Effects of High Salt Stress on Secondary Metabolite Production in the Marine-Derived Fungus Spicaria elegans

To obtain structurally novel and bioactive natural compounds from marine-derived microorganisms, the effect of high salt stress on secondary metabolite production in the marine-derived fungal strain, Spicaria elegans KLA-03, was investigated. The organism, which was isolated from marine sediment, produced different secondary metabolites when cultured in 3% and 10% saline conditions. Four characteristic metabolites, only produced in the 10% salinity culture, were isolated, and their structures were identified as (2E,2′Z)-3,3′-(6,6′-dihydroxybiphenyl-3,3′-diyl)diacrylic acid (1), aspulvinone E (2), aspochalasin E (3) and trichodermamide B (6), according to their 1D and 2D NMR spectra. Compound 1 is a new compound. High salt stress may therefore be a promising means to induce the production of new and chlorinated compounds in halotolerant fungi. Compound 1 showed moderate antibacterial activity against Pseudomonas aeruginosa and Escherichia coli with minimum inhibitory concentration (MIC) values of 0.038 and 0.767 mM, respectively.


Effect of Salt Stress on Secondary Metabolite Production
Little has been published in the literature on the metabolites produced by the fungus S. elegans, with most reports originating from our lab. A series of compounds, mainly cytochalasins and aspochalasins with antitumor activities, were isolated and identified from the cultivation of this organism at 3% salinity [2][3][4]13,14]. It has been reported that halotolerant fungi have special osmoregulatory mechanisms to regulate intracellular osmotic potential, which allows them to survive under a variety of saline conditions, including high salt conditions [15]. The secondary metabolites produced by the organism may be regulated by environmental factors such as salinity [16]. To investigate whether high salt stress could activate silent genes encoding secondary metabolites in S. elegans KLA-03, the organism was fermented at 3% and 10% salinity and the metabolites produced were monitored by HPLC. When cultured at 10% salinity, S. elegans KLA-03 was found to produce different compounds to those observed when it was cultured at 3% salinity ( Figure 2).
The main products observed under low salinity culture conditions, cytochalasins (t R 37-45 min) [8,9], were present at significantly lower levels when the organism was cultured under high salinity conditions. In contrast, the levels of other metabolites (t R 28-35 min) were significantly higher in the high salinity culture. High salinity conditions therefore induced the fungus S. elegans KLA-03 to produce much more polar metabolites. Extraction and HPLC-guided purification resulted in the isolation and identification of one new compound (1) and five known compounds (2-6), of which compounds 4 and 5 had previously been identified from the fermentation broth of this strain using 3% salinity conditions [2]. The biosynthetic pathways expressed by S. elegans KLA-03 under high salinity conditions are therefore different from those expressed under low salinity conditions. Chlorinated metabolite 6 was not produced at 3% salinity, suggesting that a halogenase enzyme may be activated under high salt conditions, and that the high concentration of chloride was exploited by S. elegans KLA-03 to synthesize chlorinated compounds. The mass of the crude EtOAc extracts obtained under the two conditions were markedly different, with 4 g obtained from a 50 L fermentation broth at 10% salinity, while 15 g was obtained from a 15 L 3% saline culture [3,4]. This suggests that high salt conditions lead to significantly lower overall metabolite productivity, as well as a change in metabolite profile.

Antimicrobial Effects of Secondary Metabolites
The antimicrobial activities of compounds 1-6 against Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans were evaluated using an agar dilution method (Table 1) [17]. New compound 1 showed moderate antibacterial activity against P. aeruginosa and E. coli. In addition, moderate antibacterial activity against E. aerogenes for compounds 2-6, and P. aeruginosa for 4 and 6 were also observed. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration at which no microbial growth could be observed.

General Experimental Procedures
UV spectra were recorded on a Beckman DU 640 spectrophotometer. IR spectra were obtained on a Nicolet NEXUS 470 spectrophotometer as KBr disks. 1 H, 13 C and DEPT NMR spectra and 2D-NMR spectra were recorded on a JEOL JNMECP 600 spectrometer using TMS as an internal standard, and chemical shifts were recorded as δ values. ESIMS was measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Analytical HPLC was performed using an ODS column (YMC-pack C18, 4.6 × 250 mm, 5 μm, 2 mL/min). TLC was performed on plates precoated with silica gel GF 254 (10-40 μm) and column chromatography (CC) was carried out using silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Sweden). Artificial sea salt used in culture media was purchased from Qingdao Marine Chemical Factory, Qingdao, China.

Fungal Material
The fungus S. elegans KLA-03 was isolated from marine sediments collected in Jiaozhou Bay, China. It was identified by Prof. C. X. Fang and preserved in the China Center for Type Culture Collection (No. CCTCCM 205049). Working stocks were prepared on potato dextrose agar slants and stored at 4 °C.

Fermentation and Extraction
The same incubation and extraction methods were used for the 10% artificial sea salt culture as those used previously for culturing with 3% sea salt [8]. In brief, the fungus S. elegans KLA-03 was grown under static conditions at 25 °C for 25 days in 180 1 L conical flasks containing a liquid medium (300 mL/flask) composed of glucose (20 g/L), peptone (5 g/L), malt extract (3 g/L), yeast extract (3 g/L), artificial sea salt (10 g/L) and tap water, after adjusting the pH of the media to 7.0. The fermented whole broth (54 L) was filtered through cheesecloth to separate the supernatant from the mycelia. The supernatant was concentrated under reduced pressure to about 5 L and then extracted three times with EtOAc to give an EtOAc extract, while the mycelia were extracted three times with acetone. The acetone was removed under reduced pressure to afford a residual aqueous solution. This aqueous solution was extracted three times with EtOAc to give a further EtOAc crude extract. The EtOAc solutions were then combined and concentrated under reduced pressure to give a final crude extract (4.0 g).

Bioassays
Antimicrobial activities against E. aerogenes, E. coli, P. aeruginosa, S. aureus and C. albicans were evaluated using an agar dilution method [17]. The tested strains were cultivated in LB agar plates for bacteria and in YPD agar plates for C. albicans, at 37 °C. Compounds 1-6 and positive controls were dissolved in 5% DMSO-H 2 O at different concentrations from 1000 to 62.5 μg/mL and then from 50 to 0.78 μg/mL, using continuous 2-fold dilution. The test solutions (5 μL) were absorbed onto paper disks (5 mm diameter) and placed on the assay plates. After 24 h incubation, zones of inhibition (mm in diameter) were recorded. The minimum inhibitory concentrations were defined as the lowest concentration at which an inhibition zone could be observed.

Conclusions
In summary, four metabolites, including a new compound and a chlorinated compound with antibacterial activity, were identified from the fungus S. elegans KLA-03 grown under high salt stress conditions. High salt stress affected the overall quantity of secondary metabolites produced and the metabolite profile. This indicates that the regulation of salinity could be a promising way to obtain new compounds and chlorinated compounds from halotolerant fungi for drug screening.