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A new diketopiperazine, glionitrin B (1), was produced using a microbial coculture of the fungus Aspergillus fumigatus KMC-901 and the bacterium Sphingomonas sp. KMK-001 that were isolated from acidic coal mine drainage. The structure of 1 was determined to be (3S,10aS)-dithiomethylglionitrin A. This structure was determined by the analyses of extensive NMR data and the circular dichroism spectra of the natural product and a semisynthetic compound derived from glionitrin A. In contrast to glionitrin A (2), glionitrin B (1) is not cytotoxic against the human prostate cancer cell line DU145. However, compound 1 caused suppression of DU145 cell invasion, producing 46% inhibition at 60 μM.

The nutrient conditions present in abandoned coal mine drainages create an extreme environment where defensive and offensive microbial interactions could be critical for survival and fitness. Coculture of a mine drainage-derived Sphingomonas bacterial strain, KMK-001, and a mine drainage-derived Aspergillus fumigatus fungal strain, KMC-901, resulted in isolation of a new diketopiperazine disulfide, glionitrin A (1). Compound 1 was not detected in monoculture broths of KMK-001 or KMC-901. The structure of 1, a (3S,10aS) diketopiperazine disulfide containing a nitro aromatic ring, was based on analysis of MS, NMR, and circular dichroism spectra and confirmed by X-ray crystal data. Glionitrin A displayed significant antibiotic activity against a series of microbes including methicillin-resistant Staphylococcus aureus. An in vitro MTT cytotoxicity assay revealed that 1 had potent submicromolar cytotoxic activity against four human cancer cell lines: HCT-116, A549, AGS, and DU145. The results provide further evidence that microbial coculture can produce novel biologically relevant molecules.