Microorganisms produce optically single bioactive natural products; the process is cheap as compared to chemical synthesis and environmentally friendly. Geodin 1 was isolated from the soft coral-derived fungus Aspergillus sp. It has a broad range of biological activities such as antiviral, antimicrobial, glucose stimulator for rat adipocytes, enhancement of fibrinolytic and cytotoxic activities, and is a subunit of the first nonpeptide and inhibitory active galanin receptor. In this work, we studied the effects of sodium chloride (NaCl) salt concentration on the production of geodin 1 and improved its yield to a multi-gram quantity through media optimization from a marine-derived fungus Aspergillus sp.
The fungal strain was cultivated at various concentrations of NaCl salt in rice medium and extracted after different intervals of time.
The yield of geodin 1 was 137.2 mg/L at optimal conditions. The optimal conditions for the high yield of geodin 1 were found as rice medium with 2.0% NaCl salt and 3 weeks incubation at room temperature.
The concentration of NaCl salt greatly affects the yield of geodin and hence its biosynthetic pathway.
Fungi play an important role in drugs discovery, producing secondary metabolites having interesting bioactivities [1, 2]. Most fungi belong to terrestrial habitats and have been studied for the bioactive secondary metabolites such as antibiotics; however, those related to the harsh marine environment are recently under extensive study for the production of bioactive natural products [3, 4]. Fungi from the depth of the sea could be studied by culturing in the same artificial environment  and those isolated from the coastal areas of the sea produce a variety of valuable bioactive secondary metabolites such as polyketides, peptides, alkaloids, and terpenoids . Fungi from the coastal regions are related either to coral or coral reefs, coastal plants such as mangroves, and rocks . Filamentous fungi synthesize commercially beneficial byproducts like wine from rice by Aspergillus oryzae, and bean curd by Monascus while the pure bioactive secondary metabolites are used as medicines such as penicillin, cephalosporin, and lovastatin produced by Aspergillusterreus and cyclosporine isolated from Tolypocladium inflatum . Furthermore, microorganisms also synthesize optically single bioactive natural products; the process is cheap and also environmentally friendly .
In our ongoing research on marine-derived fungi, we concentrate our study on a major secondary metabolite, geodin 1 (Figure 1) isolated from the soft coral Sinularia sp. (SYM-02) derived fungus Aspergillus sp. (SYM-02-005) from the South China Sea collected in 2015. It has been reported to be isolated from a filamentous fungus A. terreus , , , . Geodin 1 has interesting biological activities such as antiviral , antimicrobial , glucose stimulator for rat adipocytes , enhancement of fibrinolytic activity , cytotoxic activity , and is an essential part of the first nonpeptide, having inhibitory activity of the galanin receptor subtype GALR1 [18, 19]. In our previous study, it showed moderate cytotoxic activities against various cancer cell lines such as breast cancer (BT474), large cell lung cancer (NCI-H460), non-small cell lung cancer (H-1975), lung cancer (A549), chronic myelogenous leukemia (K562) and prostate cancer (DU145) cells .
Materials and methods
The fungal strain was cultivated in 1,000 mL Erlenmeyer flasks containing about 60 g sterilized rice medium and the respective concentrations of sodium chloride (NaCl) salt at 0.0%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 5.0%, 8.0%, 10.0%, 15.0%, 20.0%, and 30.0%. The flasks were fermented at room temperature for 1, 2, 3, 4, and 5 weeks, and extracted thrice times with ethyl acetate (EtOAc). Each crude extract was evaporated under vacuum to dryness and subjected to normal phase silica gel column chromatography (mesh 200–300), eluting with linear gradients of petroleum ether (PE) – EtOAc. Friction 4, eluted with 40% PE-EtOAc, was further subjected to the re-crystallization in methanol and dichloromethane. As a result of re-crystallization, geodin 1 was obtained in pure form as a white crystalline solid. The electrospray ionization mass spectrometry (ESI-MS) spectra were measured on Ultra Performance Liquid Chromatographic Mass Spectrometer (Waters UPLC® system using a C18 column [ACQUITY UPLC® BEH C18, 2.1 × 50 mm, 1.7 µm; 0.5 mL/min]). Nuclear magnetic resonance (NMR) spectra were recorded on an Agilent DD2 NMR spectrometer (500 MHz for 1H and 125 MHz for 13C NMR) and tetra-methyl saline (TMS) as an internal standard while deuterated chloroform (CDCl3) as a solvent. The structure of compound 1 was confirmed based on ESI-MS m/z at 398.9 [M+H]+ and NMR spectral data (Table 1) [20, 21]. The fungal strain was identified as Aspergillus sp., based on its morphology and molecular identification. The 512 base pair ITS sequence had 98% similarities with Aspergillus sp. NRRL58570 (HQ288052.1). The sequence data were submitted to GenBank with accession number KY235298 and the strain was stored in the Key Laboratory, School of Medicine and Pharmacy, Ocean University of China, Qingdao, P.R.China, with Genbank accession number KY235298.
|Positions||Data from literature||Data from experiment|
|δ C||δ H||δ C||δ H|
|7||18.7||2.57 (3H, s)||18.7||2.57 (3H, s)|
|3′||137.5||7.15 (1H, d, 1.5)||137.5||7.14 (1H, d, 1.5)|
|5′||104.4||5.83 (1H, d, 1.5)||104.4||5.82 (1H, d, 1.5)|
|7′||57.0||3.75 (3H, s)||57.0||3.75 (3H, s)|
|9′||53.1||3.71 (3H, s)||53.1||3.70 (3H, s)|
δ C , chemical shift of carbon; δ H , chemical shift of proton; NMR, nuclear magnetic resonance. aMeasured in deuterated chloroform (CDCl3).
Results and discussion
Geodin was discovered by Raistrick & Smith in 1936 from A. terreus and its structure with relative configuration was elucidated by Barton and Scott in 1958 [9, 22]. It was reported about 16 mg/L by Bizukojc and Ledakowicz  and 74.3 mg/L by Bizukojc and Pecyna  from A. terreus through lactose and glycerol as a medium. Abd Rahim et al. used a cheap industrial crude glycerol and reported about 58.9 mg/L in shake flask; they also studied the effects of viscosity, friction, and sonication and obtained about 56.9 mg/L geodin from A. terreus [12, 24]. It is for the first time to study the effect of salt concentration on the yield of geodin 1 isolated from the soft coral-derived fungus Aspergillus sp. and its yield was improved to multi-gram quantity through a cheap fermentation medium.
Broth media such as potato dextrose broth (PDB), glucose peptone yeast (GPY), Chashi, and Starch were used under shaking and unshaking conditions, containing a series of NaCl salt concentrations. However, no improvement in the yield of geodin was observed. Furthermore, rice medium having different concentrations of NaCl salt and supplements such as MgSO4, NaCOOCH3, FeSO4, CuCl2, and KCl were also studied. However, the optimal conditions for the high yield of geodin were found as rice medium with 2% NaCl salt, room temperature, static condition, and 3 weeks incubation period. NaCl salt enhances the biosynthesis of geoidin, controlling the osmotic pressure. A high concentration of NaCl salt increases osmotic pressure and thus the fungal cells expand, and hence numerous cells rupture which results in low production of metabolite . Moreover, Na+ ions are comparatively uncreative and are responsible for metabolite production . The less amount of water in the medium may interfere with physiological functions such as increasing membrane permeability and producing enzymes in the fungal cell . Similarly, as the fungal cells are delicate and therefore in a static condition, porous and less compact mediums such as rice give rise to a high yield of the respective metabolite.
The yield of compound 1 was 137.2 mg/L after 3 weeks incubation period at 25 °C at optimal fermentation medium in static condition (Figure 2), while its yield was 3.4 mg/L in broth media in a shake flask. The factor of incubation period was also studied and it was found that the yield of geodin 1 increases with time, but after 3 weeks the gradual decrease in yield occurred and disappeared after 2 months. Furthermore, in the absence of NaCl salt, no chlorinated compounds were produced as we had isolated previously . Thus, NaCl salt played an important role in the production of chlorinated secondary metabolites produced by this Aspergillus sp. SYM-02-005. Similarly, the hypothetical biosynthetic pathway  for geodin 1 was also confirmed (Figure 3).
Funding source: Program of the National Natural Science Foundation of China
Award Identifier / Grant number: 41606172
Funding source: the Fundamental Research Funds for the Central Universities
Award Identifier / Grant number: 201841004
Funding source: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University
Funding source: the Project of State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University)
Funding source: Ministry of Education of China
Award Identifier / Grant number: CMEMR2016-A06
Award Identifier / Grant number: CMEMR2016-B04
Research funding: This work was supported by the Program of the National Natural Science Foundation of China (No. 41606172), the Fundamental Research Funds for the Central Universities (No. 201841004), Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, the Project of State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China (Nos. CMEMR2016-A06 and CMEMR2016-B04).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: The authors state no conflict of interest.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: The local Institutional Review Board deemed the study exempt from review.
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