Transcript profiling of jasmonate-elicited Taxus cells reveals a ß-phenylalanine-CoA ligase.

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 ß-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. ß-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.

Synergistic effect of cyclodextrins and methyl jasmonate on taxane production in Taxus x media cell cultures.

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.

Taximin, a conserved plant-specific peptide is involved in the modulation of plant-specialized metabolism.

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.

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.

Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate.

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 8.14mg/L in control conditions to 21.48mg/L (day 12) with MeJA and 77.46mg/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.

Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate.

Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T-DNA of A. rhizogenes 9,402 and TXS, carrying both the T-DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two-stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action.

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.