A New Dioic Acid from a wbl Gene Mutant of Deepsea-Derived Streptomyces somaliensis SCSIO ZH66

The wblAso gene functions as a global regulatory gene in a negative manner in deepsea-derived Streptomyces somaliensis SCSIO ZH66. A new dioic acid (1) as well as two known butenolides (2 and 3) were isolated from the ΔwblAso mutant strain of S. somaliensis SCSIO ZH66. The structure of 1 was elucidated by a combination of spectroscopic analyses, including MS and NMR techniques. In the cell growth inhibitory evaluation, compound 3 exhibited moderate activity against the human hepatic carcinoma cell line (Huh7.5) with an IC50 value of 19.4 μg/mL, while compounds 1 and 2 showed null activity up to 100 μg/mL.


Introduction
Marine-derived bioactive compounds and their novel chemical scaffolds have been shown to be attractive starting points for drug discovery programs [1,2]. Over the last few years, the actinomycetes isolated from marine environments have attracted considerable attention, because of their great potential for producing a large diversity of bioactive compounds [3,4]. However, discovery of structurally novel secondary metabolites from microbes is becoming more and more difficult, as the rate of rediscovery of known compounds is increasing [5,6]. Recent genome sequencing revealed that actinomycetes can genetically synthesize secondary metabolites beyond those that were isolated under standard cultivation conditions [7][8][9]. However, a number of gene clusters in actinomycetes are silent or only low-expressed and thus are defined as cryptic or orphan clusters [10].
Manipulation of global regulators is an effective strategy for the activation of cryptic gene clusters [11]. In the previous study, we obtained a series of anti-MRSA (methicillin-resistant Staphylococcus aureus) α-pyrone compounds (violapyrones A-C, H, and J) from the deepsea-derived S. somaliensis SCSIO ZH66 by disruption of the negative global regulatory gene wblA so [12]. In our continuous search for significantly enhanced compounds in the ∆wblA so mutant strain, one new dioic acid (1) together with two known butenolides (2 and 3) were isolated ( Figure 1). We describe herein the isolation, structure elucidation, and biological evaluation of these accumulated compounds.

Results and Discussion
The ΔwblAso mutant of S. somaliensis SCSIO ZH66 was constructed in our previous study [12]. The fermentation broths of the wild-type and the ΔwblAso mutant strains were extracted with EtOAc, respectively, and were subsequently subjected to high-performance liquid chromatography (HPLC) analysis ( Figure 2), in which we observed three significantly enhanced peaks (1)(2)(3) in the ΔwblAso mutant compared with those in the wild-type strain at wavelengths of 200 nm. With the massive fermentation of the ΔwblAso mutant, compounds 1-3 were isolated and further identified via NMR assignments. Compound 1 was obtained as a white, amorphous solid. The molecular formula of 1 was established as C17H26O4 on the basis of HR-ESIMS data ([M + Na] + at m/z 317.1723) ( Figure S1). The structure of 1 was determined by 1D ( 1 H, 13 C) and 2D NMR (COSY, HSQC and HMBC) data ( Table 1

Results and Discussion
The ∆wblA so mutant of S. somaliensis SCSIO ZH66 was constructed in our previous study [12]. The fermentation broths of the wild-type and the ∆wblA so mutant strains were extracted with EtOAc, respectively, and were subsequently subjected to high-performance liquid chromatography (HPLC) analysis ( Figure 2), in which we observed three significantly enhanced peaks (1)(2)(3) in the ∆wblA so mutant compared with those in the wild-type strain at wavelengths of 200 nm. With the massive fermentation of the ∆wblA so mutant, compounds 1-3 were isolated and further identified via NMR assignments.
Compound 3 was isolated as a light yellow oil. The molecular formula of 3 was established as C 13 (Table S1) with those reported in [13].
Butenolides are a family of α,β-unsaturated lactones, and their saturated analogs act as signaling substances in bacteria to enhance sporulation or induce metabolite production [14]. Some butenolides have been reported to show cytotoxicity [13] or antimicrobial activities [15,16]. In the evaluation for cell growth inhibitory effects, compound 3 exhibited moderate growth inhibition against the human hepatic carcinoma cell line (Huh7.5) with an IC50 value of 19.4 μg/mL, while compounds 1 and 2 showed null activity up to 100 μg/mL. In the test of antimicrobial activity, none of these compounds showed significant activity against multi-drug resistant strains (Staphylococcus aureus CCARM 3090, Escherichia coli CCARM 1009, Enterococcus faecalis CCARM 5172, Enterococcus faecium CCARM 5203, and Salmonella typhimurium CCARM 8250).

General Experimental Procedures
HPLC was performed on an Agilent 1260 Infinity equipment with Diode Array Detector (DAD) (Agilent, Waldbronn, Germany). NMR spectra were recorded on Agilent DD2-500 spectrometers. Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δH 2.50 and δC 39.5 ppm for DMSO-d6). HR-ESIMS data were obtained on a Q-TOF Ultima Global GAA076 LC-MS spectrometer (Waters Corporation, Milford, MA, USA).

Strains and Culture Conditions
The S. somaliensis SCSIO ZH66 (CGMCC NO. 9492) was isolated from the deep-sea sediment collected at a depth of 3536 m in the South China Sea (120°0.250′ E; 20°22.971′ N) [17]. The ΔwblAso mutant was obtained as described previously [12]. The strains were grown at 30 °C on a MS (mannitol-soy flour) medium for sporulation.

Fermentation, Extraction, and Isolation of the Compounds
For each fermentation, strain spores were inoculated into 200 mL of fermentation medium (1% soluble starch, 2% glucose, 4% corn syrup, 1% yeast extract, 0.3% beef extract, 0.05% MgSO4•7H2O, 0.05% KH2PO4, 0.2% CaCO3, and 3% sea salt, pH = 7.0), which was further supplemented with 1.5% XAD-16 resin when fermenting the ΔwblAso mutant, and incubated at 30 °C, 220 rpm for 7 days. Analytical HPLC was performed on an Eclipse C18 column (5 μm, 4.6 × 150 mm) developed with a linear gradient from 20% to 70% B/A in 40 min (phase A: 0.1% formic acid in H2O; phase B: 100% acetonitrile supplemented with 0.1% formic acid). A total of 20 L of culture was made by this method. The fermentation cultures were harvested via centrifugation, and the supernatant was extracted twice with an equal volume of ethyl acetate. The combined ethyl acetate extracts were concentrated in vacuo to afford residue A. The precipitated mycelia and XAD-16 resin were extracted twice with acetone. The extracts were combined, and acetone was evaporated in vacuo to yield residue B. Both residues from fermentation broths and mycelia cakes were combined to afford crude extract (6.0 g). They were applied to reversed-phase C18 open column, eluting with a gradient eluent of 20%-100% methanol to give five fractions (Fr.1~Fr.5). Compound 1 (3.8 mg) was obtained by further separation of Fr.4 eluting with gradient solvents (phase A: 0.1% formic acid in H2O; phase B: 100% acetonitrile supplemented with 0.1% formic acid, 2 mL/min, UV detection at 200 nm) using a semi-preparative HPLC column (YMC-Pack ODS-AA C18 column, 120 Å, 250 × 10 mm, 5 μm). Fr.3 was also further subjected to semi-preparative HPLC eluting with gradient solvents to afford compounds 2 (2.9 mg) and 3 (5.5 mg).

Biological Assays
Viabilities of human hepatic carcinoma cell line (Huh7.5) were measured with a MTT assay. Briefly, logarithmically growing cells were trypsinized from culture dishes and placed into 96-well plate and cultured at 37 °C for 24 h. Cells were treated with the varying concentrations of each compound, and then 20 μL of a MTT solution (5 mg/mL) were added to each well. After incubating for 4 h, the medium was removed, and 150 μL of DMSO were added to each well to dissolve purple crystals of formazan via shaking at 260 rpm for 10 min. Absorbance was measured at 490 nm by a

General Experimental Procedures
HPLC was performed on an Agilent 1260 Infinity equipment with Diode Array Detector (DAD) (Agilent, Waldbronn, Germany). NMR spectra were recorded on Agilent DD2-500 spectrometers. Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δ H 2.50 and δ C 39.5 ppm for DMSO-d 6 ). HR-ESIMS data were obtained on a Q-TOF Ultima Global GAA076 LC-MS spectrometer (Waters Corporation, Milford, MA, USA).

Strains and Culture Conditions
The S. somaliensis SCSIO ZH66 (CGMCC NO. 9492) was isolated from the deep-sea sediment collected at a depth of 3536 m in the South China Sea (120 • 0.250 E; 20 • 22.971 N) [17]. The ∆wblA so mutant was obtained as described previously [12]. The strains were grown at 30 • C on a MS (mannitol-soy flour) medium for sporulation.

Fermentation, Extraction, and Isolation of the Compounds
For each fermentation, strain spores were inoculated into 200 mL of fermentation medium (1% soluble starch, 2% glucose, 4% corn syrup, 1% yeast extract, 0.3% beef extract, 0.05% MgSO 4 ·7H 2 O, 0.05% KH 2 PO 4 , 0.2% CaCO 3 , and 3% sea salt, pH = 7.0), which was further supplemented with 1.5% XAD-16 resin when fermenting the ∆wblA so mutant, and incubated at 30 • C, 220 rpm for 7 days. Analytical HPLC was performed on an Eclipse C18 column (5 µm, 4.6 × 150 mm) developed with a linear gradient from 20% to 70% B/A in 40 min (phase A: 0.1% formic acid in H 2 O; phase B: 100% acetonitrile supplemented with 0.1% formic acid). A total of 20 L of culture was made by this method. The fermentation cultures were harvested via centrifugation, and the supernatant was extracted twice with an equal volume of ethyl acetate. The combined ethyl acetate extracts were concentrated in vacuo to afford residue A. The precipitated mycelia and XAD-16 resin were extracted twice with acetone. The extracts were combined, and acetone was evaporated in vacuo to yield residue B. Both residues from fermentation broths and mycelia cakes were combined to afford crude extract (6.0 g). They were applied to reversed-phase C18 open column, eluting with a gradient eluent of 20%-100% methanol to give five fractions (Fr.1~Fr.5). Compound 1 (3.8 mg) was obtained by further separation of Fr.4 eluting with gradient solvents (phase A: 0.1% formic acid in H 2 O; phase B: 100% acetonitrile supplemented with 0.1% formic acid, 2 mL/min, UV detection at 200 nm) using a semi-preparative HPLC column (YMC-Pack ODS-AA C18 column, 120 Å, 250 × 10 mm, 5 µm). Fr.3 was also further subjected to semi-preparative HPLC eluting with gradient solvents to afford compounds 2 (2.9 mg) and 3 (5.5 mg).

Biological Assays
Viabilities of human hepatic carcinoma cell line (Huh7.5) were measured with a MTT assay. Briefly, logarithmically growing cells were trypsinized from culture dishes and placed into 96-well plate and cultured at 37 • C for 24 h. Cells were treated with the varying concentrations of each compound, and then 20 µL of a MTT solution (5 mg/mL) were added to each well. After incubating for 4 h, the medium was removed, and 150 µL of DMSO were added to each well to dissolve purple crystals of formazan via shaking at 260 rpm for 10 min. Absorbance was measured at 490 nm by a multi-detection microplate reader (infinite M1000 Pro, TECAN, Mannedorf, Switzerland). The 50% inhibitory concentration (IC 50 ) value was determined as the concentration that caused 50% inhibition of cell proliferation [18,19].

Conclusions
A new dioic acid (1) and two known butenolides (2 and 3) were isolated from the ∆wblA so mutant strain of deepsea-derived S. somaliensis SCSIO ZH66. In the evaluation for cell growth inhibitory effects, compound 3 showed moderate activity against the human hepatic carcinoma cell line (Huh7.5) with an IC 50 value of 19.4 µg/mL. Isolation of a novel dioic acid (1) in this study indicated that the manipulation of global regulators can be used as an effective method for the accumulation of novel secondary metabolites.