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- Alkaloid production by a Cinchona officinalis "Ledgeriana" hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseusPublication . Geerlings, A.; Hallard, D.; Martinez Caballero, A.; Cardoso, I. Lopes; Heijden, R. Van Der; Verpoorte, R.Cinchona officinalis ‘Ledgeriana’, former called Cinchona ledgeriana, hairy roots were initiated containing constitutive-expression constructs of cDNAs encoding the enzymes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) from Catharanthus roseus, two key enzymes in terpenoid indole and quinoline alkaloid biosynthesis. The successful integration of these genes and the reporter gene gus-int was demonstrated using Southern blotting and the polymerase chain reaction. The products of TDC and STR, tryptamine and strictosidine, were found in high amounts, 1200 and 1950 mg g–1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 mg g–1 dry weight, respectively. The results show that genetic engineering with multiple genes is well possible in hairy roots of C. officinalis. However, 1 year after analyzing the hairy roots for the first time, they had completely lost their capacity to accumulate alkaloids.
- Cloning of a peroxidase enzyme involved in the biosynthesis of pharmaceutically active terpenoid indole alkaloids in Catharanthus roseus (L.) G. DonPublication . Hilliou, F.; Costa, M.; Almeida, I.; Cardoso, I.L.; Leech, M.; Ros Barceló, A.; Sottomayor, M.
- Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. DonPublication . Sottomayor, M.; Cardoso, Inês Lopes; Pereira, L.G.; Ros Barceló, A.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.
- A promoter region that controls basal and elicitor-inducible expression levels of the NADPH: cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds nuclear factor GT-1Publication . Cardoso, M.I. Lopes; Meijer, A.H.; Rueb, S.; Machado, J. Queiroz; Memelink, J.; Hoge, J.H.C.NADPH:cytochrome P450 reductase (CPR) is essential for the activation of cytochrome P450 enzymes, which are involved in a wide variety of metabolic pathways in plants, including those related to defence responses. In the subtropical plant Catharanthus roseus several cytochrome P450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids (TIAs). In agreement with the importance of CPR in defence, Cpr mRNA levels in C. roseus were found to be enhanced by fungal elicitor preparations that also induce TIA biosynthesis and P450 gene expression. Here we describe the isolation of a C. roseus genomic DNA clone covering the 5¢ part of the Cpr gene and 1.6-kb of upstream sequences. Mapping of the transcription start site showed the untranslated leader sequence is approximately 280 bp long. To study the control of gene expression by the Cpr promoter, transcriptional fusions between Cpr promoter fragments and the gusA reporter gene were generated and their expression was analyzed in stably transformed tobacco plants. The Cpr promoter fragment extending from )1510 to )8, with respect to the ATG start codon, conferred basal and elicitor-inducible expression on the gusA reporter gene, strongly indicating that the Cpr gene of C. roseus is indeed controlled by this promoter region. Progressive deletion from the 5¢ end of the promoter to position )632 had little e ect on gusA expression. However, deletion to position )366 resulted in a complete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from )632 to )366 contains the main transcription-enhancing cis-regulatory sequences. Electrophoretic mobility shift assays with tobacco nuclear extracts showed that binding sites for nuclear factor GT-1 are redundant in the Cpr promoter, but absent from the downstream part of the leader sequence. The presence of strong GT-1 binding sites in the main enhancer region ()632 to )366), is suggestive of a functional role for this factor in basal expression and elicitor responsiveness of the Cpr promoter.
- Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH: cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plantsPublication . Meijer, Annemarie H.; Cardoso, Inês Lopes; Voskuilen, John Th.; de Waal, Anthony; Verpoorte, Robert; Hoge, J. Harry C.The membrane‐bound flavoprotein NADPH:cytochrome P‐450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P‐450 mono‐oxygenases, was purified from a cell suspension culture of the higher plant Catheranthus roseus. Anti‐serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P‐450 enzymes geraniol 10‐hydroxylase and trans‐cinnamate 4‐hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P‐450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P‐450 reductases from yeast and animal species. The identity of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH‐dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N‐terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P‐450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P‐450 reductase with the corresponding enzymes from yeast and animal species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P‐450‐dependent biosynthetic pathways.
- Agrobacterium-mediated transformation of the terpenoid indole alkaloid-producing plant species Tabernaemontana pandacaquiPublication . Cardoso, M.I. Lopes; Meijer, A.H.; Hoge, J.H.C.Plants of the Apocynaceae family produce a wide range of terpenoid indole alkaloids (TIAs) which have important pharmaceutical applications. Studies of the molecular mechanisms controlling TIA biosynthesis may eventually provide possibilities to improve product yield by genetic modification of plants or cell cultures. However, these studies suffer from the lack of transformation/regeneration protocols for Apocynaceae plants. We chose to study the feasibility of Agrobacterium tumefaciens-mediated transformation of Tabernaemontana pandacaqui, because of the availability of an efficient regeneration procedure for this member of the Apocynaceae family. A procedure to produce transgenic T. pandacaqui plants was established, albeit with low efficiency. Transgenic expression was demonstrated of an intron-containing β-glucuronidase reporter gene and of a gene coding for the TIA biosynthetic enzyme strictosidine synthase from Catharanthus roseus, another Apocynaceae species.