Camptothecin Yield Research Articles

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 μg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC–MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 μM) and MCF7 (3.2 μM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 μg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC–MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 μM) and UO-31 (2.2 μM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett–Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 μg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus” to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.

Effect of abiotic elicitor on the production of camptothecin from Nothapodytes nimmoniana (J. Graham) Mabb

The plant Nothapodytes nimmoniana belongs to the Icacinaceae family, and is a significant source of camptothecin (CPT), an effective quinoline alkaloid with an array of pharmacological benefits including anti-cancer, anti-HIV, antibacterial, and antioxidant etc. The elicitation method is the most effective strategy to increase and maintain the synthesis of particular compounds in vitro culture. In this study, to understand the impact of various abiotic elicitors on CPT production by using established N. nimmoniana cell suspension culture obtained from friable leaf callus grown in Murashige & Skoog basal media with sucrose and agar augmented with 2 mg/L of 2,4-dichloro phenoxy acetic acid (2,4-D). A total of six abiotic elicitors were used such as cadmium chloride, ethanol, salicylic acid, tryptophan, copper chloride, and calcium chloride. The impact of these abiotic elicitors on the CPT yield at three different concentrations by cell suspension was evaluated for up to 15 days. High-performance liquid chromatography (HPLC) was used to quantify the CPT production. A maximum of 9.43 and 6.26-fold increment was recorded in CPT production in suspension culture elicitated with 2 mM of tryptophan and 1 mM of tryptophan on the 12th day of inoculation followed by ethanol 7.5% treated cell suspension which enhanced the CPT production up to 1.44-fold increase while compared to control. The cell viability of the suspension culture treated with calcium chloride and cadmium chloride was observed in decreased biomass as per the increment in the concentration. The findings of the current study indicate the potential use of abiotic elicitation as another possible option for boosting up the synthesis of CPT in N. nimmoniana cell suspension cultures.

Production, bioprocessing and antiproliferative activity of camptothecin from Aspergillus terreus, endophyte of Cinnamomum camphora: restoring their biosynthesis by indigenous microbiome of C. camphora

Fungal producing potency of camptothecin (CPT) raise the hope for their usage to be a platform for industrial production of CPT, nevertheless, attenuation of their productivity of CPT with the subculturing and preservation is the challenge. So, screening for novel endophytic fungal isolates with a reliable CPT-biosynthetic stability was the objective. Among the isolated endophytic fungi from the tested medicinal plants, Aspergillus terreus OQ642314.1, endophyte of Cinnamomum camphora, exhibits the highest yield of CPT (89.4 μg/l). From the NMR, FT-IR and LC–MS/MS analyses, the extracted CPT from A. terreus gave the same structure and molecular mass fragmentation pattern of authentic CPT (349 m/z). The putative CPT had a significant activity against MCF7 (0.27 µM) and HEPG-2 (0.8 µM), with a strong affinity to inhibits the human Topoisomerase 1 activity (IC50 0.362 μg/ml) as revealed from the Gel-based DNA relaxation assay. The purified CPT displayed a strong antimicrobial activity for various bacterial (E. coli and B. cereus) and fungal (A. flavus and A. parasiticus) isolates, ensuring the unique tertiary, and stereo-structure of A. terreus for penetrating the microbial cell walls and targeting the topoisomerase I. The higher dual activity of the purified CPT as antimicrobial and antitumor, emphasize their therapeutic efficiency, especially with growth of the opportunistic microorganisms due to the suppression of human immune system with the CPT uses in vivo. The putative CPT had an obvious activity against the tumor cell (MCF7) metastasis, and migration as revealed from the wound healing assay. The overall yield of A. terreus CPT was maximized with the Blackett-Burman design by twofolds increment (164.8 μg/l). The CPT yield by A. terreus was successively diminished with the multiple fungal subculturing, otherwise, the CPT productivity of A. terreus was restored, and increased over the zero culture upon coculturing with C. camphora microbiome (1.5% w/v), ensuring the restoring of CPT biosynthetic potency of A. terreus by the plant microbiome-derived chemical signals “microbial communication”. This is the first report exploring the feasibility of A. terreus “endophyte of C. camphora” to be a preliminary platform for commercial production of CPT with a reliable sustainability upon uses of indigenous C. camphora microbiome.

Comprehensive analysis of OpHD-ZIP transcription factors related to the regulation of camptothecin biosynthesis in Ophiorrhiza pumila

Ophiorrhiza pumila, as a folk herb belonging to the Rubiaceae family, has become a potential source of camptothecin (CPT), which is a monoterpenoid indole alkaloid with good antitumor property. However, the camptothecin content in this herb is low, and is far from meeting the increasing clinical demand. Understanding the transcriptional regulation of camptothecin biosynthesis provides an effective strategy for improvement of camptothecin yield. Previous studies have demonstrated several transcription factors that are related to camptothecin biosynthesis, while the functions of HD-ZIP members in O. pumila have not been investigated yet. In this study, 32 OpHD-ZIP transcription factor members were genome-wide identified. Phylogenetic tree showed that these OpHD-ZIP proteins are divided into four subfamilies. Based on the transcriptome data, nine OpHD-ZIP genes were shown to be predominantly expressed in O. pumila roots, which were in line with the camptothecin biosynthetic genes. Co-expression analysis showed that OpHD-ZIP7 and OpHD-ZIP20 were potentially related to the modulation of camptothecin biosynthesis. Dual-luciferase reporter assays (Dual-LUC) showed that both OpHD-ZIP7 and OpHD-ZIP20 could activate the expression of camptothecin biosynthetic genes OpIO and OpTDC. In conclusion, this study offered the promising data for exploring the roles of OpHD-ZIP transcription factors in regulating camptothecin biosynthesis.

Cellular localisation and quantification of Camptothecin in different plant parts of Nothapodytes nimmoniana (J. Graham) Mabberley of Sri Lankan origin.

Nothapodytes nimmoniana (Icacinaceae) is a rich source of Camptothecin (CPT), an anti-cancer prodrug. Efficient extraction of CPT from various plant parts is crucial for better recovery of this pre-drug. To investigate the distribution of CPT in plant parts and to compare the methods of extraction on CPT yield to evaluate how cellular localisation affects the efficiency of extraction methods. Transverse sections of plant parts were observed under a ultraviolet (UV)-fluorescence microscope for the fluorescence that the CPT molecule emits when exposed to UV radiation. Dried plant parts were extracted using 90% methanol with ultrasonic assistance, hot ethanol (61% ethanol at 60°C), and chloroform-methanol (4:1, v/v). The CPT in plant parts were detected by thin-layer chromatography (TLC) and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Quantification was carried out by HPLC. Blue fluorescence indicated a prominent accumulation of CPT in roots compared to leaf with petiole, twigs, and stembark. This accumulation was observed in upper and lower epidermis of the leaf, isolated strands of fibres in the phloem in the petiole, and groups of idioblast cells in the cortex. The ultrasonic-assisted extraction with 90% methanol showed the highest CPT yield in the root (1.91 ± 0.02 mg/g of dry weight), followed by stembark and the least in leaves [0.02 ± 0.01 mg/g (dry weight)] irrespective of the method of extraction. However, hot ethanol extraction gave the highest CPT yield for twig and leaf, indicating the necessity of tissue-specific extraction methods for better recovery of CPT.

Production and bioprocessing of camptothecin from Aspergillus terreus, an endophyte of Cestrum parqui, restoring their biosynthetic potency by Citrus limonum peel extracts

The metabolic potency of fungi as camptothecin producer elevates their prospective use as an industrial platform for commercial production, however, the loss of camptothecin productivity by fungi with the storage and subculturing are the major obstacle. Thus, screening for endophytic fungal isolates inhabiting ethnopharmacological plants with an obvious metabolic stability and sustainability for camptothecin biosynthesis could be one of the most feasible paradigms. Aspergillus terreus ON908494.1, an endophyte of Cestrum parqui was morphologically and molecularly verified, displaying the most potent camptothecin biosynthetic potency. The chemical identity of A. terreus camptothecin was confirmed from the HPLC, FTIR and LC–MS/MS analyses, gave the same molecular structure and mass fragmentation patterns of authentic one. The purified putative camptothecin displayed a strong anticancer activity towards HepG-2 and MCF-7 with IC50 values 0.96 and 1.4 µM, respectively, with no toxicity to OEC normal cells. As well as, the purified camptothecin displayed a significant antifungal activity towards fungal human pathogen Candida albicans, Aspergillus flavus, and A. parasiticus, ensuring the unique structural activity relationships of A. terreus camptothecin, as a powerful dually active anticancer and antimicrobial agent. The camptothecin productivity of A. terreus was maximized by bioprocessing with Plackett–Burman design, with an overall 1.5 folds increment (170.5 µg/L), comparing to control culture. So, the optimal medium components for maximum yield of camptothecin by A. terreus was acid why (2.0 mL/L), Diaion HP20 (2.0 g/L), Amberlite XAD (2.0 g/L), dextrin (5.0 g/L), glucose (10.0 g/L), salicylic acid (2.0 g/L), serine (4.0 g/L), cysteine (4.0 g/L) and glutamate (10.0 g/L), at pH 6 for 15 days incubation. By the 5th generation of A. terreus, the camptothecin yield was reduced by 60%, comparing to zero culture. Interestingly, the productivity of camptothecin by A. terreus has been completely restored and over increased (210 µg/L), comparing to the 3rd generation A. terreus (90 µg/L) upon addition of methanolic extracts of Citrus limonum peels, revealing the presence of some chemical signals that triggers the camptothecin biosynthetic machinery. The feasibility of complete restoring of camptothecin biosynthetic-machinery of A. terreus for stable and sustainable production of camptothecin, pave the way for using this fungal isolate as new platform for scaling-up the camptothecin production.

Influence of plant growth regulators on morphogenic response, biomass and camptothecin production in the callus cultures of Chonemorpha fragrans (moon) Alston

In vitro morphogenic response of mature seed embryo-derived callus cultures of Chonemorpha fragrans was studied using solid and liquid Murashige and Skoog medium amended with cytokinins or their combinations with naphthalene acetic acid at 0.5 mg L-1. The plant growth regulators (PGRs) combination and concentrations tested could not stimulate organogenesis after three subcultivations of the callus cultures on the same PGRs-amended solid medium and when cultivated in the liquid but, formation of morphogenic callus was observed. Evaluation of biomass and camptothecin production showed that the PGRs influenced biomass and CPT yield of the callus cultures. The alkaloid yield of various explants of 3-4 weeks old axenic seedlings was higher in roots (0.019% CPT) followed by mature seed embryos (0.0053%), cotyledons (0.0039%), hypocotyls (0.0024%) and leaves (0.0017%), and no significant difference was observed in yield of CPT from callus induced from the various explants. Camptothecin yield of morphogenic callus cultures cultivated in liquid medium was lower than that of solid due to extracellular leaching effect of the alkaloid. Amount of synthesized CPT in the callus cultures also varied with PGR type and concentration amended in the cultivation medium, and was association with biomass production. Results of the present study suggest that callus cultures offer alternative tissue source for in vitro CPT yield enhancement through biotechnological approaches, with application in the large-scale production of the alkaloid to conserve the ever-decimated natural population of the medicinal woody climber for CPT.

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.

Variation in seedling vigour and camptothecin content of Pyrenacantha volubilis Wight: insights for domestication

As a pioneering work in the field of domestication of Pyrenacantha volubilis Wight, an anti-cancer drug (camptothecin) yielding plant, this study identifies superior seed sources of southern peninsular India for various parameters related to its growth and yield. The experiment was set up following the common garden experiment protocol in Completely Randomised Design. Laboratory extraction of camptothecin from whole seeds was carried out using High Performance Liquid Chromatography. Whereas seeds from Kizhoor population were the heaviest (test weight of 171.5 g), seedlings obtained by sowing the seeds from Villiampakkam population performed better in terms of total yield per plant (5.25 g dry weight) as well as total camptothecin yield per hectare (223.125 g). Almost three times variation was observed in the experiment between the poorest and the best performing populations. We attempt to discuss the potential for domestication, large-scale planting and sustainable harvesting of this promising and versatile new cash crop.

Biotechnological strategies for production of camptothecin from fungal and bacterial endophytes

The horrifying impact of emergence of cancer as an endemic disease worldwide associated with accelerated mortality rate caused significant boost in anticancer drug discovery. The most successful broad spectrum anti-neoplastic monoterpene indole alkaloid camptothecin (CPT) is the product of such natural product based therapeutic approach. This promising anticancer compound, currently approved against cancer, was first isolated from Camptotheca acuminata of Nyssaceae family and was reported as a potent inhibitor of eukaryotic topoisomerase I. Despite its continuous success under clinical studies, low accumulation, slow growth and over-exploitation of Camptotheca acuminata, high extraction cost, complex nature of total, semi and chemical synthesis has failed to meet the industrial demand. As an alternative bio-prospecting route, fermentation of endophytic microbial culture and co-culture of CPT producing plant cell suspension with endophytes have evolved. Endophytes are reported for their contribution in plant growth, disease resistance, stress tolerance and agricultural productivity which may have contributed to scale up the CPT production. This review sums up the current trend in endophytic microbes-based CPT biosynthesis and biotechnological strategies for optimization of CPT yield. Moreover, interaction of endophytic fungi with different host plants under experimental variables, their mode of elicitation, possibility of microbial strain improvement via application of biotechnological strategies and respective lacunas are also being elucidated.

The effects of different nitrogen sources on camptothecin content and related gene expression in Camptotheca acuminata seedlings

Camptotheac acuminata Decne is a unique tree species in China with an important secondary metabolite, camptothecin (CPT), used in the treatment of cancer. Nitrogen (N) is an important element that affects plant growth and the accumulation of CPT. Reports on the effect of N on CPT synthesis from a genetic perspective are scarce. To explore the effects of different N sources and levels on CPT synthesis in C. acuminata, two-year-old seedlings were fertilized with different concentrations of pure ammonium sulphate, source of ammonium N (NH4+–N), and potassium nitrate for nitrate N (NO3−–N). Concentrations of 2.5, 5, 7.5, and 10 g pot−1 NH4+–N and NO3−–N were used. The results showed that 7.5 g NH4+–N and NO3−–N treatments were best for growth and fresh weight of leaves. Compared with the other treatments, the CPT content, tryptophan synthase and tryptophan decarboxylase activities, and expression of the CaTSB and CaTDC1 genes under the 2.5 g NH4+–N and NO3−–N treatments peaked significantly at 30 days. However, the expression of CaTDC2 surpassed that of the other two genes at 60 days. Therefore, compared with NH4+–N source, the NO3−–N source was more beneficial for growth, and NO3−–N was better for CPT yield. Consequently, leaves of C. acuminata treated with 2.5 g NO3−–N could be harvested after 30 days to obtain maximum CPT content. CaTDC1 is more closely linked to CPT synthesis. The results of this study improved the production of CPT in C. acuminata via fertilization.

Effects of major nutrients, growth regulators and inoculum size on enhanced growth and camptothecin production in adventitious root cultures of Ophiorrhiza mungos L.

Adventitious root cultures of medicinal plants are a source of secondary metabolites of pharmaceutical importance. Ophiorrhiza mungos (Rubiaceae) contains a bioactive compound, camptothecin (CPT), which possess anticancer activity. Adventitious roots of O. mungos were induced on aseptic leaf explants and established isolated root cultures. Various culture parameters were studied and showed that 1/2th strength MS liquid media with 5.0 % (w/v) sucrose, 0.5mgL−1 IBA, 0.25mgL−1 1-Naphthaleneacetic acid (NAA) and 0.1mgL−1 Gibberellic Acid (GA3) and an inoculum size of 2gL−1 was ideal for enhanced biomass combined and CPT yield. The concentration of ammonium to nitrate at 20:40mM favoured root growth and 50:10mM favoured CPT accumulation. A significant increase in root growth was observed at 1.25mM phosphate and CPT accumulation was unaffected. Roots were grown in a modified nutrient medium (MS1) according to the results obtained with the individual culture parameters and obtained significantly high biomass and CPT production. CPT was estimated using high performance liquid chromatography (HPLC). The results revealed the effect of major nutrients, growth regulators and inoculum size on growth and CPT production. The study showed that adventitious root culture of O. mungos is a viable alternative culture method for CPT production and results are useful in the scaling up studies using suitable bioreactor systems for the large-scale production of CPT.

Should Seed Be an Alternative Source of Camptothecin From Nothapodytes nimmoniana and Chonemorpha fragrance?

The Southeast Asian forest is blessed with plant sources of anticancer molecules including camptothecin (CPT) but, the irrational harvest is among serious threat to its existence for use by the future generation. Among plant sources of CPT found in the region are Nothapodytes nimmoniana and Chonemorpha fragrance that are under heavy exploitation pressure for the alkaloid and other medicinal purposes. Under the natural conditions of forests, seeds produced by the plants show germination difficulties, and as a result lose viability few months after set. The unviable seeds can be used as an alternative source of CPT and other phytochemicals isolated from the species. In this study, evaluation of CPT yield of seeds and their parts was performed by high-performance thin layer chromatographic analysis to assess the feasible economic use of the seeds as an alternative source of the alkaloid. The results showed a manifold higher yield of CPT in seeds and their parts in N. nimmoniana over C. fragrance, suggesting seeds of the former that face more exploitation of its forest than the latter as a better alternative source of the alkaloid. The results also suggest that collection of the seed should be made before the onset of rain season. In this study, the economic implications of using seeds to isolate CPT are also discussed.

Cell line selection combined with jasmonic acid elicitation enhance camptothecin production in cell suspension cultures of Ophiorrhiza mungos L.

Ophiorrhiza mungos is a herbaceous medicinal plant which contains a quinoline alkaloid, camptothecin (CPT), an anticancer compound. A high-yielding cell line, O. mungos cell line-3 (OMC3) was selected from cell suspension cultures of O. mungos using cell aggregate cloning method and established cell suspension culture. OMC3 cell suspension produced significantly high biomass (9.25±1.3g/flask fresh weight (FW)) and CPT yield (0.095±0.002mgg-1 dry weight (DW)) compared with the original cell suspension. Inoculum size of OMC3 cell suspension culture was optimised as 14gL-1. Media optimisation has shown that 5% (w/v) sucrose and an increased ammonium/nitrate concentration of 40/20mM favoured CPT production, whereas 3% (w/v) sucrose, an ammonium/nitrate concentration of 20/40mM and 1.25mM of phosphate favoured biomass accumulation. Jasmonic acid, chitin and salicylic acid was used to elicit CPT production in the original cell suspension culture and achieved significantly high CPT production with jasmonic acid (JA) elicitation. Further, OMC3 cell suspension culture was elicited with JA (50μM) and obtained 1.12±0.08mgg-1 DW CPT and 9.52±1.4g/flask FW (190.4gL-1 FW). The combination of cell line selection and elicitation has produced 18.66-fold increases in CPT production together with significantly high biomass yield. The study is helpful in the scale-up studies of O. mungos cell suspension culture in suitable bioreactor systems for the production of CPT.

Effects of light on production of camptothecin and expression of key enzyme genes in seedlings of Camptotheca acuminate Decne

Camptotheca acuminata (C. acuminata) is utilized in preparation of drugs and as constituent in functional foods of China due to high camptothecin (CPT) content in different plant parts. Light intensity is one of the most critical factors which affect plant growth and secondary metabolites. Pot experiment was conducted to study the effect of light intensity (i.e., 100 % irradiance (control), 75 % irradiance, 50 % irradiance and 25 % irradiance) on contents of CPT, activity of enzymes and genes expression related to CPT biosynthesis of C. acuminata seedlings. The study examined total leaf biomass, CPT content, activities of tryptophan synthase (TSB) and tryptophan decarboxylase (TDC), and relative expression of TSB, TDC1, and TDC2 genes. Plants grown in 75 % irradiance possessed the greatest leaf biomass compared with 100 % light irradiance. Highest values of CPT contents were found after 60 days in plants grown in 50 % irradiance, followed by 25, 75 % and full sunlight. Furthermore, activities of TSB, TDC and relative expression of genes of TSB, TDC1, and TDC2, were significantly increased after 60 days of 50 % irradiance compared with full sunlight. Irradiance of 50 % up-regulated the expression of CPT biosynthesis-related genes and induced CPT biosynthesis. In addition to that lower or higher irradiance inhibited the expression of CPT biosynthesis-related genes and CPT biosynthesis. It is concluded that manipulating light intensity can be an effective means to achieve highest CPT yield in medicinal plants.

Enhanced camptothecin production induced by elicitors in the cell suspension cultures of Ophiorrhiza mungos Linn.

Production of camptothecin (CPT), an anticancer compound was enhanced in the cell suspension cultures of Ophiorrhiza mungos Linn. through elicitor treatment. Cell suspension culture was established using the friable callus tissues induced from the field grown leaf explants cultured in MS solid media supplemented with 3 % sucrose, 3 mg L−1 1-Naphthaleneacetic acid (NAA), 1 mg L−1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg L−1 kinetin (KIN). The callus tissues were used for establishing cell suspension culture in half-strength MS (1/2X MS) liquid media supplemented with the same hormone concentration. NAA was found to be essential for the prolific growth of O. mungos cells in suspension culture. Influence of different elicitors such as yeast extract (YE) and silver nitrate (AgNO3) on cell growth, CPT accumulation and cell viability was studied and found that YE and AgNO3 caused a significant increase in biomass and CPT yield according to their concentration, incubation time and feeding time. A maximum of 13.3-fold increment in CPT production and threefold increase in cell growth were recorded in cell cultures elicited with 50 mg L−1 YE on the 10th day of incubation. Cell growth and CPT level were found to decrease in the cultures treated with high concentration of elicitors. CPT was estimated using high performance liquid chromatography (HPLC). The results obtained in the present investigation suggest the use of elicitation as a promising alternative method to increase CPT production and cell growth in the cell suspension cultures of O. mungos.

Isolation and characterization of Paenibacillus polymyxa LY214, a camptothecin-producing endophytic bacterium from Camptotheca acuminata.

Camptothecin (CPT) is mainly produced and extracted from Camptotheca acuminata and Nothapodytes foetida for pharmaceutical use, i.e., the starting material for chemical conversion to the clinical CPT-type drugs. As the third largest plant anticancer drug, the heavy demand on CPT from global market leads to many research efforts to identify new sources for CPT production. Herein we report the isolation and characterization of a CPT-producing endophytic bacterium Paenibacillus polymyxa LY214 from Camptotheca acuminata. A 10.7μgl(-1) of CPT was presented in the fermentation broth of P. polymyxa LY214. Its CPT production decreased sharply when the strain of the 2nd generation of P. polymyxa LY214 was cultured and fermented. However, the CPT production remained relatively constant from 2.8μgl(-1) of the 2nd generation to 0.8μgl(-1) of the 8th generation of P. polymyxa LY214 under optimized fermentation conditions. A 15- to 30-fold increase of CPT yield was observed when the optimized fermentation conditions, together with the addition of putative biosynthetic precursors of CPT and adsorbent resin XAD16, were applied to ferment the strains of the 7th and 8th generation of P. polymyxa LY214. Bioinformatics analysis of the relative species of P. polymyxa LY214 indicates its potential to produce CPT, which will be helpful to decipher the mysteries of CPT biosynthesis.

Biosynthesis and biotechnological production of anti-cancer drug Camptothecin

Camptothecin (CPT) is a kind of modified monoterpene indole alkaloid firstly identified from woody plant Camptotheca acuminata, and its semisynthetic CPT analogs irinothecan and topothecan are clinically used for the treatment of various cancers throughout the world. However, the extraction of CPT from limited natural CPT-producing plant resources couldn’t meet the rapidly increasing market need. The development of plant metabolic engineering provides one alternative way to increase CPT yield by genetic manipulation, which relies on in-depth understanding of the CPT biosynthesis pathway. Several attempts have been also made to obtain CPT by biotechnological approaches such as cell suspensions, endophytic fungi, hairy roots, elicitation as well as metabolic engineering in the past decade. Here, recent advances in knowledge of biosynthesis of CPT, gene isolation, molecular regulation, production improvement and biotechnological methods are summarized and future perspectives are also discussed in this review.

Light quality modifies camptothecin production and gene expression of biosynthesis in Camptotheca acuminata Decne seedlings

The responses of plant growth and secondary metabolites to light quality are useful measurements to determine suitable habitat conditions for the cultivation of medicinal plants. The effect of light quality (white, blue, yellow, and red light) on leaf growth, camptothecin yield, the enzymatic activity, and the expression of camptothecin biosynthesis-related genes were investigated in Camptotheca acuminata seedlings. This was accomplished via measuring total leaf biomass, camptothecin content, activities of TSB and TDC, and the relative expression of TSB, TDC1, and TDC2 genes. Compared with white light, the red light treatment displayed the highest leaf biomass, while yellow light and blue light inhibited the growth of the plants. The lowest leaf biomass was found in plants under the blue light treatment. On day 45, the highest values of CPT content were observed under blue light conditions, followed by yellow light, red light, and white light. Among the four light environments, camptothecin yield was the highest in plants grown under red light. Furthermore, activities of TSB and TDC as well as the relative expression of genes of TSB, TDC1, and TDC2 were significantly increased on day 45 under blue light as compared with the white light. This suggests that blue light up-regulated the expression of camptothecin biosynthesis-related genes and induced camptothecin biosynthesis. The results indicate that red light was effective for inducing the production of camptothecin in C. acuminata seedling leaves. Manipulating light quality can be an effective means to achieve the highest camptothecin yield in medicinal plantation, but this conclusion needs to be further verified by more well-designed large-scale trials.