New Diketopiperazines from a Marine-Derived Fungus Strain Aspergillus versicolor MF180151

Six new diketopiperazines, (±)-7,8-epoxy-brevianamide Q ((±)-1), (±)-8-hydroxy-brevianamide R ((±)-2), and (±)-8-epihydroxy-brevianamide R ((±)-3), together with four known compounds, (±)-brevianamide R ((±)-4), versicolorin B (5) and averufin (6), were isolated from a marine-derived fungus strain Aspergillus versicolor MF180151, which was recovered from a sediment sample collected from the Bohai Sea, China. The chemical structures were established by 1D- and 2D-NMR spectra and HR-ESI-MS. 1 is the first sample of brevianamides with an epoxy moiety. Their bioactivities were evaluated against Candida albicans, Bacillus subtilis, Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and Bacillus Calmette-Guérin. Compounds 1–4 showed no activities against the pathogens, and compounds 5 and 6 showed moderate activities against S. aureus and methicillin-resistant S. aureus.


Characterization and Identification of the Isolated Strain MF180151
The strain MF180151 was isolated from a marine sediment sample from the Bohai Sea, China. The identification of the strain was performed based on the morphology and phylogenetic analysis.
The ITS gene region of ribosomal DNA of the strain was PCR-amplified and sequenced. By comparing the ITS sequence to GenBank, it was indicated that the strain MF180151 belonged to the genus Aspergillus and shared a highest similarity with Aspergillus versicolor (99.66%). The phylogenetic tree based on ITS gene sequence revealed that the strain MF180151 formed a distinct phylogenetic cluster with A. versicolor ( Figure 2) with a bootstrap value above 95%.

Characterization and Identification of the Isolated Strain MF180151
The strain MF180151 was isolated from a marine sediment sample from the Bohai Sea, China. The identification of the strain was performed based on the morphology and phylogenetic analysis.
The ITS gene region of ribosomal DNA of the strain was PCR-amplified and sequenced. By comparing the ITS sequence to GenBank, it was indicated that the strain MF180151 belonged to the genus Aspergillus and shared a highest similarity with Aspergillus versicolor (99.66%). The phylogenetic tree based on ITS gene sequence revealed that the strain MF180151 formed a distinct phylogenetic cluster with A. versicolor ( Figure 2) with a bootstrap value above 95%.

Characterization and Identification of the Isolated Strain MF180151
The strain MF180151 was isolated from a marine sediment sample from the Bohai Sea, China. The identification of the strain was performed based on the morphology and phylogenetic analysis.
The ITS gene region of ribosomal DNA of the strain was PCR-amplified and sequenced. By comparing the ITS sequence to GenBank, it was indicated that the strain MF180151 belonged to the genus Aspergillus and shared a highest similarity with Aspergillus versicolor (99.66%). The phylogenetic tree based on ITS gene sequence revealed that the strain MF180151 formed a distinct phylogenetic cluster with A. versicolor ( Figure 2) with a bootstrap value above 95%.

Fungal Culture and Identification
The strain MF180151 was isolated from a marine sediment sample from the Bohai Sea, China. It was incubated on potato dextrose agar (PDA) plate consisting (0.4% potato starch, 2% dextrose, and 2% agar) at 28 • C. The identification was performed based on the morphology and phylogenetic analysis. The whole genomic DNA of the strain was extracted using the E.Z.N.A. kit (Omega Bio-Tek, Norcross, GA, USA). A pair of primers (ITS4: 5"-TCCTCCGCTTATTGATATGC-3"; ITS5: 5"-GGAAGTAAAAGTCGTAACAAGG-3") was used to amplify the ITS region of MF180151. PCR amplification (50.0 µL final volume: 25 µL 2 × Taq Master Mix, 2 µL of 10 µM of each primer, 5.0 µL DNA template and 16 µL ddH 2 O) of the ITS sequence was performed on Bio-gener PCR Thermal Cycler with the initial denaturation at 95 • C for 3 min, 32 cycles of denaturation (94 • C, 15 s), annealing (60 • C, 15 s), and elongation (72 • C, 60 s), and a final elongation at 72 • C for 5 min. After multiple alignments of ITS sequence of the related species by CLUSTAL W [37], phylogenetic analysis was constructed using neighbor-joining method with bootstrap values based on 1000 replications by MEGA 5.0 [38,39].
The strain was deposited at the Institute of Microbiology, Chinese Academy of Sciences. The nucleotide sequences of ITS gene (accession number MK680178) of A. versicolor MF180151 were deposited in GenBank.

Fermentation, Extraction and Isolation
The strain MF180151 was cultured on potato dextrose agar plate at 28 • C for 7 days. Mature colonies were cut into small pieces (about 1 cm 2 ) under aseptic conditions. Then, three piece of the strain was inoculated into three 250 mL conical flasks, each containing 40 mL of liquid medium consisting of potato infusion (20%), glucose (2.0%), artificial sea salt (3.5%) and distilled artificial seawater, at 28 • C for 3 d on a rotary shaker at 160 rpm. An aliquot (5 mL) of the resultant seed culture was inoculated into twelve 1 L conical flasks, each containing solid medium consisting of rice (100 g) and artificial seawater (30 mL), and the flasks were incubated stationary for 28 d at 20 • C.
For general antimicrobial assays, a single colony which was incubated on an LB agar overnight at 37 • C was picked up and suspended in Mueller-Hinton Broth to approximately 1 × 10 4 cfu/mL. For anti-C. albicans assay, a colony of C. albicans incubated on a YPD agar plate was picked and suspended in RPMI 1640 to a concentration of 1 × 10 4 cfu/mL. A twofold serial dilution of each compound to be tested was prepared, and an aliquot of each dilution (2µL) was added to a 96-well flat-bottom microtiter plate (Greiner). Vancomycin, ciprofloxacin and ketoconazole were used as the positive control and DMSO as the negative control. An aliquot (78 µL) of suspension was then added to each well (to give final compound concentrations of 100 to 0.78 µg/mL in 2.5% DMSO) and the plate was incubated at 37 • C aerobically for 16 h. The MIC was defined as the minimum concentration of the compound that prevented visible growth of the tested bacteria. All the experiments were performed in triplicate.
The strain Bacillus Calmette-Guérin (Pasteur 1173P2) used for the anti-BCG assay was transformed with green fluorescent protein (GFP) constitutive expression plasmid pUV3583c with direct readout of fluorescence as a measure of bacterial growth. The strain was incubated to mid log phase (7 d) at 37 • C in Middle brook 7H9 broth (40 mL; Difco) supplemented with 10% OADC enrichment (Becton Dickinson), 0.05% Tween-80 and 0.2% glycerol and then diluted to an OD 600 of 0.025 with broth. Aliquots (80 µL) of the bacterial suspension were added to each well of the 96-well micro plates (clear flat-bottom), followed by adding compounds (2 µL in DMSO), which were serially twofold diluted. Isoniazid served as positive control and DMSO as negative control. The plate was incubated at 37 • C for 3 days, and GFP fluorescence was measured with Multi-label Plate Reader using the bottom read mode, with excitation at 485 nm and emission at 535 nm. MIC is defined as the minimum concentration of drug that inhibits more than 90% of bacterial growth reflected by fluorescence value.

Conflicts of Interest:
The authors declare no conflict of interest.