DNA demethylation overcomes attenuation of colchicine biosynthesis in an endophytic fungus Diaporthe

Fungal endophytes, a major component of the plant host microbiome, are known to synthesize plant-derived metabolites in vitro. However, attenuation of metabolite production upon repeated sub-culturing is a major drawback towards utilizing them as an alternative for plant-derived metabolites. In this study, we isolated Diaporthe perseae, a fungal endophyte from Gloriosa superba tubers, which showed the production of colchicine in axenic cultures. Mass spectrometry, Nuclear Magnetic Resonance spectroscopy, and tubulin polymerization assays confirmed the compound to be colchicine. Repeated sub-culturing of the endophyte for 10 generations led to a reduction in the yield of the metabolite from 55.25 μg/g to 2.32 μg/g of mycelial dry weight. Treatment of attenuated cultures with DNA methylation inhibitor 5-azacytidine resulted in increased metabolite concentration (39.68 μg/g mycelial dry weight) in treated samples compared to control (2.61 μg/g mycelial dry weight) suggesting that 5-azacytidine can induce demethylation of the fungal genome to overcome the phenomenon of attenuation of metabolite synthesis. Reduced levels of global methylation were observed upon 5-azacytidine treatment in attenuated cultures (0.41 % of total cytosines methylated) as compared to untreated control (0.78 % of total cytosines methylated). The results provide a significant breakthrough in utilizing fungal endophytes as a veritable source of plant-derived metabolites from critically endangered plants.