Taxol Production Research Articles

Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells exposed to low-energy ultrasound (US) and the signal role of NO in elicitation of plant defense responses and secondary metabolite accumulation. The US sonication (3.5–55.6 mW/cm 3 at 40 kHz fixed frequency) for 2 min induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 7 h after US sonication. The NO donor sodium nitroprusside (SNP) potentiated US-induced H 2O 2 production and cell death. Inhibition of nitric oxide synthase (NOS) activity by N ω -nitro- l-arginine ( l-NNA) or scavenging NO by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO) partially blocked the US-induced H 2O 2 production and cell death. Moreover, the NO inhibitors suppressed US-induced activation of phenylalanine ammonium-lyase (PAL) and accumulation of diterpenoid taxanes (Taxol and baccatin III). These results suggest that NO plays a signal role in the US-induced responses and secondary metabolism activities in the Taxus cells.

Effect of the plant peptide regulator, phytosulfokine‐α, 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.

Proteomic analysis reveals the spatial heterogeneity of immobilized Taxus cuspidata cells in support matrices

A proteomic approach was used to study the responses of Taxus cuspidata cells to local microenvironments in different zones of immobilized support matrices. Analysis of protein spots by 2-DE revealed significant differences in the abundance of 31 spots, 28 spots, and 23 spots in outer, middle, and central zone cells between the immobilized and suspended cells. Six of these proteins, identified by MALDI-TOF-MS, were involved in the regulation of carbohydrate, nitrogen, and sulfur metabolisms. Immobilization triggered an increase in taxol production of the immobilized cells in the middle and central zones compared to that of the suspended cells. A negative relation between taxol production and the mitotic index was observed in the cells in the immobilization support matrix. Cells in the outer zone had high mitotic index and low taxol production, while cells in the middle and central zones showed low mitotic index and high taxol production. The abundance of S-adenosylmethionine synthetase, which was identified as one of the differentially expressed proteins, was positively correlated to the cell division activity in the immobilized cell cultures.

Activation of ERK-like MAP kinase involved in regulating the cellular proliferation and differentiation of immobilized Taxus cuspidata cells

The roles of mitogen-activated protein kinase (MAPK) activation in immobilized Taxus cuspidata cells were investigated. Western blot analysis showed that the immobilized cultures enhanced the activation of extracellular signal-regulated kinase-like (ERK-like, approximately 46 kDa) MAPK in T. cuspidata cells in their lag and stationary growth phases, but reduced the ERK-like MAPK activation of the cells in their exponential growth phase compared with the freely suspended cultures. From outer to central zone in the immobilization support matrices, the down regulation of the ERK-like MAPK activation in the 20-day-old immobilized cells was accompanied by marked decrease in mitotic index, increases in the amount of lignified cells and Taxol (paclitaxel) yield. The MEK-specific inhibitor (PD98059) inhibited cell growth, increased the amount of lignified cells and the total Taxol content when inhibiting the ERK-like MAPK activation of T. cuspidata cells. These results suggest that the ERK-like MAPK activation may play important roles in regulation of cell proliferation and differentiation of immobilized T. cuspidata cells, which seem to be related to the Taxol production of the immobilized T. cuspidata cells.

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.

Taxol Biosynthesis and Molecular Genetics

Biosynthesis of the anticancer drug Taxol in Taxus (yew) species involves 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methyl erythritol phosphate pathway for isoprenoid precursor supply. Following the committed cyclization to the taxane skeleton, eight cytochrome P450-mediated oxygenations, three CoA-dependent acyl/aroyl transfers, an oxidation at C9, and oxetane (D-ring) formation yield the intermediate baccatin III, to which the functionally important C13-side chain is appended in five additional steps. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug, and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, Taxus suspension cells (induced for taxoid biosynthesis by methyl jasmonate) were used for feeding studies, as the foundation for cell-free enzymology and as the source of transcripts for cDNA library construction and a variety of cloning strategies. This approach has led to the elucidation of early and late pathway segments, the isolation and characterization of over half of the pathway enzymes and their corresponding genes, and the identification of candidate cDNAs for the remaining pathway steps, and it has provided many promising targets for genetically engineering more efficient biosynthetic production of Taxol and its precursors.

Spatio-Temporal Distributions of Metal Ions and Taxol of Taxus cuspidata Cells Immobilized on Polyurethane Foam

Distinct spatio-temporal variations of metal ions and Taxol production were observed for Taxus cuspidata cells immobilized on polyurethane foam. The Taxol content in the inner foam layer reached 215 microg g(-1) at day 30, which was 40-fold higher than that in the outer foam layer, and the Ca2+ and Mg2+ contents were 5.3 and 3.7 times higher, while the K+ content was 5.5 times lower. Thus higher intracellular Ca2+ and Mg2+ contents and lower intracellular K+ content may favor the Taxol biosynthesis in immobilized Taxus cuspidata.

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in Taxus chinensis cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicĂ­tor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.

Abnormal mitosis versus apoptosis of Taxus cuspidata induced by oleic acid in two-liquid-phase suspension cultures

The effects of oleic acid on abnormal mitosis and apoptosis of Taxus cuspidata cells were investigated, when oleic acid improved taxol production in two-liquid-phase suspension cultures. The results showed that the addition of oleic acid (4%, v/v) led to an increase of cell apoptosis and a decrease of mitotic activity. Moreover, the occurrence of some abnormal cells, chromosomal doubling, chromosomal bridge, binuclei cells and micronuclei further confirmed that oleic acid disrupted the normal mitosis of T. cuspidata cells in two-liquid-phase suspension cultures. And the frequency of total abnormal mitosis was three to four times than that of apoptosis. It is, thus, inferred that oleic acid-induced abnormal mitosis and consequent apoptosis. We found that the increases in abnormal mitosis, the increases in cell apoptosis and the decreases in mitotic index are closely relevant to the enhancement of taxol production of T. cuspidata in two-liquid-phase cultures.

The role of lipoxygenase in elicitor-induced taxol production in Taxus chinensis cell cultures

The role of lipoxygenase (LOX) in the elicitor-induced taxol production of Taxus chinensis cell cultures was studied. Fungal elicitor F 5 treatment effectively increased LOX activity and taxol production. The LOX activity inhibitor phenidone could inhibit induced LOX activity and taxol production, suggesting that LOX pathway might be involved in elicitor-induced taxol production. Methyl jasmonate (MJ) also activated LOX activity and taxol production. Phenidone could inhibit LOX activity and taxol production challenged by MJ, suggesting that exogenous MJ may induce taxol production via activating the intracellular LOX pathway. For better understanding the role of LOX in T. chinensis, some biochemical features of LOX were also investigated. Results showed that at least 49.18% LOX was associated with membrane and the molecular weight of LOX was 93 kDa.

Coexpression in yeast ofTaxuscytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.

Reactive Oxygen Species, Cell Growth, and Taxol Production of &lt;I&gt;Taxus cuspidata&lt;/I&gt; Cells Immobilized on Polyurethane Foam

Dynamic changes in reactive oxygen species (ROS) of Taxus cuspidata cells immobilized on polyurethane foam were investigated and the relation between ROS content and taxol production was discussed. Immobilization shortened the lag period of cell growth and moderately increased H2O2 and O2-* contents inside the microenvironment within the first 15 d. After 20 d, excessive production of H2O2 and O2-* was observed accompanied by marked increases in membrane lipid peroxidation and cell membrane permeability. The taxol content of immobilized cells was fourfold that of suspended cells at d 35. The addition of exogenous H2O2 barely affected malondialdehyde content and cell membrane permeability but led to an obvious accumulation of taxol. It is inferred that the intracellular and extracellular H2O2 inside the microenvironment might be one factor promoting taxol biosynthesis under the immobilization stress.

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.

Induction studies of methyl jasmonate and salicylic acid on taxane production in suspension cultures of Taxus chinensis var. mairei

The induction by methyl jasmonate (MJ) and salicylic acid (SA) of taxol and relevant taxane biosynthesis in suspension cultures of Taxus chinensis var. mairei was studied both theoretically and experimentally. The theoretical model shows that the apparent number of elicitor molecules binding with hypothetical receptor molecules for MJ in inducing taxol biosynthesis is about 75% lower than that for SA. The apparent binding constant between the elicitor and hypothetical receptor molecules for MJ is 10 orders of magnitude higher than that for SA. MJ increased taxol production more significantly than did SA as observed in our experiments. The induction model is able to predict induction efficiency of an elicitor. SA might apparently increase taxol production by blocking the biosynthesis pathway from baccatin III to cephalomannine, based on the observation that SA promotes cephalomannine production.

Induction studies of methyl jasmonate and salicylic acid on taxane production in suspension cultures of Taxus chinensis var. mairei

The induction by methyl jasmonate (MJ) and salicylic acid (SA) of taxol and relevant taxane biosynthesis in suspension cultures of Taxus chinensis var. mairei was studied both theoretically and experimentally. The theoretical model shows that the apparent number of elicitor molecules binding with hypothetical receptor molecules for MJ in inducing taxol biosynthesis is about 75% lower than that for SA. The apparent binding constant between the elicitor and hypothetical receptor molecules for MJ is 10 orders of magnitude higher than that for SA. MJ increased taxol production more significantly than did SA as observed in our experiments. The induction model is able to predict induction efficiency of an elicitor. SA might apparently increase taxol production by blocking the biosynthesis pathway from baccatin III to cephalomannine, based on the observation that SA promotes cephalomannine production.

Effect of Culture Medium, Elicitors, A Plant Growth Regulator and a Biogenetic Precursor on Taxol Production in Cell Suspension Cultures of Taxus cuspidata Variety Nana

Effect of Culture Medium, Elicitors, A Plant Growth Regulator and a Biogenetic Precursor on Taxol Production in Cell Suspension Cultures of Taxus cuspidata Variety Nana

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l−1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l−1.

Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells.

In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs. compared with the control, respectively. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor. Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.

Taxoid metabolism: Taxoid 14β-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14β-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14β-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5α-acetoxy-10β-hydroxy taxadiene and 5α,13α-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5α-acetoxy-10β-ol, but not the 5α,13α-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5α-acetoxy-10β,14β-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K m value for the taxoid substrate of about 50 μM. Because Taxol is unsubstituted at C14, the 14β-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13α,14β-hydroxy taxoids as new therapeutic agents.

Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound, methyl jasmonate and in situ solvent extraction.

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.