Biosynthesis Of Vinblastine Research Articles

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

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 (200µM) 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 (100µM) and sodium nitroprusside (200µM 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.

De novo biosynthesis of antiarrhythmic alkaloid ajmaline

The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes. Here, we show the discovery and characterization of these two elusive reductases, alongside the identification of two physiologically relevant esterases that complete the biosynthesis of ajmaline. We show that ajmaline biosynthesis proceeds with vomilenine 1,2(R)-reduction followed by its 19,20(S)-reduction. This process is further modulated by two root-expressing esterases that deacetylate 17-O-acetylnorajmaline. Expanding upon the successful completion of the ajmaline biosynthetic pathway, we engineer the de novo biosynthesis of ajmaline in Baker’s yeast.

A near-complete genome assembly of Catharanthus roseus and insights into its vinblastine biosynthesis and high susceptibility to the Huanglongbing pathogen

This study reports the assembly of a near-complete genome of Catharanthus roseus, consisting of 561.7 Mb scaffolded into 8 pseudochromosomes with a contig N50 of 24.7 Mb and a scaffold N50 of 71.1 Mb. The assembly enables the construction of a gene regulatory network of the vinblastine biosynthetic pathway and provides insights into the high susceptibility of C. roseus to the Huanglongbing pathogen.

A Catharanthus roseus Fe(II)/α-ketoglutarate-dependent dioxygenase catalyzes a redox-neutral reaction responsible for vindolinine biosynthesis

The Madagascar’s periwinkle is the model plant for studies of plant specialized metabolism and monoterpenoid indole alkaloids (MIAs), and an important source for the anticancer medicine vinblastine. The elucidation of entire 28-step biosynthesis of vinblastine allowed further investigations for the formation of other remarkably complex bioactive MIAs. In this study, we describe the discovery and characterization of vindolinine synthase, a Fe(II)/α-ketoglutarate-dependent (Fe/2OG) dioxygenase, that diverts assembly of tabersonine to vinblastine toward the formation of three alternatively cyclized MIAs: 19S-vindolinine, 19R-vindolinine, and venalstonine. Vindolinine synthase catalyzes a highly unusual, redox-neutral reaction to form a radical from dehydrosecodine, which is further cyclized by hydrolase 2 to form the three MIA isomers. We further show the biosynthesis of vindolinine epimers from tabersonine using hydrolase 2 catalyzed reverse cycloaddition. While the occurrence of vindolinines is rare in nature, the more widely found venalstonine derivatives are likely formed from similar redox-neutral reactions by homologous Fe/2OG dioxygenases.

Expression of CrMPK3 and alkaloid synthesis genes with antioxidants in callus of Catharanthus roseus in response to polyethylene glycol

To improve the yield of secondary metabolites and understand the effects of PEG-induced drought stress on physiological and biochemical characteristics and the relationship between CrMPK3 and some genes of vinblastine biosynthesis pathway, PEG (0, 2.5%, 5%, and 10%) was applied in different exposure times (24 and 72 h) on callus of two cultivars (Alba and Rosea) of Catharanthus roseus. The increase in malondialdehyde and proline contents by increasing PEG concentration in both exposure times in Alba cultivar were more than in Rosea cultivar. Also, the activity of antioxidant enzymes including catalase (CAT), guaiacol peroxidase (GOPX), superoxide dismutase (SOD), glutathione peroxidase (GPX), and ascorbate peroxidase (APX) in the Alba cultivar was higher than the Rosea cultivar. The relative expression of vinblastine biosynthesis genes including STR, DAT, and PRX1 was significantly increased in both cultivars, especially in the medium with 5% PEG. However, the change of T16H gene expression was more minor. Vinblastine content confirmed the expression results of these genes at different concentrations of PEG after 24 and 72 h in both cultivars. The relative expression of CrMPK3 and catalase genes increased significantly in all media containing PEG. The highest expression of CrMPK3 and catalase genes was obtained after 24 and 72 h at 5% PEG in both cultivars, respectively. Finally, PEG can induce the accumulation of vinblastine in calli of C. roseus through a signaling pathway in which MAPK trigger the activation of antioxidant enzymes with up-regulation of the TIAs biosynthesis genes.

Generating an EMS Mutant Population and Rapid Mutant Screening by Thin-Layer Chromatography Enables the Studies of Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus Roseus.

Decades of research on the medicinal plant Catharanthus roseus have led to the complete elucidation of the 29-step pathway for the biosynthesis of the anticancer drug vinblastine from geraniol and tryptophan precursors. Several approaches have been used to identify the enzymes involved in this iconic and remarkably complex pathway. This chapter describes the use of the classic ethyl methanesulfonate (EMS) mutagenesis to create a selfed M2 mutant population, which can be rapidly screened to select mutants with altered monoterpenoid indole alkaloid (MIA) biosynthesis with a simple, high-throughput thin-layer chromatography (TLC)-based screening strategy. This TLC-based MIA screening has led to the discovery and characterization of three enzymes responsible for vinblastine biosynthesis.

Development of a Cell Suspension Culture System for Promoting Alkaloid and Vinca Alkaloid Biosynthesis Using Endophytic Fungi Isolated from Local Catharanthus roseus.

Cell and tissue cultures of Catharanthus roseus have been studied extensively as an alternative strategy to improve the production of valuable secondary metabolites. The purpose of this study was to produce C. roseus callus and suspension cell biomass of good quality and quantity to improve the total alkaloids and bis-indole alkaloids. The young stem derived-callus of C. roseus variety Quang Ninh (QN) was grown on MS medium supplemented with 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg/L kinetin, and the growth rate increased by 67-fold after 20 days. The optimal conditions for maintaining the cell suspension culture were 150 mg/50 mL cell inoculum, a medium pH of 5.5 and a culture temperature of 25 °C. The low alkaloid content in the culture was compensated for by using endophytic fungi isolated from local C. roseus. Cell extracts of endophytic fungi—identified as Fusarium solani RN1 and Chaetomium funicola RN3—were found to significantly promote alkaloid accumulation. This elicitation also stimulated the accumulation of a tested bis-indole alkaloid, vinblastine. The findings are important for investigating the effects of fungal elicitors on the biosynthesis of vinblastine and vincristine, as well as other terpenoid indole alkaloids (TIAs), in C. roseus QN cell suspension cultures.

Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus.

Terpenoid indole alkaloid (TIA) biosynthetic pathway of Catharanthus roseus possesses the major attention in current metabolic engineering efforts being the sole source of highly expensive antineoplastic molecules vinblastine and vincristine. The entire TIA pathway is fairly known at biochemical and genetic levels except the pathway steps leading to biosynthesis of catharanthine and tabersonine. To increase the in-planta yield of these antineoplastic metabolites for the pharmaceutical and drug industry, extensive plant tissue culture-based studies were performed to provide alternative production systems. However, the strict spatiotemporal developmental regulation of TIA biosynthesis has restricted the utility of these cultures for large-scale production. Therefore, the present study was performed to enhance the metabolic flux of TIA pathway towards the biosynthesis of vinblastine by overexpressing two upstream TIA pathway genes, tryptophan decarboxylase (CrTDC) and strictosidine synthase (CrSTR), at whole plant levels in C. roseus. Whole plant transgenic of C. roseus was developed using Agrobacterium tumefaciens LBA1119 strain having CrTDC and CrSTR gene cassette. Developed transgenic lines demonstrated up to twofold enhanced total alkaloid production with maximum ninefold increase in vindoline and catharanthine, and fivefold increased vinblastine production. These lines recorded a maximum of 38-fold and 65-fold enhanced transcript levels of CrTDC and CrSTR genes, respectively.

Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus.

We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon.

Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids.

Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine-eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related.

Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling.

The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

Regulation of vinblastine biosynthesis in cell suspension cultures of catharanthus roseus

Various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase were added to a Catharanthus roseus cell culture medium to investigate the regulatory effects on tabersonine, vindoline and vinblastine biosynthesis. Hydrogen peroxide was found to be the most effective agent for enhancing the biosynthesis of tabersonine. By adding 20 μg/L hydrogen peroxide, the tabersonine concentration reached 9.02 mg/g dry weight (DW) after culturing cell suspensions for 7 days. With the addition of 30 μg/L acetyl CoA, the most vindoline (final cell content of 0.33 mg/g DW) was produced. By effective inhibition of lochnericine biosynthesis with the addition of 0.5 μmol/L benzotriazole, the cell content of vindoline was increased to 0.42 mg/g DW. An orthogonal experiment consisting of multiple regulation factors was carried out to optimize vinblastine biosynthesis. It was shown that optimal vinblastine biosynthesis was achieved by addition of 5 mg/L acetyl CoA, 20 μg/L hydrogen peroxide, 0.5 μmol/L benzotriazole, 100 mg/L tryptophan, 100 mg/L loganin and 30 mg/L cerium chloride. Under these conditions, the cell content of vinblastine reached 0.81 mg/g DW. Simultaneous changes in cell content and enzyme activities of Cytochrome P-450 monooxygenases, Deacetylvindoline-O-acetyltransferase and Peroxidase enzyme indicated that these enzymes were closely linked to vinblastine biosynthesis.

Foundations of organic chemistry: unity and diversity of structures, pathways, and reactions

Foundations of organic chemistry: unity and diversity of structures, pathways, and reactions

Improvement of Vindoline Production in a <i>Catharanthus roseus callus</i> Line

The influence of polyploidity on the accumulation of vindoline in Catharanthus roseus callus cultures was investigated. The callus line (T1) was induced from a tetraploid leaf. Total alkaloids in the callus were extracted and analyzed by LC-MS for qualitification and HPLC for quantification. Results showed that T1 callus cultures could accumulate vindoline to a high level. The highest accumulation of vindoline found in the callus was 0.11 mg g−1 DW. Our results demonstrate that polyploidity could influence the chlorophyll content or chloroplast development and improve vindoline biosynthesis in callus cultures. The T1 callus cultures also accumulated a substance which was not present in the diploid callus. The substance was preliminarily identified by LC-MS as deacetylvindoline, the direct precursor of vindoline biosynthesis. T1 callus could be used for genetic manipulation of the biosynthesis of vinblastine and vincristine, the two important antitumor drugs, and therefore, have potential commercial value.

Effect of plant growth regulators on the biosynthesis of vinblastine, vindoline and catharanthine in Catharanthus roseus

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.

Not so strictosidine

Plant cell cultures expressing a mutated form of a key alkaloid-producing enzyme could enable the biosynthesis of vinblastine and related indole alkaloids that are used to treat cancer. In addition, the technique could be used to produce new alkaloids.

Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. Don

The leaves of Catharanthus roseus (L.) G. Don produce the first natural drugs used in cancer therapy – the dimeric terpenoid indole alkaloids vinblastine and vincristine. The study of C. roseus further revealed two other terpenoid indole alkaloids with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative. The biosynthetic pathway of the medicinal alkaloids has been investigated in much detail and a number of steps are now well characterized at the enzyme and gene level and, recently, several regulatory genes have also been isolated and characterized. Since early studies of the biosynthesis of vinblastine, during the 1970s and 1980s, the dimerization reaction has attracted much attention due to its possible regulatory importance and potential application for the semi synthetic production of the dimeric alkaloids. After initial, inconclusive work suggesting the involvement of peroxidase-like enzymes, the search for the dimerization enzyme in leaf tissue detected a single dimerization activity credited to the single class III plant peroxidase present in the leaves of the plant – the basic isoenzyme CRPRX1. The enzyme was purified to homogeneity, the respective cDNA and genomic sequences were characterized, and a channeling mechanism was proposed for the peroxidase-mediated-vacuolar synthesis of the first dimeric alkaloid intermediate, α-3′,4′-anhydrovinblastine. On the other hand, the oxidation of ajmalicine into serpentine has been attributed to basic peroxidase isoenzymes localized in the vacuole of C. roseus cells. An overview of the work implying class III plant peroxidases in the biosynthesis of terpenoid indole alkaloids in C. roseus is presented here. Abbreviations: CRPRX1 –Catharanthus roseus peroxidase 1; DAB – diaminobenzidine; IEF – isoelectric focusing; UV – ultraviolet.

Studies on the Biosynthesis of Bisindole Alkaloids. The Final Stages in Biosynthesis of Vinblastine, Leurosine and Catharine

Studies on the Biosynthesis of Bisindole Alkaloids. The Final Stages in Biosynthesis of Vinblastine, Leurosine and Catharine