Detection of vinblastine and related bioactive compounds from culture extracts of endophytic fungi of Catharanthus roseus.

This study investigates the synthesis of vinblastine by endophytic fungi isolated from leaf of C. roseus. A total of 10 endophytic fungi were selected for secretion of vinca alkaloids based on the initial screening by biochemical tests and thin-layer chromatography (TLC). Out of these ten, only four fungal extracts showed positive results for presence of vinblastine at same retention time (10 min.) compared to reference compound on HPLC analysis. The detected concentration of vinblastine was maximum (17 µg/ml) in isolate no. CRL 22 followed by CRL 52, CRL 17 and CRL 28. To validate the presence of vinblastine, ultra-high-performance liquid chromatography coupled with high-resolution accurate mass spectrometry (HRMS) was employed. This analysis confirmed the presence of anhydrovinblastine, a precursor of vinblastine through the detection of molecular ions at m/z 793.4185 in extract of CRL 17. In addition to anhydrovinblastine, the intermediate compounds essential to the biosynthetic pathway of vinblastine were also detected in the extract of CRL 17. These host-origin compounds strongly suggest the presence of a biosynthetic pathway within the endophytic fungus. Based on morphological observation and sequence analysis of the ITS region of rDNA, endophytic fungi were identified as Alternaria alternata (CRL 17), Curvularia lunata (CRL 28), Aspergillus terrus (CRL 52), and Aspergillus clavatonanicus (CRL 22).

The role of the Golden2-like (GLK) transcription factor in regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Catharanthus roseus-mediated CuAl2O4 nanocomposites for evaluation of killing kinetics.

The present article portrayed on the killing kinetic of human pathogenic bacteria using bioinspired mesoporous CuAl2O4 nanocomposites (NCs). The NCs was fabricated using leaf extract of medicinal plant Catharanthus roseus (CR) as a green reducer and stabilizer. As bio-fabricated material was calcined at 800 °C and characterized by several analytical techniques like X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet-Visible Diffuse Reflectance Spectroscopy (UV-DRS), Energy Dispersive X-Ray Spectroscopy (EDS), X-Ray Photoelectron Spectroscopy (XPS), Raman, Brunauer-Emmett-Teller (BET), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) to authenticate its structure, phase, chemical bonding, chemical state, size and morphology behaviors. XRD and TEM revealed a reduced crystallite and nanoscale sizes of biosynthesized NCs. Moreover, XRD study exposed a cubic-structure of material, while transmission electron microscopy rendered an average particles size in range 10-15 nm. However, BET profile advocates a mesoporous nature of the particles. An effective biological molecular docking modulation assessed by substituting natural inhibitor by bioinspired NCs, while the protein PDB ID 4Z8D FabH as a receptor site for the present investigation. After assessment of molecular docking examination, the antibacterial activity of bioinspired NCs were performed against Staphylococcus aureus, Bacillus subtillis, Klebsiella pneumoniae and Escherichia coli using agar-well method. The broth culture method was employed on different pathogenic strains by kinetic growth assays and colony forming unit.

Crosstalk between melatonin and nitric oxide restrains Cadmium-induced oxidative stress and enhances vinblastine biosynthesis in Catharanthus roseus (L) G Don.

KEY MESSAGE: Nitric oxide functions downstream of the melatonin in adjusting Cd-induced osmotic and oxidative stresses, upregulating the transcription of D4H and DAT genes, and increasing total alkaloid and vincristine contents. A few studies have investigated the relationship between melatonin (MT) and nitric oxide (NO) in regulating defensive responses. However, it is still unclear how MT and NO interact to regulate the biosynthesis of alkaloids and vincristine in leaves of Catharanthus roseus (L.) G. Don under Cd stress. Therefore, this context was explored in the present study. Results showed that Cd toxicity (200 µM) induced oxidative stress, decreased biomass, Chl a, and Chl b content, and increased the content of total alkaloid and vinblastine in the leaves. Application of both MT (100 µM) and sodium nitroprusside (200 µM SNP, as NO donor) enhanced endogenous NO content and accordingly increased metal tolerance index, the content of total alkaloid and vinblastine. It also upregulated the transcription of two respective genes (D4H and DAT) under non-stress and Cd stress conditions. Moreover, the MT and SNP treatments reduced the content of H2O2 and malondialdehyde, increased the activities of superoxide dismutase and ascorbate peroxidase, enhanced proline accumulation, and improved relative water content in leaves of Cd-exposed plants. The scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (cPTIO) averted the effects of MT on the content of total alkaloid and vinblastine and antioxidative responses. Still, the effects conferred by NO on attributes mentioned above were not significantly impaired by p-chlorophenylalanine (p-CPA as an inhibitor of MT biosynthesis). These findings and multivariate analyses indicate that MT motivated terpenoid indole alkaloid biosynthesis and mitigated Cd-induced oxidative stress in the leaves of periwinkle in a NO-dependent manner.

Vincarostine A, a novel anti-malarial trimeric monoterpenoid indole alkaloid from Catharanthus roseus.

A novel trimeric monoterpenoid indole alkaloid, vincarostine A (1) consisting of an aspidosperma-iboga-aspidosperma type skeleton, was isolated from the whole plant of Catharanthus roseus. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and CD spectrum. Vincarostine A (1) showed anti-malarial activity.

Characterization of a CrPME indicates its possible role in determining vindoline accumulation in Catharanthus roseus leaves.

The leaf-specific Catharanthus roseus alkaloid, vindoline, is the major bottleneck precursor in the production of scarce and costly anticancer bisindoles (vincristine and vinblastine). The final steps of its biosynthesis and storage occur in the laticifers. Earlier, we have shown that vindoline content is directly related to laticifer number. Pectin remodeling enzymes, like pectin methylesterase (PME), are known to be involved in laticifer development. A search in the croFGD yielded a leaf-abundant CrPME isoform that co-expressed with a few vindoline biosynthetic genes. Full-length cloning, tissue-specific expression profiling, and in silico analysis of CrPME were carried out. It was found to possess all the specific characteristics of a typical plant PME. Transient silencing (through VIGS) and overexpression of CrPME in C. roseus indicated a direct relationship between its expression and vindoline content. Comparative analysis of transcript abundance and enzyme activity in three familial C. roseus genotypes differing significantly in their vindoline content and laticifer count (CIM-Sushil > Dhawal > Nirmal) also corroborated the positive relationship of CrPME expression with vindoline content. This study highlights the possible role of CrPME, a cell wall remodeling enzyme, in modulating laticifer-associated secondary metabolism.

Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP-F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

Catharanthus roseus intoxication mimicking acute cholangitis.

BACKGROUND: Catharanthus roseus, a Madagascar native flowering plant, is known for its glossy leaves and vibrant flowers, and its medicinal significance due to its alkaloid compounds. As a source of vinblastine and vincristine used in chemotherapy, Catharanthus roseus is also employed in traditional medicine with its flower and stalks in dried form. Its toxicity can lead to various adverse effects. We report a case of Catharanthus roseus juice toxicity presenting as acute cholangitis, emphasizing the importance of healthcare providers obtaining detailed herbal supplement histories. CASE PRESENTATION: A 65-year-old woman presented with abdominal pain, fever, anorexia, and lower limb numbness. Initial diagnosis of acute cholangitis was considered, but imaging excluded common bile duct stones. Further investigation revealed a history of ingesting Catharanthus roseus juice for neck pain. Laboratory findings showed leukocytosis, elevated liver enzymes, and hyperbilirubinemia. The patient developed gastric ulcers, possibly due to alkaloids in Catharanthus roseus. No bacterial growth was noted in blood cultures. The patient recovered after discontinuing the herbal extract. CONCLUSIONS: Catharanthus roseus toxicity can manifest as fever, hepatotoxicity with cholestatic jaundice, and gastric ulcers, mimicking acute cholangitis. Awareness of herbal supplement use and potential toxicities is crucial for healthcare providers to ensure prompt diagnosis and appropriate management. This case emphasizes the need for public awareness regarding the possible toxicity of therapeutic herbs and the importance of comprehensive patient histories in healthcare settings.

ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpene indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

CrJAT1 Regulates Endogenous JA Signaling for Modulating Monoterpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Many monoterpenoid indole alkaloids (MIAs) produced in Catharanthus roseus have demonstrated biological activities and clinical potential. However, their complex biosynthesis pathway in plants leads to low accumulation, limiting therapeutic applications. Efforts to elucidate the MIA biosynthetic regulatory mechanism have focused on improving accumulation levels. Previous studies revealed that jasmonic acid (JA), an important plant hormone, effectively promotes MIA accumulation by inducing the expression of MIA biosynthesis and transport genes. Nevertheless, excessive JA signaling can strongly inhibit plant growth, decreasing MIA productivity in C. roseus. Therefore, identifying key components balancing growth and MIA production in the JA signaling pathway is imperative for effective pharmaceutical production. Here, we identify a homolog of the jasmonate transporter 1, CrJAT1, through co-expression and phylogenetic analyses. Further investigation demonstrated that CrJAT1 can activate JA signaling to promote MIA accumulation without compromising growth. The potential role of CrJAT1 in redistributing intra/inter-cellular JA and JA-Ile may calibrate signaling to avoid inhibition, representing a promising molecular breeding target in C. roseus to optimize the balance between growth and specialized metabolism for improved MIA production.

Modulating plant-soil microcosm with green synthesized ZnONPs in arsenic contaminated soil.

Biogenic nanoparticle (NP), derived from plant sources, is gaining prominence as a viable, cost-effective, sustainable, and biocompatible alternative for mitigating the extensive environmental impact of arsenic on the interplay between plant-soil system. Herein, the impact of green synthesized zinc oxide nanoparticles (ZnONPs) was assessed on Catharanthus roseus root system-associated enzymes and their possible impact on microbiome niches (rhizocompartments) and overall plant performance under arsenic (As) gradients. The application of ZnONPs at different concentrations successfully modified the arsenic uptake in various plant parts, with the root arsenic levels increasing 1.5 and 1.4-fold after 25 and 50 days, respectively, at medium concentration compared to the control. Moreover, ZnONPs gradients regulated the various soil enzyme activities. Notably, urease and catalase activities showed an increase when exposed to low concentrations of ZnONPs, whereas saccharase and acid phosphatase displayed the opposite pattern, showing increased activities under medium concentration which possibly in turn influence the plant root system associated microflora. The use of nonmetric multidimensional scaling ordination revealed a significant differentiation (with a significance level of p < 0.05) in the structure of both bacterial and fungal communities under different treatment conditions across root associated niches. Bacterial and fungal phyla level analysis showed that Proteobacteria and Basidiomycota displayed a significant increase in relative abundance under medium ZnONPs concentration, as opposed to low and high concentrations, respectively. Similarly, in depth genera level analysis revealed that Burkholderia, Halomonas, Thelephora and Sebacina exhibited a notably high relative abundance in both the rhizosphere and rhizoplane (the former refers to the soil region influenced by root exudates, while the latter is the root surface itself) under medium concentrations of ZnONPs, respectively. These adjustments to the plant root-associated microcosm likely play a role in protecting the plant from oxidative stress by regulating the plant's antioxidant system and overall biomass.

Catharanthus roseus-assisted bio-fabricated zinc oxide nanoparticles for promising antibacterial potential against Klebsiella pneumoniae.

This study emphasized on the synthesis of zinc oxide nanoparticles (ZnO NPs) in an environmentally friendly manner from the extract of Catharanthus roseus leaves and its antibacterial assessment against the pneumonia-causing pathogen Klebsiella pneumoniae. This simple and convenient phytosynthesis approach is found to be beneficial over conventional methods, wherein plants serve as excellent reducing, capping, and stabilizing agents that enables the formation of ZnO NPs without the use of harmful chemicals. The formation of ZnO NPs was confirmed through several characterization techniques such as UV-visible spectroscopy, XRD, FT-IR, SEM, HR-TEM, and EDX. XRD analysis revealed high polycrystallinity with crystallite size of approximately 13 nm. SEM and HR-TEM revealed the hexagonal structure of ZnO NPs with the particle size range of 20-50 nm. The EDX shows the elemental purity without any impurity. Furthermore, the antibacterial efficacy by the technique of disc diffusion exhibited clear inhibition zones in ZnO NPs-treated discs. In addition, 125 µg/mL of ZnO NP concentration showed minimum inhibition by the microbroth dilution method. The potent inhibitory activity was further validated with trypan blue dye exclusion and fluorescence microscopy. Finally, SEM examination confirmed the efficient antibacterial potential of ZnO NPs through disruption of the intact morphology of Klebsiella pneumoniae.

Functional annotation of proteins in Catharanthus roseus shoot cultures under biogenic zinc nanotreatment.

Nano-interactions are well known for their positive as well as negative impacts on the morphological and physiological systems of plants. Keeping in mind, the conformational changes in plant proteins as one of the key mechanisms for stress adaptation responses, the current project was designed to explore the effect of glutathione-capped and uncapped zinc nano-entities on Catharanthus roseus shoot cultures. Zinc nanotreatment (0.05 µg/mL) significantly induced ester production in C. roseus shoots as detected by Gas Chromatography-Mass spectrometry. These nanotreated shoots were further subjected to peptide-centric nano-LC-MS/MS analysis. Mass spectrometry followed by a Heat map revealed a significant effect of zinc nanoparticles on 59 distinct classes of proteins as compared to control. Proteins involved in regulating stress scavenging, transport, and secondary metabolite biosynthesis were robustly altered under capped zinc nanotreatment. UniProt database identified majority of the localization of the abundantly altered protein in cell membranes and chloroplasts. STRING and Cytoscape analysis assessed inter and intra coordination of triosephosphate isomerase with other identified proteins and highlighted its role in the regulation of protein abundance under applied stress. This study highlights the understanding of complex underlying mechanisms and regulatory networks involved in proteomic alterations and interactions within the plant system to cope with the nano-effect.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

An overview of the ameliorative efficacy of Catharanthus roseus extract against Cd2+ toxicity: implications for human health and remediation strategies.

Rapid industrialization has led to an increase in cadmium pollution, a dangerously toxic heavy metal. Cadmium (Cd) is released into the environment through industrial processes and can contaminate air, water, and soil. This pollution poses a significant risk to human health and has become a pressing concern in many industrialized areas. Due to its extended half-life, it leads to a range of health problems, including hepato-nephritic toxicity, brain damage, and degenerative bone disorders. Intoxication alters various intracellular parameters, leading to inflammation, tissue injury, and oxidative stress within cells, which disrupts normal cellular functions and can eventually result in cell death. It has also been linked to the development of bone diseases such as osteoporosis. These adverse effects highlight the urgent need to address cadmium pollution and find effective solutions to mitigate its impact on human health. This article highlights the Cd-induced risks and the role of Catharanthus roseus (C. roseus) extract as a source of alternative medicine in alleviating the symptoms. Numerous herbal remedies often contain certain bioactive substances, such as polyphenols and alkaloids, which have the power to mitigate these adverse effects by acting as antioxidants and lowering oxidative cell damage. Research conducted in the field of alternative medicine has revealed its enormous potential to meet demands that may be effectively used in safeguarding humans and their environment. The point of this review is to investigate whether C. roseus extract, known for its bioactive substances, is being investigated for its potential to mitigate the harmful effects of cadmium on health. Further investigation is needed to fully understand its effectiveness. Moreover, it is important to explore the potential environmental benefits of using C. roseus extract to reduce the negative effects of Cd. This review conducted in the field of alternative medicine has revealed its enormous potential to meet demands that could have significant implications for both human health and environmental sustainability.

A fused hybrid enzyme of 8-hydroxygeraniol oxidoreductase (8HGO) from Gardenia jasminoides and iridoid synthase (ISY) from Catharanthus roseus significantly enhances nepetalactol and iridoid production.

MAIN CONCLUSION: The operation of 8HGO-ISY fusion enzymes can increase nepetalactol flux to iridoid biosynthesis, and the Gj8HGO-CrISY expression in Gardenia jasminoides indicates that seco-iridoids and closed-ring iridoids share a nepetalactol pool. Nepetalactol is a common precursor of (seco)iridoids and their derivatives, which are a group of noncanonical monoterpenes. Functional characterization of an 8HGO (8-hydroxygeraniol oxidoreductase) from Catharanthus roseus, a seco-iridoids producing plant, has been reported; however, the 8HGO from G. jasminoides with plenty of closed-ring iridoids remains uninvestigated. In this work, a Gj8HGO was cloned and biochemically characterized. In addition, the relatively low production of nepetalactol in plants and engineered microbial host is likely to be attributed to the fact that Cr8HGO and CrISY (iridoid synthase) are substrate-promiscuous enzymes catalyzing unexpected substrates to the undesired products. Herein, a bifunctional enzyme consisting of an 8HGO fused to an ISY was designed for the proximity to the substrate and recycling of NADP+ and NADPH cofactor to reduce the undesired intermediate in the synthesis of nepetalactol. Of four fusion enzymes (i.e., Gj8HGO-GjISY, Gj8HGO-GjISY2, Gj8HGO-GjISY4, and Gj8HGO-CrISY), interestingly, only the last one can enable cascade reaction to form cis-trans-nepetalactol. Furthermore, we establish a reliable Agrobacterium-mediated transformation system. The expression of Gj8HGO-CrISY in G. jasminoides led to a significant enhancement of nepetalactol production, about 19-fold higher than that in wild-type plants, which further resulted in the twofold to fivefold increase of total iridoids and representative iridoid such as geniposide, indicating that seco-iridoids in C. roseus and closed-ring iridoids in G. jasminoides share a nepetalactol pool. All results suggest that 8HGO and ISY can be manipulated to maximize metabolic flux for nepetalactol and iridoid production.

Vincazalidine A, a unique bisindole alkaloid from Catharanthus roseus.

A new dimeric indole alkaloid, vincazalidine A consisting of an aspidosperma type and a modified iboga type with 1-azatricyclo ring system consisting of one azepane and two piperidine rings coupled with an oxazolidine ring was isolated from Catharanthus roseus, and the structure including absolute stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Vincazalidine A induced G2 arrest and subsequent apoptosis in human lung carcinoma cell line, A549 cells.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Integrated transcriptomic and proteomic analyses uncover the early response mechanisms of Catharanthus roseus under ultraviolet-B radiation.

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are important natural source for many drugs. Ultraviolet B (UVB) radiation have been proved to have regulatory effect towards biosynthesis of TIAs, which were meaningful for boost of TIA production. To decipher more comprehensive molecular characteristics in C. roseus under UVB radiation, integrated analysis of the nuclear proteome together with the transcriptome data under UVB radiation were performed. Expression of genes related to transmembrane transporters gradually increased during the prolonged exposure to UVB radiation. Some of known TIA transporters were affected by UVB. Abundance of proteins associated with spliceosome and nucleocytoplasmic transport increased. Homologs belonging to ORCA and CrWRKY transcription factors family increased at both transcriptomic and proteomic levels. At the same time, the numbers of differential alternative splicing events between UVB-radiated and white-light-treated plants continuously increased. These results suggest that the nucleus participated in early response of C. roseus under UVB radiation, where alternative splicing events occurred and might regulate multiple pathways. Furthermore, integrative omics analysis indicates that expression of enzymes at the terminal stages of seco-iridoid pathway decreased with the prolonged radiation exposure, potentially inhibiting further rise of TIA synthesis under extended UVB exposure.

Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis.

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 µg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC-MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 µM) and UO-31 (2.2 µM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett-Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 µg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus" to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.