An Endophytic Fungus from Camptotheca acuminata That Produces Camptothecin and Analogues

Abstract

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 1H 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.

Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production

“…To establish the production pattern of CPT over successive generations, the axenic CPT-producing fungal isolates were subcultured from the first generation to its following generations by using the hyphal tip method (Kusari et al 2009). The strains of each generation were fermented, extracted, and analyzed, following the same procedure described above.…”

“…Successive subculturing of CPT-producing endophytic fungi Previous works (Kusari et al 2009;Gurudatt et al 2010) suggested that CPT production will be attenuated with subsequent successive subculturing. We performed successive subculturing of CPT-producing LY341, LY355, and LY357 to seek for CPT-producing strains with steady CPT production.…”

“…It is generally recognized that endophytes represent an important and largely untapped reservoir of unique chemical structures that have been modified through evolution (Gunatilaka 2006;Kharwar et al 2011) and exhibit the capability to produce the same functional compounds as their hosts, some examples include taxol (Stierle et al 1993(Stierle et al , 1995, podophyllotoxin , hypericin (Kusari et al 2008), and azadirachtin (Findlay et al 1997). In 2005, Puri et al reported the first discovery of CPT-producing endophytic fungus Entrophospora infrequens from the inner bark of N. foetida, which set the stage for a more comprehensive examination of other plants for the presence of CPT-producing endophytes Rehman et al 2008Rehman et al , 2009Kusari et al 2009;Shweta et al 2010Shweta et al , 2013Gurudatt et al 2010). These intriguing results have undoubtedly renewed the interests in exploring other endophytic sources of CPT and its analogues from various plant sources.…”

Antibacterial secondary metabolites from an endophytic fungus, Arthrinium sp. MFLUCC16-1053 isolated from Zingiber cassumunar

“…Endophytes may contribute to protecting their host plant from diseases by producing a diverse array of secondary metabolites such as terpenoids, palmarumycins, furandiones, phenols, dimeric anthrone and benzopyroanone (Schulz et al 2002; Porras-Alfaro and Bayman 2011). Moreover, endophytes comprise a promising rich source of potential bioactive medicinal constituents such as podophyllotoxin (Canel et al 2000), paclitaxel (Taxol) (Stierle et al 1993), hypericin (Kusari et al 2008) and camptothecin (Kusari et al 2009). Generally, medicinal plants used in traditional medicine are selected for surveying bioactive endophytic fungi due to documented beneficial applications (Kaul et al 2012; Kusari et al 2013) and thus, high likelihood of producing pharmaceutically important secondary metabolites.…”

Molecular profiling and antimicrobial potential of endophyticGliomastix polychromaCLB32 inhabitingCombretum latifoliumBlume

“…Thus, subsequent isolates can be considered as true endophytic fungi (Barnett and Hunter 1956). Earlier studies also define the successful surface sterilization process for the isolation of endophytes with no contamination of epiphytes (Kusari et al 2009; Lin et al 2010). The isolated endophytic fungus was then identified as Gliomastix sp.…”