Chemical epigenetics alters the secondary metabolite composition of guttate excreted by an atlantic-forest-soil-derived Penicillium citreonigrum

Abstract

Chemical epigenetic manipulation of Penicillium citreonigrum led to profound changes in the secondary metabolite profile of its guttate. While guttate from control cultures exhibited a relatively simple assemblage of secondary metabolites, the guttate collected from cultures treated with 50 muM 5-azacytidine (a DNA methyltransferase inhibitor) was highly enriched in compounds representing at least three distinct biosynthetic families. The metabolites obtained from the fungus included six azaphilones (sclerotiorin (1), sclerotioramine (6), ochrephilone (2), dechloroisochromophilone III (3), dechloroisochromophilone IV (4), and 6-((3E,5E)-5,7-dimethyl-2-methylenenona-3,5-dienyl)-2,4-dihydroxy-3-methylbenzaldehyde (5)), pencolide (7), and two new meroterpenes (atlantinones A and B (9 and 10, respectively)). While pencolide was detected in the exudates of both control and 5-azacytidine-treated cultures, all of the other natural products were found exclusively in the guttates of the epigenetically modified fungus. All of the metabolites from the P. citreonigrum guttate were tested for antimicrobial activity in a disk diffusion assay. Both sclerotiorin and sclerotioramine caused modest inhibition of Staphylococcus epidermidis growth; however, only sclerotioramine was active against a panel of Candida strains.

Figure 1

Guttates of solid-state P. citreonigrum cultures grown under control conditions (A) or in the presence of 50 μM 5-azacytidine (B). HPLC chromatograms (C₁₈ column using a gradient of 30–100% CH₃CN in H₂O and recorded at 210 nm) illustrating the differences between the metabolite profiles of the control (C) and 5-azacytidine-treated (D) P. citreonigrum guttates. Metabolites identified in scale-up isolation studies were used as authentic references to verify the identities of compounds 1–7 and 9 and 10 in the guttates.

Figure 2

Selected correlations obtained from ^2–3J_H-C HMBC experiment that were used to generate fragments A–F, which were critical for deducing the structure of 9a (A). Key ¹H-¹H ROESY correlations that were used to help assign the relative configuration of 9a (B).

Figure 3

Possible tautomers proposed for compound 9. The enol-keto structure 9a (upper left) was the only tautomer observed in MeOH, while the β-diketone compound 9b (lower center) was exclusively seen in chloroform. The alternative enol-keto form of 9 (upper right) was not detected by NMR under these experimental conditions.

Figure 4

ORTEP structure generated from the X-ray diffraction data for a single crystal of 9a that was obtained from MeOH.