Genomic and Metabolomic Analyses of the Marine Fungus Emericellopsis cladophorae: Insights into Saltwater Adaptability Mechanisms and Its Biosynthetic Potential

The genus Emericellopsis is found in terrestrial, but mainly in marine, environments with a worldwide distribution. Although Emericellopsis has been recognized as an important source of bioactive compounds, the range of metabolites expressed by the species of this genus, as well as the genes involved in their production are still poorly known. Untargeted metabolomics, using UPLC- QToF–MS/MS, and genome sequencing (Illumina HiSeq) was performed to unlock E. cladophorae MUM 19.33 chemical diversity. The genome of E. cladophorae is 26.9 Mb and encodes 8572 genes. A large set of genes encoding carbohydrate-active enzymes (CAZymes), secreted proteins, transporters, and secondary metabolite biosynthetic gene clusters were identified. Our analysis also revealed genomic signatures that may reflect a certain fungal adaptability to the marine environment, such as genes encoding for (1) the high-osmolarity glycerol pathway; (2) osmolytes’ biosynthetic processes; (3) ion transport systems, and (4) CAZymes classes allowing the utilization of marine polysaccharides. The fungal crude extract library constructed revealed a promising source of antifungal (e.g., 9,12,13-Trihydroxyoctadec-10-enoic acid, hymeglusin), antibacterial (e.g., NovobiocinA), anticancer (e.g., daunomycinone, isoreserpin, flavopiridol), and anti-inflammatory (e.g., 2’-O-Galloylhyperin) metabolites. We also detected unknown compounds with no structural match in the databases used. The metabolites’ profiles of E. cladophorae MUM 19.33 fermentations were salt dependent. The results of this study contribute to unravel aspects of the biology and ecology of this marine fungus. The genome and metabolome data are relevant for future biotechnological exploitation of the species.


Introduction
The genus Emericellopsis was introduced by Kingma [1] and is distributed worldwide. Species of Emericellopsis can be found in association with seaweeds in estuarine or marine habitats, but also in soils, peat, and rhizomes [2][3][4]. However, it is in saline environments that Emericellopsis species appear more frequently and thrive: macroalgae, sponges, sea and estuarine waters, marine sediments and even in soils with periodic flooding and extreme humidity and alkalinity [2][3][4][5][6][7].
It has been demonstrated that species of Emericellopsis are important sources of bioactive metabolites, such as peptaibols with antibacterial and antifungal activities [8,9]. The Norine database [10], which is dedicated to non-ribosomal peptide synthases (NRPS),
Predicted genes were functionally annotated with OmicsBox using Blast2GO [30] against NCBI's nonredundant (Nr) database, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) with an e-value threshold of 1 × 10 −3 . Proteins were classified using InterProScan and the Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG), which also contains the orthologous groups from the original COG/KOG database (euKaryotic cluster of Orthologous Groups of proteins) with an e-value of 1 × 10 −3 .

Comparative Analyses with E. atlantica
The genome of Emericellopsis cladophorae MUM 19.33 was compared with the genome of E. atlantica TS7. To evaluate the genetic and metabolic diversity of both species the information available in JGI Genome Portal database, such as genome size, GC content, CAZymes, BGCs abundance was used.

Small-Scale Fermentation and Extraction of Metabolites
A small-scale fermentation was carried out as described by Gonçalves et al. [18]. Briefly, two plugs of mycelium-colonized agar were inoculated into 1-L Erlenmeyer flasks containing 250 mL of PDB in two conditions: with and without 3% sea salt (Sigma-Aldrich, Darmstadt, Germany) with 4 replicates for each condition. The fungus was grown at 25 • C under stationary conditions for 14 days. Culture media were obtained by filtering the mycelium through sterile filter paper. Then, the culture media was filtrated with 0.45 µm cellulose membrane (GN-6 Metricel, Pall Corporation, New York, NY, USA) followed by 0.2 µm nitrate cellulose membrane (Sartorius Stedim Biotech, Gottingen, Germany) in a vacuum system. Culture media from the 4 replicates were pooled and lyophilized, and dried culture media were weighed and transferred to tubes. Next, 20 mL of cold 80% MeOH (−80 • C) was added to each tube (containing 2 g of lyophilized sample) and vortexed for 5 min. Each mixture was centrifuged at 14,000× g for 10 min at 4 • C to remove precipitated proteins. The supernatant was collected, and the extraction process was repeated once. After extraction, the methanolic extracts were filtered using a glass microfiber filter 0.47 mm (Prat Dumas, Couze-St-Front, France), evaporated in vacuo using a rotary evaporator and lyophilized.
For LC-MS, 5 replicates of dried crude extracts (100 mg) for each condition were used. Metabolite extraction was performed by adding MeOH to each of the samples allowing them to vortexed for 40 min. Then, the samples were centrifuged for 5 min at 20,000× g and 400 µL of the methanolic fraction was vacuum dried. Afterwards, 100 µL of cyclohexane/water (1/1, v/v) was added to each sample and vortexed. Each mixture was centrifuged at 20,000× g for 5 min and 90 µL of the aqueous phase was filtered on a 96-filter plate and transferred to a 96-well plate. The samples were 10× diluted in water and 10 µL was analyzed by LC-MS.
2.6. LC-MS Data Analysis, Processing, and Visualization UHPLC was performed on an ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) consisting of a binary pump, a vacuum degasser, an autosampler, and a column oven. Chromatographic separation was carried out on an ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 µm, Waters Corporation, Milford, MA, USA), and temperature was maintained at 40 • C. A gradient of solution A (99:1:0.1 water:acetonitrile:formic acid, pH 3) and solution B (99:1:0.1 acetonitrile:water:formic acid, pH 3) was used: 99% A for 0.1 min decreased to 50% A in 30 min, decreased to 30% in 5 min, decreased to 0% in 2 min. The flow rate was set to 0.35 mL min −1 , and the injection volume was 10 µL.
The UHPLC system was coupled to a Vion IMS QTOF hybrid mass spectrometer (Waters Corporation, Milford, MA, USA). The LockSpray ion source was operated in negative electrospray ionization mode under the following specific conditions: capillary voltage, 2.5 kV; reference capillary voltage, 3 kV; cone voltage, 40 V; source offset, 50 V; source temperature, 120 • C; desolvation gas temperature, 600 • C; desolvation gas flow, 800 L h −1 ; and cone gas flow, 50 L h −1 . Mass range was set from 50 to 1000 Da. The collision energy for full HDMSe was set at 6 eV (low energy) and ramped from 20 to 70 eV (high energy), intelligent data capture intensity threshold was set at 5. Nitrogen (greater than 99.5%) was employed as desolvation and cone gas. Leucin-enkephalin (250 pg µL −1 in water:acetonitrile 1:1 [v/v], with 0.1% formic acid) was used for the lock mass calibration, with scanning every 2 min at a scan time of 0.1 s. Profile data were recorded through a UNIFI Scientific Information System (Waters Corporation). Data processing was performed with Progenesis QI software v.2.4 (Waters Corporation). To understand the metabolomic fluctuations in response to sea salt, a IQR (interquartile range) filtering was applied because of a large number of significant ions, resulting in a selection of a set of 2500 ions for the data modeling. The log-transformed and pareto-scaled (normalized) LC-MS integration values of the filtered compound ions were analyzed (data not shown). Principal Component Analysis (PCA), heatmaps, and t-test on log-transformed and pareto-scaled (normalized) of the filtered ions were generated and using online MetaboAnalyst v.4.0 software [35]. Computed p-values were adjusted using the Benjamin-Hochberg False Discovery Rate (FDR) correction. Ions having an FDR < 0.01 and a log 2 fold change (FC) >2 or <−2 were considered differently expressed. For identification purposes, the fragmentation data (ESI negative) of the significant ions were selected and matched against in-house library and 44 external spectral libraries (https://mona.fiehnlab.ucdavis.edu/, accessed on 3 December 2020), using MSsearch software. For each ion, the best hit was based on a matching precursor ion (m/z < 10 ppm difference) and matching fragments (<50 ppm accuracy), generating 5 common fragments, including the precursor m/z. For each hit, the name of the matching compound followed by the collision energy used, the parent ion as a nominal mass, the chemical formula, a matching factor (MF), a reverse matching factor (RMF), and the name of the library found were obtained (File S1). File S1 contains some positive ionizations, but only ions in negative mode were considered for identification. Thus, annotation was done at level 2 of the Metabolomics Standards Initiative (MSI).

Sequencing, Assembly Data and Genomic Characteristics
General data related to the draft genome of Emericellopsis cladophorae MUM 19.33 is presented in Table 1. Briefly, the E. cladophorae genome size was estimated at 26.7 Mb, assembled in 300 contigs, with 8572 predicted genes from which 41.3% encode for hypothetical proteins, and a GC content of 54.34%.

Repetitive Sequences and of tRNAs
Repetitive sequences are classified as Dispersed Repeats (DRs) and Tandem Repeats (TRs). The total length of the 5258 DRs in E. cladophorae MUM 19.33 genome is 261,095 bp, covering 0.97% of the genome. With respect to the TRs, there are 2365 sequences with a total length of 232,036 bp covering 0.86% of the genome. 122 tRNAs were also predicted, with a total length of 10,432 bp covering 0.04% of the genome ( Table 2). Among the tRNAs, 5 are possible pseudogenes and the remaining 117 anti-codon tRNAs correspond to the 20 common amino acid codons.
Polysaccharides represent some of the most abundant bioactive substances in marine organisms representing a good resource of nutrients [39]. Thus, the presence of specific CAZymes in E. cladophorae allows the utilization of polysaccharides-chitin, starch and other marine polysaccharides such as fucoidan and ulvan present in brown and green algae is concordant with E. cladophorae being a guest of the filamentous green alga Cladophora sp. [7].

Transporter Proteins
Transport proteins are classified into five well defined classes according to the transport protein classification (TC) system [33]: channels and pores (TC 1), electrochemical potentialdriven transporters (TC 2), primary active transporters (TC 3), group translocators (TC 4), and transmembrane electron carriers (TC 5), accessory factors involved transport (TC 8) and incompletely characterized transport systems (TC 9). Emericellopsis cladophorae MUM 19.33 genome encodes transporters (2197 genes) from all the TC classes, accounting for 25.6% of the total predicted genes of E. cladophorae (Tables 3 and S4). TC 2 class accounted for 25.9% of transporters in E. cladophorae genome. This transporters' class encompass the Major Facilitator Superfamily (MFS), which can transport molecules, controlling membrane homeostasis and regulate internal pH and the stress response machinery in fungi [40]. It has been demonstrated that many of MFS transporters are required for fungi to grow under stress conditions [41] and play an important role in multidrug resistance [42]. Genes encoding transporters of glycerol, inositol, sodium, and chloride were found. Other transporters' encoding genes are related to the salt overly sensitive signaling pathway, a well-defined pathway in plants to maintain cellular ion homeostasis by restricting the accumulation of sodium [43]. It is known that at high salinity, fungi maintain osmotic balance mainly by the increased production and accumulation of glycerol or other compatible solutes, such as inositol, mannitol, arabitol, xylitol and nitrogen containing compounds (e.g., glycine, betaine) [44]. Emericellopsis cladophorae genome contains genes involved in glycerol, mannitol, inositol, sorbitol, trehalose, glycine, and betaine biosynthetic process, suggesting that E. cladophorae has adaptability mechanisms to maintain positive turgor pressure allowing the interplay of osmolyte transporters. The second most abundant transporter class in E. cladophorae is TC 1 accounted for 21% of transporters. These transporters are mainly associated to ionic homeostasis allowing rapid changes in cell physiology [45]. We identify transporters' encoding genes likely to encode for calcium channels, nucleoporins, aquaporins, among others. Aquaporins are fundamental in all living organisms for maintenance of water equilibrium and their cellular shape and turgor [46]. But to date, little is known about aquaporins in filamentous fungi. In yeasts for example, aquaporins play important roles in establishment of freeze tolerance, spore formation, and cell surface properties for adhesion [47]. We believe that some transporters play a crucial role in ions and osmolytes transport that allow marine fungi to thrive in saltwater, thereby playing roles in nutrient uptake and osmoregulation.
Ascochlorin is an isoprenoid antibiotic isolated from Acremonium egyptiacum (syn. A. sclerotigenum), previously known as the phytopathogenic fungus Ascochyta viciae [48]. Ascochlorin or related compounds have been reported to show antiviral and antitumor activities [49] and was found in some ascomycetes, including Acremonium-like or Emericellopsis species [22]. The ascochlorin cluster is constituted by eight genes: ascA (prenyltransferase), ascB (NRPS-like oxidoreductase), ascC (polyketide synthase), ascD (halogenase), ascE (P450 monooxygenase/P450 reductase), ascF (terpene cyclase), ascG (cytochrome P450) and ascR (transcription regulator). This gene architecture was found in E. cladophorae, but the ascB gene is missing ( Figure 2A). Also, near to this cluster other three genes coding for diphthamide, DNA mismatch repair and alcohol dehydrogenase were identified. This similar cluster may indicate that E. cladophorae can be producer of ascochlorin or a related compound.  Although these BGCs have been found on the genome of E. cladophorae, none of the resulting compounds were detected in metabolic analysis of the dried crude extracts (Section 3.9). In fact, it has been reported that BGCs may remain silent under laboratory culture conditions and poses a challenge for identifying marine natural products in fungi [57]. To overcome this, different fermentation culture conditions should be used to 'awake' or stimulate the expression of silent genes. Different strategies such as the use of Cephalosporins are among the most-widely used drugs for treatment of infections and belongs to the family of beta-lactam antibiotics. From the cephalosporin group, Cephalosporin C is the major source for production of 7-amino cephalosporanic acid (7-ACA) [50]. It has been demonstrated that Cephalosporin C is the major compound produced by Emericellopsis species, mainly by E. minima and E. salmosynnemata [51]. Cephalosporin C was isolated from Acremonium chrysogenum (syn. Cephalosporium acremonium) and its biosynthesis is well elucidated [52]. This cluster is comprised by cefT (MFS transporter), D-lactate dehydrogenase (ORF 3), pcbAB (ACV synthetase), pcbC (IPN synthase), cefD2 (IPN CoA epimerase), cefD1 (IPN CoA synthetase) and cefM (MFS transporter). Also, two other genes, cefEF (deacetoxycephalosporin C synthase) and cefG (acetyl CoA) were involved in cephalosporin C biosynthesis but belong to a different cluster [53]. In E. cladophorae, only four genes of Cephalosporin C cluster are present ( Figure 2B) namely pcbAB, pcbC, cefD1 and cefM, which is essential for cephalosporin biosynthesis [53]. Additionally, cefD2, cefT and D-lactate dehydrogenase lack, but another MFS transporter located near to cefM was detected, which may have the same function of cefT that is responsible for cephalosporin secretion from the cell.
Although these BGCs have been found on the genome of E. cladophorae, none of the resulting compounds were detected in metabolic analysis of the dried crude extracts (Section 3.9). In fact, it has been reported that BGCs may remain silent under laboratory culture conditions and poses a challenge for identifying marine natural products in fungi [57]. To overcome this, different fermentation culture conditions should be used to 'awake' or stimulate the expression of silent genes. Different strategies such as the use of epigenetic modifiers, of natural or chemical elicitors and the co-cultivation with other species have been used to induce the production of BGCs coded compounds [57]. This strategy, known as OSMAC (One Strain MAny Compounds) approach, which can activate many silent BGCs in microorganisms to induce the expression of more natural products and of those that are poorly expressed [58].

High-Osmolarity Glycerol (HOG) Pathway
Mitogen-activated protein kinase (MAPK) pathways have been previously identified in Saccharomyces cerevisiae, regulating diverse physiological processes such as osmoregulation and nutrient-sensing [59]. These processes are mainly controlled by the high-osmolarity glycerol (HOG) signaling pathway, allowing to adapt to external hyperosmotic stress. In marine fungi, high levels of salinity lead to osmotic and ionic stress [60]. However, it has been proposed that these organisms can maintain positive turgor pressure in a hypertonic environment, through the HOG pathway [61]. This pathway is responsible for regulation of salt efflux pumps and creation of osmolytes compatible with cellular functions [62].
The genes essential for the MAPK high osmolarity cascade were identified in the genome of E. cladophorae (Figure 3). These genes have been previously identified in S. cerevisiae [59], Candida albicans [63], Aspergillus spp. [64], Neurospora crassa [65], and Magnaporthe oryzae [59]. MAPK osmolarity cascade can be activated by osmosensors (Sho1 and Sln1) and response regulator proteins, resulting in Hog1 activation [66]. Emericellopsis cladophorae has the Sln1 and Sho1 cascades (Sln1-Ypd1-Ssk1-Ssk2-Pbs2-Hog1 and Sho1-Cdc42-Ste20(or Cla4)-Ste11-Pbs2-Hog1) to respond to changes in osmolarity in the extracellular environment. The activation of HOG-MAPK pathway ensures the accumulation of a high concentration of glycerol in the cytoplasm to reduce the osmotic pressure and prevent water losses. Also, we identified genes essential for the cascades of MAPK cell wall stress, resulting in cell wall remodeling.
lation and nutrient-sensing [59]. These processes are mainly controlled by the high-osmolarity glycerol (HOG) signaling pathway, allowing to adapt to external hyperosmotic stress. In marine fungi, high levels of salinity lead to osmotic and ionic stress [60]. However, it has been proposed that these organisms can maintain positive turgor pressure in a hypertonic environment, through the HOG pathway [61]. This pathway is responsible for regulation of salt efflux pumps and creation of osmolytes compatible with cellular functions [62].
The genes essential for the MAPK high osmolarity cascade were identified in the genome of E. cladophorae (Figure 3). These genes have been previously identified in S. cerevisiae [59], Candida albicans [63], Aspergillus spp. [64], Neurospora crassa [65], and Magnaporthe oryzae [59]. MAPK osmolarity cascade can be activated by osmosensors (Sho1 and Sln1) and response regulator proteins, resulting in Hog1 activation [66]. Emericellopsis cladophorae has the Sln1 and Sho1 cascades (Sln1-Ypd1-Ssk1-Ssk2-Pbs2-Hog1 and Sho1-Cdc42-Ste20(or Cla4)-Ste11-Pbs2-Hog1) to respond to changes in osmolarity in the extracellular environment. The activation of HOG-MAPK pathway ensures the accumulation of a high concentration of glycerol in the cytoplasm to reduce the osmotic pressure and prevent water losses. Also, we identified genes essential for the cascades of MAPK cell wall stress, resulting in cell wall remodeling.

Comparison of Genome Features between E. cladophorae MUM 19.33 and E. atlantica TS7
The genus Emericellopsis harbors 23 species described so far. Recently, Hagestad et al. [22] sequenced the first genome of an Emericellopsis species: E. atlantica strain TS7. The genome assembly and gene statistics for E. cladophorae and E. atlantica are summarized in Table 4. The E. cladophorae MUM 19.33 genome size is smaller (1.5%), has a slightly higher

Comparison of Genome Features between E. cladophorae MUM 19.33 and E. atlantica TS7
The genus Emericellopsis harbors 23 species described so far. Recently, Hagestad et al. [22] sequenced the first genome of an Emericellopsis species: E. atlantica strain TS7. The genome assembly and gene statistics for E. cladophorae and E. atlantica are summarized in Table 4. The E. cladophorae MUM 19.33 genome size is smaller (1.5%), has a slightly higher GC content (0.3%) and has 14% less genes than E. atlantica TS7. Both genomes share several conserved genes in biosynthetic gene clusters of ascochlorin, leucinostatin A/B and cephalosporin C. However, some BGCs were species specific, such as those encoding for helvolic acid, botrydial and fusaristatin A in E. atlantica, while clavaric acid, EQ-4, squalestatin S1 and (-)-Mellein in E. cladophorae.
The number of predicted genes encoding putative CAZymes of E. cladophorae is higher (2.7%) than that of E. atlantica. These differences might define the kind of carbon sources/substrates these species can use, possibly reflecting an adaption to their hosts: as mentioned above E. cladophorae was isolated from the alga Cladophora sp., and E. atlantica from the sponge Stelletta normani [22]. Such carbon sources include typically marine polysaccharides, such as agarose, alginate, carrageenan, chitin, fucoidan, laminarin, ulvan, among others [67]. Emericellopsis cladophorae contains enzymes capable to degrade these polysaccharides, such as 4 and 5 genes encoding fucosinases (GH29 and GH95) and ulvanases, respectively. These enzymes are involved in degradation of algal fucoidan and ulvan. Nineteen genes were also detected with fucose activity (GT1 and GT31) and 27 genes encoding chitinases (GH18) and 17 for chitin recognition (CBM18). The presence of specific CAZymes relating to the utilization of marine polysaccharides indicates an adaption to this environment.

Metabolome Analysis
The influence of salt on the metabolomic profile of E. cladophorae MUM 19.33, was characterized using an untargeted metabolomic approach as described above. Quintuplicate profiles were combined for each condition for comparative analysis. The full list of ions can be found in Table S6. Despite the presence of some unknow compounds, the major classes identified were polyketides, phenolic compounds, terpenes, amino acids, drugs, mycotoxins, carbohydrates, carboxylic acids, fatty acids, alkaloids, and indoles.
The scores of the PCA on all filtered compound ions clearly revealed dissimilarities in the metabolome of the salted and non-salted extracts of E. cladophorae (Figure 4). The growth of E. cladophorae grows in media with and without sea salt [7] is accompanied by a different metabolic profile, suggesting that this species has the molecular tools to survive in both media. major classes identified were polyketides, phenolic compounds, terpenes, amino acids, drugs, mycotoxins, carbohydrates, carboxylic acids, fatty acids, alkaloids, and indoles.
The scores of the PCA on all filtered compound ions clearly revealed dissimilarities in the metabolome of the salted and non-salted extracts of E. cladophorae (Figure 4). The growth of E. cladophorae grows in media with and without sea salt [7] is accompanied by a different metabolic profile, suggesting that this species has the molecular tools to survive in both media. Subsequently, statistical testing on the filtered compound ions was done using a ttest. Computed p-values were adjusted using the false discovery rate (FDR) correction. 606 ions having an FDR < 0.01 and a log2 fold change (FC) > 2 or <−2 were present in significantly different quantities: 414 and 192 ions were up-and down-regulated in the salted extracts, respectively (Table S7, Figure 5). Due to the lack of information on the MS databases (1 in-house library and MoNA), many of these compounds remain unidentified. We had already suggested [18] that sea salt induces an alteration to the metabolic profile of E. cladophorae. For example, the compounds ions annotated as ergocryptine, 2'-O-Galloylhyperin, (-)-Gallocatechin 3-gallate, N-[1-(4-methoxy-6-oxopyran-2-yl)-2-methylbutyl]acetamide, ferulic acid ethyl ester, 6-Methoxymethylone, 3'-O-Methylguanosine, heptanedioic acid, melezitose, salicylic acid, 5-Sulfosalicylic acid, N4-Acetylsulfadiazine, 5'-Iodoresiniferatoxin, and tafluprost acid were more abundant in salted medium. While the compounds annotated as maltotriose, L-N5-(1-Imino-3-pentenyl)ornithine, hymeglusin, formylciprofloxacin, epiyangambin, lactobionic acid, folinic acid, gatifloxacin, 5-Hydroxymethylcytidine, 4-Hydroxyatorvastatin lactone, and fulvestrant 9-sulfone under non-salted conditions. It is interesting to note that salt induces an increase on the quantity of metabolites produced by E. cladophorae, specifically of amino acids and peptides. It has been suggested that a selected group of physiologically compliant organic osmolytes, the compatible solutes, amasses when the environmental osmolality is raised in the cytoplasm upon hyperosmotic challenge [68].
Analysis of E. cladophorae extracts by LC-MS proved effective in detecting bioactive compounds that have been reported for their multiple activities, such as, antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and antioxidant (Table 5). Emericellopsis cladophorae produces diverse metabolites involved in carbohydrate metabolism such as guanosine, maltose, maltotriose, laminaritetraose, palatinose and others, which could be related to the fermentation medium (PDB) that was used. The main constituent of this Subsequently, statistical testing on the filtered compound ions was done using a t-test. Computed p-values were adjusted using the false discovery rate (FDR) correction. 606 ions having an FDR < 0.01 and a log 2 fold change (FC) > 2 or <−2 were present in significantly different quantities: 414 and 192 ions were up-and down-regulated in the salted extracts, respectively (Table S7, Figure 5). Due to the lack of information on the MS databases (1 inhouse library and MoNA), many of these compounds remain unidentified. We had already suggested [18] that sea salt induces an alteration to the metabolic profile of E. cladophorae. For example, the compounds ions annotated as ergocryptine, 2'-O-Galloylhyperin, (-)-Gallocatechin 3-gallate, N-[1-(4-methoxy-6-oxopyran-2-yl)-2-methylbutyl] acetamide, ferulic acid ethyl ester, 6-Methoxymethylone, 3'-O-Methylguanosine, heptanedioic acid, melezitose, salicylic acid, 5-Sulfosalicylic acid, N4-Acetylsulfadiazine, 5'-Iodoresiniferatoxin, and tafluprost acid were more abundant in salted medium. While the compounds annotated as maltotriose, L-N5-(1-Imino-3-pentenyl)ornithine, hymeglusin, formylciprofloxacin, epiyangambin, lactobionic acid, folinic acid, gatifloxacin, 5-Hydroxymethylcytidine, 4-Hydroxyatorvastatin lactone, and fulvestrant 9-sulfone under non-salted conditions. It is interesting to note that salt induces an increase on the quantity of metabolites produced by E. cladophorae, specifically of amino acids and peptides. It has been suggested that a selected group of physiologically compliant organic osmolytes, the compatible solutes, amasses when the environmental osmolality is raised in the cytoplasm upon hyperosmotic challenge [68].
Analysis of E. cladophorae extracts by LC-MS proved effective in detecting bioactive compounds that have been reported for their multiple activities, such as, antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and antioxidant (Table 5). Emericellopsis cladophorae produces diverse metabolites involved in carbohydrate metabolism such as guanosine, maltose, maltotriose, laminaritetraose, palatinose and others, which could be related to the fermentation medium (PDB) that was used. The main constituent of this medium is starch, a complex polysaccharide, widely utilized by fungi to obtain carbon and produce energy. Therefore, when the growth medium contains an excess of carbon source, fungi have the ability to accumulate carbohydrates [69]. Notably, we detected many genes involved in carbohydrate metabolism that were annotated in GO, KEGG and EggNOG databases. For example, in starch and sucrose metabolism, we identified genes encoding for glycosylases, hydrolases and cellulases, glycolysis/gluconeogenesis, pyruvate, amino and nucleotide sugars, galactose, and inositol. In addition, it was also reported in A. niger a high concentration of citric acid when grown in sugar medium [70], as we also detected in E. cladophorae. source, fungi have the ability to accumulate carbohydrates [69]. Notably, we detected many genes involved in carbohydrate metabolism that were annotated in GO, KEGG and EggNOG databases. For example, in starch and sucrose metabolism, we identified genes encoding for glycosylases, hydrolases and cellulases, glycolysis/gluconeogenesis, pyruvate, amino and nucleotide sugars, galactose, and inositol. In addition, it was also reported in A. niger a high concentration of citric acid when grown in sugar medium [70], as we also detected in E. cladophorae. According to our metabolomic analyses, a large number of amino acids and peptides was detected: Phe, Val-Leu, Pro-Ile, Leu-Gln, Try-Leu and others (Table S5). Considering the GO, KEGG and EggNOG analyses, several genes involved in the amino acid metabolism were found encoding for cysteine and methionine metabolism, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism and valine, leucine, and isoleucine biosynthesis.
The number of fungal infections-invasive candidiasis, pneumonia, aspergillosis, cryptococcal meningitis and histoplasmosis-has increased over the last years [71]. With the prevalence of antifungal resistance, it is an urgent and unmet need to develop novel and safe antifungal drugs with novel modes of action, because the few treatments available remain unsatisfactory [72]. We detected some derivative antifungal compounds in our metabolomic analyses such as 9,12,13-Trihydroxyoctadec-10-enoic acid, phosphatidylethanolamine, hymeglusin and salicylic acid. Hymeglusin was also identified in marine-derived fungus Fusarium solani, which was isolated from the mangrove sediments, with antifungal activities against tea pathogenic fungi Pestalotiopsis theae and Colletotrichum gloeosporioides [73]. According to our metabolomic analyses, a large number of amino acids and peptides was detected: Phe, Val-Leu, Pro-Ile, Leu-Gln, Try-Leu and others (Table S5). Considering the GO, KEGG and EggNOG analyses, several genes involved in the amino acid metabolism were found encoding for cysteine and methionine metabolism, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism and valine, leucine, and isoleucine biosynthesis.
The number of fungal infections-invasive candidiasis, pneumonia, aspergillosis, cryptococcal meningitis and histoplasmosis-has increased over the last years [71]. With the prevalence of antifungal resistance, it is an urgent and unmet need to develop novel and safe antifungal drugs with novel modes of action, because the few treatments available remain unsatisfactory [72]. We detected some derivative antifungal compounds in our metabolomic analyses such as 9,12,13-Trihydroxyoctadec-10-enoic acid, phosphatidylethanolamine, hymeglusin and salicylic acid. Hymeglusin was also identified in marine-derived fungus Fusarium solani, which was isolated from the mangrove sediments, with anti-fungal activities against tea pathogenic fungi Pestalotiopsis theae and Colletotrichum gloeosporioides [73].
Meir and Osherov [74] suggested that vitamin biosynthesis could be antifungal targets. Several genes from E. cladophorae were annotated as encoding for metabolism of cofactors and vitamins, particularly of vitamin B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), B6 (pyridoxine), B7 (biotin) and B9 (folate). Compounds as (-)-Riboflavin and pantothenic acid were also detected in metabolomic analysis. In fungi, these vitamins are important for cellular processes as iron homeostasis [74]. Several filamentous fungi and yeasts, such as Aspergillus niger, A. terreus, A. flavus, Penicillium chrysogenum, and Fusarium and Candida spp. were reported as natural flavin producers capable of synthesizing riboflavin [75]. A recent study has shown that Emericellopsis alkalina produces the antimicrobial peptides Emericellipsins A-E. These compounds have a strong activity against drug-resistant pathogenic fungi (Aspergillus niger, A. terreus, A. fumigatus, Candida albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilopsis, Cryptococcus neoformans and Cryp. laurentii), thus indicating its ability act against aspergillosis and cryptococcosis [8]. In a recent study, Gonçalves et al. [18] did not observe antifungal activity against Candida spp. in E. cladophorae extracts, a close relative to E. alkalina [7]. The observed effects may be related to the fermentation culture conditions or extractions methods. Kuvarina et al. [8] used an alkaline medium (pH 10.5) containing malt and yeast extract with ethyl acetate as solvent to obtain the crude extracts, while Gonçalves et al. [18] used PDB and methanol. Additionally, Hagestad et al. [22] used 11 different fermentation culture media and observed different bioactivities profiles, indicating the likelihood of expression of different compounds.
Antibiotic resistance is a major public health concern and a most serious challenges of our time. In this regard, finding effective solutions to address this problem is crucial. Antibacterial compounds were detected in metabolomic analyses, mainly NovobiocinA and N4-Acetylsulfadiazine. Emericellopsis cladophorae genome contains genes involved, not only in the biosynthesis of novobiocin, but also in monobactam, streptomycin, carbapenem, penicillin and cephalosporin biosynthesis. Although these molecules were not detected, we cannot discard the presence of derivative compounds. Apart from biosynthetic gene cluster of Cephalosporin C, the genes mentioned above were also detected in genome of E. atlantica [22]. However, only penicillins and cephalosporins have been registered as natural products from Emericellopsis [76].
The crude extract of E. cladophorae contains metabolites used in chemotherapy, such as daidzein, daunomycinone, isoreserpin, 3,4-dihydroxycinnamic acid and flavopiridol. To our knowledge, this is the first time that these compounds have been described in a fungus, with exception of 3,4-dihydroxycinnamic acid, which was identified from Pycnoporus cinnabarinus [77]. Additionally, rohitukine, a precursor of flavopiridol, was isolated from F. proliferatum, F. oxysporum and F. solani [78]. Hence, the discovery of novel drugs with an increased efficacy for the treatment of different cancers is vital. Therefore, E. cladophorae could be an interesting candidate to produce compounds used for anticancer therapy.

Conclusions
This study unveils the genome and the metabolome of the algae-associated fungus, E. cladophorae strain MUM 19.33. The genome sequence analysis includes many hypothetical proteins, which is directly related to the lack of sequencing data. Sequencing marine fungal genomes will increase data availability, which can be used as templates for further sequencing analysis. Furthermore, marine fungal genome sequencing allows us to unveil the full biosynthetic potential of compounds with medical, pharmaceutical, and biotechnological applications. Genome sequencing also allows us to disclose fungal biology and specific genomic signatures. To understand the adaptability of fungi to marine environment, an in-depth study is necessary to properly access the function of specific CAZymes related to marine polysaccharides. Our findings also underline that the metabolites produced by E. cladophorae were responsive to sea salt. Fungal species present in marine environment are expected to have adapted for tolerance to high sodium and chloride concentrations. However, the involvement of these transporters in adaptability mechanisms to osmotic stress in marine fungi is unclear. Also, additional transcriptome and gene expression analyses will help enhance the integrity of this study.
Emericellopsis cladophorae is capable of producing a range of antifungal derived compounds, among others. Further studies using are required to confirm the production of these metabolites unraveling the full potential of E. cladophorae. Also, different culture media, alternative extractions methods and genetic engineering experiments are essential in the future to produce, isolate, and characterize putatively novel compounds.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof8010031/s1, Table S1: Gene annotation, Table S2: Carbohydrate active enzymes prediction, Table S3: Secreted proteins, Table S4: Transporter's prediction, Table S5: Biosynthetic Gene Clusters, Table S6: Full list of compounds, Table S7: List of the significantly differential compounds, File S1: matched spectral library compounds.  Data Availability Statement: This Whole-Genome Shotgun project has been deposited in the Gen-Bank database under the accession number JAGIXG000000000. The genome raw sequencing data and the assembly reported in this paper is associated with NCBI BioProject: PRJNA718178 and BioSample: SAMN18524397 within GenBank. The SRA accession number is SRR14127580. Data generated or analyzed during this study are included in this published article and its supplementary information files.