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Nature Chemical Biology Aug 2023Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells...
Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.
Topics: Catharanthus; Plants, Medicinal; Multiomics; Indole Alkaloids; Antineoplastic Agents; Monoterpenes; Gene Expression Regulation, Plant; Plant Proteins
PubMed: 37188960
DOI: 10.1038/s41589-023-01327-0 -
Molecules (Basel, Switzerland) Feb 2015Catharanthus roseus is a medicinal plant belonging to the family Apocynaceae which produces terpenoid indole alkaloids (TIAs) of high medicinal importance. Indeed, a... (Review)
Review
Catharanthus roseus is a medicinal plant belonging to the family Apocynaceae which produces terpenoid indole alkaloids (TIAs) of high medicinal importance. Indeed, a number of activities like antidiabetic, bactericide and antihypertensive are linked to C. roseus. Nevertheless, the high added value of this plant is based on its enormous pharmaceutical interest, producing more than 130 TIAs, some of which exhibit strong pharmacological activities. The most striking biological activity investigated has been the antitumour effect of dimeric alkaloids such as anhydrovinblastine, vinblastine and vincristine which are already in pre-, clinical or in use. The great pharmacological importance of these indole alkaloids, contrasts with the small amounts of them found in this plant, making their extraction a very expensive process. To overcome this problem, researches have looked for alternative sources and strategies to produce them in higher amounts. In this sense, intensive research on the biosynthesis of TIAs and the regulation of their pathways has been developed with the aim to increase by biotechnological approaches, the production of these high added value compounds. This review is focused on the different strategies which improve TIA production, and in the analysis of the beneficial effects that these compounds exert on human health.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Antioxidants; Antiviral Agents; Catharanthus; China; Humans; Hypoglycemic Agents; Plant Extracts; Plant Shoots; Plants, Medicinal; Secologanin Tryptamine Alkaloids
PubMed: 25685907
DOI: 10.3390/molecules20022973 -
Journal of the American Chemical Society Sep 2019A new triarylaminium radical cation promoted coupling of catharanthine with vindoline is disclosed, enlisting tris(4-bromophenyl)aminium hexachlororantimonate (BAHA, 1.1...
A new triarylaminium radical cation promoted coupling of catharanthine with vindoline is disclosed, enlisting tris(4-bromophenyl)aminium hexachlororantimonate (BAHA, 1.1 equiv) in aqueous 0.05 N HCl/trifluoroethanol (1-10:1) at room temperature (25 °C), that provides anhydrovinblastine in superb yield (85%) with complete control of the newly formed quaternary C16' stereochemistry. A definition of the scope of aromatic substrates that participate with catharanthine in the BAHA-mediated diastereoselective coupling reaction and simplified indole substrates other than catharanthine that participate in the reaction are disclosed that identify the key structural features required for participation in the reaction, providing a generalized indole functionalization reaction that bears little structural relationship to catharanthine or vindoline.
Topics: Amines; Cations; Free Radicals; Molecular Structure; Stereoisomerism; Vinblastine; Vinca Alkaloids
PubMed: 31442047
DOI: 10.1021/jacs.9b06968 -
FEBS Letters May 1998An H2O2-dependent enzyme capable of coupling catharanthine and vindoline into alpha-3',4'-anhydrovinblastine (AVLB) was purified to apparent homogeneity from...
An H2O2-dependent enzyme capable of coupling catharanthine and vindoline into alpha-3',4'-anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS-PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.
Topics: Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Horseradish Peroxidase; Kinetics; Molecular Weight; Plant Leaves; Plants, Medicinal; Vinblastine; Vinca Alkaloids
PubMed: 9654153
DOI: 10.1016/s0014-5793(98)00551-1 -
Plant Physiology Feb 2008Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The...
Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.
Topics: Alkaloids; Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Base Sequence; Catharanthus; Cloning, Molecular; Gene Expression Regulation, Plant; Models, Molecular; Molecular Sequence Data; Peroxidases; Phylogeny; Plant Leaves; Vacuoles
PubMed: 18065566
DOI: 10.1104/pp.107.107060 -
Frontiers in Plant Science 2019contains a variety of monoterpenoid indole alkaloids (MIAs), among which bisindole alkaloids vinblastine and vincristine are well-known to have antitumor effects and...
contains a variety of monoterpenoid indole alkaloids (MIAs), among which bisindole alkaloids vinblastine and vincristine are well-known to have antitumor effects and widely used in clinical treatment. However, their contents in is extremely low and difficult to meet market demands. Therefore, it is of great significance to study the transcriptional regulation mechanism of MIAs biosynthesis for high yielding of bisindole alkaloids in . Studies have shown that MIAs biosynthesis in has complex temporal and spacial specificity and is under tight transcriptional regulation, especially bisindole alkaloids. In this study, an AP2/ERF transcription factor CrERF5 was selected by RNA-seq of organs, and its full-length sequence was cloned and characterized. CrERF5 responds to both ethylene and JA signals and is localized in the nucleus. CrERF5 could activate the transcriptional activity of the TDC promoter. Transient overexpressing CrERF5 in petals caused a significant increase of the expression levels of key genes in both the upstream and downstream pathways of MIAs biosynthesis while silencing resulted in a decrease of them. Accordingly, the contents of bisindole alkaloids anhydrovinblastine and vinblastine, monoindole alkaloids ajmalicine, vindoline, and catharanthine were strongly enhanced in -overexpressing petals while their contents decreased in -silenced plants. These results suggested that CrERF5 is a novel positive ethylene-JA-inducible AP2/ERF transcription factor upregulating the MIAs biosynthetic pathway leading to the bisindole alkaloids accumulation.
PubMed: 31379908
DOI: 10.3389/fpls.2019.00931 -
PloS One 2012In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy...
In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of (1)H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.
Topics: Alkaloids; Catharanthus; Gene Expression Regulation, Plant; Indoles; Iridoids; Magnetic Resonance Spectroscopy; Metabolomics; Plant Proteins; Plants, Genetically Modified; Polymerase Chain Reaction; Signal Transduction; Transcription Factors
PubMed: 22916202
DOI: 10.1371/journal.pone.0043038 -
Metabolic Engineering Communications Jun 2023With over 3000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important...
With over 3000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from (Madagascar's periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme -acetylstemmadenine oxidase in (baker's yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also reported the simultaneous integration of ∼18 kb MIA biosynthetic gene cassettes as single copies into four genomic loci of baker's yeast by CRISPR-Cas9, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs.
PubMed: 36569379
DOI: 10.1016/j.mec.2022.e00215 -
Frontiers in Plant Science 2018is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and...
Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic .
is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in . Here, to overcome this limitation and enhance MIAs levels in , bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by -mediated transformation. Both transient and stable overexpression of and co-expression of + significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in but not in + transgenic lines that correlated with transcript levels of peroxidase-1 () involved in coupling of vindoline and catharanthine into 3',4'-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T) lines exhibited comparable transcript and metabolite levels to that of T lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in through transgenic approaches.
PubMed: 30034406
DOI: 10.3389/fpls.2018.00942