Screening and selection of camptothecin producing endophytes from Nothapodytes nimmoniana.

Endophytic fungi with the ability to produce plant based secondary metabolites are a potential alternative for producing the host plant metabolite and to prevent natural plants from extinction. To isolate a high metabolite yielding endophytic strain from plants, hundreds of endophytic strains are screened and tested for product yield separately under axenic state, before shortlisting the potential endophyte, which involves huge time consumption. In this study, strategies for screening and selection of high camptothecin yielding endophytes from their natural habitat were proposed. A correlation was built between the camptothecin yield in the explants and the endophytes isolated from them. In addition, camptothecin yield was compared between the endophytes isolated from young and matured plants. Further, camptothecin producers and non-producers strains were compared for their tolerance toward camptothecin. The study indicates that high camptothecin yielding endophytes were isolated from high yielding explants and younger plants and they were more tolerant to camptothecin in comparison to non-camptothecin yielding endophytes. Thus, choosing a young and high yielding explant for endophyte isolation, and use of camptothecin as a selective agent in the growth medium, can be instrumental in screening and selection of high camptothecin yielding endophytes from nature in relatively less time.

Sustainable production of camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana.

Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host-plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 µg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.