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Chelliah Jayabaskaran - One of the best experts on this subject based on the ideXlab platform.
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evaluation of spore inoculum and confirmation of pathway genetic blueprint of t13αh and dbat from a taxol producing endophytic fungus
Scientific Reports, 2020Co-Authors: Balabhadrapatruni V S K Chakravarthi, Satpal Singh, Subban Kamalraj, Vijai Kumar Gupta, Chelliah JayabaskaranAbstract:Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and Baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L-1 and 128.3 µg L-1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylBaccatin III-10-β-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and Baccatin III.
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Biochemical insights into the recombinant 10-deacetylBaccatin III-10-β-O-acetyltransferase enzyme from the Taxol-producing endophytic fungus Lasiodiplodia theobromae
Fems Microbiology Letters, 2019Co-Authors: Kamalraj Subban, Chelliah JayabaskaranAbstract:10-deacetylBaccatin III-10-beta-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylBaccatin III into Baccatin III, as shown by liquid chromatography-mass spectroscopy. The mass spectra of Baccatin III were identical to the authentic Baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors.
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inhibition of cancer cell proliferation and apoptosis inducing activity of fungal taxol and its precursor Baccatin III purified from endophytic fusarium solani
Cancer Cell International, 2013Co-Authors: Balabhadrapatruni V S K Chakravarthi, Ramanathan Sujay, Gini C Kuriakose, Anjali A Karande, Chelliah JayabaskaranAbstract:Background Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor Baccatin III in liquid grown cultures J Biosci 33:259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal Baccatin III of F. solani isolated from T. celebica.
R. M. Cusido - One of the best experts on this subject based on the ideXlab platform.
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Development of a hazel cell culture-based paclitaxel and Baccatin III production process on a benchtop scale
Journal of Biotechnology, 2015Co-Authors: Ana Gallego, Javier Palazon, R. M. Cusido, Mercedes Bonfill, Nicole Imseng, Regine Eibl, Elisabeth MoyanoAbstract:The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1. μM coronatine (Cor) and 100. μM methyl jasmonate (MeJA).Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28gDCW/L and 5.69gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin® Bioreactors 50, where the highest taxane content (8583.3μg/L) was obtained when 1μM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT® B plus and UniVessel® SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel® SU resulted in a total taxane content of 6246.1μg/L, a 10-fold increase compared with shake flask experiments.
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The relationship between TXS, DBAT, BAPT and DBTNBT gene expression and taxane production during the development of Taxus baccata plantlets
Plant Science, 2011Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Alain Goossens, R. M. CusidoAbstract:Abstract Taxol and related taxane accumulation in plants is regulated by the expression of genes involved in their biosynthesis. Although the metabolic pathway leading to taxol has been almost completely elucidated, comparatively little is known about the rate-limiting steps and their regulation. In this paper we report on a study of taxane production in Taxus baccata plantlets grown in vitro for 1 year. The relationship between taxane patterns and the expression of genes encoding the enzymes taxadiene synthase (TXS), 10-deacetylBaccatin III-10β- O -acetyltransferase (DBAT), Baccatin III 13- O -(3-amino-3-phenylpropanoyl) transferase (BAPT) and 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT), involved in early and late steps of the taxane pathway, has been considered. A far higher content was found in the aerial part of the plantlets than in the roots. The most abundant taxane in the aerial parts was 10-deacetylBaccatin III, which increased as the plantlets grew, indicating a low conversion to Baccatin III and taxol. In contrast, the levels of 10-deacetylBaccatin III in the roots remained lower than those of taxol. These results correlated with transcript accumulation of the studied genes, since in the aerial parts the expression of DBAT , which codes for the enzyme that converts 10-deacetylBaccatin III into Baccatin III, did not increase with the age of plantlets, unlike that of TXS , BAPT and DBTNBT , suggesting that this gene controls a rate-limiting step in the taxane biosynthetic pathway. The lower taxane levels found in the roots also correlated with gene expression, since only the early pathway gene TXS was induced in this organ during the 1-year growth period.
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an approach to the molecular mechanism of methyl jasmonate and vanadyl sulphate elicitation in taxus baccata cell cultures the role of txs and bapt gene expression
Biochemical Engineering Journal, 2010Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabeth Moyano, Oscar Exposito, R. M. CusidoAbstract:Abstract Before the biotechnological production of the anticancer compound taxol can be improved, the mechanism that regulates its biosynthetic pathway needs to be further understood. In this paper we have studied the effect of methyl jasmonate (MeJ) and vanadyl sulphate (VS) on the taxane pattern and transcript profile of two key genes involved in taxol biosynthesis, txs and bapt, in a selected Taxus baccata cell line. Our results showed that MeJ significantly activated both taxol and Baccatin III production (4- and 3.6-fold, respectively), whereas VS only enhanced taxol production (also 4-fold). At the same time, MeJ treatment clearly increased the expression of the txs and bapt genes but the presence of VS in the medium only increased bapt gene expression. The results suggest that the elicitation conditions assayed are good strategies to improve taxol production, and that the elicitors have different and probably interfering mechanisms of action in taxol biosynthesis. To our knowledge, this is the first time that a relationship between the expression of the txs and bapt genes and the taxane profile has been shown in T. baccata cell cultures.
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Paclitaxel and Baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata
Biologia Plantarum, 2007Co-Authors: Mercedes Bonfill, R. M. Cusido, S. Bentebibel, E. Moyano, J. Palazón, R. Eibl, M. T. PiñolAbstract:The effects of 100 and 200 µM methyl jasmonate (MJA) on cell proliferation and paclitaxel and Baccatin III production were investigated in free and alginate immobilized cells of Taxus baccata growing in a selected product formation culture medium. The greatest accumulation of paclitaxel (13.20 mg dm^−3) and Baccatin III (4.62 mg dm^−3) occurred when 100 µM MJA was added to the culture medium of cells entrapped using a 1.5 and 2.5 % alginate solution. The effects of different treatments on the viability of cultured cells and their capacity to excrete both taxanes into the surrounding medium were considered.
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source of isopentenyl diphosphate for taxol and Baccatin III biosynthesis in cell cultures of taxus baccata
Biochemical Engineering Journal, 2007Co-Authors: R. M. Cusido, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Oscar Exposito, Teresa M PinolAbstract:Abstract To achieve a better understanding of the metabolism and accumulation of taxol and Baccatin III in cell cultures of Taxus , three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and Baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and Baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and Baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.
Javier Palazon - One of the best experts on this subject based on the ideXlab platform.
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improved effects of polyethylene glycol on the growth antioxidative enzymes activity and taxanes production in a taxus baccata l callus culture
Plant Cell Tissue and Organ Culture, 2019Co-Authors: Marziyeh Sarmadi, Javier Palazon, Naser Karimi, Alireza Ghassempour, Mohammad Hossein MirjaliliAbstract:Application of abiotic stress-inducing elicitors is one of the effective strategies in order to increase the production of secondary metabolites in plant tissues. Here, the effect of in vitro polyethylene glycol (PEG)-induced drought stress on morphological and biochemical changes as well as the production of paclitaxel and 10-deacetyl Baccatin III in a Taxus baccata callus culture was investigated. The results showed that increased PEG concentration in the culture medium enhanced the levels of H2O2 and the membrane lipid peroxidation in the calli. The activity of catalase and guaiacol peroxidase as antioxidant responses were increased significantly in all PEG concentrations. At 6% of PEG concentration, the levels of fresh weight (FW) and dry weight (DW) in the calli were declined significantly by dropping in the amount of water content in the calli and rising the level of oxidative stress, and the morphological changes occurred following the increased browning index of the calli. However, the PEG at low to moderate concentrations reduced the accumulation and oxidation of phenolic compounds in the calli through the absorption of phenolic compounds and the reduction in the activity of the polyphenol oxidase. As a result, improved growth and viability of the calli resulted in the enhanced levels of FW and DW at the concentrations of 1% up to 3% (w/v) PEG. The highest contents of 10-deacetyl Baccatin III and taxol were obtained at concentrations of 2% and 3% PEG, respectively. The concentration of 3% PEG increased the production of taxol by 2.5 times more than the control. In conclusion, PEG can be proposed as an effective elicitor in the production of taxanes in the Taxus cell and tissue cultures.
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Development of a hazel cell culture-based paclitaxel and Baccatin III production process on a benchtop scale
Journal of Biotechnology, 2015Co-Authors: Ana Gallego, Javier Palazon, R. M. Cusido, Mercedes Bonfill, Nicole Imseng, Regine Eibl, Elisabeth MoyanoAbstract:The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1. μM coronatine (Cor) and 100. μM methyl jasmonate (MeJA).Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28gDCW/L and 5.69gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin® Bioreactors 50, where the highest taxane content (8583.3μg/L) was obtained when 1μM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT® B plus and UniVessel® SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel® SU resulted in a total taxane content of 6246.1μg/L, a 10-fold increase compared with shake flask experiments.
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The relationship between TXS, DBAT, BAPT and DBTNBT gene expression and taxane production during the development of Taxus baccata plantlets
Plant Science, 2011Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Alain Goossens, R. M. CusidoAbstract:Abstract Taxol and related taxane accumulation in plants is regulated by the expression of genes involved in their biosynthesis. Although the metabolic pathway leading to taxol has been almost completely elucidated, comparatively little is known about the rate-limiting steps and their regulation. In this paper we report on a study of taxane production in Taxus baccata plantlets grown in vitro for 1 year. The relationship between taxane patterns and the expression of genes encoding the enzymes taxadiene synthase (TXS), 10-deacetylBaccatin III-10β- O -acetyltransferase (DBAT), Baccatin III 13- O -(3-amino-3-phenylpropanoyl) transferase (BAPT) and 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT), involved in early and late steps of the taxane pathway, has been considered. A far higher content was found in the aerial part of the plantlets than in the roots. The most abundant taxane in the aerial parts was 10-deacetylBaccatin III, which increased as the plantlets grew, indicating a low conversion to Baccatin III and taxol. In contrast, the levels of 10-deacetylBaccatin III in the roots remained lower than those of taxol. These results correlated with transcript accumulation of the studied genes, since in the aerial parts the expression of DBAT , which codes for the enzyme that converts 10-deacetylBaccatin III into Baccatin III, did not increase with the age of plantlets, unlike that of TXS , BAPT and DBTNBT , suggesting that this gene controls a rate-limiting step in the taxane biosynthetic pathway. The lower taxane levels found in the roots also correlated with gene expression, since only the early pathway gene TXS was induced in this organ during the 1-year growth period.
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an approach to the molecular mechanism of methyl jasmonate and vanadyl sulphate elicitation in taxus baccata cell cultures the role of txs and bapt gene expression
Biochemical Engineering Journal, 2010Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabeth Moyano, Oscar Exposito, R. M. CusidoAbstract:Abstract Before the biotechnological production of the anticancer compound taxol can be improved, the mechanism that regulates its biosynthetic pathway needs to be further understood. In this paper we have studied the effect of methyl jasmonate (MeJ) and vanadyl sulphate (VS) on the taxane pattern and transcript profile of two key genes involved in taxol biosynthesis, txs and bapt, in a selected Taxus baccata cell line. Our results showed that MeJ significantly activated both taxol and Baccatin III production (4- and 3.6-fold, respectively), whereas VS only enhanced taxol production (also 4-fold). At the same time, MeJ treatment clearly increased the expression of the txs and bapt genes but the presence of VS in the medium only increased bapt gene expression. The results suggest that the elicitation conditions assayed are good strategies to improve taxol production, and that the elicitors have different and probably interfering mechanisms of action in taxol biosynthesis. To our knowledge, this is the first time that a relationship between the expression of the txs and bapt genes and the taxane profile has been shown in T. baccata cell cultures.
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source of isopentenyl diphosphate for taxol and Baccatin III biosynthesis in cell cultures of taxus baccata
Biochemical Engineering Journal, 2007Co-Authors: R. M. Cusido, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Oscar Exposito, Teresa M PinolAbstract:Abstract To achieve a better understanding of the metabolism and accumulation of taxol and Baccatin III in cell cultures of Taxus , three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and Baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and Baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and Baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.
Mercedes Bonfill - One of the best experts on this subject based on the ideXlab platform.
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Development of a hazel cell culture-based paclitaxel and Baccatin III production process on a benchtop scale
Journal of Biotechnology, 2015Co-Authors: Ana Gallego, Javier Palazon, R. M. Cusido, Mercedes Bonfill, Nicole Imseng, Regine Eibl, Elisabeth MoyanoAbstract:The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1. μM coronatine (Cor) and 100. μM methyl jasmonate (MeJA).Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28gDCW/L and 5.69gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin® Bioreactors 50, where the highest taxane content (8583.3μg/L) was obtained when 1μM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT® B plus and UniVessel® SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel® SU resulted in a total taxane content of 6246.1μg/L, a 10-fold increase compared with shake flask experiments.
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The relationship between TXS, DBAT, BAPT and DBTNBT gene expression and taxane production during the development of Taxus baccata plantlets
Plant Science, 2011Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Alain Goossens, R. M. CusidoAbstract:Abstract Taxol and related taxane accumulation in plants is regulated by the expression of genes involved in their biosynthesis. Although the metabolic pathway leading to taxol has been almost completely elucidated, comparatively little is known about the rate-limiting steps and their regulation. In this paper we report on a study of taxane production in Taxus baccata plantlets grown in vitro for 1 year. The relationship between taxane patterns and the expression of genes encoding the enzymes taxadiene synthase (TXS), 10-deacetylBaccatin III-10β- O -acetyltransferase (DBAT), Baccatin III 13- O -(3-amino-3-phenylpropanoyl) transferase (BAPT) and 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT), involved in early and late steps of the taxane pathway, has been considered. A far higher content was found in the aerial part of the plantlets than in the roots. The most abundant taxane in the aerial parts was 10-deacetylBaccatin III, which increased as the plantlets grew, indicating a low conversion to Baccatin III and taxol. In contrast, the levels of 10-deacetylBaccatin III in the roots remained lower than those of taxol. These results correlated with transcript accumulation of the studied genes, since in the aerial parts the expression of DBAT , which codes for the enzyme that converts 10-deacetylBaccatin III into Baccatin III, did not increase with the age of plantlets, unlike that of TXS , BAPT and DBTNBT , suggesting that this gene controls a rate-limiting step in the taxane biosynthetic pathway. The lower taxane levels found in the roots also correlated with gene expression, since only the early pathway gene TXS was induced in this organ during the 1-year growth period.
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an approach to the molecular mechanism of methyl jasmonate and vanadyl sulphate elicitation in taxus baccata cell cultures the role of txs and bapt gene expression
Biochemical Engineering Journal, 2010Co-Authors: Miriam Onrubia, Javier Palazon, Mercedes Bonfill, Elisabeth Moyano, Oscar Exposito, R. M. CusidoAbstract:Abstract Before the biotechnological production of the anticancer compound taxol can be improved, the mechanism that regulates its biosynthetic pathway needs to be further understood. In this paper we have studied the effect of methyl jasmonate (MeJ) and vanadyl sulphate (VS) on the taxane pattern and transcript profile of two key genes involved in taxol biosynthesis, txs and bapt, in a selected Taxus baccata cell line. Our results showed that MeJ significantly activated both taxol and Baccatin III production (4- and 3.6-fold, respectively), whereas VS only enhanced taxol production (also 4-fold). At the same time, MeJ treatment clearly increased the expression of the txs and bapt genes but the presence of VS in the medium only increased bapt gene expression. The results suggest that the elicitation conditions assayed are good strategies to improve taxol production, and that the elicitors have different and probably interfering mechanisms of action in taxol biosynthesis. To our knowledge, this is the first time that a relationship between the expression of the txs and bapt genes and the taxane profile has been shown in T. baccata cell cultures.
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Paclitaxel and Baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata
Biologia Plantarum, 2007Co-Authors: Mercedes Bonfill, R. M. Cusido, S. Bentebibel, E. Moyano, J. Palazón, R. Eibl, M. T. PiñolAbstract:The effects of 100 and 200 µM methyl jasmonate (MJA) on cell proliferation and paclitaxel and Baccatin III production were investigated in free and alginate immobilized cells of Taxus baccata growing in a selected product formation culture medium. The greatest accumulation of paclitaxel (13.20 mg dm^−3) and Baccatin III (4.62 mg dm^−3) occurred when 100 µM MJA was added to the culture medium of cells entrapped using a 1.5 and 2.5 % alginate solution. The effects of different treatments on the viability of cultured cells and their capacity to excrete both taxanes into the surrounding medium were considered.
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source of isopentenyl diphosphate for taxol and Baccatin III biosynthesis in cell cultures of taxus baccata
Biochemical Engineering Journal, 2007Co-Authors: R. M. Cusido, Javier Palazon, Mercedes Bonfill, Elisabet Moyano, Oscar Exposito, Teresa M PinolAbstract:Abstract To achieve a better understanding of the metabolism and accumulation of taxol and Baccatin III in cell cultures of Taxus , three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and Baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and Baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and Baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.
Torsten Binscheck - One of the best experts on this subject based on the ideXlab platform.
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Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-DeacetylBaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
2016Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten BinscheckAbstract:This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid–liquid extrac-tion under alkaline conditions followed by LC–MS–MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 3 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, Baccatin III, 10-deacetylBaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohy-droxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantita-tive evaluation revealed taxoid concentrations ranging from 4.5 to 132 mg/L (stomach content), 1 to 200 mg/L (urine), <0.5 to 12 mg/ L (cardiac blood), <0.5 to 7.3 mg/L (femoral blood), and 4.9 to 290 mg/L (bile). In brain tissue, none of these taxoids could be detected (<0.5 mg/L). In urine, after enzymatic hydrolysis, the con-centration of 3,5-dimethoxyphenol (3,5-DMP) was 23,000 mg/L. The alkaloidal diterpenoids were found in all postmortem samples. The newly developed LC–MS–MS method enables the identifica-tion of alkaloidal and non-alkaloidal diterpenoids and 3,5-dimethox-yphenol in human body fluids and tissues for the confirmation of accidental or intentional poisonings with yew plant material
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Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-DeacetylBaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
Journal of Analytical Toxicology, 2012Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten BinscheckAbstract:This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid-liquid extraction under alkaline conditions followed by LC-MS-MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 × 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, Baccatin III, 10-deacetylBaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohydroxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantitative evaluation revealed taxoid concentrations ranging from 4.5 to 132 µg/L (stomach content), 1 to 200 µg/L (urine), Language: en
