Review on Camptothecin Producing Medicinal Plant: Nothapodytes Nimmoniana

Nothapodytes nimmoniana is an endangered medicinal plant widely distributed throughout the Western Ghats of India. The plant contains camptothecin (CPT) which is renowned anticancer drugs. Though, CPT found in many plant species but maximum amount of CPT has been reported from N. nimmoniana. Due to very good source of CPT, this plant has been explored for its Phytochemical, Biotechnological and Pharmacological aspects. Looking to the huge global demand for CPT, overexploitation of N. nimmoniana, unplanned deforestation, and lowest production of CPT from intact plant, reduction of seed germination, high market cost and not have economically feasible process of production has optimistic us to investigate this plant in a systematic manner. The proposed article can be utilized for the establishment of extraction methods and analytical protocol for CPT. Also, Bioreactors production of CPT using high yielding cell line of N. nimmoniana. The pharmacological data will be applicable for discovery of new Drug and development lead to novel compounds which are safe and effective.

Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila

Background The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.

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.

Transcriptome-wide identification, characterization, and phylogenomic analysis of cytochrome P450s from Nothapodytes nimmoniana reveal candidate genes involved in the camptothecin biosynthetic pathway

The plant Nothapodytes nimmoniana is an important source of camptothecin (CPT), an anticancer compound widely used in the treatment of colorectal, lung, and ovarian cancers. CPT is biosynthesized by the combination of the seco-iridoid and indole pathways in plants. The majority of the biosynthetic steps and associated genes still remain unknown. Certain reactions in the seco-iridoid pathway are catalyzed by cytochrome P450 enzymes. Hence, identifying transcriptionally active cytochrome P450 genes becomes essential in the elucidation of the CPT biosynthetic pathway. Here, we report the identification of 94 cytochrome P450s from the assembled transcriptomic data from leaf and root tissues of N. nimmoniana. The identified cytochrome P450 genes were full length and possessed all four conserved characteristic signature motifs of cytochrome P450 genes. Phylogenetic analysis of the protein sequences revealed their evolution and diversification and further categorized them into A-type (52.12%) and non-A-type (47.87%) cytochrome P450s. These 94 sequences represent 38 families and 63 subfamilies of cytochrome P450s. We also compared the transcriptional activity of identified cytochrome P450s with the expression of their homologs in the CPT-producing plant Ophiorrhiza pumila. Based on expression profiles and quantitative PCR validation, we propose NnCYP81CB1 and NnCYP89R1 as candidate cytochrome P450 genes involved in camptothecin biosynthesis in N. nimmoniana.

Sustainable Production of Camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana

ABSTRACTA total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of Nothapodytes 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.