De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.
Abstract:Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Nothapodytes nimmoniana… Show more
“…The molecular structure of each compound was proven by its specific fragmentation patterns and structural information. Some recent studies have indicated that two alternative seco-iridoid pathways are proposed to be participated in plant MIA biosynthesis, either through multiple isomeric intermediates of the methyl ester of strictosidine or the carboxylic acid strictosidinic acid [31]. Our results were coincident with the conclusion that strictosidinic acid was the key MIA precursor and the central intermediate in CPT biosynthesis in C. acuminata .…”
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.
“…The molecular structure of each compound was proven by its specific fragmentation patterns and structural information. Some recent studies have indicated that two alternative seco-iridoid pathways are proposed to be participated in plant MIA biosynthesis, either through multiple isomeric intermediates of the methyl ester of strictosidine or the carboxylic acid strictosidinic acid [31]. Our results were coincident with the conclusion that strictosidinic acid was the key MIA precursor and the central intermediate in CPT biosynthesis in C. acuminata .…”
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.
“…Nothapodytes nimmoniana (Icacinales) also produces camptothecin, though is more closely related to Gentianales than Cornales, and the pathway appears to proceed using the typical Gentianales route via secologanin. 135 Icaninales and Gentianales may share an MIA origin, and this is supported by characterisation of an active secologanin synthase from N. nimmoniana (NnCYP72A1). 136…”
Plant alkaloids are central to both modern and traditional medicines. This review describes the progress made towards understanding the metabolic origins of these compounds, focussing on the common features of different biosynthetic pathways.
“…Furthermore, this study is in continuation to our recent endeavour related to development of comprehensive transcriptome resource of N. nimmoniana to identify putative pathway genes, cytochromes related to CPT biosynthesis and transcription factors. It also entailed artificial microRNA (aMIR) mediated suppression of Nn CYP76B6 for suggesting its role in CPT biosynthesis [36]. Tissue-specific chemoprofiling revealed secologanin as a central intermediate of seco-iridoid pathway in N. nimmoniana in contrast to secologanic acid in C. accuminata [36, 37].…”
Section: Introductionmentioning
confidence: 99%
“…It also entailed artificial microRNA (aMIR) mediated suppression of Nn CYP76B6 for suggesting its role in CPT biosynthesis [36]. Tissue-specific chemoprofiling revealed secologanin as a central intermediate of seco-iridoid pathway in N. nimmoniana in contrast to secologanic acid in C. accuminata [36, 37]. Fig.…”
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.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1912-x) contains supplementary material, which is available to authorized users.
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