An Endophytic Fungus from Camptotheca acuminata That Produces Camptothecin and Analogues

The pentacyclic quinoline alkaloid camptothecin (1) is a potent antineoplastic agent. Two of its analogues, 9-methoxycamptothecin (2) and 10-hydroxycamptothecin (3), exhibit similar potency but do not have the potential therapeutic drawbacks produced by unmodified 1. We have established methodology for the isolation and unequivocal identification and characterization of a novel endophytic fungus isolated from the inner bark of the medicinal plant Camptotheca acuminata, which produced 1-3 in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified by its morphology and authenticated by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Camptothecin (1) and its analogues were identified by H-1 NMR spectroscopy and LC-HRMS and confirmed by comparison with authentic standards. The production pattern of the metabolites over seven successive subculture generations of this endophyte was studied. A sharp attenuation in the production of 1 and 2 was observed from the first-through to the seventh-generation subculture. Therefore, these results offer a caution as to the possibility of using endophytic fungi as alternate sources of plant secondary metabolite production. Further studies have been initiated on the analysis of the upstream metabolic intermediates to understand the steps at which the production of the metabolites in question is constrained.