Effect of the culture medium and biotic stimulation on taxane production in Taxus globosa Schltdl in vitro cultures

Taxus globosa is the only species of the Taxus genus that grows in Mexico. In this study, callus cultures from leaves and young shoots of T. globosa were established in Gamborg’s B5 medium supplemented with 2,4-dichlorophenoxiacetic acid (2 mg/L), kinetin (0.5 mg/L) and gibberellic acid (0.25 mg/L). Callus growth and taxane production were evaluated using two culture media: Woody Plant Medium and Gamborg’s B5 supplemented with picloram (2 mg/L), kinetin (0.1 mg/L) and gibberellic acid (0.5 mg/L). The effect of the inoculum size (50, 100 and 150 g FW/L) and culture media (Woody Plant Medium and Gamborg’s B5) with and without the presence of methyl jasmonate (100 μM) on T. globosa cell suspensions was assessed. Taxane analysis revealed that the calli in Gamborg’s B5 produced taxol (50 μg/g DW), baccatin III, 10-deacetyl baccatin III and 10-deacetyl taxol. Woody Plant Medium also induced the production of taxol, although to a lesser extent. The optimum inoculum size was 50 g FW/L. In cell suspension cultures, both media had a significant effect on taxane production when supplemented with methyl jasmonate. In Woody Plant Medium, at day 14, a total concentration of 197.999 μg/L of taxol, 160.622 μg/L of baccatin III, 633.724 μg/L of 10-deacetyl baccatin III and 229.611 μg/L 10-deacetyl taxol were obtained, with total excretion of baccatin III and 10-deacetyl taxol to the culture medium. In Gamborg’s B5, cephalomanine was obtained at a concentration of 91.428 μg/L without elicitation, and all taxanes were excreted to the medium to a variable extent.     

One step more towards taxane production through enhanced Taxus propagation

We have developed a high-yielding procedure for the in vitro propagation of juvenile material of Taxus baccata involving a combination of seed handling and culture on WP culture medium supplemented with sucrose (2%), activated charcoal (0.5%) and BAP (22.19 mM) for 30 days, followed by 40 days on hormone-free medium. Shoot apical ends should be decapitated to obtain propagation rates up to 12- to 18-fold per subculture period (70 days). In this way the high genetic variability of the juvenile material can be used in the most productive way. In addition to producing large numbers of yew plants (difficult to get by traditional methods), this procedure allows the fast screening of individuals for their taxane content. A negative correlation between growth and secondary metabolite content was found for paclitaxel. The positive correlation with 10-deacetyl baccatin III accumulation reflects once more the commercial viability of using 10-deacetyl baccatin III extraction as an alternative to taxane production, but this time opening up the possibility of selecting genotypes with both characteristics: fast growth and high productivity. Received: 12 July 1999 / Revision received: 22 November 1999 / Accepted: 22 November 1999     

Development of a culture sub-population induction model: signaling pathways synergy and taxanes production by Taxus canadensis

Cell cultures of Taxus canadensis were subjected to exogenously applied ethylene (ET) hormone and methyl jasmonate (MJ) elicitation in factorial design experiments. Levels of extracellular taxanes, including paclitaxel, were used with principal component analysis for fault detection and real-coded genetic algorithms for parameter optimization to construct a culture sub-population induction model. Culture sub-populations were identified by the model as (1) uninduced, (2) induced to unilateral function of the ET-signaling pathway, and (3) induced to cooperation between jasmonic acid (JA)- and ET-signaling pathways. Comprehensive model results suggested greater rates of cellular induction (resulting in exogenous taxane production) by ET gas as opposed to MJ elicitation. However, cellular induction of ET-signaling pathway genes increased the rate of induction of JA-signaling pathway genes by orders of magnitude. In addition, model results showed that induction of genes leading to extracellular production of the simple taxane 10-deacetylbaccatin III was regulated by the unilateral ET-signaling pathway. However, it was suggested that further processing of this simple taxane to complex taxane structures, such as paclitaxel, required further gene induction by the JA-signaling pathway. Thus, production rate constants of exogenous complex taxanes were predicted to be an order of magnitude lower than that for the simple taxane 10-deacetylbaccatin III. The fraction of the cell culture sub-population displaying unilateral ET-signaling pathway gene induction was found inversely proportional to levels of MJ elicitation. When coupled with simple non-growth product models, levels of all extracellular taxanes were effectively predicted using the culture sub-population induction model.     

Manipulation by culture mixing and elicitation of paclitaxel and baccatin III production in Taxus baccata suspension cultures

Summary Experiments were carried out with Taxus baccata cell lines showing different paclitaxel-producing capacities (between 1.74 and 19.91 mgl^−1) when growing in a selected product-formation medium that specifically stimulated the production of taxane to the detriment of cell growth. Through mixing low-, medial- and high-producing lines, it could be observed that paclitaxel productivity in the resulting mixed lines was clearly higher than the mean productivity of the individual lines before mixing. This suggests that culture components generated by high-producing individual lines within the population might induce paclitaxel production. Although the accumulation of paclitaxel and baccatin III was higher when 100 μM methyl jasmonate was added to the subcultures of the mixed lines, the results indicate that exogenously applied methyl jasmonate was not the first factor to stimulate taxane production. The possible effects of methyl jasmonate elicitation and paclitaxel accumulation on cell viability are also considered.     

Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of taxol and baccatin III in cell cultures of Taxus, three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐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 β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐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.     

Sex influences the taxanes content in Taxus baccata

Like other species of the genus Taxus, European yew trees contain taxanes, including paclitaxel (T) and its precursor 10-deacetylbaccatin III (10-DAB). Taxanes are one of the most effective anticancer drugs. This study was undertaken to describe the levels and patterns of taxane variation in the Sudetian region (SW Poland). Paclitaxel (T) and 10-deacetylbaccatin III (10-DAB) concentrations were analysed in five populations. Needles and twigs were analysed from 60 individuals (30 males and 30 females) in each population. In addition, morphometric measurements were taken in the populations to obtain light intensity coefficients (specific leaf area, SLA). High variability in the taxane contents at both intra and interpopulational levels was found. Nevertheless, females had a significantly higher taxane content compared to males. Because taxanes are carbon-based secondary metabolites, females have higher rate of gas exchange of females compared to males. This was probably an adaptation to greater reproductive effort incurred by females. In this regard, female individuals seem to be better for selecting elite cultivars with a higher taxane production. The relationship between light intensity and taxane content was not significant. Shading, important for optimizing crop production, should not reduce the concentration of taxanes.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

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. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Variation of taxane content in needles of Taxus x media cultivars with different growth characteristics

Needles from 17 different Taxus x media cultivars, belonging to 4 groups showing different growth characteristics, were analyzed using high performance liquid chromatography for their content of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel (Taxol). The 4 Taxus x media cultivar groups were: 1.) medium to fast growing and upright form; 2.) slow growing and upright form; 3.) fast growing and spreading form; and 4.) slow growing and spreading form. The purpose of this study was to identify yew cultivars of fast growth rate, upright growth and high taxane content in their needles. The highest content of paclitaxel was found in 'Coleana' of group 1 (378 microg/g of the extracted dry weight). Three cultivars in group 1, 'Coleana', 'Stovekenii' and 'Hicksii', make good candidates for taxane extraction because of their high paclitaxel and 10-deacetylbaccatin III content, fast biomass accumulation and upright growing form. They are also good starting materials to develop alternative methods for the production of paclitaxel and its analogous compounds through modern biotechnology approaches.     

Effect of the plant peptide regulator, phytosulfokine-alpha, on the growth and Taxol production from Taxus sp. suspension cultures

Phytosulfokine-alpha (PSK-alpha) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-alpha was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-alpha supplementation on production of Taxol (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-alpha supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-alpha supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-alpha added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-alpha (100 nM) and methyl jasmonate (MeJA, 100 microM) elicitation was observed, resulting in Taxol level of 35.3 +/- 2.1 mg/L or 1.83 +/- 0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture.     

Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures

The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.     

Taxane diterpenoids from the seeds of Chinese yew Taxus chinensis

The taxoid chinentaxunine has been isolated from the seeds of Chinese yew Taxus chinensis, and its structure determined on the basis of spectral and chemical methods. In addition, the known taxol C, paclitaxel, 10-deacetyl taxol A, 10-deacetyl-7-epitaxol, 10-deacetyl-10-oxo-7-epi-taxol, taxinine M, taxchinin A, 10-deacetyl taxinine B and taxuspine X were also isolated and identified from this source.     

Screening of the needles of different yew species and cultivars for paclitaxel and related taxoids

The needles of several yew species and cultivars were analysed by high-pressure liquid chromatography for paclitaxel, 10-deacetylpaclitaxel, cephalomannine, baccatin III, 10-deacetylbaccatin III and brevifoliol. About 750 samples were collected from five different locations in the Netherlands and the UK. The results of this screening show a large variation in taxane content between the different species and cultivars. The content of paclitaxel and 10-deacetylbaccatin III varied from 0 to 500 micrograms/g and 0 to 4800 micrograms/g dried needles, respectively. Brevifoliol was found in a very high concentration in Taxus brevifolia. 10-Deacetylpaclitaxel, cephalomannine and baccatin III were found in concentrations ranging from 0 to 500 micrograms/g dried needles.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.