Bioactive Metabolites from the Mariana Trench Sediment-Derived Fungus Penicillium sp. SY2107

Mariana Trench sediments are enriched in microorganisms, however, the structures and bioactivities of their secondary metabolites are not very known. In this study, a fungus Penicillium sp. SY2107 was isolated from a sample of Mariana Trench sediment collected at a depth of 11000 m and an extract prepared from the culture of this fungus in rice medium showed antimicrobial activities. Chemical investigation on this active extract led to the isolation of 16 compounds, including one novel meroterpenoid, named andrastone C. Structure of the new compound was elucidated based on high-resolution electrospray ionization mass spectroscopy (HRESIMS) data, extensive nuclear magnetic resonance (NMR) spectroscopic analyses and a single crystal X-ray diffraction. The crystal structure of a known meroterpenoid andrastone B was also reported in this study. Both andrastones B and C exhibited antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans with minimum inhibitory concentration (MIC) values in a range from 6 to 13 μg/mL.


Introduction
Marine natural products are important sources for the discovery of novel bioactive agents and drug leads [1][2][3][4][5]. However, the vast majority of these reported marine natural products are obtained from the shallow-water samples and only circa 2% are isolated from the deep-sea organisms [6][7][8]. This statistic contrasts significantly with that of the knowledge that 95% of the Earth's seas are greater than 1000 m deep and the main reason for this disparity is the limitations in obtaining deep-sea organisms [7,8]. With the developments in technology to access deep-sea organisms, more and more deep-sea natural products have been reported [7][8][9].
The deep-sea organisms under extreme conditions have had to make significant biochemical and physiological adaptations for survival, which results in the modifications of both gene regulation and metabolic pathways to produce metabolites with unique structures and bioactivities that differ from those produced by the shallow-water organisms [7,8]. It was reported that about 75% of deep-sea natural products possess biological activity, about 40% were drug-like, and two/three were within Known Drug Space (KDS) [8]. For example, the marine obligate Salinospora actinomycetes are found in tropical and subtropical marine sediments at the depth of up to 1100 m [10,11]. The genus Salinispora has become a robust model for natural product research and the secondary metabolites reported to date from the Salinospora actinomycetes are predominantly new, including salinosporamide A, a second-generation proteasome inhibitor [12]. Salinosporamide A is currently termed as marizomib under investigation in malignant glioma and relapsed-refractory multiple myeloma [13,14]. termed as marizomib under investigation in malignant glioma and relapsed-refractory multiple myeloma [13,14].
Mariana Trench sediments are enriched in microorganisms [15,16], however, the structures and bioactivities of their secondary metabolites are not very known and need to be explored. During the course of our ongoing project for the discovery of novel bioactive agents from marine microorganisms [17][18][19][20][21], a fungus strain SY2107 was isolated from a sediment sample collected from the Mariana Trench at depth of 11000 m. The extract prepared from the culture of this isolated hadal fungus in rice medium showed activities in inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans. Chemical investigation on this active extract resulted in the isolation of 16 compounds (1-16, Figure 1), including one novel antimicrobial meroterpenoid, named andrastone C (1). Herein, we describe the isolation, structure elucidation, and bioactive evaluation of these isolated marine natural products.

Results and Discussion
The hadal fungus SY2107 ( Figure S1, Supplementary Materials) was identified as Penicillium sp. SY2107 according to the result from its internal transcribed spacer (ITS) rDNA sequence (552 bp, Figure S2) analysis, which was 100% match to those of several Penicillium fungi (Table S1). An extract prepared from the culture of strain SY2107 in rice medium was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-16.

Results and Discussion
The hadal fungus SY2107 ( Figure S1, Supplementary Materials) was identified as Penicillium sp. SY2107 according to the result from its internal transcribed spacer (ITS) rDNA sequence (552 bp, Figure S2) analysis, which was 100% match to those of several Penicillium fungi (Table S1). An extract prepared from the culture of strain SY2107 in rice medium was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-16.

C, Type 1 H (J in Hz)
No. 13  correlation of H-7 with C-16 was observed, the downfield chemical shifts at C 91.9 for C-7 and C 185.3 for C-16 suggested a five-membered ether ring for E, which was confirmed by the crystal structure ( Figure 2) of 1 obtained from a single crystal X-ray diffraction.  Table 1. 13 C and 1 H NMR data of andrastone C (1, in dimethylsulfoxide-d6).

C, type 1 H (J in Hz)
No. 13 (Figure 2). Based on the foregoing evidences, compound 1 was elucidated as a new analogue of andrastone B (2), named andrastone C. Its 13 C and 1 H NMR data (Table 1) were assigned based on the heteronuclear multiple quantum correlation (HMQC), COSY, HMBC, and NOE correlations. To the best of our knowledge, andrastone C is the first example of this type of meroterpenoids with a five-membered ether ring (ring E).
Compounds 1-16 were tested for their antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans by the micro-broth dilution method [39]. Vancomycin (an antibiotic against MRSA), gentamicin (an antibiotic against both Gram-positive and negative bacteria), and amphotericin B (an antifungal drug) were used as positive controls. The results ( Table 2) showed that both andrastones C (1) and B (2) had antimicrobial activities with minimum inhibitory concentration (MIC) values of 8 and 9 µg/mL against MRSA, 8 and 12 µg/mL against E. coli, and 13 and 6 µg/mL against C. albicans, respectively. Known compounds 3-12 also showed antimicrobial activities against MRSA, E. coli, and C. albicans with MIC values in a range from 9 to 22 µg/mL, while compounds 13-16 only showed antibacterial activities against MRSA and E. coli with MIC values of 22-38 µg/mL. Compounds 1-3 were also evaluated for their activities in inhibiting the proliferation of human glioma U251 and U87MG cells using the sulforhodamine B (SRB) assay [40]. Doxorubicin (DOX, a chemotherapeutic drug) was used as a positive control. (Z)-N-(4-hydroxystyryl)formamide (3) exhibited antiproliferative activity against U251 and U87MG cells with IC 50 values of 17.0 ± 2.9 and 39.8 ± 1.6 µM, respectively. Both andrastones C (1) and B (2) showed no antiproliferative activity at a concentration of 50 µM.

Isolation and Identification of Strain SY2107
Strain SY2107 was isolated from a sediment sample, which was collected from the Mariana Trench at depth 11000 m on November, 2018. Briefly, the sediment was air dried at 28 • C for 7 days and the dried sample (1.0 g) was diluted with sterile water to make dilutions of 10 −2 , 10 −3 , and 10 −4 g/mL. Each dilution (200 µL) was covered on the surface of ten different media of B, BY, D, DY, E, EY, ISP2, ISP2Y, ISP4, and ISP4Y in Petri dishes and then incubated at 28 • C for 14 days. The single pure colony of SY2107 was picked from the 10 −2 g/mL suspension in ISP2Y solid medium and then transferred to another ISP2Y solid medium plate. After growth for another 7 days at 28 • C, the single colony (SY2107) that grew well was transferred onto an ISP2Y solid medium slant and stored at 4 • C for further study.
The strain SY2107 was identified by internal transcribed spacer (ITS) rDNA sequence analysis conducted by Legenomics (Hangzhou, China). The ITS rDNA sequence of strain SY2107 was compared to those in the GenBank using nucleotide BLAST (Basic Local Alignment Search Tool) and the rDNA sequence data of strain SY2107 has been deposited in GenBank with accession number MT355647. The strain Penicillium sp. SY 2107 was preserved at the Laboratory of Institute of Marine Biology and Pharmacology, Ocean College, Zhoushan Campus, Zhejiang University, China.

Scale Up Culture of Strain SY2107
Pure colony of strain SY2107 from the ISP2Y solid medium slant was inoculated into a 500 mL Erlenmeyer flask, which contained 250 mL ISP2Y liquid medium and then incubated for 3 days in a shaker (180 rpm, 28 • C) to produce seed broth. The seed broth (10 mL) was then transferred into rice medium (40 g rice and 60 mL artificial seawater) in 500 mL Erlenmeyer flask and then all flasks were incubated at 28 • C for 30 days in a static state. A total of 200 cultured flasks were prepared for this study.

Isolation of Compounds 1-16
The culture of strain SY2107 in rice medium in each flask was extracted with EtOAc (250 mL) three times. The combined EtOAc extract was dried in vacuo to give an extract (70 g). This extract was fractionated on a column (160 × 10 cm) of silica gel (1200 g) eluting with a mixture (1000 mL) of cyclohexane and EtOAc in different ratios (10:1, 51, 2: 1, 1:1, and 1:2) to give five fractions fractions A-E. Similarly, fraction C was fractionated on a CT-30 column (280 × 30 mm, 10 µm; UV detection: 210 nm; mobile phase: MeOH/H 2 O, 50/50; flow rate: 10 mL/min) by a CXTH LC-3000 HPLC system to afford parts C 1 and C 2 . Compounds 12 (6.7 mg, t R 19.7 min) and 15 (12.1 mg, t R 25.7 min) were obtained from parts C 1 and C 2 , respectively, by HPLC purification using the Zorbax SB-C 18 column with UV detection of 210 nm and mobile phase of 55% MeOH/H 2 O at flow rate of 1.0 mL/min.

Antimicrobial Active Assay
The antimicrobial activities of all isolated compounds against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans were tested by the micro-broth dilution method as describe in the previous study [37] with little modification. Vancomycin, gentamicin, and amphotericin B were used as positive controls. Briefly, 96-well plates were used to make dilutions of the tested compounds. The first serial dilution was followed to get a broad range of concentration for each compound. Initial concentration was 200 µg/mL and then by serial dilution with 50% DMSO, other concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 µg/mL were achieved. The final volume was 200 µL. After that 2 µL from 10 8 cfu/mL of culture was added and the plates were incubated at 37 • C for 12 h overnight. Minimum inhibitory concentration (MIC) that inhibited the growth of microorganisms and minimum bactericidal concentration (MBC) that completely killed microorganisms were recorded. Finally, based on results obtained, specific concentrations of each compound were prepared to get more accurate values of MIC and MBC.

Antiproliferative Active Assay
The Sulforhodamine B (SRB) assay [38] was applied to evaluate the activity of the tested compounds in inhibiting the proliferation of human glioma U251 and U87MG cells. Doxorubicin (DOX) was used as a positive control. Human glioma U251 and U87MG cells were cultured in DMEM (Dulbecco s Modified Eagle Medium, Gibco) and MEM (Minimum Essential Medium, Gibco) and with 10% FBS (Fetal Bovine Serum, PAA Laboratories Inc.), respectively. All cells were incubated in a 5% CO 2 humidified incubator at 37 • C and the cultured cells after the third generation were used for the experiments.

Conclusions
One novel meroterpenoid, named andrastone C, and fifteen known compounds with diverse structural classes, were discovered and characterized from the culture of a Mariana Trench sediment-associated fungus Penicillium sp. SY2107 in rice medium. Andrastone C and most of the known compounds showed antimicrobial activities against MRSA, E. coli, and C. albicans. (Z)-N-(4-hydroxystyryl) formamide exhibited antiglioma activity. Data from this study enriched the chemical and bioactive diversities of the secondary metabolites from the Mariana Trench-sourced microorganisms.