Highly oxidized ergosterols and isariotin analogs from an entomopathogenic fungus, Gibellula formosana, cultivated in the presence of epigenetic modifying agents
Graphical abstract
Introduction
Epigenetic modifying agents, such as histone deacetylase (HDAC) and DNA methyltransferase inhibitors, have been introduced as effective tools for inducing transcription of silent biosynthetic gene clusters to obtain a variety of secondary metabolites.1, 2 Recently, a few studies have shown the isolation of novel fungal secondary metabolites using HDAC or DNA methyltransferase inhibitors,2, 3 and we have also demonstrated the potential for applying these inhibitors to entomopathogenic fungi by obtaining tryptophan analogs from Torrubiella luteorostrata,4 an aromatic polyketide glycoside from Cordyceps indigotica,5 dihydrobenzofurans showing ligand activity toward cannabinoid receptors (CB1 and CB2) and an alkylated polyketide from Cordyceps annullata.6 On the other hand, studies on the effect of the concomitant addition of an HDAC inhibitor and a DNA methyltransferase inhibitor on fungal secondary metabolite production have not been reported. Most recently, we have firstly identified significant changes in the secondary metabolites produced in the medium of Isaria tenuipes cultivated in the presence of both SBHA (an HDAC inhibitor) and RG-108 (a DNA methyltransferase inhibitor) and succeeded in the isolation of a novel skeletal polyketide,7 showing the usefulness of concomitantly adding both inhibitors. Continuing our search for novel secondary metabolites from entomopathogenic fungi using both types of inhibitor, we found that the cultivation of Gibellula formosana with SBHA 1 mM and RG-108 1 mM resulted in a marked increase in the production of the secondary metabolites compared with those of the fungi cultivated without inhibitors or with either SBHA or RG-108 (Fig. 1). G. formosana yielded two new highly oxidized ergosterols, formosterols A (1) and B (2), bearing unique cis-22,23-epoxide side chains and five new isariotin analogs, 12′-O-acetylisariotin A (4), 1-epi-isariotin A (5), and isariotins K–M (6–8), together with 22,23-epoxy-3,12,14,16-tetrahydroxyergosta-5,7-dien-11-one (formosterol C) (3), isariotins A (9), C (10), and E (11), TK-57-164A (12),8, 9 and beauvericin (13).10 Here, we describe the isolation and structural analysis of seven new compounds. The stereochemistry of formosterol C (3), which remained to be solved, is also reported.
Section snippets
Results and discussion
G. formosana was cultivated in YM medium (60 mL) containing both SBHA (1 mM) and RG-108 (1 mM), either SBHA (1 mM) or RG-108 (1 mM), or no chemicals. The EtOAc extracts of the culture media were analyzed by reversed-phase HPLC. The HPLC chromatograms showed that many compounds were clearly abundant in the extract obtained from the cultivation in the presence of both inhibitors compared with the extracts obtained from the other three cultivations (Fig. 1). The fermentation was scaled up (15 L)
Conclusion
The concomitant addition of both SBHA (1 mM) and RG-108 (1 mM) to the culture medium of G. formosana significantly enhanced the secondary metabolite production. The compounds induced production of two types of natural product, highly oxidized ergosterols and isariotin analogs. Previously, isariotins A–J were isolated from I. tenuipes, an entomopathogenic fungus, as minor constituents, along with a major compound, beauvericin (13).8, 9, 13 The culture medium of G. formosana cultivated without
General experimental procedures
Analytical TLC was performed on silica gel 60 F254 (Merck). Column chromatography was carried out on silica gel 60 (70–230 mesh, Merck). NMR spectra were recorded on JEOL ECA-600. Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative to tetramethylsilane (δH 0.00) and residual solvent signals (δC 77.0) for CDCl3, (δH 3.30)/(δC 49.0) for CD3OD as internal standards. Mass spectra were measured on JEOL JMS-700 and JMS-DX303. UV spectra were recorded on a JASCO-V-550
Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research (No. 23710248 and 23590582) from the Ministry of Education, Science, Sports and Culture of Japan; A-STEP (FS-stage) (No. AS231Z01347G) from Japan Science and Technology Agency (JST); the Uehara Memorial Foundation; Japan Association for Chemical Innovation.
References and notes (13)
- et al.
Tetrahedron Lett.
(2012) - et al.
Org. Lett.
(2012) - et al.
J. Nat. Prod.
(2007) - et al.
J. Nat. Prod.
(2004) - et al.
J. Ind. Microbiol. Biotechnol.
(2009) - et al.
Org. Biomol. Chem.
(2008)et al.Org. Biomol. Chem.
(2009)et al.J. Nat. Prod.
(2010)
Cited by (0)
- †
These two authors contributed equally to this work.