Isolation and Structure Elucidation of New Metabolites from the Mariana-Trench-Associated Fungus Aspergillus sp. SY2601

Fungi are important resource for the discovery of novel bioactive natural products. This study investigated the metabolites produced by Mariana-Trench-associated fungus Aspergillus sp. SY2601 in EY liquid and rice solid media, resulting in the isolation and structure determination of 28 metabolites, including five new compounds, asperindopiperazines A–C (1–3), 5-methoxy-8,9-dihydroxy-8,9-deoxyaspyrone (21), and 12S-aspertetranone D (26). Structures of the new compounds were elucidated based on extensive NMR spectral analyses, HRESIMS data, optical rotation, ECD, and 13C NMR calculations. The new compound 12S-aspertetranone D (26) exhibited antibacterial activity against both methicillin-resistant Staphylococcus aureus and Escherichia coli with MIC values of 3.75 and 5 μg/mL, respectively.


Introduction
Marine fungi are important resources for the discovery of novel bioactive natural products and drug lead compounds [1][2][3][4][5].Among them, Aspergillus fungi have been proved to be one of the most abundant novel bioactive compound producers [3][4][5].It was reported that a total of 512 new marine-derived natural products were isolated from Aspergillus fungal origins from 1992 to 2014, of which 36% exhibited diverse bioactivities [3].Recent updates indicated that 361 new secondary metabolites were identified from the Aspergillus fungi from 1915 to 2020.Since then, more and more novel bioactive natural products have been continuously isolated from marine-derived Aspergillus species, including p-terphenyl derivatives of asperterphenyls A-N from A. sp.SCSIO41315 [6], cyclopentapeptides of pseudoviridinutans A-F from A. pseudoviridinutans TW58-5 [7], and indoloquinazoline alkaloids of clavutoines A-U from A. clavutus LZD32-24 [8].
The Mariana Trench is well known for being the deepest site in the Earth's oceans, and a number of investigations showed that the Mariana Trench is rich in microorganisms [9][10][11][12].Previously reported metabolites from the Mariana Trench microorganisms included phenazines [13,14], aniline-tetramic acids [15], phenylbutenote and nocapyrone [16], and n-acetylglutaminyl glutamine amide and desferrioxamine B [17].Obviously, the diversity of chemical structures and bioactivities of the metabolites produced by the Mariana Trench microorganisms is unclear.
Recently, we have conducted chemical investigations on the metabolites of several Mariana-Trench-associated microorganisms, leading to the isolation and structure elucidation of number of novel compounds, such as streptothiazolidine A, streptodiketopiperazines A and B, and (S)-1-(3-ethylphenyl)-1,2-ethanediol [18].Streptothiazolidine A and streptodiketopiperazines A and B had antifungal activity against Candida albicans [18].In current study, we further investigated the metabolites produced by a Mariana-Trenchderived fungus Aspergillus sp.SY2601 cultured in EY liquid and rice solid media, resulting in the isolation and identification of twenty-eight metabolites (1-28, Figure 1), including five new compounds, asperindopiperazine A-C (1-3), 5-methoxy-8,9-dihydroxy-8,9-deoxyaspyrone (21), and 12S-aspertetranone D (26).Herein, we report the culture of strain SY2601 and the isolation, structure elucidation, and bioactive evaluation of these isolated compounds.

Results and Discussion
The isolated strain SY2601 (Figure S1, Supplementary Materials) was assigned as Aspergillus sp.SY2601 based on its ITS rDNA sequence (Figure S2), which was an over 99.8% match to those of eleven Aspergillus species (Table S1).The extracts prepared from the large-scale cultures of strain SY2601 in EY liquid and rice solid media were separated by column chromatography, followed by HPLC purification, to afford compounds 1-28.

Results and Discussion
The isolated strain SY2601 (Figure S1, Supplementary Materials) was assigned as Aspergillus sp.SY2601 based on its ITS rDNA sequence (Figure S2), which was an over 99.8% match to those of eleven Aspergillus species (Table S1).The extracts prepared from the large-scale cultures of strain SY2601 in EY liquid and rice solid media were separated by column chromatography, followed by HPLC purification, to afford compounds 1-28.

Isolation and Identification of Strain SY2601
The strain SY2601 was isolated from sediment obtained from the Mariana Trench at a depth of 5842 m, as per the described procedure in our previous publication [18] by using ten different solid media (B, BY, CA, CAY, D, DY, E, EY, SC, SCY).The signal-purified colony of SY2601 was obtained on the E medium coated with a 10 -3 g/mL sample suspension.
The strain SY2610 was identified by comparing its ITS rDNA sequence (accession number: OR646740) with the data of the GenBank.The ITS rDNA sequence analysis was performed by Legenomics (Hangzhou, China).The strain Aspergillus sp.SY2601 can be obtained from the Laboratory of Institute of Marine Biology and Pharmacology, Ocean College, Zhoushan Campus, Zhejiang University, China.

Mass Culture of Strain SY2601 in EY Liquid and Rice Solid Media
For strain SY1601 cultured in EY medium, a pure colony of the strain SY2601 picked from the E slant medium was transferred into a 250 mL EY liquid medium in a 500 mL Erlenmeyer flask and incubated at 28 • C for 3 days with shaking (180 rpm) to obtain a seed broth.The 5 mL seed broth was further transferred into 250 mL of an EY liquid culture medium in a 500 mL Erlenmeyer flask, and then statically incubated at 28 • C for 30 days.A total of 300 bottle cultures (75 L) were prepared for this study.
For strain SY2610 cultured in rice solid medium, the above prepared seed broth (5 mL) was transferred into a rice medium (40 g rice, 60 mL of 25 g/L sea salt solution) in a 500 mL Erlenmeyer flask and then incubated at 28 • C for 24 days.A total of 200 bottles of rice medium cultures were prepared for this study.
Compounds 13, 14, and 16-28 were isolated from the cultures of strain SY 2601 in rice medium.Each of the rice cultures in the 200 bottles was extracted by EtOAc three times (each 200 mL).The combined EtOAc extract solution was dried under reduced pressure to give a crude extract (100.75 g).

Optical Rotation Calculations
Optical rotation (OR) calculations were conducted as per our previously described method [36].

ECD Calculations
The previously described method [18] was used for ECD calculations.

13 C NMR Calculations
13 C NMR calculations were carried out referring to our previous publications [18].

Antimicrobial Activity Assay
The micro-broth dilution method as described in the previous study [42] was applied to determine the antimicrobial activities of all tested compounds against the growth of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans.Gentamicin and amphotericin B were used as positive controls.

Conclusions
Chemical investigation on the metabolites produced by the Mariana-Trench-derived fungus Aspergillus sp.SY2601 in both EY liquid and rice solid media resulted in the isolation and identification of twenty-eight metabolites, including five new compounds, asperindopiperazines A-C (1-3), 5-methoxy-8,9-dihydroxy-8,9-deoxyaspyrone (21), and 12S-aspertetranone D (26).12S-aspertetranone D (26) had activity in inhibiting the growth of methicillin-resistant S. aureus and E. coli.The twenty-eight isolated compounds belong to different structural types of indolyl diketopiperazines, diketopiperazines, merosesquiterpenoids, putative meroterpenoid, nitrobenzoyl sesquiterpenoids, peptides, aspyrones, and phenols.The data obtained from current study show that the fungus Aspergillus sp.SY2601 is able to produce abundance metabolites, which enriched the diversity of structures and bioactivities of the metabolites produced by the Mariana-Trench-associated microorganisms.

Figure 4 .
Figure 4. COSY and key NOE correlations of 12S-aspertetranone D (26), the experimental ECD spectrum of 12S-aspertetranone D (26), and the calculated ECD curves of the two model molecules of 26a and 26b at the b3lyp/6-311+g (d, p) level in MeOH.

Figure 4 .
Figure 4. COSY and key NOE correlations of 12S-aspertetranone D (26), the experimental ECD spectrum of 12S-aspertetranone D (26), and the calculated ECD curves of the two model molecules of 26a and 26b at the b3lyp/6-311+g (d, p) level in MeOH.

Author
Contributions: X.-Y.L. and Z.Z.conceived and designed the experiments; C.S. performed the chemical and bioactive experiments; Y.H., X.L. and C.S. carried out the OR and ECD calculations and prepared the data; N.W. and Z.Z.analyzed the data, wrote, and revised the paper.All authors have read and agreed to the published version of the manuscript.Funding: This study was financially supported by the Basic Public Welfare Research Project of Zhejiang Province (No. LGF21H300005), the Hainan Provincial Joint Project of Sanya Yazhou Bay