Javier Palazón

Steroidal Lactones from Withania somnifera, an Ancient Plant for Novel Medicine

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 C28-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 Alzheimers disease. The most recent attempts in biotechnological production of withanolides are also discussed.

Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants

Abstract Transformation of Nicotiana tabacum cv. Xanthi leaf sections with the pPCV002-ABC (rol genes A, B and C together under the control of their own promoter) or pPCV002-CaMVC (rol gene C alone under the control of the CaMV 35S promoter) construction present in trans-acting Agrobacterium tumefaciens vectors yielded several transgenic root lines. The two types (rolABC and rolC) of transgenic root lines were examined for their nicotine productivity in relation to growth rate and the amount of rolC gene product measured with specific antibodies. In all cases, the changes in the amount of this polypeptide were positively correlated with the capacity of the transgenic roots to grow and produce nicotine. Both capacities were greatly increased when the rolA, rolB and rolC genes were present together, which demonstrates that the activity of the three rol-gene-encoded functions is synergistic. Consistent observations were also made in the corresponding regenerated plants.

Biotechnological production of taxol and related taxoids: current state and prospects.

Taxol is one of the most effective anti-cancer drugs ever developed. The natural source of taxol is the inner bark of several Taxus species, but it accumulates at a very low concentration and with a prohibitively high cost of extraction. Another problem is that the use of inner bark for taxol production implies the destruction of yew trees. For all these reasons, the growing demand for taxol greatly exceeds the supply that can be sustained by isolation from its natural source and alternative sources of the drug are being sought. Although taxol has been prepared by total synthesis, the process is not commercially viable. Taxol can also be semisynthetically produced via the conversion of baccatin III or 10-deacethylbaccatinIII found in Taxus needles but the cost and difficulty of the extraction process of the semisynthetic precursors are also very high. The most promising approach for the sustainable production of taxol and related taxoids is provided by plant cell cultures at an industrial level. Taxol is currently being clinically used against different tumour processes but due to the difficulty of its extraction and formulation, as well as the growing demand for the compound, new taxol analogues with improved properties are being studied. In this revision we discuss current research in the design of new taxol-related compounds, the chemical structure/anti-cancer activity relationship and new formulations of the drug. We also consider the optimizing strategies to improve taxol and related taxoid production in cell cultures, as well as the current knowledge of taxol metabolism, all of which are illustrated with examples, some of them from our own research.

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.

Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories

Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM) production (the “Plant Cell Factory” concept). Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.

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

Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants

Abstract 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 aux 1 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 significant 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.