Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea

The diversity of symbiotic fungi derived from two marine sponges and sediment collected off Obhur, Jeddah (Saudi Arabia), was investigated in the current study. A total of 23 isolates were purified using a culture-dependent approach. Using the morphological properties combined with internal transcribed spacer-rDNA (ITS-rDNA) sequences, 23 fungal strains (in the majority Penicillium and Aspergillus) were identified from these samples. The biological screening (cytotoxic and antimicrobial activities) of small-scale cultures of these fungi yielded several target fungal strains which produced bioactive secondary metabolites. Amongst these isolates, the crude extract of Aspergillus terreus strain S020, which was cultured in fermentation static broth, 21 L, for 40 days at room temperature on potato dextrose broth, displayed strong antimicrobial activities against Pseudomonas aeruginosa and Staphylococcus aureus and significant antiproliferative effects on human carcinoma cells. Chromatographic separation of the crude extract by silica gel column chromatography indicated that the S020 isolate could produce a series of chemical compounds. Among these, pure crystalline terrein was separated with a high yield of 537.26 ± 23.42 g/kg extract, which represents the highest fermentation production of terrein to date. Its chemical structure was elucidated on the basis of high-resolution electrospray ionization mass spectrometry (HRESIMS) or high-resolution mass spectrometry (HRMS), 1D, and 2D NMR spectroscopic analyses and by comparison with reported data. The compound showed strong cytotoxic activity against colorectal carcinoma cells (HCT-116) and hepatocellular carcinoma cells (HepG2), with IC50 values of 12.13 and 22.53 µM, respectively. Our study highlights the potential of A. terreus strain S020 for the industrial production of bioactive terrein on a large scale and the importance of future investigations of these strains to identify the bioactive leads in these fungal extracts.


Sponge Samples
Surface sterilization of the sponge samples was performed. The sponge samples were disinfected with 5% sodium hypochlorite, followed by 70% ethanol [55]. The inner tissues of sponge materials were cut into pieces of approximately 2 cm 3 and homogenized aseptically with sterile, artificial seawater. Three dilutions (1:10, 1:100, and 1:1000) of the resulting homogenate were made with sterile seawater.

Sediment Samples
The deep-sea sediment sample was homogenized aseptically with 15 mL of sterile water, and the resulting solution was diluted by a serial dilution method.
For fungal cultivation, 90 µL of each diluted homogenate was transferred onto plates of each of the following media in triplicate (HiMedia Laboratories, Mumbai, India): Czapek-Dox yeast agar medium (CYE), malt agar medium (ME), and Sabouraud dextrose agar medium (SD). All media were supplemented with an antibiotic (0.25% chloramphenicol) and 2% NaCl. According to Wei's morphological criteria [56], the fungal isolates were identified morphologically on agar plates after 7-14 days incubation at 29 • C. A series of purification and subculture steps were carried out to obtain purified fungal isolates, and photos were taken of each pure isolate ( Figure 1).

Sediment Samples
The deep-sea sediment sample was homogenized aseptically with 15 mL of sterile water, and the resulting solution was diluted by a serial dilution method.
For fungal cultivation, 90 µL of each diluted homogenate was transferred onto plates of each of the following media in triplicate (HiMedia Laboratories, Mumbai, India): Czapek-Dox yeast agar medium (CYE), malt agar medium (ME), and Sabouraud dextrose agar medium (SD). All media were supplemented with an antibiotic (0.25% chloramphenicol) and 2% NaCl. According to Wei's morphological criteria [56], the fungal isolates were identified morphologically on agar plates after 7-14 days incubation at 29 °C. A series of purification and subculture steps were carried out to obtain purified fungal isolates, and photos were taken of each pure isolate ( Figure 1).

DNA Genome Extraction from Pure Subcultured Fungal Isolates
The distinct, pure fungal isolates described above were subcultured in Sabouraud dextrose liquid medium for 3-7 days at 29 °C. The mycelia were separated by filtration and dried using a freeze dryer. According to the manufacturer's instructions, the fungal DNA extraction of the resulting tissues was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The integrity of the extracted DNA was checked and confirmed by gel electrophoresis.

Internal Transcribed Spacer-rDNA (ITS-rDNA) Fragments Amplification, Sequencing, and Phylogenetic Analysis of Fungal Isolates
Using the primers ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [57], the genomic DNA of the fungal isolates was used as the template to amplify fungal ITS-rDNA fragments. For preliminary identification, the sequences of fungal ITS-rDNA regions were compared with related sequences at the National Center for Biotechnology Information (NCBI) as previously described [58][59][60].

Fermentation and Preparation of Extracts of the Fungal Isolate S020
A. terreus strain S020 was cultured under static conditions at room temperature in 2 L Erlenmeyer flasks containing 500 mL of potato dextrose liquid culture medium (PDB). After 40 days of cultivation, 21 L of whole broth was filtered through cheesecloth to separate the culture broth from the mycelia. The broth was extracted three times with ethyl acetate (EtOAc), while the mycelia were extracted three times with methanol (MeOH). Both EtOAc and MeOH extracts showed a similar TLC pattern and so were combined and concentrated to generate a crude extract (17.49 g) for further separation.

Isolation and Purification of Terrein
The total crude extract (17.49 g) of A. terreus strain S020 was subjected to silica gel column chromatography (CC) using gradient elution of n-hexane, CHCl3, and MeOH at a flow rate of about 20 mL/min. Fractions of 100 mL were collected and examined by TLC; similar fractions were combined and evaporated under reduced pressure to obtain fractions 1-10. Fraction 5 (CHCl3 fraction, 13.50 g) was subjected to silica gel CC with CHCl3-MeOH gradient elution to afford seven further fractions. Of these, the bioactive fractions eluted with CHCl3-MeOH (20:1) were concentrated to yield a terrein precipitate (9.20 g, purity 85%) which was finally purified by HPLC (XDB-C18 Zorbax, 5 µm, 250 mm × 4.6 mm) using 20% CH3CN/H2O at a flow rate of 1 mL/min and UV detection at 281 nm.

DNA Genome Extraction from Pure Subcultured Fungal Isolates
The distinct, pure fungal isolates described above were subcultured in Sabouraud dextrose liquid medium for 3-7 days at 29 • C. The mycelia were separated by filtration and dried using a freeze dryer. According to the manufacturer's instructions, the fungal DNA extraction of the resulting tissues was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The integrity of the extracted DNA was checked and confirmed by gel electrophoresis.

Internal Transcribed Spacer-rDNA (ITS-rDNA) Fragments Amplification, Sequencing, and Phylogenetic Analysis of Fungal Isolates
Using the primers ITS1 (5 -TCCGTAGGTGAACCTGCG-3 ) and ITS4 (5 -TCCTCCGCTTATT GATATGC-3 ) [57], the genomic DNA of the fungal isolates was used as the template to amplify fungal ITS-rDNA fragments. For preliminary identification, the sequences of fungal ITS-rDNA regions were compared with related sequences at the National Center for Biotechnology Information (NCBI) as previously described [58][59][60].

Fermentation and Preparation of Extracts of the Fungal Isolate S020
A. terreus strain S020 was cultured under static conditions at room temperature in 2 L Erlenmeyer flasks containing 500 mL of potato dextrose liquid culture medium (PDB). After 40 days of cultivation, 21 L of whole broth was filtered through cheesecloth to separate the culture broth from the mycelia. The broth was extracted three times with ethyl acetate (EtOAc), while the mycelia were extracted three times with methanol (MeOH). Both EtOAc and MeOH extracts showed a similar TLC pattern and so were combined and concentrated to generate a crude extract (17.49 g) for further separation.

Isolation and Purification of Terrein
The total crude extract (17.49 g) of A. terreus strain S020 was subjected to silica gel column chromatography (CC) using gradient elution of n-hexane, CHCl 3 , and MeOH at a flow rate of about 20 mL/min. Fractions of 100 mL were collected and examined by TLC; similar fractions were combined and evaporated under reduced pressure to obtain fractions 1-10. Fraction 5 (CHCl 3 fraction, 13.50 g) was subjected to silica gel CC with CHCl 3 -MeOH gradient elution to afford seven further fractions. Of these, the bioactive fractions eluted with CHCl 3 -MeOH (20:1) were concentrated to yield a terrein precipitate (9.20 g, purity 85%) which was finally purified by HPLC (XDB-C18 Zorbax, 5 µm, 250 mm × 4.6 mm) using 20% CH 3 CN/H 2 O at a flow rate of 1 mL/min and UV detection at 281 nm.
Terrein was characterized as follows: Colorless crystal needles (

Preparation of the Extracts of Isolates S001-S023
The fungal strains S001-S023 were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of the corresponding liquid media (Table 1), incubated at 29 • C, and continuously shaken at 150 rounds per minute (rpm) in an orbital shaker for 14 days. After incubation, 50 mL of EtOAc was added to each flask and left overnight to stop cell growth. The mycelia were separated by filtration, and the filtrate was extracted three times (3 × 50 mL) with EtOAc. The organic portion (combined extracts) was evaporated under vacuum, and the residues obtained were washed with water and then taken to dryness to obtain colored crude extracts (broth extract). Other crude extracts (mycelia extract) were obtained by extraction of mycelia with MeOH and evaporation of the solvent under a vacuum. The resulting EtOAc and MeOH extracts were lyophilized and stored for biological screening.

Antimicrobial and Cytotoxic Activities of Fungal Extracts of Isolates S001-S023
The crude extracts of the broth and mycelial biomass were subjected to antimicrobial activity assessment against four pathogenic microorganisms: P. aeruginosa (ATCC27853), B. subtilis (ATCC6633), S. aureus (ATCC25923) and C. albicans (ATCC76615). Assays were performed by placing 50 µL of the test extract solution (2 mg/mL, DMSO) into each hole on the plates and allowing solutions to stand overnight into an incubator at an appropriate temperature. Activity is indicated by the presence of a clear zone of growth inhibition surrounding the holes. Inhibition zones were measured in mm, and the results are reported in Table 1.
The cytotoxic activity of the test extracts on MCF-7, HCT-116, and HepG2 carcinoma cells was tested using the sulforhodamine B (SRB) assay, as previously described in our studies [54], and the results are presented in Table 2.

Diversity of Culturable Fungal Strains Derived from the Marine Samples
The cultivation of fungal strains from two marine sponges (Stylissa carteri and Hyrtios erectus) and deep-sea sediment of the Red Sea yielded a total of 36 isolates. Based on morphological traits as well as DNA analysis of the ITS regions [57,61], the redundant strains were excluded, and 23 distinct, pure isolates, were identified (S001-S023; Table 3, Figure 1). The strains from Penicillium spp. and Aspergillus spp. accounted for a large proportion of the total isolates. Twenty-three isolates were identified on the basis of morphological traits at the genus and species levels via genomic DNA extraction and sequencing analysis. The phylogenetic tree of fungal strains ( Figure 2) represents those fungi that are easily cultivable and could be recovered when culture-dependent techniques are applied [62]. Representative fungal isolates of these strains that have been previously cultured from marine samples include sponges, algae, cnidarians, and sea grasses [63]. The marine invertebrate symbiotic fungi have been reported as a rich source of bioactive secondary metabolites, such as polyketides, with antimicrobial and/or antitumor activities [14,[62][63][64]. Our study revealed that the diversity of symbiotic fungal isolates (Penicillium, 6 strains; Aspergillus, 17 strains; Pleosporaceae, 1 strain) obtained from the marine samples was high.

Large-Scale Production and Purification of Terrein from A. terreus Strain S020
The culture broth and mycelia of A. terreus strain S020 were extracted using organic solvents, and the combined extracts were fractionated using silica gel column chromatography. The compound terrein was finally purified using a C18 semi-preparative HPLC column.

Structure Elucidation of the Isolated Terrein
Terrein (Figure 3) was isolated and purified as pale yellow crystal needles. The molecular formula, C8H10O3, was determined to be m/z 154.06 by HRESIMS at m/z 155.0698 [M + H]⁺ and m/z 177.0529 [M + Na]⁺. Interpretation of NMR and HRMS data ( Figures S4-S8) suggested that the compound isolated was terrein, as previously reported [28,42].

Large-Scale Production and Purification of Terrein from A. terreus Strain S020
The culture broth and mycelia of A. terreus strain S020 were extracted using organic solvents, and the combined extracts were fractionated using silica gel column chromatography. The compound terrein was finally purified using a C18 semi-preparative HPLC column.

Structure Elucidation of the Isolated Terrein
Terrein (Figure 3) was isolated and purified as pale yellow crystal needles. The molecular formula,  (Figures S4-S8) suggested that the compound isolated was terrein, as previously reported [28,42].

Large-Scale Production and Purification of Terrein from A. terreus Strain S020
The culture broth and mycelia of A. terreus strain S020 were extracted using organic solvents, and the combined extracts were fractionated using silica gel column chromatography. The compound terrein was finally purified using a C18 semi-preparative HPLC column.  Figures S4-S8) suggested that the compound isolated was terrein, as previously reported [28,42].

Antimicrobial Activities of the Fungal Extracts
All 23 fungal isolates were cultured on a small scale, and the crude extracts of their broth and mycelia were tested for antimicrobial activity (against P. aeruginosa ATCC27853, B. subtilis ATCC6633, S. aureus ATCC25923 and C. albicans ATCC76615) using the agar diffusion assay [65]. Fungal isolate extracts displayed different levels of antimicrobial activities against at least one pathogen (Table 1). It is worth pointing out that the extracts of most fungal strains displayed exceptionally high antibacterial activities against P. aeruginosa and S. aureus (inhibition diameters more than 15 mm). Amongst these, the extract of fungal strain S013 exhibited high activity against C. albicans, while other extracts were inactive (Table 1).
From our investigation in this study, we found that extracts of some fungal broths and/or mycelia have antimicrobial effects and that different fungal strains could secrete intracellular and extracellular bioactive metabolic products [66]. Because of the efficacy of these extracts against P. aeruginosa and S. aureus, these extracts might have the potential to serve as drug leads to treat a wide variety of diseases. The results of the antimicrobial assay revealed that fungi derived from the marine source isolated in this study might be a prolific source of active compounds, which may hold potential as antibacterial and antifungal natural compounds.

Antiproliferative Activities of the Fungal Extracts
All 23 fungal isolates were cultured on a small scale, and the crude extracts of their fungal broth and mycelia were tested for antiproliferative activity against MCF-7, HepG2, and HCT-116 cell lines using the SRB-U assay [67]. The extracts showed variable antiproliferative activity against the cell lines under investigation. Amongst these, extracts of S004, S006, S016, S017, and S020 presented the most promising antiproliferative profile (IC 50 values of < 50 µg/mL) ( Table 2). The present study revealed the diversity of the antiproliferative potential of marine fungal extracts and, hence, demonstrated their strong potential to produce cytotoxic compounds. Marine fungi and their purified extracts have been shown to be good producers of antiproliferative and cytotoxic compounds. This property could be attributed to different classes of secondary metabolites, as reported previously [20,[25][26][27][68][69][70][71].

Cytotoxic Activity of the Isolated Compound Terrein
The cytotoxic effect of terrein (Table 4) against HCT-116 and HepG2 cancer cell lines (concentration range 0.01-100 µM) was assessed using the SRB-U assay [67]. The compound displayed strong antiproliferative activity against the two cell lines under investigation, with IC 50 values of 12.13 µM and 22.53 µM for HCT-116 and HepG2 cells, respectively. Doxorubicin was used as a standard cytotoxic control. Doxorubicin positive cytotoxic control, presented as the mean ± SD; n = 3.

Conclusions
In conclusion, a total of 23 symbiotic fungi distributed among 3 genera were identified and isolated from marine sponges and sediment collected off Obhur (Saudi Arabia). The biological screening of small-scale cultures of these fungi yielded several target fungal strains which produced secondary bioactive metabolites. Amongst these isolates, the chromatographic separation of the crude extract of A. terreus strain S020 by silica gel column chromatography led to the isolation of pure crystalline terrain, with a high yield of 537.26 ± 23.42 g/kg dried crude extract; this represents the highest fermentation production of terrein to date. The chemical structure was elucidated on the basis of HRMS, 1D, and 2D NMR spectroscopic analyses, and by comparison with reported data. The isolated compound, terrein, showed strong cytotoxic activity against colorectal carcinoma cells and hepatocellular carcinoma cells, with IC 50 values of 12.13 and 22.53 µM, respectively. Our study contributes to the understanding of fungal diversity and provides the basis for future investigations of these symbionts with respect to identifying and purifying the bioactive leads in these fungal extracts. This study also describes an efficient approach for producing bioactive terrein in a very high yield. Currently, there is great demand for the development of new drugs to combat the emergence of bacterial resistance to traditional antibiotics.

Conflicts of Interest:
All contributing authors declare no conflict of interest to disclose, whether financial or of any other nature.