Chromosome-level genome assembly of Ophiorrhiza pumila reveals the evolution of camptothecin biosynthesis.

Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49 Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes' evolution post γ whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

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.

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.

Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS.

Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in Camptotheca acuminata, the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the present study, a 70% aqueous acetonitrile was verified as an optimal extraction solvent for microwave-assisted extraction (MAE) of metabolites by spiking experiments. Based on multi-objective optimization, the best extraction conditions of a solid-liquid ratio of 1:20, microwave power of 230 W, and a time of 4 min were achieved using a full factorial 34 experimental design. Crude extracts obtained from the shoot apex of C. acuminata using MAE have been qualitatively profiled by high-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS/MS) and a HPLC triple quadrupole-MS (HPLC-TSQ-MS) analysis was conducted for their metabolite content in different tissues. CPT, and ten related metabolites and their isomers, including tryptamine, loganic acid, secologanic acid, strictosidinic acid, strictosamide, strictosamide epoxide, strictosamide diol, strictosamide ketolactam, pumiloside, and deoxypumiloside, were detected and tentatively identified. Scanning electron microscopy (SEM) imaging of the shoot apex demonstrated that severe cell disruption was evident after intensified extraction processes. The study showed the difference of metabolite profiles and the enhancement of metabolite content after microwave-pretreated techniques, and the established MAE procedure is an effective methodology to preserve valuable metabolite compounds for analysis.

Possible clues for camptothecin biosynthesis from the metabolites in camptothecin-producing plants.

The plant derived camptothecin (CPT) is a pentacyclic pyrroloquinoline alkaloid with unique antitumor activity. Successive discoveries of new CPT-producing plants occurred in recent years due to market demands. The scattered distribution among angiosperms drew researchers' attention. The aim of this review is to appraise the literature available to date for CPT distribution and the phytochemistry of these CPT-producing plants. Metabolite comparative analyses between the plants were also conducted for tracking of possible clues for CPT biosynthesis. Forty-three plant species in total were reported to possess CPT-producing capability, and one hundred twenty-five alkaloids classified into three major categories are summarized herein. Metabolite comparative analysis between these plants suggests the probability that the formation of the central intermediate for CPT biosynthesis has multiple origins. A more complete biogenetic reasoning for CPT and its structural homolog was delineated based on this fragmentary phytochemical evidence from a chemical point of view. Furthermore, an in-house compound database was constructed for further metabolomic analysis.

Camptothecin-producing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin.

BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook. (Icacinanceae). The bacterial isolates were purified and analyzed for production of CPT by ESI-MS/MS and NMR analysis. Bacterial identity was established based on the morphology and 16s rRNA sequence analysis. Crude extracts of the bacterial endophytes were evaluated for their cytotoxicity using colon cancer cell lines. The role of plasmid in the production of CPT was studied by purging the plasmid, using acriflavine, as well as reconstituting the bacteria with the plasmid. RESULTS: Four bacterial isolates, Bacillus sp. (KP125955 and KP125956), Bacillus subtilis (KY741853) and Bacillus amyloliquefaciens (KY741854) were found to produce CPT in culture. Both based on ESI-MS/MS and NMR analysis, the identity of CPT was found to be similar to that produced by the host plant. The CPT was biologically active as evident by its cytotoxicity against colon cancer cell line. The production of CPT by the endophyte (Bacillus subtilis, KY741853) attenuated with sub-culture. A likely role of a plasmid in the production of CPT was established. A 5 kbp plasmid was recovered from the bacteria. Bacterial isolate cured of plasmid failed to produce CPT. CONCLUSION: Our study implies a possible role of a plasmid in the production of CPT by the endophytic bacteria and opens up further work to unravel the exact mechanisms that might be involved.

New indole glucosides as biosynthetic intermediates of camptothecin from the fruits of Camptotheca acuminata.

Six new indole glucosides (1-6) and fifteen known alkaloids (7-21) were isolated from the fruit of Camptotheca acuminata. The planar structures of 1-6 were determined on the basis of spectroscopic data analysis and their absolute configurations were established by CD. The isolated indole glucosides showed a clear biosynthetic pathway of camptothecin (7), which started from tryptamine and secologanin and was proposed by synthetic chemists previously. Particularly, compound 1 supplemented the process of the transformation from pumiloside (20) or 3-epi-pumiloside (21) to camptothecin (7). In addition, camptothecin 10-O-ß-D-glucopyranoside (13) and norcamptothecin (17), synthesized in the early structural modification of 7, were first isolated from the natural resources. The new compounds 1-6 were screened for their in vitro cytotoxicity but they did not show any exciting result.

Multiple shoot cultures of Ophiorrhiza rugosa var. decumbens Deb and Mondal--a viable renewable source for the continuous production of bioactive Camptotheca alkaloids apart from stems of the parent plant of Nothapodytes foetida (Wight) Sleumer.

Camptotheca alkaloids were isolated from multiple shoot cultures of O. decumbens (0.056% dry weight) and stems of N. foetida. The cytotoxicity of the extracts and products were tested in a panel of five cell lines. Crude extract from O. decumbens (Cr-Od) and N. foetida (Cr-Nf) showed more potent cytotoxic activity as compared to the isolated camptothecin from O. decumbens (CPT-Od) and N. foetida (CPT-Nf). CPT isolated from shoot cultures contained biological activity suggesting the possibility of using this system of O. decumbens as a renewable source for the production of camptotheca alkaloids. 9-Methoxy camptothecin (9-mCPT), isolated from N. foetida, was a very effective cytotoxic agent as compared to Cr-Nf or CPT-Nf. The IC50 of 9-mCPT was 0.84, 0.32, and 0.35 µg/ml for A549, MCF7 and Jurkat cell lines and >3 µg/ml for U937. Viability assays using MTT dye were further confirmed by assessing extent of apoptosis in these cells. These findings suggest that shoot cultures of O. decumbens offer a rich alternative plant source for the anticancer compound, CPT and 9-mCPT is a more potent compound in N. foetida as compared to CPT.

Isolation of endophytic bacteria producing the anti-cancer alkaloid camptothecine from Miquelia dentata Bedd. (Icacinaceae).

Camptothecine (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this activity, several semi-synthetic derivatives of CPT are in clinical use against ovarian and small lung cancers. Together with its derivatives, CPT is the third largest anti-cancer drug in the world market. CPT is produced by several plant species belonging to the Asterid clade. In the recent past, several studies have reported the production of CPT by endophytic fungal associates of some of these plant species. In this paper, we report the production of CPT by endophytic bacteria isolated from Miquelia dentata Bedd. (Icacinaceae). Besides CPT, the bacteria also produced 9-methoxy CPT (9-MeO-CPT), in culture, independent of the host tissue. The chemical nature of CPT and 9-MeO-CPT was determined by LC-MS and ESI-MS/MS analysis, and was shown to be similar to that produced by the host tissue. One of the bacterial isolates examined, showed indications of attenuation of CPT production through sub-culture. This is the first report of production of CPT by endophytic bacteria. The identity of the bacteria was ascertained by Gram staining and 16s rRNA sequencing. We discuss the possible mechanisms that might be involved in the synthesis of CPT by endophytic bacteria.

Endophytic fungi from Miquelia dentata Bedd., produce the anti-cancer alkaloid, camptothecine.

Camptothecine (Campothecin, CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Several semi-synthetic derivatives of CPT are in clinical use against ovarian, small lung and refractory ovarian cancers. While CPT is produced by several plant species belonging to the Asterid clade, in recent years, efforts have been made to isolate endophytic fungi from some of these plants as possible alternative sources of CPT. In this study we report the isolation of three endophytic fungi from fruit and seed regions of Miquelia dentata (Icacinaceae), that produce CPT, 9-methoxy CPT (9-MeO-CPT) and 10-hydroxy CPT (10-OH-CPT). All the three fungi identified as, Fomitopsis sp. P. Karst (MTCC 10177), Alternaria alternata (Fr.) Keissl (MTCC 5477) and Phomposis sp. (Sacc.) produced CPT, 9-MeO-CPT and 10-OH-CPT in mycelial mats in shake flasks containing potato dextrose broth. Methanolic and ethyl acetate extracts of these fungal species were cytotoxic to colon and breast cancer cell lines. We discuss these results in the context of the recent interest in endophytic fungi as possible alternative sources of plant secondary metabolites.

[Application and prospect of fungi elicitors in fermentation industry].

Fungal elicitors are a group of chemicals that can stimulate the secondary metabolite production in plants and microbial cells. After being recognized, it could enhance the expression of related genes through the signal-transduction pathway; regulate the activity of the enzyme involved in the biosynthesis of secondary metabolites. In recent years, the inducible mechanism of fungal elicitors has been studied deeply worldwide. Meanwhile, it has acquired wide concern in the area of biological industry, especially in the fermentation industry. This paper addresses the application and prospect of fungal elicitors in the secondary metabolites of plant and microbial cells.

Production of camptothecin by hairy roots and regenerated transformed shoots of Ophiorrhiza rugosa var. decumbens.

Camptothecin (CPT), the derivatives of which are used clinically for the treatment of metastatic colon cancer, is isolated from intact plants that can be subjected to environmental fluctuations. In vitro cultures may be an alternate and continuous source for year-round production of CPT. Since CPT production by undifferentiated cell cultures is low, differentiated tissues such as root cultures may be a viable alternate source for CPT production. Hairy roots were induced in Ophiorriza rugosa, a source of CPT, using Agrobacterium rhizogenes strain LBA9402. The hairy roots, when cultured in light, showed spontaneous regeneration of shoots. Analysis of CPT levels in the hairy roots and in vitro-grown transformed shoots revealed 0.009% d.w. and 0.012% d.w., respectively.

Correlations between camptothecin and related metabolites in Camptotheca acuminata reveal similar biosynthetic principles and in planta synergistic effects.

Camptothecin (CPT), 9-methoxycamptothecin (9-MeO-CPT), and 10-hydroxycamptothecin (10-OH-CPT) are potent antineoplastic metabolites. We analyzed these metabolites in Camptotheca acuminata sampled from Germany and China, using LC-MS/MS and LC-ESI-HRMS/MS, coupled with chemometrics. Multivariate analysis revealed that fresh stems of C. acuminata from China had the highest comprehensive metabolite load. Significant positive correlations of CPT with 9-MeO-CPT and 10-OH-CPT were observed by Kruskal's multidimensional scaling and principal component analysis. Linear discriminant analysis and hierarchical agglomerative cluster analysis revealed that C. acuminata from China was separated from others. These positive correlations indicate that these metabolites are biosynthesized similarly and operate synergistically in planta.

Correlation of camptothecin-producing ability and phylogenetic relationship in the genus Ophiorrhiza.

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.

Comparative studies and identification of camptothecin produced by an endophyte at shake flask and bioreactor.

The fungus showing homology with Nodulisporium by 28S ribosomal gene sequencing, which has been discovered as an endophyte on medicinal plant Nothapodytes foetida, was found to produce 45 and 5.5 microg of camptothecin (CPT) per gram of mycelia at bioreactor and at shake flask, respectively, which was further quantified and characterised by various spectroscopic analyses.

An endophytic Neurospora sp. from Nothapodytes foetida producing camptothecin.

The medicinal plant, Nothapodytes foetida contains a number of important alkaloids like camptothecin (an anticancer drug molecule) but its concentration is less to meet the existing demand of this important molecule, so in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as potential source of anticancer drug lead compound i.e. camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of precursor anticancer drug molecule in future at large scale.

Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer.

A novel camptothecin-producing endophytic fungus viz., Entrophospora infrequens was isolated from an important Indian medicinal plant Nothapodytes foetida. The present study reports evaluation ofbioactivities of two novel extracts viz., chloroform (CEEI) and methanolic (MEEI) extracts of Entrophospora infrequens with respect to their immunomodulatory potential in vitro and in vivo (in Balb/c mice). The endophyte E. infrequens was found to synthesize camptothecin, which tested positive in CEEI. The immunomodulatory potential of CEEI and MEEI was compared with standard camptothecin (CPT). Doses of the chloroform extract (CEEI) ranging from 12.5-100 mg/kg body weight, significantly (p < 0.05) stimulated the humoral and cell-mediated immune responses in a dose-dependent manner. MEEI on the other hand significantly (p < 0.05) stimulated the delayed type hypersensitivity (DTH) reaction (by nearly 80%), plaque forming cell (PFC) assay (33%), phagocytic response (38%) and haemagglutination antibody (HA) titre [IgM by 79.07% and IgG by 62.05%] at a dose of 12.5 mg/kg body weight. The present study is the first report of the immunomodulatory potential of this neoteric camptothecin-producing endophyte from Nothapodytes foetida.

[Improved camptothecin production by cell lines of Camptotheca acuminata].

The concentration of camptothecin was determined in different tissues of Camptotheca acuminata seedling. The concentrations of camptothecin in new leaves and roots were significantly higher than in other tissues. However, the concentration of camptothecin declined with leaves becoming old. The induction of callus and cell suspension cultures from younger leaves of Camptotheca acuminata was observed. Cell lines were selected with improved camptothecin production as 0.02%.

Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds.

Camptothecin derivatives are clinically used anti-tumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA). We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin. The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH:cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.