Biotechnological approaches for the production of camptothecin.

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved . KEY POINTS: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production.

[Advances in paclitaxel biosynthesis and transcriptional regulation mechanisms].

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots.

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

Chlorophyllides: Preparation, Purification, and Application.

Chlorophyllides can be found in photosynthetic organisms. Generally, chlorophyllides have a-, b-, c-, d-, and f-type derivatives, and all chlorophyllides have a tetrapyrrole structure with a Mg ion at the center and a fifth isocyclic pentanone. Chlorophyllide a can be synthesized from protochlorophyllide a, divinyl chlorophyllide a, or chlorophyll. In addition, chlorophyllide a can be transformed into chlorophyllide b, chlorophyllide d, or chlorophyllide f. Chlorophyllide c can be synthesized from protochlorophyllide a or divinyl protochlorophyllide a. Chlorophyllides have been extensively used in food, medicine, and pharmaceutical applications. Furthermore, chlorophyllides exhibit many biological activities, such as anti-growth, antimicrobial, antiviral, antipathogenic, and antiproliferative activity. The photosensitivity of chlorophyllides that is applied in mercury electrodes and sensors were discussed. This article is the first detailed review dedicated specifically to chlorophyllides. Thus, this review aims to describe the definition of chlorophyllides, biosynthetic routes of chlorophyllides, purification of chlorophyllides, and applications of chlorophyllides.

Phytochemical, Antiplasmodial, Cytotoxic and Antimicrobial Evaluation of a Southeast Brazilian Brown Propolis Produced by Apis mellifera Bees.

Seven phenolic compounds (ferulic acid, caffeic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside), a flavanonol (7-O-methylaromadendrin), two lignans (pinoresinol and matairesinol) and six diterpenic acids/alcohol (19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid and isopimaric acid) were isolated from the hydroalcoholic extract of a Brazilian Brown Propolis and characterized by NMR spectral data analysis. The volatile fraction of brown propolis was characterized by CG-MS, composed mainly of monoterpenes and sesquiterpenes, being the major α-pinene (18.4 %) and ß-pinene (10.3 %). This propolis chemical profile indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia might be its primary plants source. The brown propolis displayed significant activity against Plasmodium falciparum D6 and W2 strains with IC50 of 5.3 and 9.7 µg/mL, respectively. The volatile fraction was also active with IC50 of 22.5 and 41.8 µg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside showed IC50 of 3.1 and 1.0 µg/mL against D6 and W2 strains, respectively, while communic acid showed an IC50 of 4.0 µg/mL against W2 strain. Cytotoxicity was determined on four tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methylaromadendrin, and isopimaric acid showed an IC50 range of 1.8-0.78 µg/mL, 7.3-100 µg/mL, and 17-18 µg/mL, respectively, against the tumor cell lines but they were not cytotoxic against normal cell lines. The crude extract of brown propolis displayed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus, and P. aeruginosa at 29.9 µg/mL, 178.9 µg/mL, and 160.7 µg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans at 53.0 µg/mL. The compounds 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 7-oxodehydroabietic acid were active against C. neoformans, and caffeic and communic acids were active against methicillin-resistant Staphylococcus aureus.

Triptolide: pharmacological spectrum, biosynthesis, chemical synthesis and derivatives.

Triptolide, an abietane-type diterpenoid isolated from Tripterygium wilfordii Hook. F., has significant pharmacological activity. Research results show that triptolide has obvious inhibitory effects on many solid tumors. Therefore, triptolide has become one of the lead compounds candidates for being the next "blockbuster" drug, and multiple triptolide derivatives have entered clinical research. An increasing number of researchers have developed triptolide synthesis methods to meet the clinical need. To provide new ideas for researchers in different disciplines and connect different disciplines with researchers aiming to solve scientific problems more efficiently, this article reviews the research progress made with analyzes of triptolide pharmacological activity, biosynthetic pathways, and chemical synthesis pathways and reported in toxicological and clinical studies of derivatives over the past 20 years, which have laid the foundation for subsequent researchers to study triptolide in many ways.

Current advances of endophytes as a platform for production of anti-cancer drug camptothecin.

Camptothecin (CPT), a well-known monoterpenoid indole alkaloid with broad-spectrum anti-cancer activity, is produced from plants and endophytes. In view of the limitations of plants as sources of camptothecin in productivity and efficiency, endophytes serve as the fast growth, high cost-effectiveness, good reproducibility, and feasible genetic manipulation, so they have the potential to meet the huge market demand of the pharmaceutical industry. In this review, we summarized the isolation, identification and fermentation of CPT-producing endophytes, as well as the biosynthesis, extraction and detection of camptothecin from endophytes. Among them, we put emphasis on increasing the production of camptothecin in endophytes through different strategies such as changing the proportion of carbon, nitrogen and phosphate source, adding the precursors, elicitors or adsorbent resin, utilizing co-culture fermentation or fermenter culture. However, cell subculture and metabolic reprogramming affect the expression of camptothecin biosynthetic genes in CPT-producing endophytes, which poses a challenge to the industrial production of camptothecin. Therefore, it will be useful to gain insights through the review of these researches and provide alternative approaches to develop economical, eco-friendly and reliable natural products.

The Research Progress of Taxol in Taxus.

BACKGROUND: Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. OBJECTIVE: In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. RESULTS: The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. CONCLUSION: Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

Research progress on the source, production, and anti-cancer mechanisms of paclitaxel.

Paclitaxel, a tetracyclic diterpenoid compounds, was firstly isolated from the bark of the Pacific yew trees. Currently, as a low toxicity, high efficiency, and broad-spectrum natural anti-cancer drug, paclitaxel has been widely used against ovarian cancer, breast cancer, uterine cancer, and other cancers. As the matter of fact, natural paclitaxel from Taxus species has been proved to be environmentally unsustainable and economically unfeasible. For this reason, researchers from all over the world are devoted to searching for new ways of obtaining paclitaxel. At present, other methods, including artificial cultivation of Taxus plants, microbial fermentation, chemical synthesis, tissue and cell culture have been sought and developed subsequently. Meanwhile, the biosynthesis of paclitaxel is also an extremely attractive method. Unlike other anti-cancer drugs, paclitaxel has its unique anti-cancer mechanisms. Here, the source, production, and anti-cancer mechanisms of paclitaxel were summarized and reviewed, which can provide theoretical basis and reference for further research on the production, anti-cancer mechanisms and utilization of paclitaxel.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Nanoelicitor based enhancement of camptothecin production in fungi isolated from Ophiorrhiza mungos.

In the study, endophytic fungi isolated from Ophiorrhiza mungos were screened for camptothecin (CPT) biosynthetic potential by high performance liquid chromatography (HPLC). Among the 16 fungi screened, OmF3, OmF4, and OmF6 were identified to synthesize CPT. Further LC-MS analysis also showed the presence of CPT specific m/z of 349 for the extracts from OmF3, OmF4, and OmF6. However, the fragmentation masses with m/z of 320, 305, 277 and 220 specific to the CPT could be identified only for the OmF3 and OmF4. These CPT producing fungi were further identified as Meyerozyma sp. OmF3 and Talaromyces sp. OmF4. The cultures of these two fungi were then supplemented with nanoparticles and analyzed for the quantitative enhancement of CPT production by LC-MS/MS. From the result, Meyerozyma sp. OmF3 was found to produce 947.3 ± 12.66 µg/L CPT, when supplemented with 1 µg/mL zinc oxide nanoparticles and the same for uninduced parental strain OmF3 was only 1.77 ± 0.13 µg/L. At the same time, Talaromyces sp. OmF4 showed the highest production of 28.97 ± 0.37 µg/L of CPT when cultured with 10 µg/mL silver nanoparticles and the same for uninduced strain was 1.19 ± 0.24 µg/L. The observed quantitative enhancement of fungal CPT production is highly interesting as it is a rapid and cost effective method. The study is remarkable due to the identification of novel fungal sources for CPT production and its enhancement by nanoparticle supplementation.

Towards the Microbial Production of Plant-Derived Anticancer Drugs.

Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs.

Production of plant-derived anticancer precursor glucoraphanin in chromosomally engineered Escherichia coli.

Glucoraphanin is a methionine-derived glucosinolate that imparts numerous health-benefits with broad bioactivity. Low amounts in plant tissues and high cost of extraction have limited the production of glucoraphanin. Metabolic engineering in heterologous microorganisms is an attractive approach to achieve efficient production of valuable natural products. In this study, a microbial fermentation process for glucoraphanin production was demonstrated. The engineered bacterial strain stably expressed 10 allogeneic enzymes in E. coli chromosome, including nine heterologous genes from Arabidopsis and Brassica and one from fungus Neurospora crassa, which could produce the specialized glucosinolate compound glucoraphanin with a titer of 0.675 µg/L by fermentation from glucose. The cofactor supplements and individual gene overexpression for glucoraphanin production were also investigated. This work highlights the possibility of supplying specialized plant glucosinolates by microbial fermentation process, instead of chemical extraction. Additionally, the limiting step enzyme, UDP-glucose-thiohydroximate glucosyltransferase, identified in this study also laid a foundation for further optimizing the glucoraphanin-producing cell factory.

High-effective biosynthesis of baccatin â ¢ by using the alternative acetyl substrate, N-acetyl-d-glucosamine.

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

Mechanistic biosynthesis of SN-38 coated reduced graphene oxide sheets for photothermal treatment and care of patients with gastric cancer.

The graphene-based nanomaterials have been measured as a most promising nanomaterials in the various fields. Graphene oxide having attractive and effective attention in the modified of medicine. Also, graphene oxide is one of the distinct bio-chemical properties with minimum cytotoxicity compared to the other nanomaterials. Up to till date, gastric treatments with reduced graphene oxide not studied so far. In this report, 7-ethyl- 10-hydroxycamptothecin (SN-38) coated graphene oxide synthesized (SN-rGO) effectively and are characterized using various analytical methods. The hydroxyl and carbonyl gatherings of oxidized SN38 will in general ingest onto GO by means of hydrogen bond arrangement with their leftover oxygen functionalities and offers steadiness to SN38. The morphological analyses showed the foldable fields of SN38-rGO NPs with acquire transparency, thin sheets and the crumpled structures. Further the photothermal efficiency and cytotoxicity of the SN-rGO were examined by MTT assay using two NCI-N87 and SGC-791 gastric carcinoma cells. In addition, the morphological changes were examined through the live and dead cells and nuclear staining biochemical techiniques and apoptosis were evaluated through flow cytometry analysis. Our result's suggested that the SN-38 coated reduced graphene oxide can be used for the photothermal treatment of gastric carcinoma for the future nanotechnology cancer therapies without using functionalized polymeric nanoparticles.

Rewiring cellular metabolism for heterologous biosynthesis of Taxol.

Taxol is one of the anticancer drugs synthesized naturally in the evergreen Taxus brevifolia forest tree belonging to the yew family (Taxaceae) growing on the Pacific. There are reportedly evidence for treating ovarian, breast and lung cancers through this drug given its unique structural and functional features. Extraction of this drug from yew trees bark is one of the most common ways of producing this drug, but 3000 trees are needed to obtain a kilogram of Taxol. Hence, further attention has recently been attracted to the metabolic engineering strategies, including, engineering cellular metabolism of microorganisms and their optimization. Accordingly, the present paper article was aimed to review recent advances in elevating the production and commercialization of Taxol through metabolic engineering techniques.

Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae).

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Iron oxide nanoparticles: a novel elicitor to enhance anticancer flavonoid production and gene expression in Dracocephalum kotschyi hairy-root cultures.

BACKGROUND: Dracocephalum kotschyi Boiss. is a valuable source of rosmarinic acid (RA) and methoxylated hydroxyflavones (such as xanthomicrol and cirsimaritin) with antioxidative and antiplatelet effects and with antiproliferative potential against various cancer cells. The extensive application of nanotechnology in hairy root cultures is a new sustainable production platform for producing these active constituents. In the present study, hairy roots derived from 4-week-old leaves and Agrobacterium rhizogenes strain ATCC15834 were used to investigate the impact of various concentrations of iron oxide nanoparticles (Fe NPs) in two elicitation time exposures (24 and 48 h) on growth, antioxidant enzyme activity, total phenolic and flavonoid content (TPC and TFC), and some polyphenols. Gene expression levels of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) were also analyzed. RESULTS: Iron nanoparticles enhanced biomass accumulation in hairy roots. The treatment time and Fe NP dosage largely improved the activity of antioxidant enzymes, TPC and TFC. The highest RA (1194 µg g-1 FW) content (9.7-fold), compared to controls, was detected with 24 h of exposure to 75 mg L-1 Fe NP, which was consistent with the expression of pal and ras genes under the influence of elicitation. The xanthomicrol, cirsimaritin, and isokaempferide content was increased 11.87, 3.85, and 2.27-fold, respectively. CONCLUSION: Stimulation of D. kotschyi hairy roots by Fe NPs led to a significant increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and xanthomicrol. © 2019 Society of Chemical Industry.

Metabolic and transcriptional analyses in response to potent inhibitors establish MEP pathway as major route for camptothecin biosynthesis in Nothapodytes nimmoniana (Graham) Mabb.

BACKGROUND: Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production. RESULTS: In present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40-57% and 64-71.5% respectively, while 4.61-7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7-11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect. CONCLUSIONS: Metabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production.