The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), ... more The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.
In Vitro Cellular & Developmental Biology - Plant, 2015
ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerabl... more ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerable interest, particularly for in vitro cell cultures. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves, and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability, and costs. Statistical analysis (ANOVA) revealed that 1-naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol, and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold-temperature-based method to maintain a stock of calli using half-strength Murashige and Skoog solid medium, concluding that up to 5 mo at 4°C is optimal to ensure white friable calli upon regrowth at 25°C.
Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plan... more Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert b-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. b-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.
We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl... more We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the
Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant... more Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures.
Small peptides play important roles in the signalling cascades that steer plant growth, developme... more Small peptides play important roles in the signalling cascades that steer plant growth, development and defence, and often crosstalk with hormonal signalling. Thereby, they also modulate metabolism, including the production of bioactive molecules that are of high interest for human applications. Yew species (Taxus spp.) produce diterpenes such as the powerful anticancer agent paclitaxel, the biosynthesis of which can be stimulated by the hormone jasmonate, both in whole plants and cell suspension cultures. Here, we identified Taximin, as a gene encoding a hitherto unreported, plant-specific, small, cysteine-rich signalling peptide, through a transcriptome survey of jasmonate-elicited T. baccata suspension cells grown in two-media cultures. Taximin expression increased in a coordinated manner with that of paclitaxel biosynthesis genes. Tagged Taximin peptides were shown to enter the secretory system and localize to the plasma membrane. In agreement with this, the exogenous application of synthetic Taximin peptide variants could transiently modulate the biosynthesis of taxanes in T. baccata cell suspension cultures. Importantly, the Taximin peptide is widely conserved in the higher plant kingdom with a high degree of sequence conservation. Accordingly, Taximin overexpression could stimulate the production of nicotinic alkaloids in Nicotiana tabacum hairy root cultures in a synergistic manner with jasmonates. In contrast, no pronounced effects of Taximin overexpression on the specialized metabolism in Medicago truncatula roots were observed. This study increases our understanding of the regulation of Taxus diterpene biosynthesis in particular and plant metabolism in general. Ultimately, Taximin might increase the practical potential of metabolic engineering of medicinal plants.
The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its pro... more 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 11.54 gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28 gDCW/L and 5.69 gDCW/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 (8583.3 μg/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 6246.1 μg/L, a 10-fold increase compared with shake flask experiments.
Plant cell factories constitute an alternative source of high added value phytochemicals such as ... more Plant cell factories constitute an alternative source of high added value phytochemicals such as the anticancer drug taxol (generic name paclitaxel), biosynthesized in Taxus spp. The growing demand for taxol and its derivatives, due to a specific action mechanism and the scarcity of the taxane ring in nature, has made this group of compounds one of the most interesting targets for biotechnological production. This review is focused on recent advances in the production of taxol and related taxanes in Taxus baccata, the taxol-producing European yew, using cell suspension culture technology. The review contains a brief description of the botany and phytochemistry of T. baccata, as well as the chemical structure of taxol and the molecular requirements for its anticancer effects. After a short overview of taxol production at an industrial level, the review focuses on taxol biosynthesis in plant cells and the attempts to produce taxol in T. baccata cell cultures, giving particular emphasis to the optimization steps that have improved production, and including the most recently developed new tools. Finally, the future prospects for the biotechnological production of taxol are also discussed.
We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the eff... more We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.
Small callus pieces excised from a selected 1-year-old stable callus line of Taxus baccata were g... more Small callus pieces excised from a selected 1-year-old stable callus line of Taxus baccata were grown on B5 solid medium supplemented with either phenylalanine (1 mM) or the elicitor compound VSO 4 (0.05 or 0.1 mM). Under these conditions, callus growth and the production of Taxol ® and baccatin III were tested during 8 weeks of culture. Growth was enhanced by the presence of both phenylalanine and VSO 4 . The elicitor compound VSO 4 (0.05 and 0.1 mM) moreover clearly increased Taxol ® and baccatin III production, especially at the end of the culture. The specific activity of PAL in callus pieces cultured under the different conditions studied, is also considered. In addition, liquid cultures were prepared from the selected callus line in order to study the effect of the VSO 4 addition to the culture medium on Taxol ® and baccatin III production. The presence of this elicitor (VSO 4 ; 0.05 mM), increased Taxol ® and baccatin III production (by a factor of 2.5 and 3.6, respectively) from 5.2 mg g -1 DW to 13.1 mg g -1 DW for Taxol ® and from 4.4 mg g -1 DW to 16.0 mg g -1 DW for baccatin III in the T. baccata cell suspension cultures. It also increased their release into the medium by a factor of 3.2 and 2.2, respectively
Plant Cell, Tissue and Organ Culture (PCTOC), 2011
Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammat... more Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammatory properties. Its bioactive compounds are ursane-type triterpene saponins known as centellosides. With the aim of increasing the biotechnological production of these compounds, C. asiatica cell suspensions were established and treated with two concentrations (100 and 200 lM) of methyl jasmonate (MeJA). The maximum centelloside production was observed in the stationary growth phase, reaching 0.16 mg g-1 dry weight (DW) at day 25 of the culture in the control and 1.11 mg-1 g DW at day 15 in the MeJA-elicited cultures. The elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. Reverse transcription polymerase chain reaction (RT-PCR) analysis of the b-amyrin synthase gene (CabAS, the specific oxidosqualene cyclase that leads to centelloside formation) showed higher levels of expression in the elicited cultures than in the control. The maximum content of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis. In the cultures elicited with 200 lM MeJA, the centelloside production did not increase compared to the control. Both elicitor concentrations decreased the content of phytosterols. Thus, MeJa elicitation in this type of culture was dose-dependent and its inducing role was apparent at low concentrations. Keywords Asiaticoside Á Cell suspension cultures Á Centella asiatica Á Centellosides Á Madecassoside Á Methyl jasmonate
Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with ... more Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes, induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux1 gene of A. rhizogenes was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 25±60% of the root cultures established showing typical hairy morphology. This fact suggests a signi®cant role of aux genes in the morphology of transformed roots. Inoculation of leaf sections with A. tumefaciens strain C58 C1 carrying the pRiA4TR-(deletion of aux genes) did not produce roots with callus-like morphology.
Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to... more Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.
Withania somnifera, commonly known as Ashwagandha, is an important medicinal plant that has been ... more Withania somnifera, commonly known as Ashwagandha, is an important medicinal plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years. In view of its varied therapeutic potential, it has also been the subject of considerable modern scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C 28 -steroidal lactone triterpenoids built on an intact or rearranged ergostane framework, in which C-22 and C-26 are appropriately oxidized to form a six-membered lactone ring. In recent years, numerous pharmacological investigations have been carried out into the components of W. somnifera extracts. We present here an overview of the chemical structures of triterpenoid components and their biological activity, focusing on two novel activities, tumor inhibition and antiangiogenic properties of withaferin A and the effects of withanolide A on Alzheimer's disease. The most recent attempts in biotechnological production of withanolides are also discussed.
Hyoscyamine-6b-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopola... more Hyoscyamine-6b-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.
Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received m... more Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA-or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.
The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), ... more The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.
In Vitro Cellular & Developmental Biology - Plant, 2015
ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerabl... more ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerable interest, particularly for in vitro cell cultures. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves, and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability, and costs. Statistical analysis (ANOVA) revealed that 1-naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol, and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold-temperature-based method to maintain a stock of calli using half-strength Murashige and Skoog solid medium, concluding that up to 5 mo at 4°C is optimal to ensure white friable calli upon regrowth at 25°C.
Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plan... more Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert b-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. b-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.
We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl... more We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the
Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant... more Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures.
Small peptides play important roles in the signalling cascades that steer plant growth, developme... more Small peptides play important roles in the signalling cascades that steer plant growth, development and defence, and often crosstalk with hormonal signalling. Thereby, they also modulate metabolism, including the production of bioactive molecules that are of high interest for human applications. Yew species (Taxus spp.) produce diterpenes such as the powerful anticancer agent paclitaxel, the biosynthesis of which can be stimulated by the hormone jasmonate, both in whole plants and cell suspension cultures. Here, we identified Taximin, as a gene encoding a hitherto unreported, plant-specific, small, cysteine-rich signalling peptide, through a transcriptome survey of jasmonate-elicited T. baccata suspension cells grown in two-media cultures. Taximin expression increased in a coordinated manner with that of paclitaxel biosynthesis genes. Tagged Taximin peptides were shown to enter the secretory system and localize to the plasma membrane. In agreement with this, the exogenous application of synthetic Taximin peptide variants could transiently modulate the biosynthesis of taxanes in T. baccata cell suspension cultures. Importantly, the Taximin peptide is widely conserved in the higher plant kingdom with a high degree of sequence conservation. Accordingly, Taximin overexpression could stimulate the production of nicotinic alkaloids in Nicotiana tabacum hairy root cultures in a synergistic manner with jasmonates. In contrast, no pronounced effects of Taximin overexpression on the specialized metabolism in Medicago truncatula roots were observed. This study increases our understanding of the regulation of Taxus diterpene biosynthesis in particular and plant metabolism in general. Ultimately, Taximin might increase the practical potential of metabolic engineering of medicinal plants.
The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its pro... more 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 11.54 gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28 gDCW/L and 5.69 gDCW/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 (8583.3 μg/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 6246.1 μg/L, a 10-fold increase compared with shake flask experiments.
Plant cell factories constitute an alternative source of high added value phytochemicals such as ... more Plant cell factories constitute an alternative source of high added value phytochemicals such as the anticancer drug taxol (generic name paclitaxel), biosynthesized in Taxus spp. The growing demand for taxol and its derivatives, due to a specific action mechanism and the scarcity of the taxane ring in nature, has made this group of compounds one of the most interesting targets for biotechnological production. This review is focused on recent advances in the production of taxol and related taxanes in Taxus baccata, the taxol-producing European yew, using cell suspension culture technology. The review contains a brief description of the botany and phytochemistry of T. baccata, as well as the chemical structure of taxol and the molecular requirements for its anticancer effects. After a short overview of taxol production at an industrial level, the review focuses on taxol biosynthesis in plant cells and the attempts to produce taxol in T. baccata cell cultures, giving particular emphasis to the optimization steps that have improved production, and including the most recently developed new tools. Finally, the future prospects for the biotechnological production of taxol are also discussed.
We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the eff... more We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.
Small callus pieces excised from a selected 1-year-old stable callus line of Taxus baccata were g... more Small callus pieces excised from a selected 1-year-old stable callus line of Taxus baccata were grown on B5 solid medium supplemented with either phenylalanine (1 mM) or the elicitor compound VSO 4 (0.05 or 0.1 mM). Under these conditions, callus growth and the production of Taxol ® and baccatin III were tested during 8 weeks of culture. Growth was enhanced by the presence of both phenylalanine and VSO 4 . The elicitor compound VSO 4 (0.05 and 0.1 mM) moreover clearly increased Taxol ® and baccatin III production, especially at the end of the culture. The specific activity of PAL in callus pieces cultured under the different conditions studied, is also considered. In addition, liquid cultures were prepared from the selected callus line in order to study the effect of the VSO 4 addition to the culture medium on Taxol ® and baccatin III production. The presence of this elicitor (VSO 4 ; 0.05 mM), increased Taxol ® and baccatin III production (by a factor of 2.5 and 3.6, respectively) from 5.2 mg g -1 DW to 13.1 mg g -1 DW for Taxol ® and from 4.4 mg g -1 DW to 16.0 mg g -1 DW for baccatin III in the T. baccata cell suspension cultures. It also increased their release into the medium by a factor of 3.2 and 2.2, respectively
Plant Cell, Tissue and Organ Culture (PCTOC), 2011
Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammat... more Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammatory properties. Its bioactive compounds are ursane-type triterpene saponins known as centellosides. With the aim of increasing the biotechnological production of these compounds, C. asiatica cell suspensions were established and treated with two concentrations (100 and 200 lM) of methyl jasmonate (MeJA). The maximum centelloside production was observed in the stationary growth phase, reaching 0.16 mg g-1 dry weight (DW) at day 25 of the culture in the control and 1.11 mg-1 g DW at day 15 in the MeJA-elicited cultures. The elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. Reverse transcription polymerase chain reaction (RT-PCR) analysis of the b-amyrin synthase gene (CabAS, the specific oxidosqualene cyclase that leads to centelloside formation) showed higher levels of expression in the elicited cultures than in the control. The maximum content of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis. In the cultures elicited with 200 lM MeJA, the centelloside production did not increase compared to the control. Both elicitor concentrations decreased the content of phytosterols. Thus, MeJa elicitation in this type of culture was dose-dependent and its inducing role was apparent at low concentrations. Keywords Asiaticoside Á Cell suspension cultures Á Centella asiatica Á Centellosides Á Madecassoside Á Methyl jasmonate
Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with ... more Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes, induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux1 gene of A. rhizogenes was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 25±60% of the root cultures established showing typical hairy morphology. This fact suggests a signi®cant role of aux genes in the morphology of transformed roots. Inoculation of leaf sections with A. tumefaciens strain C58 C1 carrying the pRiA4TR-(deletion of aux genes) did not produce roots with callus-like morphology.
Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to... more Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.
Withania somnifera, commonly known as Ashwagandha, is an important medicinal plant that has been ... more Withania somnifera, commonly known as Ashwagandha, is an important medicinal plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years. In view of its varied therapeutic potential, it has also been the subject of considerable modern scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C 28 -steroidal lactone triterpenoids built on an intact or rearranged ergostane framework, in which C-22 and C-26 are appropriately oxidized to form a six-membered lactone ring. In recent years, numerous pharmacological investigations have been carried out into the components of W. somnifera extracts. We present here an overview of the chemical structures of triterpenoid components and their biological activity, focusing on two novel activities, tumor inhibition and antiangiogenic properties of withaferin A and the effects of withanolide A on Alzheimer's disease. The most recent attempts in biotechnological production of withanolides are also discussed.
Hyoscyamine-6b-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopola... more Hyoscyamine-6b-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.
Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received m... more Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14 mg/L in control conditions to 21.48 mg/L (day 12) with MeJA and 77.46 mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA-or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.
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