Untapped Potential of Marine-Associated Cladosporium Species: An Overview on Secondary Metabolites, Biotechnological Relevance, and Biological Activities

The marine environment is an underexplored treasure that hosts huge biodiversity of microorganisms. Marine-derived fungi are a rich source of novel metabolites with unique structural features, bioactivities, and biotechnological applications. Marine-associated Cladosporium species have attracted considerable interest because of their ability to produce a wide array of metabolites, including alkaloids, macrolides, diketopiperazines, pyrones, tetralones, sterols, phenolics, terpenes, lactones, and tetramic acid derivatives that possess versatile bioactivities. Moreover, they produce diverse enzymes with biotechnological and industrial relevance. This review gives an overview on the Cladosporium species derived from marine habitats, including their metabolites and bioactivities, as well as the industrial and biotechnological potential of these species. In the current review, 286 compounds have been listed based on the reported data from 1998 until July 2021. Moreover, more than 175 references have been cited.


Introduction
The marine environment covers approximately 70% of the Earth's surface and represents an enormous pool of biodiversity resources [1][2][3]. Marine microorganisms possess the potential for several biotechnological and industrial applications and play an important ecological role [4,5]. The last decades have witnessed numerous studies in the natural metabolites derived from marine creatures or their associated microorganisms [6][7][8]. Marine-derived fungi consist of a wide range of parasites, saprotrophs, symbionts, epiphytes, and endophytes [9,10]. They can be obtained from various marine samples such as algae, seagrasses, corals, sponges, ascidians, crustaceans, bivalves, fishes, and inorganic matter [11,12]. Jones et al. reported 530 marine taxa in 321 genera, which included 12 Basidiomycota (nine genera), 94 asexual morphs (61 genera), and 424 Ascomycota (251 genera) [13]. In 2011, the number of marine fungi was estimated to be 10,000 to 12,500 species based on substrates and geographical locations [14]. Currently, 1901 species have been listed on the marine fungi website, in 769 genera, 88 orders, 226 families, 22 classes, and seven phyla [15]. They are acknowledged as a rich source of novel metabolites with unique structural features, bioactivities, and biotechnological applications that attracted the attention of many biologists and chemists [16]. Cladosporium (Cladosporiaceae) is one of the largest genera of dematiaceous hyphomycetes [17]. Cladosporium species are frequent airborne molds, which can be isolated from almost every environment and geographic location, because their small conidia are easily dispersed [18][19][20][21]. C. herbarum, C. cladosporioides, and C. sphaerospermum are its three major species [22]. It comprises many important plant pathogens causing stem rots and leaf spots such as C. fulvum is the causal agent of tomato leaf mold [23,24]. Some species are also known as common contaminants in clinical laboratories and cause allergic lung diseases [25][26][27][28]. Some species have been reported as endophytes and possessed a positive influence, for example, C. sphaerospermum isolated from Glycine max roots which can promote its growth [29]. Several species were linked to allergic rhinitis and respiratory arrest in asthmatic patients, and some are described as a cause of opportunistic phaeohyphomycosis, including subcutaneous and deep infections in humans and animals [30,31]. Some species are fungicolous that possess a potential for biological control in agriculture and forestry [32,33]. Moreover, many Cladosporium species have the potential to be used in various industrial processes [34,35]. Marine-associated Cladosporium species have attracted considerable interest because of their ability to produce a wide array of metabolites, including macrolides, pyrones, phenolics, alkaloids, diketopiperazines, terpenes, sterols, quinones, lactones, and tetramic acid derivatives. These metabolites possess versatile bioactivities such as anticancer, antimicrobial, antiviral, insecticidal, antifouling, anti-malarial, anti-hyperlipidemic, and α-glucosidase and protein tyrosine phosphatase inhibiton [36][37][38][39][40][41][42]. It has been shown that these species have significant impacts on biotechnology, ecosystems, and food production. They are a wealthy source of enzymes such as pectinases, agarases, carrageenases, xylanases, laccases, peroxidases, tannases, invertases, cellulases, and reductases that have wide biotechnological influences in developing eco-friendly technologies in the pulp and paper industry, food and feed industries, biomasses and contaminants bioremediation and biodegradation, and generation chemicals and liquid fuels [11,12,[43][44][45][46][47][48][49][50]. The main goal of this review is the focus on the reported research in Cladosporium species derived from a marine habitat, including the structures and bioactivities of the reported metabolites, as well as the industrial and biotechnological potential of these species (Tables 1 and 2). This work covers the studies that have appeared in literature from 1998 until July 2021. The structures and bioactivities of reported metabolites from Cladosporium species have been highlighted. Furthermore, the biotechnological and industrial potential of Cladosporium species has been summarized. We hope that this work can provide knowledge that can help for the dereplication and bioactivities evaluation of these marine-associated Cladosporium species. The present data were collected through the search on the various databases, including Web of Knowledge, ScienceDirect, SCOPUS, Taylor & Francis, Wiley Online Library, PubMed, JACS, Springer, and Google Scholar.

Importance of Marine Associated Cladosporium Species
Recently, cold-active microbial enzymes have attracted a great attention, and they are preferred to the thermophilic and mesophilic enzymes due to the reduction in the energy expenditure and costs of processing accompanied by industrial heating steps [51]. Many marine-associated Cladosporium species display noticeable enzyme production capacity. Many of these enzymes are exclusively produced at low temperature and high salt concentrations. Therefore, they play a substantial ecological role in lignin-cellulosic materials decomposition in the marine environment. Besides, these enzymes can be utilized in various biotechnological applications and allow the performance of industrial processes even in harsh conditions. In this review, the biotechnological and industrial relevance of Cladosporium species has been highlighted.
The polycyclic aromatic hydrocarbons (PAHs) are volatile pollutants that can cause various environmental pollutions such as oceanic and freshwater contamination, which can take place during storage, use, or transportation of crude oil and its products. PAHs inhalation or ingestion through contaminated food and airborne contaminants leads to serious health disorders such as endocrine disruption, cancer, and reproductive and birth problems [52]. Therefore, introducing marine-adapted microorganisms to increase the PAH-biodegradation rate is an important approach to reduce PAHs concentration in the contaminated regions. Investigation of the PAH biodegradation potential of various marine-derived fungi revealed that Cladosporium sp. CBMAI 1237 had a great potential for bioremediation and biodegradation of PAHs (e.g., anthracene, anthrone, anthraquinone, acenaphthene, phenanthrene, fluorene, pyrene fluoranthene, and nitropyrene) even in a non-marine environment [44].                         Pectinases are hydrolytic enzymes that are accountable for the hydrolysis of pectins. They are commonly found in fungi, bacteria, and plants. They have remarkable importance in the food industry such as vegetables and fruits processing, wine production, and olive oil extraction, as well as coffee, cocoa, and tea fermentation. They are utilized in the beverage industry to produce high yields due to improving clarification and pressing of concentrated fruit juices [103]. Bastos et al. purified pectinase enzymes PG and PME from C. cladosporioides using the Buescher and Furmanski procedure after 10-day incubation and precipitation with (NH 4 ) 2 SO 4 and benzoate buffer at pH 4.0 [49].
Agarases and carrageenases can decompose algal biomass, producing carrageenans and agars that are the major components of the red algae cell wall. Furthermore, agarases hydrolyze agar, resulting in oligosaccharides that are employed as food additives with beneficial influences on human health [104,105]. Additionally, carrageenases are used to obtain carrageenans that have varied industrial applications as emulsifying, thickening, and gelling agents in the preparation of food, as well as bioactivities such as anti-tumor, antiviral, antithrombotic, immunomodulatory, anticoagulant, and antioxidant [106]. Cladosporium sp. isolated from the Antarctic macroalgae Ascoseira mirabilis and Georgiella confuens produced agarase that may have industrial importance in the extraction of agar or its byproducts such as bioactive galactose and oligosaccharides exist in the algal biomass to be utilized as substrates of 3rd generation bioethanol [11].
Xylan, the main component of hemicelluloses in the plant cell walls, represents about one-third of all renewable organic carbon on earth. Xylanases hydrolyze xylan to oligosaccharides that are further degraded to xylose. The latter is utilized for xylitol and bioethanol production. Xylanases have remarkable biotechnological influence in developing ecofriendly technologies in the pulp and paper industry and in food and feed industries, and for generating chemicals and liquid fuels from lignocellulose [107][108][109]. The coldactive xylanases have notable applications in bioremediation and food and textile and industries [110]. Cladosporium sp. isolated from Antarctic marine sponge had high xylanase potential when grown on wheat bran and pure xylans at lower temperatures that is a feature of cold-active enzymes [48]. Therefore, cold-active xylanases preparations from Cladosporium sp. could be convenient for many biotechnological processes, utilizing moderate-to low-temperature processes, especially those in food industries [48]. Gil-Durán et al. purified and characterized XynA, a cold-active endo-xylanase from Cladosporium sp. derived from Antarctic sponge. XynA is highly active on xylans with high arabinose content. Moreover, it is the most thermolabile endo-xylanase reported from filamentous fungus. Therefore, it could be a good alternative in some biotechnological operations to avoid heating, thereby reducing the costs [45].
The three main lignin-hydrolyzing enzymes that have great potential for industrial applications are LiP (lignin peroxidase), MnP (manganese-dependent peroxidase), and Lac (laccase) [111]. LiP is a high oxidant heme protein that oxidizes non-phenolic and phenolic substrates. MnP is a H 2 O 2 -dependent glycoprotein that needs Mn 2+ for oxidizing aromatic dyes and mono-aromatic phenols [112]. Laccase is multi-copper oxidase, which oxidizes aromatic amines and catalyzes the O 2 reduction to H 2 O [111]. C. cladosporioides CBMAI 857 isolated from the Brazilian cnidarian Palythoa variabilis produced ligninolytic enzymes (LiP, MnP, and Lac) with particular response to the various conditions of salinity and carbon sources. It possessed high values of MnP and laccase activities under salinity (12.5% and 23% w/v, respectively), indicating the potential use of this fungus for industrial applications and bioremediation of high-salt contaminated sites [50].
RBBR (Remazol Brilliant Blue R) and polymeric dyes decolorization has been assigned as an effective screening method for the fungi ability to degrade recalcitrant pollutants, including aromatic compounds such as PAHs. It was demonstrated that marine-derived fungi are often more effective than terrestrial fungi in treating various colored effluents because they are better adapted to perform under extreme conditions such as high salinity [113]. C. cladosporioides CBMAI-857 associated with the coral Palythoa caribaeorum was tested for its RBBR decolorizing potential. It had efficient dye decolorization potential (93%) after 12 days in both liquid and solid media [114]. Further, Cladosporium sp. associated with the seagrass Posidonia oceanica produced tannases and ligninolytic enzymes at high salt concentrations. Its laccase and peroxidase activity was evident by the degradation of RBBR and Amaranth Red dyes [12,115].
Invertase is a β-fructo-furanosidase that catalyzes sucrose conversion into fructose and glucose, giving invert syrup. This invert syrup is utilized in the beverage and food industries as a humectant in non-crystallizing creams, candies, artificial honey, and jam preparation [116]. Molasses is a sugar solution that is obtained as a co-product of sugar production. Due to its high sucrose content and low cost, it is utilized as an invertase production substrate to produce industrially valuable substances [117]. However, it contains melanoidins, which are dark brown pigments. Its discharge in the soil prohibits seed germination and decreases manganese availability and soil alkalinity. Furthermore, it blocks photosynthesis and sunlight penetration in the aquatic system [118]. Therefore, its removal from molasses-based wastewater is potentially important for environmental safety. Taskin et al. reported that C. herbarum ER-25 possessed a high invertase potential and removed melanoidins from molasses through bio-adsorption and biodegradation mechanisms by Lac and MnP in the non-sterilized medium than in sterilized one at 5.5 pH and 20 • C. Therefore, this cold-adapted fungus can be used for molasses de-colorization [46].
Cellulose is a main component of the plant material that is abundantly utilized for the production of alternative liquid fuels such as bioethanol. C. sphaerospermum obtained from deteriorated seaweed Ulva through SSF (solid-state fermentation) produced cellulase that had saccharification potential of seaweed biomass using green seaweed Ulva fasciata. Therefore, this cellulase can be utilized for saccharification of cellulosic feedstock for bioethanol production from marine macro-algal feedstock [47].
Biocatalysis is an eco-friendly process for renewable raw materials and clean energy production and for the remediation of environmental contaminants [119]. Recently, the synthesis of industrial and chemically interesting complex molecules using biocatalysts, including enzymes and whole-cell systems is a grown-research field. Reductases have been utilized for various substrates reduction such as aldehydes, carboxylic acid derivatives, ketones, nitro compounds, and nitriles [119,120]. Furthermore, it has been reported that microorganisms' whole cells are a potential source for new enzymes used in carbonylated compounds reduction [121]. Knoevenagel condensation is a very useful synthetic tool for functionalization, as well as for increasing the carbon chains that is applied for the synthesis of intermediates polymers and various bioactive organic compounds [122]. Birolli et al. reported that the bio-reduction of Knoevenagel adducts between cyanoacetamide and aromatic aldehydes was achieved in considerable yields with whole-cells of Cladosporium sp. CBMAI 1237 isolated from Dragmacidon reticulatum, revealing the existence of ene-reductases [43]. Additionally, C. cladosporioides CBMAI-857 isolated from the Brazilian cnidarian Palythoa caribaeorum catalyzed the asymmetric bio-reduction of 1-(4-methoxyphenyl)ethanone to 1-(4-methoxyphenyl)ethanol [123]. Moreover, the spongeassociated C. cladosporioides CBMAI-857 catalyzed the enantio-selective bio-reduction of different aromatic ketones at pH 7.0 and 32 • C [124].

Secondary Metabolites and Bioactivities of Marine-Associated Cladosporium Species
Marine-associated Cladosporium species are rich with diverse types of metabolites with varied structural features such as macrolides, fatty acids, pyrones, phenolics, alkaloids, diketopiperazines, terpenes, sterols, quinones, lactones, and tetramic acid derivatives. Their classification was carried out here according to the chemical nature. During our search, it was found that some of the reported metabolites had the same structures and molecular formulae with different nomenclature. On the other hand, some metabolites had the same names with different structures. Moreover, some metabolites did not have names, thus they are named here using the AUPAC system for nomenclature. Herein, the reported secondary metabolites from Cladosporium species, as well as their bioactivities have been discussed (Tables 1 and 2).

Tetramic Acid Derivatives
Tetramic acids are five-membered heterocycles with a pyrrolidine-2,4-dione core that are formed by the fusion of polyketide units and amino acid [125]. The tertarmic acid moiety is commonly present as 3-acyl or 4-O-alkyl ether derivatives [126]. These structures can be characterized as simple heterocycles or more complex systems possibly containing long chains or fused polycyclic skeletons [127]. They are found in varied natural metabolites and isolated from various terrestrial and marine species, such as bacteria, sponges, and fungi [127,128]. They exhibited a wide range of bioactivities: cytotoxic, antimicrobial, antiulcer, and antiviral [125]. Note that 30 tetramic acid derivatives have been reported from marine-derived Cladosporium species, 28 (93.3%) of them are from C. sphaerospermum.

Butanolides and Butenolides
Butanolides and butenolides are five-membered γ-lactones which may also be regarded as furan derivatives. They are an important class of structural motifs often encountered in various natural metabolites and synthetic targets [140]. They have an impressive range of bioactivities including antibiotic, antitumor, and anticancer that are intimately connected to their relative and absolute configurations [141].

Tetralones (Napthalenones)
Tetralones comprise a bicyclic aromatic hydrocarbon and a ketone and are regarded as benzo-fused cyclohexanone derivatives. They played a substantial role as a starting material for the synthesis of a range of synthetic heterocyclic compounds and pharmaceuticals due to their potential reactivity and suitability [142]. Additionally, they are precursors of many natural metabolites and their derivatives. They have been used in the synthesis of therapeutically functional compounds such as antibiotics, acetylcholinesterase inhibitors, antidepressants, and antitumor alkaloids [142,143].

Perylenequinones
Perylenequinones comprise a class of natural products characterized by an oxidized pentacyclic core. They are dark-colored pigments isolated from diverse sources such as mold species, plants, and aphids [146]. They reported to have anthelmintic, photoactivity, antiviral and antitumor [146].

Naphthalene Derivatives
Naphthalenes are a class of arenes containing two ortho-fused benzene rings that have been reported from plants, liverworts, fungi, and insects [148]. Their derivatives exhibited anti-inflammatory, antimicrobial, antioxidant, anti-protozoal, cytotoxic, and anti-platelet aggregation activities [148].
Silber et al. reported the isolation of malettinins A-C (178-180), along with the new metabolite, malettinin E (181) from Cladosporium sp. strain KF501 isolated from the German Wadden Sea (Figure 22). These metabolites have dihydropyran/tropolone structures connected to a furan ring. The configuration of 181 was determined by the single-crystal X-ray diffraction method. Interestingly, this was the first report for tropolones isolation from genus Cladosporium. They were evaluated for antimicrobial activity towards X. campestris, B. subtilis, S. epidermidis, C. albicans, and Trichophyton rubrum using the microplate assay. Note that 178-181 exhibited weak antifungal potential towards Trichophyton rubrum (IC 50

Bioactivities of Cladosporium Species Extracts
Ding et al. stated that Cladosporium sp. isolate N5 associated with Porphyra yezoensis red alga did not produce any pathogenic symptoms in the reinfection assay. Further, its EtOAc extract displayed no lethality to A. salina and had a moderate antimicrobial activity which indicated that Cladosporium sp. had no toxicity to the aquatic ecosystem and could be applied as a biocontrol agent [59]. In the disc diffusion method, Cladosporium sp. EIODSF 008 EtOAc extract exhibited significant antibacterial potential towards E. coli, M. luteus, and B. subtilis (conc. 100 µg/disc) [57]. The EtOAc extract of Cladosporium sp. EN-S01 isolated from Sargassum cinereum brown algae showed anticancer activity towards MCF-7, HeLa, and DU-145 cell lines (IC 50 8.46, 9.87, and 98.03 µg/mL, respectively). The extract had greater cytotoxic activity and anti-proliferative towards MCF-7 and HeLa cell lines than towards DU-145 [157]. Moreover, the EtOAc extract of C. cladosporioides KT384175 isolated from the seaweed Sargassum wightii possessed remarkable antioxidant potential that was comparable to ascorbic acid, as well as significant Fe 3+ reducing power that could be referred to its phenolic contents. Moreover, it revealed anti-angiogenic potential as evidenced by the decrease in the number and length of blood vessel branches on CAM (chick chorioallantoic membrane) in-vivo in the CAM assay. Further, C. cladosporioides extract (conc. 1.0 mg/mL) had lower wound healing potential than thalidomide (conc. 1.0 µg/mL) in the in vitro scratch assay using MCF-7 cells [158]. The sea water-derived fungus Cladosporium sp. F14 can produce antifouling and antibiotic metabolites in the existence of xylose or glucose. Significantly, it showed higher antibiotic activity towards M. luteus, P. piscida, Rhodovulum sp., Ruegeria sp., V. fluvialis, and V. harveyi in the existence of a sugar carbon source than in its absence in the disc diffusion assay, even though the fungal cells were well-grown under both conditions. Moreover, it possessed antifouling potential as it reduced the attachment of B. neritina (bryozoan larvae) in the larval settlement assay [159]. The gold nanoparticles synthesized from C. cladosporioides isolated from the seaweed S. wightii possessed noticeable antimicrobial potential towards E. coli MTCC-118, B. subtilis MTCC-441, S. aureus MTCC-7443, P. aeruginosa MTCC-424, and A. niger MTCC-281 with the highest growth inhibition towards S. aureus (IZD 12 mm) and least activity against B. subtilis (IZD 9.5 mm), compared to ampicillin (IZDs 15 and 12 mm, respectively) in the well diffusion method. Furthermore, they also had significant antioxidant potential comparable to ascorbic acid in the DPPH assay and moderate effectiveness in reducing power assay [160]. Ameen et al. reported that the AgNPs synthesized from C. halotolerans biomass isolated from the marine debris collected around Tarout Island showed a significant free radical scavenging effect (%inhibition 78% within 30 min incubation) in the DPPH assay. Moreover, it exhibited cytotoxic potential towards MCF-7 (IC 50 34.27 µL/mL), compared to cisplatin (IC 50 17.69 µL/mL) in the MTT assay, as well as an antifungal effect against A. niger (%inhibition 70 and 45% at conc. 1000 and 500 ppm, respectively) in the broth dilution method [161].
From the comprehensive review of the available literature, it was noticed that C. phlei (causal agent of Timothy leaf spot disease) and C. cucumerinum (causal agent of scab disease of many Cucurbitaceae plants) were isolated mainly from plant sources [162][163][164][165]. These species produced perylenequinone derivatives as major metabolites which are responsible for pigmentation and discolorations of the leaves [162,165]. Additionally, cotylenins, plant growth regulators were isolated from an unidentified Cladosporium species [166][167][168][169][170]. However, tetralones, seco-acids, macrolides, diketopiperazines, alkaloids, and tetramic acid derivatives were reported mainly from marine-associated Cladosporium species.

Conclusions
Numerous structurally diverse biometabolites are discovered from marine-derived fungi that represent a rich library for the development of drug lead. Marine-associated Cladosporium species are of biotechnological and industrial relevance and could be considered as substantial enzyme producers. Their enzymes are active in harsh conditions such as extremely low temperatures and high salinity. Therefore, they can be utilized in various industrial and biotechnological applications. Besides, these species were found to be a wealthy pool covering a wide array of metabolites with various bioactivities. Over the past 22 years, 286 metabolites have been separated from marine-associated Cladosporium species isolated from various marine samples, including mangrove, sediment, sponges, corals, gorgonians, algae, bivalves, hydroids, and others ( Figure 32).
The results revealed that alkaloids, macrolides, tetramic acid and pyrone derivatives, and phenolics are the major metabolites reported from this marine-associated fungal species (Figure 34). They could be privileged and useful candidates for chemists and biologists to design structurally novel and pharmacologically important compounds for various diseases.  Although the structural diversity of these metabolites, they were insufficiently evaluated for their bioactivities. Most of them had been assessed for their antimicrobial, cytotoxicity, antiviral, and insecticidal activities ( Figure 35). Figure 36 illustrated the prominent activities of each class of secondary metabolites. However, there are limited studies that focus on the mechanism of action of these metabolites. Many of the tested metabolites possessed no noticeable efficacy in some of the tested activities. Therefore, estimation of other potential bioactivities and derivatization of these metabolites, as well as the mechanistic and in vivo studies of the active metabolites should clearly be the target of future research.

Strategies for Activating Silencing Gene Clusters
Growing evidence has revealed that the activation of silent gene clusters has the potential to significantly enhance the discovery of new natural metabolites of high-therapeutic leads. Different strategies to awake the silent biosynthetic gene clusters of Cladosporium species such as co-cultivation of organisms and elicitors epigenetic, as well as, modifiers can be applied [171][172][173][174]. The production of secondary metabolites (SMs) is affected by cultivation media, environment, and conditions [171,175]. Therefore, manipulating the culture conditions can improve the outputs from living organisms. Small changes in the growth media composition can induce not only variation in the amount of SMs, but also the production of a completely different pattern of molecules [171][172][173]. OSMAC (one strain many compounds) is a form of strain improvement that summarized the ability of single strains to produce different compounds when growing under different conditions e.g., aeration rate, media composition, type of culturing vessel, or a combination of these factors [174][175][176]. Challenging the fungi with external cues or chemicals has been shown to enhance the SMs production. Antibiotics have been widely reported as elicitors that can activate a broad spectrum of silent BGCs [171][172][173][174]. The co-cultivation of strains of the same or different species has been shown to represent a promising strategy for the activation of silent BGCs that enhances the production of SMs and discovery of new bioactive SMs [171,172,177]. Activation of silent biosynthetic gene clusters (BGCs) by quorum sensing class of signaling molecule is another strategy that has been shown to dramatically increase SMs production [171][172][173]. Engineering strains to circumvent the regulatory systems has the potential to free silent BGCs from their locked-in state and result in a significantly enhancement of SMs production. This can be done through various ways such as ribosome and polymerase engineering, an awakening of the genes encoding transcriptional regulatory proteins, and deletion or deactivation of the suppressor proteins. Another approach is the insertion of inducible artificial promoters to drive the expression of the silent genes [171,172,178]. Modulating epigenetic control also plays a role in the expression of silent gene clusters linked to natural product expression [173,179].

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