Improved accumulation of ajmalicine and tetrahydroalstonine in Catharanthus cells expressing an ABC transporter. J Plant Physiol

Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, PO Box 9502, Leiden 2333 CC, The Netherlands.
Journal of plant physiology (Impact Factor: 2.56). 05/2009; 166(13):1405-12. DOI: 10.1016/j.jplph.2009.02.015
Source: PubMed


The biosynthetic pathway of monoterpenoid indole alkaloids in Catharanthus roseus is located throughout various membranes at both the cellular and intercellular levels. ATP-binding cassette (ABC) transporters are known to export vincristine and vinblastine from human cancer cells. It has recently been shown that ABC transporters are also involved in the transport of various monoterpenoid alkaloids in Catharanthus roseus cells. Over-expression of an ABC transporter in this plant might therefore affect the regulation of the alkaloid biosynthetic pathway. CjMDR1, an ABC transporter gene originally isolated from Coptis japonica, was expressed in Catharanthus roseus cell cultures. Cells showing a positive PCR signal of the transgene in both cDNA and genomic DNA samples were subject to transport studies using selected substrates. Unexpectedly, transport of the isoquinoline alkaloid berberine, the main substrate of CjMDR1 transporter in Coptis japonica, was not affected as compared with control and wild-type Catharanthus cells. On the other hand, the endogenous alkaloids ajmalicine and tetrahydroalstonine were accumulated significantly more in Catharanthus roseus cells expressing CjMDR1 in comparison with control lines after feeding these alkaloids.

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Available from: Barbora Pomahacová, Jan 21, 2015
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    • "The yeast system, which has been adapted for the functional characterization of many plant alkaloid transporters, offers the following advantages: i) it is easy to implement with standard laboratory equipment, ii) it is a eukaryotic system and therefore suitable for functionally expressing plant proteins, and iii) it is capable of measuring the activities of both primary and secondary transporters, which transport substrate by directly using energy of ATP or PPi hydrolysis or using electrochemical gradients of protons or ions, respectively, and so should be a first choice for such examinations (Shitan et al. 2013b). As shown for CjABCB1, CrTPT2 and NtNUP1, altering the expression of alkaloid transporters influences the accumulation, and sometimes also the production, of relevant alkaloids (Hildreth et al. 2011; Pomahacova et al. 2009; Shitan et al. 2005; Yu and De Luca 2013). However, the mechanistic details underlying these phenomena remain unclear. "
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    ABSTRACT: Plants produce a multitude of secondary metabolites, including alkaloids with biological activities, and many alkaloids have been used for medicinal purposes. The biosynthetic enzymes and genes involved in alkaloid metabolic pathways exhibit divergent localizations, implying that alkaloid metabolites, including pathway products and intermediates, travel from organelle to organelle, cell to cell, and organ to organ. Biochemical studies have indicated that specific transporters move these metabolites. Indeed, molecular and cellular approaches have identified alkaloid transporters of the ATP-binding cassette (ABC) protein, multidrug and toxic compound extrusion (MATE), and purine permease (PUP) families. Interestingly, some of these transporters were found to be required for the efficient biosynthesis of alkaloids in plants. Here, we provide an updated inventory of alkaloid transporters and discuss the possibility of genetically manipulating the expression of these transporters to increase the accumulation of valuable alkaloid compounds.
    Plant Biotechnology 12/2014; 31(5):453-463. DOI:10.5511/plantbiotechnology.14.1002a · 0.87 Impact Factor
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    • "A series of genes encoding MIA biosynthetic enzymes (anthranilate synthase alpha subunit, Asα; 1-deoxy-D-xylulose synthase, DXS; tryptophan decarboxylase, TDC; strictosidine synthase, STR; strictosidine beta-glucosidase, SGD; geraniol 10-hydroxylase, G10H; desacetoxyvindoline 4-hydroxylase, DAT; apoplastic peroxidase, CrPrx; secologanin synthase, SLS) have been characterized, cloned and overexpressed alone or in combination in cell cultures and hairy roots of C. roseus in the past decade [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Transformation of transcription factors (ORCA2 and ORCA3) [16], transporters (ATP-binding cassette transporter, ABC) [17] and heterologous genes (Bcl-2 Associated X protein, Bax) [18] were also performed in C. roseus cells and hairy roos. Genetic modification is a good approach to study the regulation of MIA biosynthesis and to improve the production of targeted MIAs in C. roseus, thus lower the production costs for these very expensive clinical medicines. "
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    ABSTRACT: 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.
    PLoS ONE 08/2012; 7(8):e43038. DOI:10.1371/journal.pone.0043038 · 3.23 Impact Factor
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    • "Thus, over-expression of an ABC transporter may think to affect the regulation of the alkaloid biosynthetic pathway. CjMDR1, an ABC transporter gene originally isolated from Coptis japonica, was inserted and expressed in C. roseus cell cultures, which showed enhanced levels of ajmalicine and tetrahydroalstonine in comparison with control lines (Pomahacová et al. 2009). "
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    ABSTRACT: Catharanthus roseus (L.) G. Don. is a well known medicinal plant. It produces several phyto-compounds many of which show anticancerous properties. The yields of these compounds are however, very low. In this present article, the current development of secondary metabolite synthesis in C. roseus involving biotechnology has been reviewed keeping in mind the various basic fac-tors that influence yield. The importance of cell culture, the role of culturing conditions and other approaches aiming at higher production of alkaloids have been discussed. The genes encoding important enzymes, proteomics, expressed sequence tag and transcription factors in relation to alka-loid yield have also been summarized in order to under-stand the regulatory mechanisms of C. roseus.
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