Hidden Treasure: Halophilic Fungi as a Repository of Bioactive Lead Compounds

The pressing demand for novel compounds to address contemporary health challenges has prompted researchers to venture into uncharted territory, including extreme ecosystems, in search of new natural pharmaceuticals. Fungi capable of tolerating extreme conditions, known as extremophilic fungi, have garnered attention for their ability to produce unique secondary metabolites crucial for defense and communication, some of which exhibit promising clinical significance. Among these, halophilic fungi thriving in high-salinity environments have particularly piqued interest for their production of bioactive molecules. This review highlights the recent discoveries regarding novel compounds from halotolerant fungal strains isolated from various saline habitats. From diverse fungal species including Aspergillus, Penicillium, Alternaria, Myrothecium, and Cladosporium, a plethora of intriguing molecules have been elucidated, showcasing diverse chemical structures and bioactivity. These compounds exhibit cytotoxicity against cancer cell lines such as A549, HL60, and K-562, antimicrobial activity against pathogens like Escherichia coli, Bacillus subtilis, and Candida albicans, as well as radical-scavenging properties. Notable examples include variecolorins, sclerotides, alternarosides, and chrysogesides, among others. Additionally, several compounds display unique structural motifs, such as spiro-anthronopyranoid diketopiperazines and pentacyclic triterpenoids. The results emphasize the significant promise of halotolerant fungi in providing bioactive compounds for pharmaceutical, agricultural, and biotechnological uses. However, despite their potential, halophilic fungi are still largely unexplored as sources of valuable compounds.


Introduction
Environments with extreme conditions have typically been considered as malicious to be inhabited by organisms.The discovery of extremophiles changed this belief, and numerous micro-organisms, including the members of archaea, prokarya, and eukarya, have been reported in natural habitats with very high heat [1], cold, extreme pH, or high salinity.Those organisms exclusively growing in hypersaline conditions are called saltloving organisms or halophiles.Hypersaline conditions are found in several locations across the globe, including littoral and arid regions as well as artificial salterns developed for mining salts [2].Under hypersaline conditions, a water-limiting situation is generated due to the chemical binding of water to salt molecules.These water-limiting conditions prohibit the majority of life forms from growing in hypersaline environments; on the other hand, these are favorable conditions for halophiles to flourish.Most of the identified and studied extremophiles are archaea; however, other micro-organisms, such as bacteria, algae, and fungi, are also found in extreme environments.This wide variety of extremophilic organisms attracts the scientific community to investigate their uniqueness, which may enable inferring the evolutionary process of stress tolerance in other living forms.For the first time, the presence of eukaryotic fungi was reported in the hypersaline environment of the Dead Sea by [3] and in salterns by [4].Since then, numerous novel species, including those once considered merely food contaminants, have been identified in hypersaline environments worldwide [5].Researchers predominantly concentrate on bacteria, archaea, and algae when exploring extremophiles and extremozymes from saline environments, overlooking filamentous fungi, which remain largely unexplored [6].However, extremophilic fungi hold significant promise in uncovering novel compounds given that fungi contribute over 40% of the active compounds derived from microorganisms [7][8][9][10][11][12][13][14].This review aims to elucidate the ecological niche and adaptive strategies of halophilic fungi in natural hypersaline ecosystems, along with the pharmaceutical potential of their bioactive metabolites for prospective drug development.

Physiology of Halophilicity in Fungi
Fungi living under hypersaline conditions possess a specialized physiology that enables them to flourish in such conditions that are considered harsh for other organisms.The hypersaline environment imposes two major limitations inhibiting the growth of organisms: (1) high osmolarity and (2) ionic toxicity due to the high ionic concentration.The hypersaline conditions lead to high osmolarity, resulting in very low water activity.This low water activity is generated due to the bonding of water molecules (H 2 O) with salt (NaCl).Thus, water activity is one of the most determinative factors for the growth of fungal life under hypersaline conditions.The mitigation strategies of halophiles to survive and grow in hypersaline environments mainly include maintaining a low water potential condition in comparison to their surrounding environment.These mitigation strategies involve several physiological mechanisms, such as selective modification of the fluidity of the plasma membrane, generation and accumulation of compatible solutes, and the high osmolarity glycerol (HOG) pathway [15][16][17].

Ecology and Biodiversity of Halophilic Fungi
Similar to other abiotic factors such as temperature, water, and light, salinity also imposes deleterious effects on the growth and development of organisms.The water activity of saline environments defines the extent of adverse impacts on organisms; however, some fungi belonging to different phylogenetic origins growing in hypersaline conditions showed their halotolerant and halophilic nature [18].Research indicates that halophilic fungi thrive in various hypersaline environments worldwide, including regions like Slovenia, Romania, Thailand, China, India, Brazil, and others [19][20][21][22].These fungi have been found in habitats with diverse salinity levels, such as marine environments (sea water, marine plants, and mangroves) [23], solar salterns used for salt production through seawater evaporation [24], natural salt lakes, salt mines [25,26], saline soil, salt deserts, sebkhas (resulting from salt lake evaporation, characterized by a range of soluble salts) [27], and high-salt-content foods and fermented products [28].Kirk et al. [18] noted that, among the 106 existing orders of fungi, 10 were identified as tolerant to low water activity (a w ).Halophilic or halotolerant characteristics were observed in certain orders, including Wallemiales, Trichnosporales, and Sporidiales within Basidiomycota, and Capnodiales, Eurotiales, and Dothideales within Ascomycota.Various yeast species such as Rhodotorula, Debaryomyces, Aureobasidium, and Trichosporum, along with filamentous fungi like Cladosporium, Scopulariopsis, Alternaria, and species of Aspergillus and Penicillium, were described as halotolerant.Halophilic species were identified within genera such as Wallenia, Hortea, Phaetotheca, and Trimmatostroma [29,30].Several studies have consistently reported the presence of numerous fungi in hypersaline water and environments across different continents [4].Fungi from orders including Dothideales, Capnodiales, and Eurotiales of Ascomycota, as well as Wallemiales and Tremellales of Basidiomycota, demonstrated halotolerance, with black yeasts being particularly prominent in the hypersaline water of salterns [31].These yeasts have melanized cell walls, enabling them to withstand high-salt-stress conditions [32].Notably, among black yeasts, Hortaea werneckii is widely distributed across saline environments, thriving optimally at 3.0-4.5M NaCl [5].Wallemia ichthyophaga (Basidiomycetes) stands out as a genuinely halophilic fungus requiring a minimum of 10% NaCl for growth [33,34].

Conclusions and Perspective
Within the captivating domain of extreme environments, where salinity reigns supreme, a fascinating assembly of organisms arise as resilient conquerors-halotolerant and halophilic fungi.Residing in saline habitats, these fungi defy the odds, thriving in conditions that test the fundamental essence of life.It has been theorized that such extreme

Conclusions and Perspective
Within the captivating domain of extreme environments, where salinity reigns supreme, a fascinating assembly of organisms arise as resilient conquerors-halotolerant and halophilic fungi.Residing in saline habitats, these fungi defy the odds, thriving in conditions that test the fundamental essence of life.It has been theorized that such extreme environments, characterized by high salt concentrations, could awaken dormant genes and activate unique biosynthesis pathways, potentially leading to the production of structurally distinctive and biologically active secondary metabolites.Consequently, these environments serve as fertile grounds for the discovery of novel compounds and enzymes.The ability of extremophiles to conduct biochemical reactions under extreme conditions offers them a distinct advantage in the production of fuels and chemicals.
Recent advancements in biotechnology have significantly enhanced our understanding of halophilic fungi and their potential applications through the development of innovative methods for characterizing bioactive compounds.Techniques such as metabolomics, genomics, and bioinformatics have enabled researchers to delve deeper into the intricate metabolic pathways of these fungi, uncovering novel bioactive compounds with promising implications across various fields, including biotechnology, agriculture, and medicine.By deciphering the genomes of these organisms, researchers can lay the groundwork for uncovering new biochemical processes, developing innovative applications and products, and gaining insights into the mechanisms by which organisms overcome abiotic stresses.By leveraging advanced analytical tools and multiomics, scientists have overcome challenges associated with studying halophilic fungi, such as difficulties in cultivation and metabolite extraction.Researchers have found that the multiomics approach provides a cost-effective, comprehensive, structured, and interactive overview of biological mechanisms to explore the spectrum from key transcriptional players in the regulation of secondary metabolite biosynthesis and its epigenetic control to approaches for the detection of new gene clusters and substances by genome mining, metagenomics/metatranscriptomics, and metabolomics to the use of secondary metabolite profiles in fungal chemotaxonomy [59].
For instance, Zhou et al. utilized transcriptomics to investigate the expression of the genes involved in the bioactive compound biosynthesis in the medicinal fungi Sanghuang.Their study demonstrated how the multiomics approach offers a cost-effective and comprehensive understanding of biological mechanisms, ranging from transcriptional regulation to genome mining and metabolomics analysis for the detection of new gene clusters and substances [60].Similarly, Gonçalves et al. employed untargeted metabolomics coupled with genome sequencing to explore the chemical diversity of Emericellopsis cladophorae MUM 19.33, revealing a rich repertoire of genes encoding various enzymes, transporters, and secondary metabolite biosynthetic gene clusters.This integrated approach shed light on the resilience mechanisms of fungi against harsh environmental conditions, including the biosynthesis of osmolytes and ion transport systems [61].Additionally, Gómez et al. pioneered the use of multiomics, specifically transcriptomics and metabolomics, to compare the saturation and optimal concentrations of salt for halophilic Aspergillus sydowii fungi.This interdisciplinary collaboration provided valuable insights into the adaptation mechanisms of halophilic fungi to saline environments [62].Overall, the integration of multiomics and interdisciplinary collaboration is crucial for fully exploring the potential of halophilic fungi for bioactive metabolites.These innovative approaches not only facilitate the identification and isolation of bioactive compounds but also contribute to a deeper understanding of the ecological functions and survival strategies of halophilic fungi.
This review delves deeply into the intricacies of salt-loving fungi, probing their unique adaptations and revealing the diverse array of secondary metabolites that hold the potential to revolutionize our understanding of biology and medicine.For instance, the halotolerant fungal strain Aspergillus variecolor B-17, sourced from sediments in the Jilantai salt field, Inner Mongolia, China, has unveiled several intriguing compounds.Variecolorquinones A-B exhibited cytotoxic activity against cancer cell lines, while Variecolorins A-L displayed radical-scavenging properties.Similarly, Variecolortides A-C demonstrated cytotoxic and radical-scavenging activity.Pennicitrinone C and penicitrinol B showcased radical-scavenging abilities.The halotolerant fungus Alternaria raphanin THW-18, isolated from a sea salt field in China, produced cerebrosides and a diketopiperazine alkaloid with antimicrobial activity.Aspergillus sclerotiorum PT06-1 yielded cyclic hexapeptides and cyclic tripeptides with antifungal and cytotoxic properties.Moreover, the halotolerant fungal strain Aspergillus flocculosus PT05-1 generated compounds including a derivative of ergosterol and a red pyrrole pigment with cytotoxic and antimicrobial effects.Similarly, Wallemia sebi PXP-89 produced a cyclopentapyridine alkaloid with antimicrobial efficacy.Compounds such as N-acetyl-2,4,10,17-tetrahydroxyheptadecylamine and Nacetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine exhibited cytotoxicity against cancer cells, while others like perylenequinone showed promise against plant pathogenic fungi.Halotolerant fungi such as Penicillium chrysogenum PXP-55 and Myrothecium sp.GS-17 unveiled cerebrosides, alkaloids, and polyketides with antimicrobial activity.A variety of novel compounds with antimicrobial and cytotoxic properties were also discovered from Penicillium notatum B-52, Aspergillus terreus PT06-2, Aspergillus ochraceus LCJ11-102, and Penicillium sp. from Wadi El-Natrun, Egypt.
In summary, these findings underscore the vast industrial potential of halotolerant fungi, offering a rich source of bioactive compounds for pharmaceutical, agricultural, and other industrial applications.Further exploration and development of these compounds could lead to valuable products with diverse commercial uses.Despite the myriad of advantages and vast potential they offer, the mycobiota of saline environments remain largely unexplored, suggesting that numerous biomolecules with exceptional properties may still lie concealed within.Hence, further research on halophilic fungi is imperative to fully harness their potential.