelium of microscopic fungi, the fruiting bodies of macroscopic fungi (mushroom), as well as lichens [46]. The variety of anthraquinones results from the presence of different substituents, like -OH, -CH3, -OCH3, -CH2OH, etc. as well as the reduction of carbonyl groups or double bonds in the benzene ring. Anthraquinones produce a yellow color, whereas the substituents produce various hues of the molecules ranging through yellow, orange, red, brown, and violet [47]. The most widely distributed variants found among fungi are 1, 8-dihydroxy and 1, 5, 8- or 1, 6, 8- trihydroxy anthraquinone derivatives [48]. However, substituents at other positions of the ring have been recently identified. For example, chrysophanol is a trihydroxyanthraquinone with a

methyl substituent at C-3. It has shown antiviral and anti-inflammatory activities. Both chrysophanol and helminthosporin have shown inhibitory activity against cholinesterase with helminthosporin showing permeability across the blood-brain barrier [49]. Cholinesterase is a key enzyme involved in the development of Alzheimer's disease. Most anthraquinone-producing fungi produce a mixture of pigments of the quinone class. *Aspergillus crisfafus*, for instance, has been found to synthesize as many as 15 pigments related to anthraquinone. A strain of *Curvularia lunata* has been characterized that produces a mixture of three anthraquinone derivatives: chrysophanol, helminthosporin, and cynodontin, with cynodontin (1, 4, 5, 8-tetrahydroxy-3-methylanthraquinone) comprising more than 70% of the mixture and showing potential as a pharmaceutical with antifungal activity [44].

Chrysophanol, helminthosporin, and cynodontin are all hydroxyanthraquinones (HAQNs), derivatives of anthraquinone via the substitution of hydrogen atoms by hydroxyl groups plus the addition of other functional groups. Anthraquinones including HAQNs have been found to be produced by such fungi as *Aspergillus* spp., *Eurotium* spp., *Fusarium* spp., *Dreschlera* spp., *Penicillium* spp., *Emericella purpurea*, *Culvularia lunata*, *Mycosphaerella rubella*, *Microsporum* spp., etc. [48].

Filamentous fungi, such as *Penicillium* and *Aspergillus*, synthesize HAQNs through the polyketide pathway. This process has been employed for the production of natural food-grade colorants [50]. Apart from their usage as colorants in the food and cosmetics industries, they have been found to possess antiviral activities. For instance, emodin has been extracted from *Penicillium citrinum, P. islandicum*, *Aspergillus glaucus* and *A. variecolor* [51,52] and aloe-emodin from *Penicillium oxalicum* [53]. Emodin and its congener aloe-emodin are known antiviral agents [54]. Aspergilol H and I, HAQNs produced by *Aspergillus versicolor*, have been reported to have antiviral effects against HSV-1 [55]. Notably, HAQNs are able to absorb visible light and display colour, with the colours varying based on the position and number of the hydroxyl substituents in the different rings [56]. HAQNs are relatively stable and they exhibit superior brightness in comparison to azo pigments.

Naphthoquinone is a class of organic compounds structurally related to naphthalene. Naphthoquinones have very significant pharmacological activities against bacterial, fungal, viral, and insect pathogens and pests. In addition, they have anti-inflammatory, and antipyretic properties. *Fusarium* spp. have shown capable of producing a diversity of naphthoquinones with a broad spectrum of biological activities [57,58]. For instance, a compound produced by *Fusarium fujikuroi* and other fungi, bikaverin, a red heterotetracyclic pigment with the chemical structure of 6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione, has shown anti-neoplastic activity, suggesting its potential as a pharmaceutical agent against lymphoma, carcinoma, and sarcoma, etc. [59–62].

#### *2.4. Azaphilones*

Azaphilones are a structurally variable group of fungal secondary metabolites (polyketide derivatives) [63], namely pigments with a highly oxygenated pyrone-quinone bicyclic core usually known as isochromene and a chiral quaternary centre [64,65]. Both a pyrone-quinone backbone and a chiral quaternary centre (Figure 2) are essential for compounds to be classified as azaphilones [66]. Azaphilone is so designated because of its ready insertion of nitrogen. For example, ammonia readily converts the pyran oxygen of monascorubrin into the violet-colored nitrogen analog monascorubramine (Figure 3) [67].

The azaphilone pigments are produced by a plethora of the ascomycetous and basidiomycetous fungi, particularly the former [68], including the genera *Aspergillus*, *Penicillium*, *Chaetomium*, *Talaromyces*, *Pestalotiopsis*, *Phomopsis*, *Emericella*, and *Epicoccum,* as well as *Monascus* and *Hypoxylon*, where they give rise to the yellow, red, or green hues of fruiting bodies and/or mycelia [69].

Fungal azaphilones, structurally bearing the isochromane-like ring, possess a broad-spectrum of biological activitiesincludingmonoamine oxidaseinhibition, phospholipase A2inhibition, tumor promotion suppression, gpl20-CD4 binding inhibition, and acyl-CoA: cholesterolacyltransferase (AGAT) inhibition. However, the relationships between the structures of azaphilones, their spectrum of activities, and the corresponding mechanistic modes of action remain elusive [66].

**Figure 2.** Azaphilonoid scaffold.

**Figure 3.** Azaphilone pigments derived from the yeast *Monascus*.

Monascin and ankaflavin (Figure 4B) are two yellow pigments of the azaphilonoid group. The fungal genus *Monascus*-derived monascin and ankaflavin have been found to potently inhibit preadipocyte differentiation, and promote lipolysis of mature adipocytes [70]. Furthermore, these two pigments are able to reduce blood lipids as well as lower the synthesis and accumulation of triglycerides (TG) [71]. For example, Lee et al. (2013) investigated the effects of eight-week administrations of monascin and ankaflavin on obesity factors using obese rats fed with a high-fat diet [72]. Their work illustrated the involvement of these two *Monascus* azaphilones in the modulation of preadipocyte differentiation, lipogenesis, and lipid absorption, highlighting their potential in the development of hypolipidemic drugs.

Monacolin K (viz. lovastatin) is a statin compound and previously thought to be a hypolipidemic component of *Monascus*-fermented products. While earlier studies suggested anti-obesity effects of monacolin K (Figure 4A), recent investigations indicated that monascin and ankaflavin (Figure 4B), rather than monacolin K, were likely the major players in the *Monascus*-mediated amelioration of diabetes and fatty liver [73].

**Figure 4.** Chemical structure of monacolin K, monascin and ankaflavin. (**A**) Monacolin K; (**B**) Monascin and ankaflavin.

Indeed, there is mounting evidence that a long-term high-dosage administration of monacolin K might lead to numerous side effects such as rhabdomyolysis and reduction in the level of coenzyme Q<sup>10</sup> (CoQ10). Furthermore, although monacolin K reduces the serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels, it also substantially reduces high-density lipoprotein cholesterol (HDL-C) levels. On the other hand, Lee et al. (2010) showed that monascin and ankaflavin could increase HDL-C levels significantly while imposing no damage on the liver or kidneys [68]. In addition, monascin has been shown to protect the liver from chemical damage via its anti-inflammatory effect [74]. Other work with monascin has indicated that it significantly inhibits peroxynitrite (ONOO−; PN), and ultraviolet light B (UVB)-induced skin carcinogenesis [75].

− Together, the above results suggest that monascin and ankaflavin possess hypolipidemic, anti-atherosclerosis, antioxidative, and anti-inflammatory activities, thereby rendering them promising compounds that may be of value in the prevention and treatment of cardiovascular diseases [73].

Apart from *Monascus* species, the production of azaphilone pigments by other fungal species has also been documented [76]. For instance, chaetoviridins, a class of azaphilones synthesized by *Chaetomium globosum*, have been found to have strong antifungal activities. To date, four chaetoviridins, chaetoviridin A—D, have been characterized. Studies with mice have demonstrated that chaetoviridin A is able to suppress tumor progression via 12-O-tetradecanoylphorbal-13-acetate in two-phase carcinogenesis [77]. Independent work by Tomoda et al. revealed that chaetoviridin A and B might exert inhibitory effects on cholesteryl ester transfer protein [78].

#### *2.5. Other Fungal Pigments*

The pigment sclerotiorin has been isolated from cultures of *Penicillum sclerotiorum* 2AV2, which was recovered from Amazonian soil [6]. Sclerotiorin (Figure 5) has been found to have a variety of biological activities. Some of the biological activities most pertinent to human health include inhibitory effects on aldose reductase [79], endothelin receptor binding activity [80], antimicrobial effects including antifungal activity [17,81], apoptosis-triggering actions towards HCT-116 cancer cells [82], and the inhibition of integrase and protease of HIV-1 [83].

Studies on *Penicillum chrysogenum* have identified a polyketide synthase (PKS) gene required for the biosynthesis of sorbicillinoid, a yellow pigment [84]. The authors demonstrated that a highly reducing PKS enzyme, encoded by Pc21g05080 (*pks13*), is indispensable for the production of polyketide precursors such as sorbicillinol and dihydrosorbicillinol as well as their derivatives such as bisorbicillinoids. PKSs are also involved in the biosynthesis of sorbicillinoid in *Acremonium chrysogenum*, the fungal species used in the industrial production of cephalosporin C, an antibiotic in current clinical use [85].

etoviridin A―D, have been characterized. Studies with mice have demonstrated

**Figure 5.** Chemical structure of the pigment sclerotiorin produced by *Penicillum sclerotiorum* 2AV2.

#### **3. Biosynthesis of Fungal Pigments**

So far, four major pathways have been identified as responsible for the biosynthesis of the main fungal pigments, including the polyketide synthetic pathway, the shikimate pathway, the terpenoid synthetic pathway, and the nitrogen-containing metabolite pathway.

#### *3.1. Polyketide Synthetic Pathways*

The polyketide synthetic pathway is involved in producing many fungal pigments with human health relevance. For example, there is increasing evidence that fungal pigments such as melanin, quinones, flavins, ankaflavin, and azaphilones, all relevant to human health [5,6], involve the polyketide synthesis pathway [86,87]. This pathway has been studied in diverse fungal species, including those in the genera *Monascus, Fusarium*, *Alternaria*, and *Epicoccum* [64]. The core polyketide synthetic pathway is shown in Figure 6. Fungal pigments are produced by this pathway via repetitive Claisen condensations of an acyl-coenzyme A (CoA) starting unit with malonyl-CoA elongation units in a manner analogous to fatty acid biosynthesis. The polyketide pathway generates either aromatic ketides or fatty acids. The extending chain of the aromatic ketides is stabilized by cyclization reactions and partial reduction. However, in the fatty acids, the carbonyl groups of the chain are reduced prior to the addition of the next C<sup>2</sup> group. One of the major differences between the two metabolic routes is that polyketides have varied degrees of β-keto processing. Specifically, some are not or only partially reduced, giving rise to the formation of (poly-)cyclic aromatic compounds. On the other hand, they may be significantly reduced, yielding linear or macrocyclic, non-aromatic carbon skeletons.

**Figure 6.** Mechanisms of fungal polyketide biosynthesis.

The synthesized fungal polyketides can vary in their chain length, the degree of β

carrier protein (ACP) domains. Further, optional β

architecture and the presence or absence of additional β

Aromatic ketides of this pathway include tetra-, hepta-, octa- and higher number ketides. Fungi possess a variety of pigments of the octaketide origin based on the anthra-9,10-quinone backbone with both rings being substituted. Anthraquinones are representatives of this type of pigment. In many cases anthraquinones are found in fungi in the corresponding colourless reductive states (anthranol, anthrone, anthrahydroquinone, and oxanthrone derivatives) that may be present in the forms of diverse glycoside conjugants. However, many naturally occurring, coloured anthraquinones are oligomers made by the coupling of two or more anthraquinone molecules. These oligomers differ in the number and positions from which monomers and amino acids are ligated to generate a diversity of fungal pigments [88,89].

A large number of enzymes have been identified in the fungal polyketide synthesis pathway. These include a core set of enzymes, including ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains. Further, optional β-keto processing steps may be catalyzed by keto reductase (KR), dehydratase (DH) and enoyl reductase (ER) domains. Other optional ancillary domains involve cyclase (CYC) [90] and methyl transferase (MT) activities [91]. Based on their architecture and the presence or absence of additional β-keto processing domains, fungal PKSs are categorized into non-reducing or aromatic (NR-PKS), partially reducing (PR-PKS), and highly reducing PKS (HR-PKS).

The synthesized fungal polyketides can vary in their chain length, the degree of β-keto processing, and cyclization. Moreover, the tremendous structural diversity of polyketides is further obtained from derivatization of the polyketide carbon backbone by alkylation, acylation, and oxygenation, and by post-PKS modification or tailoring. Genome sequence analyses so far have revealed that all genes essential for fungal polyketide biosynthesis are clustered, including the PKS genes, genes encoding enzymes associated with tailoring, as well as regulatory genes [92].

While most fungal polyketide pigments are synthesized as aromatic ketides, the biosynthesis of azaphilones uses both the polyketide pathway and the fatty acid synthesis pathway. The polyketide pathway assembles the main polyketide chain of the azaphilone pigments from acetic acid (the starter unit) and five malonic acid molecules (the chain extender units) in a conventional way to generate the chromophore structure. The fatty acid synthesis pathway produces a medium-chain fatty acid (octanoic or hexanoic acid) that is then bound to the chromophore by a transesterification reaction [93,94].

#### *3.2. Shikimate Pathways*

Aromatic amino acids act as the precursor of various fungal secondary metabolites such as pigments and vitamins [95]. In the fungal phylum *Basidiomycota*, tyrosine is the precursor of a distinct class of pigments, the betalains, solely present in the genera of *Amanita* and *Hygrocybe* [96,97]. In the phylum *Ascomycota*, tyrosine-derived pigments, such as melanin and tyrosine betaine, are thought to contribute to stress tolerance (e.g., temperature, radiation) [98,99] and pathogenicity [100,101].

The shikimate pathway is present in the prokaryotes, microbial eukaryotes and plants studied. However, it is absent in those animal species investigated [102]. This pathway links carbohydrate metabolism to the biosynthesis of aromatic compounds. Metabolically, the shikimate pathway is a seven-step route utilized by fungi for the biosynthesis of aromatic amino acids like phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), as well as para-aminobenzoic acid, via the central intermediates shikimic and chorismic acids [86]. The first step involves the condensation of the glycolytic intermediate phosphoenol pyruvate and pentose phosphate pathway intermediate erythrose-4-phosphate to yield a seven-carbon heterocyclic compound, 3-deoxy-D-arabinose-heptulosonate-7-phosphate derivative (DAHP). The second step involves the generation of a highly substituted cyclohexane derivative, 3-dehydroquinate, by the replacement of the exocyclic C<sup>7</sup> of DAHP by the ring oxygen. The remaining five steps involve the introduction of a side chain and two of the three double bonds that convert this cyclohexane into a benzene ring, the core of aromatic amino acids [5]. The metabolic routes may vary for different classes of pigments.

The pigments produced via the shikimate pathway are generally water-soluble phenolic compounds, including terphenyls and pulvinic acids. Studies of *p*-terphenyls as a family of the mushroom pigments were initiated in 1877. Isolation of polyporic acid, atromentin and thelephoric acid represented the inception of the chemical investigation of fungal pigments. The elucidation of the structures of polyporic acid and atromentin by Kögl is recognized as a milestone in organic chemistry [103]. It has been demonstrated that some terphenyls display biological activities, such as immunosuppressive, antithrombotic, anticoagulant, neuroprotective, 5-lipoxygenase inhibitory (for the treatment of inflammatory bowel disease), and cytotoxic activities [104].

Many fungi have evolved pathways utilizing the aromatic products of shikimate metabolism (Figure 7). The initial steps in the biogenesis of *p*-terphenyls are the well-known reactions of primary metabolism flowing from shikimate to chorismic acids and subsequently to arylpyruvic acids. Experiments involving the feeding of <sup>13</sup>C- and <sup>14</sup>C-labeled precursors to fungal cultures revealed that *p*-terphenyls are assembled by initial condensation between two molecules of unbranched phenylpropanoid precursors, either phenylpyruvic acid or phenylalanine. Previous studies also revealed the involvement of 4-hydroxyphenylpyruvic acids or tyrosine in the initial condensation during the biosynthesis of terphenylquinones such as atromentin [105]. Atromentin is known as a key intermediate for further conversions, for instance, to more highly hydroxylated terphenylquinones and pulvinic acids [106,107].

**Figure 7.** Shikimate pathway leading to biosynthesis of *p*-terphenyls.

group (with β and ε rings being the most frequently introduced) into at least one of the ends of the

#### *3.3. Terpenoid Synthetic Pathways*

Biochemically, the carotenoids are terpenoids [15]. Accordingly, the synthesis of carotenoids utilizes the terpenoid synthetic pathways deriving from the condensation of C<sup>5</sup> isoprene units [108]. The common terpenoid precursor is isopentenyl pyrophosphate (IPP), which is synthesized either via the mevalonate pathway (generated from hydroxymethylglutaryl coenzyme A, HMG-CoA), or via the non-mevalonate pathway (generated from the condensation of pyruvate and glyceraldehyde 3-phosphate [109]. So far, available research has demonstrated that fungal IPP is produced via the mevalonate pathway, whereas in bacteria and photosynthetic species IPP is produced via the non-mevalonate pathway. The early biosynthetic steps involve the sequential additions of IPP (isoprene, C5) units to yield geranyl pyrophosphate (GPP, C10), farnesyl pyrophosphate (FPP, C15), and geranylgeranyl pyrophosphate (GGPP, C20) [108,109]. The initial compound possessing the typical aliphatic carotenoid-like structure is 15-cis-phytoene (a colorless molecule), consisting of a symmetrical polyene chain generated via the condensation of two GGPP units catalyzed by phytoene synthase (Figure 8). The light-absorbing characteristics of carotenoids are attributed to the presence of a chromophore, which is comprised of an array of conjugated double bonds via the catalysis of desaturases. Different subsequent chemical modifications, usually the introduction of a cyclic end group (with β and ε rings being the most frequently introduced) into at least one of the ends of the molecule, and/or oxidative reactions (for instance, hydroxylation, carboxylation, epoxidation, esterification, etc.), may contribute to the huge arsenal of carotenoids [108].

Simplified scheme of the biosynthetic pathways for β **Figure 8.** Simplified scheme of the biosynthetic pathways for β-carotene (black arrows) and the astaxanthin and neurosporaxanthin (blue arrows) from GPP.

#### *3.4. Nitrogen-Containing Metabolite Pathways*

– –N moiety was derived from an α

Chalciporone (Figure 9) is a type of 2H-azepine alkaloid pigment produced by the mushroom *Chalciporus piperatus* (Basidiomycetes). This pigment is believed to act as a deterrent to insects and other predators and thereby can potentially protect the mushroom. The biosynthesis of chalciporone was illustrated in 2001 [110]. In their experimentations, the radioactive labelled sodium [U-<sup>13</sup>C2] acetate was

not incorporated into chalciporone by *C. piperatus*. In contrast, the supplementation of a mixture of [U-<sup>13</sup>C]-labelled fats led to chalciporone, in which seven intact acetate units were incorporated (*ca.* 10 atom % enrichment) between C<sup>3</sup> and C16. However, the enrichment of the C<sup>1</sup> methyl group and the adjacent carbon (C2) was not observed during this experiment, suggesting that the CH3–CH–N moiety was derived from an α-amino acid. This was confirmed by the observation that administration of a mixture of [U-<sup>13</sup>C]-labelled amino acids to *C. piperatus* gave rise to the enrichment of both C<sup>1</sup> and C<sup>2</sup> and of all the other carbon signals in the spectrum of chalciporone [107]. The work clearly demonstrated that the carbon backbone of chalciporone is generated from an amino acid plus seven acetate (=malonate) units. – –N moiety was derived from an α

Simplified scheme of the biosynthetic pathways for β

**Figure 9.** Chalciporone.

The wood-rotting fungus *Pycnoporus cinnabarinus* (Basidiomycetes) can produce a red pigment, cinnabarinic acid (Figure 10), via oxidative dimerization of the precursor 3-hydroxyanthranilic acid in sporocarps as well as in culture broth [111]. This reaction is catalyzed by laccase and necessary for the production of antibacterial compounds by the fungus. Cinnabarinic acid shows inhibitory effects towards several Gram-positive bacteria of the *Streptococcus* genus. It is known that cinnabarinic acid shares structural homology with the antibiotic group of actinomycins (e.g., actinomycin D) produced by *Streptomyces* spp., having two cyclic pentapeptides linked to the phenoxazinone chromophore.

**Figure 10.** Cinnabarinic acid.

3.9 μg per ml, respectively [109]. Two indole pigments were isolated as free radical scavengers from the fruiting bodies of the mushroom *Agrocybe cylindracea* [112]. Based on spectroscopic data, they were identified as 6-hydroxy-1*H*-indole-3-carboxaldehyde and 6-hydroxy-1*H*-indole-3-acetamide (Figure 11). 6-hydroxy-1*H*-indole-3-acetamide is an amide derivative of 6-hydroxyindole-3-acetic acid. A previous study identified that 6-hydroxyindole-3-acetic acid could be produced through the transformation of indole-3-acetic acid by *Aspergillus niger* [113]. These two indolic compounds were found to possess potent inhibitory activity on lipid peroxidation in rat liver microsomes, with IC<sup>50</sup> values of 4.1 and 3.9 µg per ml, respectively [109].

—β

3.9 μg per ml, respectively [109].

**Figure 11.** The indolic compounds 6-hydroxy-1H-indole-3-carboxaldehyde (**left**) and 6-hydroxy-1H-indole-3-acetamide (**right**) isolated from the fruiting bodies of *Agrocybe cylindracea* (middle, insert image).

#### **4. Interaction of Fungal Pigments with the Host Immune System**

—β One of the most interesting defense mechanisms of fish may involve non-specific immune responses that have evolved due to their long-term associations with fungi [114]. Specifically, the prevalent colonization of *Rhodotorula mucilaginosa* (a red yeast) in the fish gastrointestinal (GI) tract might have contributed to the stimulation of non-specific mechanisms of host immunoprotection as well as the maturation of the host GI tract [115]. Spectrophotometric analysis in wild fish (*Abramis brama*, *Rutilus rutilus*, *Perca fluviatilis*) indicated the presence of a set of three pigments—β-carotene, torularhodin, and torulene, which are produced by the commensal *Rh. mucilaginosa*. In addition to acting as scavengers of free radicals, these fungus-derived pigments are hypothesized to play a role in the immunostimulation of the host via enhanced activation of T lymphocytes, supporting the action of macrophages [116] or antimicrobial activity [115].

For decades, an accumulating body of evidence has revealed that carotenoids or their derivatives are involved in the activation of thymocites [117], the expression of immune-associated genes [118], and the increase in membrane fluidity [119], which represent vitally important functions in mounting immune responses in animals. Notably, the immunostimulatory effects of carotenoids are thought to be distinct from their antioxidant property [116]. Earlier investigations indicated that β-carotene and the non-provitamin A carotenoids are able to enhance cell-mediated and humoral immune responses in mammals [116]. It is not surprising that β-carotene-producing red yeast *Rh. mucilaginosa* might be beneficial to its hosts, in this case the wild fish, presumably through immuno-stimulation.

Given that (i) immunostimulatory effects of carotenoids in animals are thought to be independent of their antioxidant property [116], and (ii) innate immunity relies on effectors which produce cytotoxic molecules that may not only kill pathogens, but also harm host tissues, the mechanisms underlying the roles of carotenoid pigments in the well-being of animals need to be elucidated. Although the functionality of dietary antioxidants in invertebrate immunity is not fully understood, it has been found in vertebrates that carotenoids can scavenge cytotoxic radicals formed during the immune response. Carotenoids may consequently decrease the self-harming cost of immunity. A positive correlation between the effectiveness of the innate immune defense and the levels of circulating carotenoid might therefore be expected. In accordance with this hypothesis, a study using the amphipod species *Gammarus pulex* showed that the maintenance and use of the prophenoloxidase system were highly correlated with the carotenoid level in the haemolymph within the natural populations of this crustacean [120].

The enhanced immunity by carotenoids has also been demonstrated in mammals [121] where mice administered *Rhodotorula glutinis-*derived carotenoid could survive for 2 weeks post lethal-dose challenges of pathogenic *Pseudomonas aeruginosa* and *C. albicans.* This result points to the potential use of fungal carotenoids as food additives or dietary supplements to boost the human immune response against microbial infections.

Like carotenoids, melanins are ubiquitously produced by fungi, particularly pathogenic fungi such as *Cryptococcus neoformans*, *Aspergillus fumigatus*, and *Candida albicans*. As described previously, melanins in fungal pathogens contribute to fungal pathogenesis towards mammalian cells [10]. The melanin pigments contribute to antigen masking to circumvent recognition by host immune system and to the survival of fungal pathogens against phagocytosis [122]. For example, phagocytosed *C. neoformans* produces melanin to protect against the oxidative environment inside the phagolysosome [122]. For *A. fumigatus*, it is believed that melanins protect the conidia against reactive oxygen species, mask the recognition of various *A. fumigatus* pathogen-associated molecular patterns (PAMPs) by the host, inhibit macrophage apoptosis and phagolysosome fusion, and attenuate the host immune response. The protective effect of melanin might also be due to its role in modifying the surface properties of conidia and vegetative cells by altering the charge and hydrophobicity, thereby reducing the effectiveness of the immunological functions of the host [123]. For example, in contrast to the wild type conidia, melanin-deficient mutant conidia can activate human DCs (dendritic cells) and subsequent cytokine production, leading to their elimination by the host immune system.

The protective effects of melanin for fungal pathogens have also been demonstrated for autophagy. Autophagy plays an important role in host immunity to microbial pathogens. The autophagy system targets pathogens in phagosomes, promotes phagosome maturation and prevents pathogen escape into the cytosol. Autophagy protein LC3 is believed to be a key player during phagocytosis [124]. During *Aspergillus* germination, exposure of PAMPs enables the activation of host LC3-associated phagocytosis (LAP), which promotes the killing and eradication of fungi. Shielding PAMPs from detection by pattern recognition receptors is a predominant strategy adopted by fungi for evasion from host immunity. Recent studies with *A. fumigatus* have demonstrated that LAP activation also requires the removal of fungal cell wall melanin, based on the genetic, biochemical or biological (germination) data [125]. Fungal melanin is known to inhibit the NADPH oxidase-dependent activation of LAP via excluding the p22phox subunit from the phagosome. Therefore, LAP blockade is a general trait of melanin pigments [126]. Physiologically, melanin-triggered LAP blockade is able to enhance fungal virulence.

#### **5. Medical Relevance of Fungal Pigments**

In extreme environments, fungi having pigmented cell walls in their spores and mycelia are able to tolerate dehydration–hydration cycles, thermal fluctuations, toxic metal-contamination, and high ultraviolet light (UV) radiation better than the moniliaceous fungi, which are unpigmented [127]. A remarkable example is the black fungus *Cryomyces antarcticus*, an extremophile initially recovered from Antarctic deserts and later found to be able to survive in the outer part of the International Space Station [38]. The wide geographical distribution of pigmented fungal species and their extraordinary ability to survive in hostile environments suggest that fungal pigments may be of value in a variety of fields, including radioprotection and biomedical applications.

In addition to their broad distribution and their ability to synthesize secondary metabolites such as medically significant pigments, many fungi are readily grown in lab conditions. The prospect of large-scale production attracts interest from both the pharmaceutical industry and fundamental science. The scope of focus includes species from a wide range of environments of marine origin, soil, as well as endophytic fungi from terrestrial and marine flora and endolichenic origin [127,128].

#### *5.1. Medical Roles of Melanins*

Recent studies with fungi capable of producing melanin (also called melanotic fungi) have revealed a wide spectrum of functions for this kind of pigment, ranging from drought and radiation resistance to increased virulence in fungal pathogens. For example, melanotic fungi are present in diverse environments including but not limited to radiation-contaminated soils (e.g., the Chernobyl nuclear power station and the surrounding soils), Antarctic deserts, ancient cave paintings, and spacecraft, etc. In fact, melanized fungi, including spores of *Aspergillus niger*, *Cladosporium herbarum*, *Ulocladium chartatum*, *Basipetospora halophile*, etc., have been reported to be able to grow on the Mir Space Station and International Space Station. Some edible mushrooms (Basidiomycetes) are rich in

melanins as well. Melanins contain persistent free radical centers, as detected by electron paramagnetic resonance (EPR) spectroscopy. These free radical centers allow melanins acting as antioxidants to scavenge free radicals and to protect the fungi from oxidative stress and damage by ionizing radiation in both atmospheric and terrestrial environments [129]. In a recent study [130], Pacelli et al. compared the effects of densely-ionizing deuterons and sparsely ionizing X-rays on two melanized fungal species, namely the fast-growing pathogenic basiodiomycete *Cryptococcus neoforman*s and the slow-growing Antarctic rock-inhabiting *Cryomyces antarcticus* to their non-melanized counterparts. *C. antarcticus* showed more resistance to deuterons than *C. neoformans*, and similar resistance to X-rays was observed for both species. Melanin offered protection against high-dose (1.5 kGy) deuterons for both *C. neoformans* and *C. antarcticus* (*p* < 10−<sup>4</sup> ). In addition, melanin protected *C. antarcticus* (*p* < 10−<sup>4</sup> ) and probably *C. neoformans* against X-rays (0.3 kGy). The use of both XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)25-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide) and MTT (2-(4,5-dimethyl-2-thiazolyl)23,5-diphenyl-2H-tetrazolium bromide) assays in parallel helped define the location of the melanin-mediated electron transfer in the cells. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained unchanged or decreased. The opposite was observed with levels of ATP (the indicator of the metabolic activity of cells): upon exposure to deuterons, ATP decreased in melanized strains, but not in non-melanized ones. Larger and more distinct differences in both XTT and ATP were found in *C. neoformans* than in *C. antarcticus*. Further research by Pacelli et al. revealed that *C. antarcticus* could produce both 1,8-dihydroxynaphthalene (DHN) and L-3,4- dihydroxyphenylalanine (L-DOPA) melanins, which likely contribute to its ability to thrive under diverse stress conditions [131]. Together, these results indicate for the first time that melanin could protect both fast- and slow-growing fungi from high-dose radiation (deuterons in this case) under physiological conditions [130]. Indeed, the remarkable ability of melanin to confer resistance to radiation led to further research that revealed that melanized fungi could harvest radiation energy for their growth and reproduction. Broadly speaking, such an ability has both fundamental ecological significance and practical implications. Ecologically, the ability of melanotic fungi to make use of electromagnetic radiation for their physiological processes helps us understand energy flows in the biosphere. Practically, the understanding paves the way for potentially developing melanin-based radiation resistant materials and equipment. In astrobiology and space travel, melanin represents a new type of material with which to experiment for space travel [132].

The unique stress-resistance properties of *C. antarcticus*, a heavily melanized black fungus in the class Dothideomycetes of the Ascomycota phyla, were further demonstrated by its ability to survive not only the Antarctic desert but also the harsh outer space environment and cosmic radiation exposure [38,130]. Indeed, the mechanisms for how fungal melanin molecules confer resistance to extreme heat/cold stresses and high-dosage radiations have gained wide attention from scientists [133,134]. One of the protection mechanisms is the chelating properties of melanins towards biologically damaging free radicals generated by the physical and chemical stresses. These results are inspiring biomedical engineering in healthcare and in radiation- and thermal-protective applications [135].

Aside from melanized microscopic fungi such as yeasts and molds, melanized macroscopic mushrooms are also attracting increasing attentions. One such mushroom is the medicinal mushroom *Inonotus obliquus* (also called Chaga), a basidiomycete. This mushroom grows mainly on birch trees and is broadly distributed across the northern hemisphere in Europe, Asia, and North America. Long used in folk medicine, Chaga contains massive amounts of melanin pigments. Several research groups have reported the therapeutic potential of Chaga's bioactive pigments in countering the proliferation of cancer cells, and diabetes mellitus [136,137]. The pigments extracted from *I. obliquus* contain both phenolic compounds such as melanins, and triterpenoids such as inotodiol. Interestingly, the water-soluble component of melanin complexes of *I. obliquus* possesses insulin-sensitizing and hypoglycemic activity [138], consistent with their anti-diabetic functions. In addition, aqueous extracts

of *I. obliquus* lowered the viability of human hepatoma (HepG2) cell lines in a dose-dependent fashion in vitro [139]. Subsequent studies revealed that the intraperitoneal administration of aqueous extracts of *I. obliquus* could inhibit tumor growth in vivo in mice implanted with melanoma B16-F10 cells at a dose of 20 mg/kg/d for 10 d [140]. Similarly, another study of melanin from *I. obliquus* showed its suppressive effect on the proliferation of HeLa 229 tumor cells [141]. Together with the above beneficial effects, the discovery of water-soluble melanin from *I. obliquus* in protecting DNA from carcinogenic damage [142] is also accelerating the preclinical studies of *I. obliquus* pigments. Results from such studies could generate significant medical and economic benefits in an environmentally friendly way (green chemistry).

As shown above, melanins have many potential benefits to humans. However, their presence in pathogenic fungi can also cause detrimental effects to humans both directly as human fungal pathogens or indirectly as plant and animal fungal pathogens. For example, in the human fungal pathogens *C. neoformans* [143], *Aspergillus fumigatus* [144], *Paracoccidioides brasiliensis* [145], *Penicillium marne*ff*ei* [146], *Fonsecaea pedrosoi* [147], and *Sporothrix schenckii* [148], melanin is an important virulence factor and contributes to drug resistance. Recently identified as a part of host innate immunity, platelets could be activated by exposure to *Aspergillus fumigatus*. In *A. fumigatus*, a mutant lacking melanin exhibited decreased platelet-stimulating activity, and the platelet activating effect can be mimicked by "melanin ghosts" [149], large isolated melanin molecules. Interestingly, melanins in *Fonsecaea monophora,* a common causal agent of chromoblastomycosis, were able to reduce Th1 cytokines and to elevate Th2 cytokines secreted by macrophages [150], likely by regulating the MAPK signalling pathway of macrophages [151], pointing out that melanins interacting with the macrophages might contribute to the immune evasion of fungal pathogen. In these species, melanin protects the invading fungal pathogen against oxidative stress within phagocytes, interferes with host cell signaling and autophagy, and masks recognition of fungal cells by the host immune system. Our understanding should benefit the development of therapeutic targets for treating mycosis in humans.

#### *5.2. Medical Roles of Other Pigments*

#### 5.2.1. Anti-Tumor Activities

Carcinogenesis is a prolonged and multi-phase process that involves initiation, promotion, progression, and metastasis as an outcome of an unbalanced cell proliferation and cell apoptosis /death [152]. Anti-proliferative agents are often associated with anti-mutagenic and hence anti-tumor activities.

The majority of the anticancer chemotherapeutics prescribed nowadays, such as alkylating agents, antibiotics, compounds targeting the cell cycle (microtubules, G1/S/G/M checkpoint proteins), and topoisomerase inhibitors, are cytotoxic compounds. These compounds are developed to kill carcinoma cells/tissues more effectively than normal cells/tissues because they generally target the more rapidly dividing tumor cells. However, this may bring about side effects on some actively dividing normal healthy cells, like bone marrow cells, hair follicles and gastrointestinal epithelial cells. Notwithstanding the problems associated with the application of cytotoxic drugs, bioassays testing cytotoxicity toward a panel of cancer cell types represent a reliable approach to screen natural products with anticancer potential, which has led to the successful discovery of anti-cancer pharmaceuticals like paclitaxel and camptothecin [153]. Table 1 summarizes the known fungal pigments with antitumor activities. These pigments are produced by fungi from a variety of species and ecological niches, including species in the genus *Monascus*, endophytic fungi, marine fungi, mushrooms, and fungi residing in special habitats. Together, they have demonstrated activity against a variety of cancer cell lines.

*J. Fungi* **2020**, *6*, 280


#### **Table 1.** Fungal pigments with antitumor activities.


**Table 1.** *Cont.*

† rubropunctatin & monascorubin. <sup>ξ</sup> Matrix metalloproteinase-3 (MMP-3). # a variety of human cancer cell lines: A549 (lung adenocarcinoma), HepG2 (heptoblasoma), HEp-2 (laryngeal carcinoma), WiDr (colon adenocarcinoma), MGC-803 (gastric adenocarcinoma), HO8910 (ovarian cancer), HL-60 (promyelocytic leukemia), HCT-116 (colon cancer), MDA-MB-435 (breast cancer), Y79 (retinoblastoma), LOX IMVI (melanoma), SK-MEL-2 (skin melanoma), HCT-15 (colon adenocarcinoma).

#### 5.2.2. Anti-Biofilm Activity

Melanin from the wood-ear edible fungus *Auricularia auricula* has been demonstrated to display distinct anti-biofilm effects towards *Escherichia coli* K-12, *Pseudomonas aeruginosa* PAO1, and *P. fluorescens* P-3, respectively, while no inhibitory activities were observed on bacterial growth [181]. Zhu et al. demonstrated that pigments from *A. auricula* can repress the production of violacein, a quorum-sensing signal for biofilm formation in the reporter strain *Chromobacterium violaceum* CV026 [182]. It is believed that the ability to form biofilm constitutes the predominant virulence factor for bacterial pathogens, as the biofilm formation is known to account for 80% of bacterial infections in humans [183]. Furthermore, biofilms represent a major cause of nosocomial infections, especially related to the emergence of multi-drug resistant strains, thereby contributing to refractory infectious diseases in humans. Thus, the quest of novel compounds that may effectively inhibit these biofilm-forming bacteria has been a hot topic in both pharmaceutical and clinical settings.

#### 5.2.3. Photosensitizers

Perylenequinones (PQs) represent a class of fungal pigments with a signature 3,10-dihydroxy-4,9-perylene-quinone chromophore. PQs are known as reactive oxygen species (ROS)-generating photosensitizers in medical and agricultural settings. Consequently, PQs have attracted significant attention [184]. Indeed, PQs-producing bambusicolous fungal species *Shiraia bambusicola* has long been used in Chinese folk medicine to treat stomach ache, rheumatic pain, as well as some dermatologic disorders like vitiligo and psoriasis [185]. Interestingly, PQs have also been extracted from fungal fruiting bodies, where a diverse microbial community typically resides. At present, how the PQs are produced in fungal fruiting bodies is not known [186].

Due to the complexity and difficulty of the chemical synthesis of PQs [187], mushroom fruiting bodies have been the major source for the supply of PQs. However, recent work by Ma et al. revealed that *Pseudomonas fulva* SB1 isolated from the fruiting body of the fungus *Shiraia bambusicola* was able to boost the production of fungal PQs, including hypocrellins A, C (HA and HC), and elsinochromes A–C (EA, EB and EC). The results revealed that following two days of co-cultures, *Shiraia* mycelial cultures exhibited the highest production of HA, about 3.2-fold of that in axenic culture. The co-culture might have led to the elicitation of fungal conidiation and the formation of compacted fungal pellets, as compared to axenic culture. Furthermore, the bacterial co-culture might have up-regulated the expression of polyketide synthase gene (PKS) and activated genes of the ATP-binding cassette (ABC) transporter as well as major facilitator superfamily transporter (MFS) for PQ exudation [186].

#### 5.2.4. Cholesterol-Lowering and/or Anti-Atherosclerotic Agents

In view of the association of obesity with an elevated risk of developing diabetes as well as cardiovascular disorders, the search for the cholesterol-lowering compounds has attracted much attention from chemists, biologists, pharmacists and medical practitioners. Monascin and ankaflavin, the two yellow pigments from *Monascus* spp., are found to have a remarkable antiobesity activity in a 3T3-L1 preadipocyte model of rat [71]. Their hypolipidemic effects were attributed to: (i) the inhibition of the differentiation and lipogenesis of preadipocytes by downregulating CCAT/enhancer-binding protein β (C/EBPβ) expression and its downstream peroxisome proliferator-activated receptor γ (PPARγ) and CCAT/enhancer-binding protein α (C/EBPα) expressions, and (ii) the inhibition of lipogenesis by increasing lipase activity and decreasing heparin releasable lipoprotein lipase (HR-LPL) activity [72]. Notably, the actions of monascin and ankaflavin do not resemble that of monacolin K (viz. lovastatin), a well-known cholesterol-lowering drug, which elevates creatine phosphokinase (CPK) activity, known to be a rhabdomyolysis marker [72].

Epidemiological studies have revealed a potential role of carotenoids in preventing cardiovascular disease (CVD) [8]. Reductions of low density lipoprotein (LDL) oxidation and of oxidative stress during plaque formation were thought to account for their effects. An association has been found between low serum lycopene level and an increased risk of atherosclerotic vascular events in middle-aged men [188]. Recent studies have demonstrated that fungal species such as *Blakesleea trispora* and *Rhodotorula* can produce lycopenes [189], thereby indicating their potential roles in the production of pharmaceuticals, food additives, or nutritional supplements that could be beneficial for patients with CVD.

#### 5.2.5. Promising Anti-Alzheimer Agents

As one of the most common forms of dementia, Alzheimer's disease (AD) results in huge economic, emotional, and healthcare costs to individuals, families, and societies throughout the world. At present, there is a relative shortage of therapies available to treat AD. The aggregation of the microtubule-associated protein tau is suspected to be a seminal event in AD, thereby reducing/eliminating tau aggregation represents a potential therapeutic target for AD treatment and prevention. Paranjape et al. screened *Aspergillus nidulans* secondary metabolites for their capability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol [190]. An aggregation inhibitor, asperbenzaldehyde, was identified. Asperbenzaldehyde is an intermediate in azaphilone biosynthesis. The authors further examined 11 azaphilone derivatives to determine their inhibitory activities against tau aggregation in vitro. All compounds examined were able to inhibit tau filament assembly to some degree, and four of the eleven compounds exhibited a pronounced activity of disassembling pre-formed tau aggregates in a dose-dependent fashion. of dementia, Alzheimer's disease (AD) results in huge

Similarly, a detailed investigation into the metabolites of the Mediterranean sponge *Tethya aurantium*-associated fungus *Bartalinia robillardoides* strain LF550 led to the isolation and identification of new chloroazaphilones, helicusin E, isochromophilone X and isochromophilone XI, along with the known pigment helicusin A [191]. Based on the bioassays of these chloroazaphilones, helicusin A (Figure 12) was found to be a potent acetylcholinesterase inhibitor with an IC<sup>50</sup> value of 2.1 µM. Given that inhibition of acetylcholinesterase is widely considered a therapeutic target for ameliorating AD [192], helicusin A is seen as a promising lead compound to develop a potential novel pharmaceutical regime against AD. value of 2.1 μM. Given

**Figure 12.** Structure of helicusin A, a pigment from marine fungus *Bartalinia robillardoides*.

We should note that the anti-Alzheimer activities of the above-mentioned pigments have only come from in vitro studies. Such in vitro results are different from the fungal pigments already in clinical use, such as the cholesterol-lowering and/or anti-atherosclerotic agent monascin as described

Alzheimer's disease d

in 5.2.4. However, the preliminary results as demonstrated by fungal pigments are showing great promise as anti-Alzheimer's disease drugs.

#### 5.2.6. Anti-Inflammatory Activity

Hsu et al. (2011) reported the isolation of an azaphilonidal derivative monaphilone A as well as ankaflavin from the fermented products of *Monascus purpureus* NTU 568 [193]. Further studies showed that ankaflavin and monaphilone A decreased lipopolysaccharide (LPS)-induced inflammatory responses including production of nitrite, and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in murine macrophage RAW 264.7 cells, pinpointing the anti-inflammatory effects of these two fungal pigments.

#### 5.2.7. Antimicrobial Activities

A number of fungal pigments have shown significant antimicrobial activities [194,195]. Table 2 summaries the results from a few representative studies over the last decade. These pigments were from a variety of fungi living in diverse ecological niches, including marine environments, soil, and plants. The pigment-producing fungi are also broad, including both unicellular forms such as *Rhodotorula glutinis* and filamentous fungi such as *Monascus ruber* and species in the genera *Aspergillus, Fusarium,* and *Penicillium* (Table 2). Furthermore, most of these fungal pigments demonstrated broad activities against different groups of microorganisms, including plant and human fungal pathogens as well as both Gram-positive and Gram-negative bacterial pathogens. Of special note are pigments chaetoviridide A and B from the deep sea-derived filamentous fungus *Chaetomium* sp. NA-S0-R1 and pigments penicilones B−D from *Penicillium janthinellum* strain HK106 isolated from mangrove soil that showed strong activities against the methicillin-resistant *Staphylococcus aureus* (MRSA) [196,197].

#### 5.2.8. Others

Aside from the above-described roles of fungal pigments in human health, other human health related roles have also been reported. Those included antioxidative, cytotoxic, and immunosuppressive roles. Below are brief descriptions of those studies.

Anti-oxidative properties are commonly found among fungal pigments. The conjugated polyene chain in the carotenoids provides chemical reactivity against oxidizing compounds and free radicals that may otherwise result in damage to cellular functions. Apart from the anti-oxidative actions of carotenoids previously reviewed elsewhere [15], investigation of the Chinese medicinal fungus *Phellinus igniarius* has led to the isolation and identification of three fungal pigments derived from pyrano[4,3-c]isochromen-4-one, phelligridins H, I, and J [198]. These orange or yellow pigments all showed antioxidant activity, as evidenced by inhibiting rat liver microsomal lipid peroxidation with IC<sup>50</sup> values of 4.8, 3.7, and 6.5µM, respectively, for phelligridins H, I, and J. In addition, phelligridins H and I inhibited protein tyrosine phosphatase 1B (PTP1B), while phelligridin J exhibited cytotoxic activity against four human cancer cell lines, A2708 (ovary cancer), A549 (lung cancer), Bel-7402 (hepatoma) and HCT-8 (colon cancer) [198]. As PTP1B is an important player in cell proliferation, differentiation, and malignancy, being involved, for instance, in the development of breast cancer, lung cancer, and esophageal squamous cell carcinoma [199,200]. The above findings highlight the potential of these novel pigments as antitumor agents.


#### **Table 2.** Antimicrobial activities of fungal pigments.

, MRSA: Methicillin-resistant *Staphylococcus aureus*; 2 , MIC: minimum inhibitory concentrations.

The macula of human eye contains two carotenoids, lutein and zeaxanthin. Based on the NIH Eye Disease Case-Control Study [208], the dietary intake of antioxidants such as carotenoids is highly correlated to the incidence of age-related macular degeneration (AMD), demonstrating a statistically significant linear trend associating the reduction of risk with increasing consumption of carotenoids. It is widely recognized that these carotenoids may protect the macula from light-induced damage and scavenge free radicals generated in the photoreceptors [8].

Four new azaphilones, namely longirostrerones A−D, along with three known sterols, have been isolated from extracts of the Thai soil fungus *Chaetomium longirostre* [209]. Longirostrerones A−D exhibited potent cytotoxicity against KB (human epidermoid carcinoma of the mouth) cell lines with IC<sup>50</sup> of 1.04, 1.52, 0.23, and 6.38 µM, respectively. Among these azaphilones under study, longirostrerone C showed significant cytotoxicity against KB (IC<sup>50</sup> = 0.23 µM), close to the control drugs doxorubicine and ellipticine. Moreover, longirostrerones A showed strong inhibitory effects on human breast adenocarcinoma MCF7 and small-cell lung cancer NCI-H187 cancer cell lines with IC<sup>50</sup> values of 0.24 and 3.08 µM, respectively. Apart from the antitumor activities shown above, longirostrerones A−C also displayed antimalarial activity against *Plasmodium falciparum* with the IC<sup>50</sup> values ranging from 0.62 to 3.73 µM.

An early report revealed the pronounced immunosuppressive activities of monascin and ankaflavin, two yellow oligoketide pigments isolated from the mycelium of *Monascus purpureus* [210]. By inhibiting murine T-splenocyte proliferation, monascin and ankaflavin could interact with regulatory mechanisms of the immune system and thus suppress its function, indicating that the two *Monascus* pigments might serve as promising immunosuppressants.

#### **6. Structure–Activity Relationship (SAR) Studies of Fungal Pigments**

The structure–activity relationships have been investigated for several groups of fungal pigments. For example, the presence of a halogen atom at C-5, a proton at C-8 and a diene moiety in the C-3 side chain of the 6-oxoisochromane ring in azaphilones are necessary for gp120-CD<sup>4</sup> binding, as exemplified by isochromophilones and their derivatives [211]. Furthermore, (an) electrophilic ketone(s) and/or enone(s) at both C-6 and C-8 in the 6-oxoisochromane ring are indispensable for the activity of cholesteryl ester transfer protein (CETP) inhibition, which is found in the azaphilone pigments such as chaetoviridin B, sclerotiorin, and rotiorin, etc. [78].

In addition to the above-mentioned bioactivities, studies with murine macrophage cells RAW 264.7 have demonstrated that the inhibition of NO production relies on the structure of azaphilones [212]. For instance, spiro-derivatives daldinins C, E, and F (structures shown in Figure 13) only exert weak inhibition on NO production. In contrast, azaphilones bearing a lactone ring such as multiformin D and sassafrins A–C (Figure 13), are shown to exert stronger inhibitory actions on NO production than daldinins. Entonaemin A, rubiginosins A and B (Figure 13) are all azaphilones where an orsellinic acid moiety is attached to the bicyclic core via an ester linkage. However, since only rubiginosin A displays strong activity in this subclass of azaphilones, the location of the orsellinic acid moiety must not influence the activity of the compounds. Nonetheless, the presence of an acetyl group (such as in the case of rubiginosin A) is indispensable for an elevation of activity. Azaphilones with a fatty acid side chain linked to a bicyclic azaphilone core via an ester bond, like rubiginosin C, cohaerins A and B (Figure 13), demonstrated only weak activities. The most potent inhibitors of NO production seem to be dimeric azaphilones, such as rutilins A and B (Figure 13). Overall, while the acetyl group is necessary for inhibition, the location of orsellinic acid does not seem to change their activities [212]. Taken together, these studies showed that the inhibitory effects of azaphilones are substantially fortified by the number of orsellinic acid moieties in the molecule and the presence of conjugated double bonds in dimeric compounds [212].

'

**Figure 13.** Structures of azaphilones.

Previous studies have shown that the antioxidant properties of the carotenoids are tightly correlated with their chemical structure. Both the polyene chain and these other structural features influence the chemical properties (e.g., redox properties) of the carotenoids [213]. For example, the structure–activity relationship was investigated by Rodrigues and his colleagues for fifteen carotenoids, showing that the

opening of the β-ionone ring and the increase of chromophore extension in the carotenoid structure could be the predominant factors giving rise to the elevated capacity of peroxyl radical scavenging [214]. Specifically, the conjugated polyene chromophore gives rise to not only the light absorption properties, and thus hues, but also the chemical properties of the compound and hence light-harvesting and photoprotective activity. In addition, the polyene chain is the main determinant responsible for antioxidant role of carotenoids due to its chemical reactivity with free radicals, and oxidizing agents. Furthermore, carotenoids are found to be in precise locations and orientations within subcellular structures in vivo. Thus, their chemical and physical traits are strongly affected by other molecules in their vicinity (particularly proteins as well as membrane lipids), and vice versa [215]. Indeed, structural characteristics, such as size, shape, and polarity, are critical to the capability of a carotenoid to fit precisely into its molecular milieu to allow for its functionality. The role of carotenoids in influencing cell membrane-associated molecular processes by modifying the structure, properties, and stability of these membranes may constitute a significant aspect of their beneficial effects on human health.

Apart from their antioxidant actions, studies with marine carotenoids have unveiled the relationship between structure and anti-obesity activity. The key structure responsible for anti-obesity activity is thought to be the carotenoid end of the polyene chromophore bearing an allenic bond and two hydroxyl groups [216].

#### **7. Conclusions and Further Prospects**

Fungal pigments have primarily been known for their extensive application as colorants in the food, cosmetic and textiles industries for the past several decades. However, their applications other than as colorants are very broad, including as cholesterol-lowering drugs, exemplified by monacolin K, a clinically used drug also called lovastatin [217], and various anti-microbial and antitumor agents. Unexpected functions of new pigments as well as known ones are continuously being revealed. Fungal pigments have attracted significant interests from the pharmaceutical industry as sources of potential drugs to fight many life-threatening diseases such as cardiovascular disorders, Alzheimer's disease, human carcinomas (hepatoma, breast, lung, colorectal, gastric, pancreatic, leukemia, hematopoietic, renal cell, and other cancers), infectious diseases, and parasitic diseases such as malaria. Since many fungal pigments have shown potential relevance to human health, there is increasing interest in understanding their mechanisms of action to pave the way for the future development of therapeutics against both acute and chronic diseases. Among the fungal pigments, melanin stands out for two reasons: (i) melanin confers excellent protection from such stressors as radiation, thermal fluctuations and drought, thereby giving an insight into the development of radiation-protective agents and devices for the human exploration into the outer space; (ii) melanin is known to contribute to pathogenesis and drug resistance in pathogenic fungi, and consequently represents an important target for the treatment of recalcitrant fungal infection in humans.

Thanks to advancements in microbiological, biochemical, genetic and genomics technologies and the development of high-throughput bioassay systems as well as the bioinformatics analyses, there are several potentially fruitful areas of research on fungal pigments. First, there is a diversity of fungal pigments produced by evolutionarily distant fungi. At present, the origins for the genes and the regulatory pathways governing their synthesis are largely unknown. Phylogenomic and transcriptomic studies should provide novel insights in this area. Second, a standardized test system and/or protocol should be established for screening the bioactivity of fungal pigments for both in vitro and in vivo assays, including appropriate positive and negative controls. Third, the structure–activity relationships are unknown for most fungal pigments. Finally, while the focus of our review is on colored fungal pigments, fungal metabolites that impact pigment productionunpub in both fungi and other organisms, including precursors and regulators for and metabolic products of pigments but they themselves are not colored, represent an additional source of fungal metabolites that have also been explored for human health benefits. One such metabolite in this category is kojic acid, a compound obtained from the filamentous fungus *Aspergillus oryzae* [218]. While colorless itself, kojic acid can

inhibit tyrosinase which is essential for the synthesis of melanin in human skin and, consequently, it has been broadly used in dermatological applications [219]. With the increasing demand from the pharmaceutical industry, fungal pigments and their related products are expected to be an excellent resource for the design and development of novel therapeutic molecules in the future.

**Author Contributions:** L.L. and J.X. conceived and delineated manuscript organization; L.L. wrote the original draft; J.X. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Natural Science and Engineering Research Council of Canada (2020-531998) and McMaster University (to J.X.), and by a China Scholarship Council fellowship (to L.L.).

**Acknowledgments:** We thank Heather Yoell for helpful suggestion and proofreading.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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