A chromosome-level Camptotheca acuminata genome assembly provides insights into the evolutionary origin of camptothecin biosynthesis.

Camptothecin and its derivatives are widely used for treating malignant tumors. Previous studies revealed only a limited number of candidate genes for camptothecin biosynthesis in Camptotheca acuminata, and it is still poorly understood how its biosynthesis of camptothecin has evolved. Here, we report a high-quality, chromosome-level C. acuminata genome assembly. We find that C. acuminata experiences an independent whole-genome duplication and numerous genes derive from it are related to camptothecin biosynthesis. Comparing with Catharanthus roseus, the loganic acid O-methyltransferase (LAMT) in C. acuminata fails to convert loganic acid into loganin. Instead, two secologanic acid synthases (SLASs) convert loganic acid to secologanic acid. The functional divergence of the LAMT gene and positive evolution of two SLAS genes, therefore, both contribute greatly to the camptothecin biosynthesis in C. acuminata. Our results emphasize the importance of high-quality genome assembly in identifying genetic changes in the evolutionary origin of a secondary metabolite.

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.

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

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

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.

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

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

The biosynthesis of camptothecin derivatives by Camptotheca acuminata seedlings.

10-hydroxycamptothecin and 9-methoxycamptothecin, naturally occurring camptothecin derivatives, are reportedly present in Camptotheca acuminata with a powerful cytotoxic effect and strong antitumor activity. In this paper, we studied the derivatization reaction of camptothecin catalyzed by C. acuminata seedlings. HPLC traced the reaction between exogenous camptothecin and C. acuminata seedlings. The results showed that the exogenous camptothecin was converted into 10-hydroxycamptothecin and 9-methoxycamptothecin by the tender roots and stems of C. acuminata seedlings, which would be a new method for the synthesis of two camptothecin derivatives.

Distribution of camptothecin biosynthetic intermediates and identification the rate-limiting step of camptothecin biosynthesis.

Two key biosynthetic intermediates (pumiloside and strictosamide) of camptothecin were isolated. A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed to determine four main alkaloid compounds (pumiloside, strictosamide, camptothecin and 10-hydroxycamptothecin) and estimate two minor compounds (deoxypumiloside, 9-methoxycamptothecin) simultaneously in different parts of Camptotheca acuminata, with a good linearity and R2 > 0.999 for all curves. The results indicated that there was a positive correlation between the two key intermediates (strictosamide and pumiloside) and camptothecin in vivo. The speculation that the root was the synthetic position of camptothecin in vivo was confirmed. The rate-limiting step of camptothecin biosynthesis was estimated the step from pumiloside to deoxypumiloside based on its concentration fall sharply.

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.

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.

Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.

Camptothecin is a high-value anti-cancerous compound produced in many taxonomically unrelated species. Its biosynthesis involves a complex network of pathways and a diverse array of intermediates. Here, we report the functional characterization and regulation of secologanin synthase (NnCYP72A1), a cytochrome P450 involved in camptothecin biosynthesis from Nothapodytes nimmoniana. It comprises an open reading frame of 1566 bp in length. Heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays using loganin as substrate confirmed the formation of secologanin. In planta transient overexpression analysis of NnCYP72A1 resulted in 4.21- and 2.73-fold increase in transcript levels of NnCYP72A1 on days 3 and 6 respectively. Phytochemical analysis of transformed tissues revealed ~ 1.13-1.43- and 2.02-2.86-fold increase in secologanin and CPT accumulation, respectively. Furthermore, promoter analysis of NnCYP72A1 resulted in the identification of several potential cis-regulatory elements corresponding to different stress-related components. Methyl jasmonate, salicylic acid, and wounding treatments resulted in considerable modulation of mRNA transcripts of NnCYP72A1 gene. Chemical analysis of elicitor-treated samples showed a significant increase in CPT content which was concordant with the mRNA transcript levels. Overall, the functional characterization and overexpression of NnCYP72A1 may plausibly enhance the pathway intermediates and serve as prognostic tool for enhancing CPT accumulation.

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.

Bifunctional Cytochrome P450 Enzymes Involved in Camptothecin Biosynthesis.

Camptothecin (CAM) is a well-known, complex, plant-derived antitumor monoterpenoid indole alkaloid (MIA). Featuring a unique pentacyclic pyrroloquinoline scaffold, CAM is biosynthetically distinct from the other known MIAs, such as antitumor vincristine and vinblastine. Herein, CaCYP72A565 and CaCYP72A610 enzymes involved in the biosynthesis of the monoterpenoid moiety of CAM were cloned from CAM-producing  Camptotheca acuminata. Heterologous overexpression and functional characterization assays showed that CaCYP72As catalyzes two consecutive reactions, the stereoselective hydroxylation at C-7 of 7-deoxyloganic acid and the subsequent carbon-carbon (C-C) bond cleavage between C-7 and C-8 of iridoid glucoside, to generate the intramolecular cyclopentane ring-opening secoiridoid glucoside. Comparative metabolite profiling analyses suggested that C. acuminata synthesizes loganic acid, secologanic acid, and strictosidinic acid as its MIA carboxylic acid intermediates. CaCYP72As are novel bifunctional enzymes that catalyze stereoselective hydroxylation and subsequent C-C bond cleavage reactions to give a ring-opening product with two functional groups, an aldehyde and a double bond.

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.

A bZIP transcription factor, CaLMF, mediated light-regulated camptothecin biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) has powerful biological activities and its analogs, irinothecan and topothecan, are effective anti-cancer drugs for clinical therapy. Camptothecin was first isolated from Camptotheca acuminata and its low accumulation in planta limits drug supply in the market. Previous works have confirmed that many environmental factors and plant hormones/elicitors could regulate CPT biosynthesis, but only light irradiance has a negative effect on CPT production in C. acuminata. Although light irradiance has been identified as a negative CPT biosynthesis regulator in C. acuminata for many years, the mechanisms of this regulation are still unknown. In order to search possible signal components involved in the process of light-regulated CPT biosynthesis, coexpression analysis was carried out according to the transcriptome database of Camptotheca above-ground green tissues. From coexpression analysis, a light-responsive bZIP transcription factor, CaLMF (Light-Mediated CPT biosynthesis Factor), was identified and further investigations showed that overexpression of CaLMF down-regulated the expression of CPT biosynthesis genes and decreased the accumulation of CPT in leaves, while light-regulated expression of CPT biosynthesis genes and CPT production were abolished in CaLMF silencing leaves under shading treatment. Our results show that CaLMF is a significant light signaling component, which mediates light-regulated CPT biosynthesis in C. acuminata.

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.

De novo transcriptome assembly and characterization of the 10-hydroxycamptothecin-producing Xylaria sp. M71 following salicylic acid treatment.

In the present study, we identified genes that are putatively involved in the production of fungal 10-hydroxycamptothecin via transcriptome sequencing and characterization of the Xylaria sp. M71 treated with salicylic acid (SA). A total of 60,664,200 raw reads were assembled into 26,044 unigenes. BLAST assigned 8,767 (33.7%) and 10,840 (41.6%) unigenes to 40 Gene Ontology (GO) annotations and 108 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. A total of 3,713 unigenes comprising 1,504 upregulated and 2,209 downregulated unigenes were found to be differentially expressed between SA-induced and control fungi. Based on the camptothecin biosynthesis pathway in plants, 13 functional genes of Xylaria sp. M71 were mapped to the mevalonate (MVA) pathway, suggesting that the fungal 10-hydroxycamptothecin is produced via the MVA pathway. In summary, analysis of the Xylaria sp. M71 transcriptome allowed the identification of unigenes that are putatively involved in 10-hydroxycamptothecin biosynthesis in fungi.

De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin.

Camptotheca acuminata is 1 of a limited number of species that produce camptothecin, a pentacyclic quinoline alkaloid with anti-cancer activity due to its ability to inhibit DNA topoisomerase. While transcriptome studies have been performed previously with various camptothecin-producing species, no genome sequence for a camptothecin-producing species is available to date. We generated a high-quality de novo genome assembly for C. acuminata representing 403 174 860 bp on 1394 scaffolds with an N50 scaffold size of 1752 kbp. Quality assessments of the assembly revealed robust representation of the genome sequence including genic regions. Using a novel genome annotation method, we annotated 31 825 genes encoding 40 332 gene models. Based on sequence identity and orthology with validated genes from Catharanthus roseus as well as Pfam searches, we identified candidate orthologs for genes potentially involved in camptothecin biosynthesis. Extensive gene duplication including tandem duplication was widespread in the C. acuminata genome, with 2571 genes belonging to 997 tandem duplicated gene clusters. To our knowledge, this is the first genome sequence for a camptothecin-producing species, and access to the C. acuminata genome will permit not only discovery of genes encoding the camptothecin biosynthetic pathway but also reagents that can be used for heterologous expression of camptothecin and camptothecin analogs with novel pharmaceutical applications.

Enhanced production of camptothecin and biological preparation of N 1-acetylkynuramine in Camptotheca acuminata cell suspension cultures.

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.