M. Teresa Piñol

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene

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.

Ginsenoside production in different phenotypes of Panax ginseng transformed roots.

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.

Relation between the amount of rolC gene product and indole alkaloid accumulation in Catharanthus roseus transformed root cultures

Summary Transformed root lines of Catharanthus roseus were established after the inoculation of aseptic stem segments with Agrobacterium rhizogenes strain A4, and cultured in a hormone-free B5/2 solid medium. In these root cultures, in addition to the changes in the ethylene accumulated, the amount of rol C gene product was determined using specific antibodies in order to investigate their relation to growth rate, morphology and the indole alkaloid production of the cultured roots. Our results showed the presence, in all transformed root lines considered, of vindoline and catharanthine, the two monomer precursors for the enzymatic synthesis of the antineoplastic drugs vinblastine and vincristine. In the culture conditions of our experiment, roots with thin morphology that reached the highest rol C gene product amount showed lower growth rate and higher alkaloid production when compared with those with thick morphology and lowest rol C gene product amount. The effect of the protein encoded by this gene on ethylene production is also discussed.

Improved Paclitaxel and Baccatin III Production in Suspension Cultures of Taxus media

A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L−1) and baccatin III (2.56 mg L−1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 μg g−1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N‐benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two‐stage culture for cell suspension was carried out using a 5–l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L−1 of paclitaxel and 56.03 mg L−1 of baccatin III were obtained after 8 days of culture in the production medium.

Production of Taxol ® and baccatin III by a selected Taxus baccata callus line and its derived cell suspension culture

Abstract 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 VSO4 (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 VSO4. The elicitor compound VSO4 (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 VSO4 addition to the culture medium on Taxol® and baccatin III production. The presence of this elicitor (VSO4; 0.05 mM), increased Taxol® and baccatin III production (by a factor of 2.5 and 3.6, respectively) from 5.2 μg g−1 DW to 13.1 μg g−1 DW for Taxol® and from 4.4 μg g−1 DW to 16.0 μg 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

Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of paclitaxel and baccatin III in cell cultures of Taxus, inhibitors of the early steps in the terpenoid pathway were applied to a cell suspension culture of Taxus baccata: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway, and mevinolin as an inhibitor of the mevalonate branch. Synthesis of both taxanes in the cell suspension was first increased when cultured in the product formation medium supplemented with methyljasmonate (100 microM). The product formation medium was selected after assaying 24 different culture media. When fosmidomycin (200 microM) was added to the product formation medium together with the elicitor, the accumulation of paclitaxel and baccatin III was reduced by up to 3.0 and 1.5 times, respectively, whereas the inhibitory effect of mevinolin (1 microM) was only clearly exerted in the case of paclitaxel. Under the conditions of our experiment, we conclude that in the synthesis of both taxanes, the non-mevalonate pathway is the main source of the universal terpenoid precursor isopentenyl diphosphate (IPP).

Influence of calcium ion-concentration in the medium on tropane alkaloid accumulation in Datura stramonium hairy roots

Abstract Hairy root cultures of Datura stramonium were established after the infection of stem sections with Agrobacterium rhizogenes A4, and cultured in phytohormone free B 5 liquid medium containing different calcium concentrations in order to investigate the relationship between Ca 2+ -dependent peroxidase activity and hyoscyamine accumulation. Putrescine:SAM N -methyltransferase (PMT) activity and the levels of its mRNA were determined. Although calcium concentrations below that present in B 5 medium (1.0 mM) did not affect hairy root line growth, they significantly reduced root peroxidase activity which is probably involved in the degradation of secondary products. Lower calcium concentrations also reduced the hyoscyamine synthesis ability of the hairy root lines. This loss of hyoscyamine production was concurrent with the loss of PMT activity, which is supported by Northern blot analysis performed on the total RNA fraction using cDNA corresponding to PMT of tobacco as probe.

Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures

To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200mgL(-1)). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200mgL(-1) exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.

Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses

Panax ginseng calluses were cultured for 5 weeks on solid MS medium supplemented with kinetin 0.46 mM (0.1 mg l−1) and 2 mg l−1 of 2,4-D (9.05 mM), IBA (9.98 mM) or NAA (10.74 mM). In the conditions studied, 2,4-D inhibited the organogenic capacity of the calluses, whereas IBA or NAA increased this capacity. IBA induced the formation of a high number of buds and roots, but the roots were thin and necrotized. Calluses grown with NAA produced fewer buds and roots than those grown in IBA medium, but the roots were thick and showed good growth. The highest ginsenoside content was found in root forming calluses grown in the presence of NAA.In calluses forming roots or buds, 2,4-D, NAA and especially IBA increased the Rb group of ginsenosides rather than that of the Rg group.

Influence of elicitors on taxane production and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Taxus media cells

Abstract The present study examined and compared the influence of elicitation with arachidonic acid, methyl jasmonate and vanadyl sulfate on the accumulation of paclitaxel and related taxanes in a cell suspension of Taxus media . The yields of baccatin III, 10-deacetylpaclitaxel and paclitaxel were significantly increased in the presence of methyl jasmonate (up to 3-fold, 5-fold and 12-fold, respectively), whereas those of 10-deacetylbaccatin III and cephalomannine were significantly increased in the presence of vanadyl sulfate (up to 40-fold) and arachidonic acid (up to 4-fold), respectively. The production of these taxanes was not only affected by the nature of the elicitor tested but also by the growth stage of the cells. At the same time, although the elicitors caused a considerable decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) activity, we found that this decrease did not affect taxane accumulation.