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.

内生真菌是植物宿主微生物组的主要组成部分，已知可在体外合成植物源代谢物。然而，重复传代培养时代谢物产生的减弱是利用它们作为植物源代谢物替代品的主要缺点。在这项研究中，我们从嘉兰块茎中分离出了Diaporthe perseae ，一种内生真菌，表明在无菌培养物中可以产生秋水仙碱。质谱、核磁共振波谱和微管蛋白聚合分析证实该化合物是秋水仙碱。内生菌重复传代10代导致代谢物产量从55.25μg/g菌丝干重降低至2.32μg/g菌丝干重。与对照（2.61 μg/g 菌丝干重）相比，用 DNA 甲基化抑制剂 5-氮杂胞苷处理减毒培养物导致处理样品中代谢物浓度（39.68 μg/g 菌丝干重）增加，表明 5-氮杂胞苷可诱导真菌基因组克服了代谢物合成减弱的现象。与未处理的对照（总胞嘧啶甲基化的 0.78%）相比，在减毒培养物中，经 5-氮杂胞苷处理后观察到整体甲基化水平降低（总胞嘧啶甲基化的 0.41%）。这些结果为利用真菌内生菌作为极度濒危植物的植物源代谢物的名副其实的来源提供了重大突破。