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.

Discovery of a novel flavonol O-methyltransferase possessing sequential 4'- and 7-O-methyltransferase activity from Camptotheca acuminata Decne.

The biosynthetic route for flavonol in Camptotheca acuminata has been recently elucidated from a chemical point of view. However, the genes involved in flavonol methylation remain unclear. It is a critical step for fully uncovering the flavonol metabolism in this ancient plant. In this study, the multi-omics resource of this plant was utilized to perform flavonol O-methyltransferase-oriented mining and screening. Two genes, CaFOMT1 and CaFOMT2 are identified, and their recombinant CaFOMT proteins are purified to homogeneity. CaFOMT1 exhibits strict substrate and catalytic position specificity for quercetin, and selectively methylates only the 4'-OH group. CaFOMT2 possesses sequential O-methyltransferase activity for the 4'-OH and 7-OH of quercetin. These CaFOMT genes are enriched in the leaf and root tissues. The catalytic dyad and critical substrate-binding sites of the CaFOMTs are determined by molecular docking and further verified through site-mutation experiments. PHE181 and MET185 are designated as the critical sites for flavonol substrate selectivity. Genomic environment analysis indicates that CaFOMTs evolved independently and that their ancestral genes are different from that of the known Ca10OMT. This study provides molecular insights into the substrate-binding pockets of two new CaFOMTs responsible for flavonol metabolism in C. acuminata.

Discovery of unique CYP716C oxidase involved in pentacyclic triterpene biosynthesis from Camptotheca acuminata.

Dozens of triterpenes have been isolated from Camptotheca acuminata, however, triterpene metabolism in this plant remains poorly understood. The common C28 carboxy located in the oleanane-type and ursane-type triterpenes indicates the existence of a functionally active triterpene, C28 oxidase, in this plant. Thorough mining and screening of the CYP716 genes were initiated using the multi-omics database for C. acuminata. Two CYP716A (CYP716A394 and CYP716A395) and three CYP716C (CYP716C80-CYP716C82) were identified based on conserved domain analyses and hierarchical cluster analyses. CYP716 microsomal proteins were prepared and their enzymatic activities were evaluated in vitro. The CYP716 classified into the CYP716C subfamily displays ß-amyrin oxidation activity, and CYP716A displays α-amyrin and lupeol oxidation activity, based on gas chromatography-mass spectrometry analyses. The oxidation products were determined based on their mass and nuclear magnetic resonance spectrums. The optimum reaction conditions and kinetic parameters for CYP716C were determined, and functions were verified in Nicotiana benthaminana. Relative quantitative analyses revealed that these CYP716C genes were enriched in the leaves of C. acuminata plantlets after 60 d. These results indicate that CYP716C plays a dominant role in oleanane-type triterpene metabolism in the leaves of C. acuminata via a substrate-specific manner, and CYP716A is responsible for ursane- and lupane-type triterpene metabolism in fruit. This study provides valuable insights into the unique CYP716C-mediated oxidation step of pentacyclic triterpene biosynthesis in C. acuminata.

Hybrid molecularly imprinted polymers for targeted separation and enrichment of 10-hydroxycamptothecin in Camptotheca acuminata Decne.

The molecularly imprinted polymer was synthesized using 3-aminopropylthiosilane-methacrylic acid monomer (APTES-MAA) as the functional monomer and 10-hydroxycamptothecin (HCPT) as the template, based on computer simulation. The hybrid molecularly imprinted polymers (HMIPs) were characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, particle size measurement, scanning electron microscopy and energy dispersive X-ray spectroscopy. It has been shown that HMIPs are irregularly shaped and porous, with particle sizes ranging mainly from 130 to 211 nm. At 298 K, the HMIPs exhibit a maximum adsorption capacity of 8.35 mg·g-1 for HCPT and demonstrate good adsorption specificity (α = 5.38). The pseudo-second-order reaction mechanism suggests that the equilibrium adsorption capacity of HCPT on HMIPs is 8.11 mg·g-1. Finally, HCPT was successfully separated and enriched from the extract of Camptotheca acuminata Decne. seeds using HMIPs.

An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Camptotheca acuminata in Sichuan, China.

Camptotheca acuminata (C. acuminata), is belongs to a monotypic genus endemic to southwestern China and listed as the first class national protected plant in China in 1999 (Wen, et al. 2020). Camptothecin, isolated from the wood and bark of C. acuminata Decne, which exhibits clinical effects in various cancer treatments (Pommier, et al. 2006; Kang, et al. 2021). In October 2021, we investigated leaf spot disease occurrence on C. acuminata (FigS1.A) with 80% incidence in Beichuan County, Sichuan Province of China. Leaf symptoms were randomly distributed on the adaxial surfaces and consisted of punctate spots of alternating light gray and dark brown in the early stage of onset (FigS1. B, C). As the disease progressed, these spots expanded irregularly shaped regions of necrotic tissue, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. Infected tissues from symptomatic leaves disinfected in 75% ethanol for 45 s, and with 0.1% HgCl2 for 1 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 µg/ml each). Plates were incubated for 3 days at 25°C. Then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA medium for subculture. Aerial hyphae had a cotton-like appearance with white to pale gray color (FigS1.D). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1E), with 1-6 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 10.0-50.9 µm in length and 5.6-11.8 µm in width (n = 20) (FigS1E, G). On the basis of conidial and cultural characteristics, the fungus was consistent with those of members of the Alternaria genus (Simmons, 2008). To confirm this tentative identification, DNA was extracted from isolate XS9, the internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), partial RNA polymerase II largest subunit (RPB2) genes were amplified with primers pairs ITS1/ITS4 (White et al.1990), GDF/GDR (Templeton et al.), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al. 1990; Liu et al. 1999), Bt-2a/Bt-2b (Glass and Donaldson 1995) respectively. The resulting sequences were deposited in GenBank (ITS, OP113690; GAPDH, OP120953; TEF, OP120952; RPB2, OP120954). Further phylogenetic analyses of isolate XS9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. Pathogenicity identification of isolate XS9 was carried out on the detached leaves. The pure agar plugs (as control) or spraying water on the leaf surface were inoculated on detached leaves, the controls remained healthy after 8 days (FigS1.H-J). but the leaves inoculated with other the mycelium plugs (Fig S1K, L) or the conidia suspension (2×105 conidia/mL) of isolate XS9 was sprayed on the detached leaves (Fig S1M, N), both showed brown necrotic lesions that are similar to the symptoms observed in the field. The pathogen was reisolated and confirmed to be A. brassicae. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on C. acuminata in China. Leaf spot disease causes the branches and leaves of camptotheca acuminata to wither and even the whole plant to die. To ensure the protection of the irreplaceable species, effective measures should be taken to prevent the spread of the leaf spot disease.

Functional characterization of a catalytically promiscuous tryptophan decarboxylase from camptothecin-producing Camptotheca acuminata.

Tryptophan decarboxylases (TDCs) are a group of pyridoxal 5'-phosphate-dependent enzymes involved in the enzymatic conversion of tryptophan into tryptamine, a critical biogenic amine. We herein mined and cloned a TDC-encoding gene, CaTDC3, from camptothecin-producing plant Camptotheca acuminata. The intact CaTDC3 was heterologously overexpressed in Escherichia coli and the recombinant CaTDC3 was purified to homogeneity. High-performance liquid chromatography (HPLC)-diode array detector (DAD) and high resolution mass spectrometry (HRMS) data analyses of the CaTDC3-catalyzed reaction mixture confirmed the catalytically decarboxylative activity of CaTDC3. CaTDC3 shows strict stereoselectivity for L-tryptophan. Homology modeling and molecular docking implied CaTDC3's recognition of L-tryptophan derivatives and analogs. Substrate scope investigations revealed that the appropriate substituent groups on the indole ring, i.e., hydroxylated and halogenated L-tryptophans, could be recognized by CaTDC3 and the decarboxylation reactions generated the corresponding tryptamines. The Cß -methyl-L-tryptophans were decarboxylated by CaTDC3 efficiently. 1-Thio-L-tryptophan, the NH group of the indole ring replaced by an S atom, could be decarboxylated by CaTDC3. CaTDC3 catalyzed the decarboxylation of 7-aza-L-tryptophan, an N displacement of the C on the aromatic ring, to afford 7-aza-tryptamine. L-Kynurenine, an L-tryptophan degradation product, could be decarboxylated by CaTDC3. The present works uncover a catalytically promiscuous TDC and the TDC is a versatile decarboxylase in synthetic biology for specialized pharmaceutically important substances.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

Integrative Analysis of Elicitor-Induced Camptothecin Biosynthesis in Camptotheca acuminata Plantlets Through a Combined Omics Approach.

Treatments with abiotic elicitors can efficiently induce the accumulation of specialized metabolites in plants. We used a combined omics approach to analyze the elicitation effects of MeJa, AgNO3, and PEG on camptothecin (CPT) biosynthesis in Camptotheca acuminata plantlets. Untargeted analyses revealed that treatments with MeJa, AgNO3, and PEG significantly inhibited the photosynthetic pathway and promoted carbon metabolism and secondary metabolic pathways. The CPT levels increased by 78.6, 73.3, and 50.0% in the MeJa, AgNO3, and PEG treatment groups, respectively. Using C. acuminata plantlets after elicitation treatment, we mined and characterized 15 new alkaloids, 25 known CPT analogs and precursors, 9 iridoid biosynthetic precursors, and 15 tryptamine biosynthetic precursors based on their MS/MS fragmentation spectra. Using 32 characterized genes involved in CPT biosynthesis as bait, we mined 12 prioritized CYP450 genes from the 416 CYP450 candidates that had been identified based on co-expression analysis, conserved domain analysis, and their elicitation-associated upregulation patterns. This study provides a comprehensive perspective on CPT biosynthesis in C. acuminata plantlets after abiotic elicitation. The findings enable us to elucidate the previously unexplored CYP450-mediated oxidation steps for CPT biosynthesis.

Valuable alkaloids content is preserved in Camptotheca acuminata and Morus alba grown in trace elements contaminated soil.

Phytoextraction of trace elements (TE) using woody species is an economically challenging soil remediation approach because of the long time needed. Yet, some trees contain alkaloids that can be exploited along structural components to enhance biomass value. As alkaloids are thought to be involved in plant defence mechanisms, we hypothesized that potentially hostile phytoremediation conditions could increase their level. Camptothecin in Camptotheca acuminata and 1-deoxynojirimycin in Morus alba were measured from trees grown in a field in presence of Cu, Pb and Zn all together, and from M. alba grown in a greenhouse in presence of Cd or other abiotic stressors (NaCl and bending). The trees did not extract TE in the field, but M. alba stems accumulated Cd in the greenhouse experiment, with no consequence on stomatal conductance and leaves pigments concentration. Camptothecin and 1-deoxynojirimycin concentrations were preserved under all experimental conditions, as was biomass yield, and phenolics were slightly increased in M. alba exposed to TE. This study provides evidence that valuable and persistent alkaloids and phenolics can be extracted from trees facing phytoremediation-associated stresses, without a negative impact on their quantity and on biomass yield. Such products could generate a sustainable stream of revenues during phytoremediation.

There is scarce data on tree alkaloid content and scarcer data on how it is affected by exposure to trace elements in a phytoremediation context. We provide evidence that the content of two specific alkaloids is not altered in Morus alba and Camptotheca acuminata exposed to moderate to elevated levels of contaminating trace elements. The manuscript introduces the use of M. alba for phytoremediation in the Americas and is the first to propose the use of C. acuminata on trace element contaminated sites to produce camptothecin, a valuable anticancer alkaloid.

P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata.

Divergent terpene indole alkaloid (TIA) pathways in Catharanthus roseus and Camptotheca acuminata generate vinblastine and vincristine, and camptothecin, respectively. In contrast to Catharanthus which feeds secologanin (from methylated loganin) into its species-specific late pathway, Camptotheca feeds secologanic acid (from unmethylated loganic acid) into its late pathway. Having identified putative Camptotheca secologanic acid synthases (SLASs) and cytochrome P450 reductases (CPRs) in transcriptome databases, we have demonstrated that two P450s, CYP72A564 and CYP72A565, are capable of utilizing both loganic acid and loganin to generate secologanic acid and secologanin. We have extended the previous report of these activities by CYP72A565 and CYP72A610 (Yang et al., 2019) by demonstrating that both Arabidopsis CPRs (ATR1, ATR2) couple with these CYP72A proteins in yeast microsomal assays and that purified Camptotheca CPR1 couples with them in in vitro reconstitution assays. Kinetic analyses of purified full-length Camptotheca SLASs have indicated that both process loganic acid with nearly identical catalytic rates and efficiencies as measured by their kcat and kcat/KM. In contrast, CYP72A564 processes loganin with two-fold greater efficiency than CYP72A565 correlating with the former's 3-fold greater affinity for loganin. The closely-related CYP72A730 does not bind or process either compound. Molecular modeling of these three proteins and comparisons with Catharanthus secologanin synthase (SLS) have identified key differences that likely determine their SLAS versus SLS selectivities. Our ability to reconstitute these SLAS/SLS activities provides valuable tools for further examinations of the residues involved in substrate recognition and determinations of their unusual mechanism of C-C bond scission.

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.

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.

Two classes of cytochrome P450 reductase genes and their divergent functions in Camptotheca acuminata Decne.

Cytochrome P450 monooxygenases (CYP450s) and their auxiliary cytochrome P450 reductases (CPRs) are important and commonly involved in the biosynthesis of camptothecin (CPT). To better understand the possible functions of CPRs in planta, we first isolated two CaCPRs genes from Camptotheca acuminata. Sequence analysis revealed the presence of common conserved FMN-, FAD- and NADPH-binding motifs in putative CaCPR1/CaCPR2 proteins. The two CaCPR paralogs were assigned to the Class I and Class II of CPRs, respectively, according to phylogenetic tree. The recombinant CrCYP72-CaCPR1 and CrCYP72-CaCPR2 enzymes and their substrate bioconversion rates of 23.09% and 35.23% demonstrated that both CaCPRs could support the enzyme activities of CrSLS1. Gene silencing of CaCPRs by VIGS led to downregulation of CaCPR1/CaCPR2 expression by 50-67%, accompanied with 10-15% slight decrease and 57-63% dramatic reduction for CaCPR1 and CaCPR2 individually in CPT accumulations. Moreover, CaCPR1/CaCPR2 displayed almost omnipresent expression patterns across Camptotheca tissues. While in comparison to constitutive expression of CaCPR1 gene, CaCPR2 and CYP450 genes were all dramatically phytohormone-induced expressed in leaves which were main tissues for isoprenoid and CPT biosynthesis. Our results suggested that, in Camptotheca seedlings, CaCPR2 had a distinct function from CaCPR1 that was clearly involved in the inducible specialized metabolism for CPT biosynthesis.

Aspernolide A Inhibits the Proliferation of Human Laryngeal Carcinoma Cells through the Mitochondrial Apoptotic and STAT3 Signaling Pathways.

Aspernolide A, a butyrolactone secondary metabolite, was purified from the endophytic fungus Cladosporium cladosporioides derived from roots of Camptotheca acuminata Decne. In this study, the antitumor activity and mechanisms of aspernolide A on human laryngeal cancer Hep-2 and TU212 cells were studied by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, morphological observation and Western blotting. The results showed that aspernolide A significantly inhibited the proliferation of Hep-2 and TU212 cells in dose- and time-dependent manners. Morphological changes of apoptotic cells could be observed under an inverted microscope, such as irregular margins, decreased adherence ability and chromatin condensation. The expressions of Bax, Caspase-9, Caspase-3 and PARP (poly ADP-ribose polymerase) increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, the expression of the phosphorylation of STAT3 decreased with the increase of dosage, suggesting that the apoptotic mechanism might be related to the STAT3 signaling pathway. All these conclusions indicated that aspernolide A has the potential anti-laryngocarcinoma effects.

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.

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism.

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides.

One-step targeted accumulation and detection of camptothecin analogues from fruits of Camptotheca acuminata Decne using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry.

Camptothecins, a kind of monoterpene-quinoline alkaloids from Camptotheca acuminata Decne, have long attracted much attention worldwide as an anti-cancer drug. However, there is still a lack of effective methods for the accumulation and discovery of camptothecin analogues from botanic resources for camptothecin-based drug research. This work develops a one-step method for the targeted accumulation, quick detection, and identification of camptothecin analogues from C. acuminata fruit using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (bilayer-SPE-UHPLC-Q-TOF-MS/MS). The bilayer-SPE cartridge, with polyamide (PA) as the upper layer and octadecyl silane (ODS) as the lower layer, was designed for the removal of flavonoid and ellagic acid impurities and the enrichment of camptothecins for further MS analysis. Subsequently, the mass spectrometry fragmentations, especially multistage retro-Diels-Alder cleavage, were summarized based on the MS/MS data of 10 reference camptothecins. The UHPLC-Q-TOF-MS/MS conditions were optimized, and the MS/MS data of the potential camptothecin analogues in the bilayer-SPE enriched fractions were analyzed. A total of 30 camptothecin analogues, including 15 new compounds, were identified from the fruit according the fragmentation pathways of the reference standards. The proposed structure of peak 20 was confirmed using its NMR data through rapid enrichment and purification. Overall, the bilayer-SPE enrichment and reliable mass spectrometry fragmentation in our work could provide an effective and simple method for the exploration of the biosynthesis pathway and metabolomics of camptothecin analogues.

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.

Comparative and Phylogenetic Analyses of the Complete Chloroplast Genomes of Three Arcto-Tertiary Relicts: Camptotheca acuminata, Davidia involucrata, and Nyssa sinensis.

The Arcto-Tertiary relict genera, Camptotheca, Davidia, and Nyssa represent deep lineages in the asterid order Cornales. Recent phylogenetic studies suggested that these genera should be placed in a newly circumscribed family, Nyssaceae. However, because these analyses were based upon a few genes, it is prudent and necessary to examine further evidence before adopting this taxonomic treatment. In this study, we determined the complete chloroplast (cp) genomes of Camptotheca acuminata, Davidia involucrata, and Nyssa sinensis. Their cp genomes ranged from 156,672 to 158,409 bp, which included 115 genes, and their genome features were highly similar to those of other species within the order Cornales. The phylogenetic relationships among the genera Camptotheca, Davidia, Nyssa, and 23 related taxa in the asterids were analyzed based on 73 protein-coding genes from the cp genomes. All of the previously recognized major clades (namely Cornales, Ericales, Campanulids, and Lamiids) in the asterids, as well as their relationships, were recovered with robust support. A clade including the genera Davidia, Nyssa, Camptotheca, and Diplopanax, was resolved as a well-supported monophyletic group, which was fully separated from the family Cornaceae by the family Hydrangeaceae. Our results provide novel evidence to support the acceptance of the family Nyssaceae outlined by the updated Angiosperm Phylogeny Group.