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Robert Verpoorte - One of the best experts on this subject based on the ideXlab platform.
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Proteome analysis of the medicinal plant Catharanthus roseus
Planta, 2005Co-Authors: Denise I. Jacobs, Robert Van Der Heijden, Marco Gaspari, Jan Van Der Greef, Robert VerpoorteAbstract:A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus . The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids Strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of Strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of Strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.
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effects of alkaloid precursor feeding on a camptotheca acuminata cell line
Plant Physiology and Biochemistry, 2002Co-Authors: Andrea Silvestrini, Barbara Monacelli, Gabriella Pasqua, Bruno Botta, Robert Van Der Heijden, Robert Verpoorte, Robert VerpoorteAbstract:Abstract To better understand the biosynthesis of Camptotheca acuminata alkaloids, the effect on camptothecin production of feeding with potential precursors of biosynthesis was studied (i.e., tryptamine and loganin combined, secologanin, and Strictosidine). Two key enzymes in alkaloid biosynthesis 〚i.e., tryptophan decarboxylase (TDC; EC 4.1.1.28) and Strictosidine synthase (STR; EC 4.3.3.2)〛 were also studied. The analyses were conducted using a C. acuminata CG1 cell line that does not produce alkaloids, which could be useful in better understanding the biosynthetic pathway and in identifying possible limiting factors. The activity of TDC was 5 pkat mg–1; the activity of STR was 1.1 pkat mg–1. Feeding with Strictosidine revealed that this precursor is easily biotransformed by two enzymes (i.e., a hydroxylase and a dehydrogenase) in hydroxyStrictosidine and didehydroStrictosidine, but camptothecin was never detected. The indole pathway and the low level of STR activity could be limiting factors in the production of camptothecin in the cell line used.
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Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin
Plant Cell Tissue and Organ Culture, 2002Co-Authors: E. Lucumi, Robert Verpoorte, D. Hallard, R. Van Der Heijden, A. Vera, Robert VerpoorteAbstract:A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but Strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, Strictosidine, 10-hydroxy Strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied Microbiology and Biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Robert Verpoorte, Johan Memelink, R. Van Der Heijden, F J Redondo, Robert VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, R. Van Der Heijden, M. M. L. Ibañez, Robert VerpoorteAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting Strictosidine to cathenamine. The biosynthetic pathway toward Strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of Strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
Johan Memelink - One of the best experts on this subject based on the ideXlab platform.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied Microbiology and Biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Robert Verpoorte, Johan Memelink, R. Van Der Heijden, F J Redondo, Robert VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied microbiology and biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Johan Memelink, R. Van Der Heijden, F J Redondo, R VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, R. Van Der Heijden, M. M. L. Ibañez, Robert VerpoorteAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting Strictosidine to cathenamine. The biosynthetic pathway toward Strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of Strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
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An assay for secologanin in plant tissues based on enzymatic conversion into Strictosidine
Phytochemical Analysis, 1998Co-Authors: D. Hallard, Soren Rosendal Jensen, Adriana Contin, Robert Van Der Heijden, Robert Verpoorte, Emilia M. Tomas Jiménéz, Wim Snoeijer, M. Inês Lopes Cardoso, Giancarlo Pasquali, Robert Verpoorte, Johan MemelinkAbstract:The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and in cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding Strictosidine, in a reaction catalysed by the enzyme Strictosidine synthase (STR; E.C. 4.3.3.2). Subsequently, the formation of Strictosidine is quantified by high performance liquid chromatography (HPLC). STR was isolated from transgenic Nicotiana tabacum cells expressing a cDNA-derived gene coding for STR from Catharanthus roseus. The high specificity of STR for secologanin, in combination with a sensitive and selective HPLC system, allows a simple extraction of secologanin from plant tissue. The detection limit of this method is 15 ng secologanin. Using this assay, secologanin contents were determined in tissues of various plant species; Lonicera xylosteum hairy roots were found to contain 1% of secologanin on a dry weight basis. # 1998 John Wiley & Sons, Ltd. Phytochem. Anal. 9, 162‐167, 1998
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Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and Strictosidine synthase cDNAs from Catharanthus roseus produce Strictosidine upon secologanin feeding
Plant cell reports, 1997Co-Authors: D. Hallard, Robert Verpoorte, Giancarlo Pasquali, Johan Memelink, R. Van Der Heijden, M. I. Lopes Cardoso, Robert Verpoorte, J. H. C. HogeAbstract:A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and Strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher Strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced Strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of Strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No Strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and Strictosidine release by the cells.
Arjan Geerlings - One of the best experts on this subject based on the ideXlab platform.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied Microbiology and Biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Robert Verpoorte, Johan Memelink, R. Van Der Heijden, F J Redondo, Robert VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied microbiology and biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Johan Memelink, R. Van Der Heijden, F J Redondo, R VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, R. Van Der Heijden, M. M. L. Ibañez, Robert VerpoorteAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting Strictosidine to cathenamine. The biosynthetic pathway toward Strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of Strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
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New Strictosidine β-glucosidase from Strychnos mellodora
Plant Physiology and Biochemistry, 2000Co-Authors: Viviane Brandt, Robert Van Der Heijden, Arjan Geerlings, Robert Verpoorte, Monique Tits, Clément Delaude, Luc AngenotAbstract:Abstract A stable Strictosidine glucosidase was isolated from dried powdered material of Strychnos mellodora . The kinetic parameters K m and V max of the purified glucosidases from Catharanthus roseus and S. mellodora towards Strictosidine, dolichantoside and palicoside were determined and compared.
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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 roseus.
Plant cell reports, 1999Co-Authors: Arjan Geerlings, D. Hallard, R. Van Der Heijden, A. Martinez Caballero, I. Lopes Cardoso, Robert VerpoorteAbstract: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 μg g–1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 μg 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.
R. Van Der Heijden - One of the best experts on this subject based on the ideXlab platform.
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Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin
Plant Cell Tissue and Organ Culture, 2002Co-Authors: E. Lucumi, Robert Verpoorte, D. Hallard, R. Van Der Heijden, A. Vera, Robert VerpoorteAbstract:A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but Strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, Strictosidine, 10-hydroxy Strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied Microbiology and Biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Robert Verpoorte, Johan Memelink, R. Van Der Heijden, F J Redondo, Robert VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied microbiology and biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Johan Memelink, R. Van Der Heijden, F J Redondo, R VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, R. Van Der Heijden, M. M. L. Ibañez, Robert VerpoorteAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting Strictosidine to cathenamine. The biosynthetic pathway toward Strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of Strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
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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 roseus.
Plant cell reports, 1999Co-Authors: Arjan Geerlings, D. Hallard, R. Van Der Heijden, A. Martinez Caballero, I. Lopes Cardoso, Robert VerpoorteAbstract: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 μg g–1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 μg 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.
Robert Verpoorte - One of the best experts on this subject based on the ideXlab platform.
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Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin
Plant Cell Tissue and Organ Culture, 2002Co-Authors: E. Lucumi, Robert Verpoorte, D. Hallard, R. Van Der Heijden, A. Vera, Robert VerpoorteAbstract:A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but Strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, Strictosidine, 10-hydroxy Strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.
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Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
Applied Microbiology and Biotechnology, 2001Co-Authors: Arjan Geerlings, Adriana Contin, Robert Verpoorte, Johan Memelink, R. Van Der Heijden, F J Redondo, Robert VerpoorteAbstract:A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for Strictosidine synthase (STR) and Strictosidine β-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of Strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells Strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the Strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
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An assay for secologanin in plant tissues based on enzymatic conversion into Strictosidine
Phytochemical Analysis, 1998Co-Authors: D. Hallard, Soren Rosendal Jensen, Adriana Contin, Robert Van Der Heijden, Robert Verpoorte, Emilia M. Tomas Jiménéz, Wim Snoeijer, M. Inês Lopes Cardoso, Giancarlo Pasquali, Robert Verpoorte, Johan MemelinkAbstract:The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and in cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding Strictosidine, in a reaction catalysed by the enzyme Strictosidine synthase (STR; E.C. 4.3.3.2). Subsequently, the formation of Strictosidine is quantified by high performance liquid chromatography (HPLC). STR was isolated from transgenic Nicotiana tabacum cells expressing a cDNA-derived gene coding for STR from Catharanthus roseus. The high specificity of STR for secologanin, in combination with a sensitive and selective HPLC system, allows a simple extraction of secologanin from plant tissue. The detection limit of this method is 15 ng secologanin. Using this assay, secologanin contents were determined in tissues of various plant species; Lonicera xylosteum hairy roots were found to contain 1% of secologanin on a dry weight basis. # 1998 John Wiley & Sons, Ltd. Phytochem. Anal. 9, 162‐167, 1998
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The inoculum size triggers tryptamine or secologanin biosynthesis in a Catharanthus roseus cell culture
Plant Science, 1998Co-Authors: Adriana Contin, Robert Van Der Heijden, Robert Verpoorte, Hens J.g. Ten Hoopen, Robert VerpoorteAbstract:Abstract A Catharanthus roseus cell line was characterized for the accumulation of secologanin, tryptophan, tryptamine, Strictosidine and ajmalicine, as well as for the activities of the enzymes geraniol 10-hydroxylase, tryptophan decarboxylase and Strictosidine synthase. The effects of the inoculum size on these parameters were discussed. The increase of the inoculum size from 40–160 g of fresh weight per liter of medium favored the accumulation of secologanin and alkaloids. A coordinated regulation of geraniol 10-hydroxylase and tryptophan decarboxylase by inoculum size seems to contribute to the effects observed.
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Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and Strictosidine synthase cDNAs from Catharanthus roseus produce Strictosidine upon secologanin feeding
Plant cell reports, 1997Co-Authors: D. Hallard, Robert Verpoorte, Giancarlo Pasquali, Johan Memelink, R. Van Der Heijden, M. I. Lopes Cardoso, Robert Verpoorte, J. H. C. HogeAbstract:A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and Strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher Strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced Strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of Strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No Strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and Strictosidine release by the cells.
