Effect of Culture Conditions on Metabolite Production of Xylaria sp.

Seeking a strategy for triggering the cryptic natural product biosynthesis to yield novel compounds in the plant-associated fungus Xylaria sp., the effect of culture conditions on metabolite production was investigated. A shift in the production of five known cytochalasin-type analogues 1–5 to six new α-pyrone derivatives, xylapyrones A–F (compounds 6–11), from a solid to a liquid medium was observed. These compounds were identified by analysis of 1D and 2D NMR and HRMS data. Compounds 1–3 showed moderate cytotoxicity against HepG2 and Caski cancer cell lines with IC50 values ranging from 25 to 63 μM and compounds 4–11 were found to be inactive, with IC50 values >100 μM.


Introduction
Natural products from microorganisms are a vital source for innovative therapeutic agents and drug leads [1]. Unfortunately, the high rediscovery rate of known compounds in traditional screening methods has completely frustrated researchers [2]. Secondary metabolism of microbes is regulated by large amounts of genes encoding biosynthetic enzymes and therefore a variety of secondary metabolites should be produced [3,4]. In fact, only a minority of pathway genes is expressed under standardized laboratory conditions and many valuable compounds are overlooked. In order to exploit the full metabolic potential of microorganisms, many regulatory strategies to activate cryptic pathways to facilitate the discovery of new natural products through modification of culture conditions [5], external cues [6], stress [7], co-cultures [8] and genomic approaches [9] were described in the literature. In our approach, by altering easily accessible culture conditions from a solid to a liquid medium, a shift in the production of reported cytochalasin-type analogues 1-5 to hitherto unknown α-pyrone derivatives 6-11 was observed in the plant-associated fungus Xylaria sp. This paper describes the isolation, structure elucidation, and cytotoxic activities of the isolated compounds 1-11 ( Figure 1).
Compound 9 was obtained as a white amorphous solid. Its HREIMS exhibits a peak at m/z 228.0991 [M] + (calcd for 228.0998), indicating a molecular formula C11H16O5 with one more oxygen atom than 8 [C11H16O4]. The NMR spectroscopic data ( Table 2 and Supporting Information) indicated that they were structurally related, and that they differed in their oxidation status. Detailed analysis of the 1 H-and 13 C-NMR spectra revealed that the methylene unit at δH 2.44 and δC 33.1(CH2) of 8 was replaced by an oxygenated methine unit at δH 4.23 (1H, brs) and δC 69.5(CH) in 9. Unexpectedly, no HMBC correlation from H1-7 (δH 4.23) to any other carbon signal was observed, so the location of the hydroxyl group was deduced to be C-7 according to the COSY correlations from OH-7 (δH 5.60) via H1-7 (δH 4.23) to H2-8 (δH 1.65 and 1.54) and a strong HMBC correlation from H2-8 (δH 1.54) to C-6 (δC 168.3) (Figure 2). Consequently, the structure of 9 was identified as shown in Figure 1 and named xylapyrone D.
The structures of xylapyrone E (10) and xylapyrone F (11) were determined straightforwardly as they were very similar to those of 8. Their NMR data ( Table 2 and Supporting Information) and HREIMS suggested the difference of their structures is that side-chains attached to C-6 in 8, 10 and 11 are pentanol, butanol and propanol, respectively.

Cytotoxic Properties
Isolated compounds 1-11 were evaluated for in vitro cytotoxicity against two cancer cell lines HepG2 and Caski. Mitomycin was employed as positive control. Of the tested compounds, compounds 1-3 showed moderate cytotoxicity, with IC50 values ranging from 25 to 63 μM, and compounds 4-11 were found to be inactive, with IC50 values >100 μM ( Table 3). The most cytotoxic compound is cytochalasin E (1). Cytochalasin-type compounds have been reported as promising lead compounds for anticancer drug discovery that reduce the proliferation of different cancer cell lines such as P388, A549 and KB cells [10,11]. α-Pyrone natural products are widespread in Nature and have a broad spectrum of biological activities [13]. The literature [14][15][16] has suggested that structural variations of the side chains attached to α-pyrones ring may make significant differences to the cytotoxicity, which is worth further investigation.

Isolation and Identification of the Strain
The endophytic fungus Xylaria sp. BM9 was isolated from a piece of fresh tissue from the inner part of a medicinal plant leaf of Saccharum arundinaceum Retz., collected from the Yichang (Hubei Province, China) in April 2011. The fungus was deposited as Xylaria sp. BM9 (GenBank accession numbers KC184129) at the Hubei Key Laboratory of Natural Products Research and Development, College of Chemistry and Life Sciences, China Three Gorges University, Yichang, China.

Seed and Mass Cultures of the Strain
The fungus Xylaria sp. BM9 was maintained on potato dextrose agar. Agar plugs containing the fungal strain were inoculated in 500 mL Erlenmeyer flasks, each containing 200 mL of potato dextrose broth. Flask cultures were incubated at 28 °C on a rotary shaker at 130 rpm for 3 days as seed culture.
Liquid fermentation was performed by the following procedure: each of the seed cultures (200 mL) was transferred into 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth. These flasks were incubated at 28 °C on a rotary shaker at 130 rpm for 14 days. After fermentation, the culture (25 L) was centrifuged to yield the supernatant and a mycelial cake. The supernatant was extracted three times with an equal volume of EtOAc, the extracts were combined and solvent was removed under reduced pressure. The mycelial cake was immersed in 2 L of acetone and the organic layers were collected and removed under reduced pressure. Two residues were combined for purification.
Solid fermentation was carried out in 500 mL Erlenmeyer flasks containing 40 g rice, each flask was inoculated with the seed cultures (5.0 mL) and incubated at 25 °C for 30 days. The fermented material was soaked in EtOAc, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract.

Cytotoxicity Test
The cancer cell lines CaSki and HepG2 were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Science (Shanghai, China). All cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 25 mM HEPES buffer, 2 mmol/L L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated in a humidified atmosphere of 5% CO2 at 37 °C. Cells (1 × 10 4 /well) were seeded in supplemented culture medium (100 μL/well) in a 96-well plate and incubated for 24 h. The medium was then replaced with a test compound-containing medium, and the cells were further incubated for 48 h. All experiments were run in parallel with controls (0.1% DMSO without test compounds) and the cell viabilities were evaluated by MTT assays. The absorbance of formazan formed was measured at 570 nm by a microplate reader. The concentrations resulting in 50% inhibition of cell proliferation/survival (IC50) as measured by this assay are given in Table 3. Each experiment was repeated three times.

Conclusions
The Xylaria genus is an ubiquitous filamentous fungus, often isolated from marine environments and terrestrial sources, that can produce various types of secondary metabolites, including typical cytochalasins [17], terpenoids [18], benzofurans [19], xanthones [20], and cyclopeptides [21]. Long-chain α-pyrones are widespread in Nature and most of the PKS genes have been involved in the biosynthesis of their derivatives from acetate units [22,23]. A few papers have reported α-pyrone metabolites from Xylaria sp. Pukachaisirikul et al. described the isolation of one known α-pyrone derivative [24]. In this paper, five known cytochalasin-type analogues 1-5 were isolated as the major metabolites from a solid rice medium culture of Xylaria sp. BM9. However, applying liquid culture conditions to Xylaria sp. BM9 resulted in the identification of six new α-pyrone derivatives, xylapyrones A-F (compounds 6-11), with different biogenetic origin, in which its PKS genes for the biosynthesis of complex liquids were actived. Xylapyrones A-F were evaluated for in vitro cytotoxicity against two cancer cell lines (Hep-G2 and Caski) and found to be inactive. Further bioassay evaluation of the compounds against fungi and other targets are ongoing, so it is an issue that we don't have sufficient amounts of these compounds to determine the absolute stereochemistry of compounds 6, 7 and 9.