Taxol Production Research Articles

Kinetics of taxol production and nutrient use in suspension cultures of Taxus cuspidata in shake flasks and a Wilson-type bioreactor

Suspension cultures of Taxus cuspidata were grown in shake flasks and Wilson-type reactors where bubbled air provided both agitation and mixing. Taxol titers of 22 mg l −1 were achieved for both configurations in 20 days for a volumetric productivity of 1.1 mg l −1 day −1. This productivity is many-fold higher than reported for other Taxus sp. suspension cultures. Taxol was released to the extracellular medium as it was produced with little intracellular retention (⩽10%). Taxol production occurred during the last seven days of the cultivation period and was not growth-associated. Although the same taxol titers could be obtained in both reactor types, nutrient uptake rates were faster in the Wilson-type reactor than in shake flasks. Formation of a growth ring in the Wilson-type reactor reduced measured cell mass yields.

Taxol partitioning in two-phase plant cell cultures of Taxus brevifolia

An accumulation phase for taxanes was evaluated for its effect on growth and production kinetics in Taxus brevifolia cell cultures. The second phase, tricaprylin, is an eight-carbon triglyceride that favorably partitions taxol secreted into the medium. Cell growth was generally unaffected by the presence of tricaprylin and a moderate increase in taxol synthesis over a 4-week period was observed. Adjustments of the medium pH can be used to obtain significantly higher partitioning (at higher pH), but this adversely affects the cell productivity with a net loss in taxol production for pH values greater than 7.5. Nevertheless, in situ liquid partitioning provides a clean reservoir for taxane separation in otherwise normal cell culture that can also be easily sampled and replaced during a semi-batch or continuous culture.

Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models.

Suspension cultures of Taxus cuspidata were challenged with various concentrations and combinations of methyl jasmonate and ethylene. Taxol productivity increased 19-fold when T. cuspidata suspension cultures were exposed to 5 ppm ethylene and 10 microM methyl jasmonate. This increase was 15-fold when either 0 or 10 ppm ethylene was combined with 10 microM methyl jasmonate. The induction of taxol occurred within 51 h after elicitation and would reduce fermentation times and costs. Ethylene concentration at 50 ppm had an inhibitory effect on taxol production but not on phosphate uptake rate, suggesting independent regulation of taxol and physiological functions of the cell. A simple induction model is proposed to explain the action and effects of both ethylene and methyl jasmonate with regard to receptor binding and regulatory systems in plants.

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1−1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1−1) and low (<0.1 mg 1−1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1−1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1−1.

Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana.

Pestalotiopsis microspora was isolated from the inner bark of a small limb of Himalayan yew, Taxus wallachiana, and was shown to produce taxol in mycelial culture. Taxol was identified by spectroscopic and chromatographic comparisons with authentic taxol. Optimal taxol production occurred after 2-3 weeks in still culture at 23 degrees C. [14C]Acetate and [14C]phenylalanine served as precursors for fungal [14C]taxol. These observations on P. microspora are discussed in relation to the biological importance of taxol production by fungi in general.

Taxol Production in Nodule Cultures of Taxus

The in vitro synthesis of secondary compounds from plants is one source of scarce and valuable phytopharmaceuticals. Often, some level of cellular or tissue differentiation is needed for the biosynthesis of many of these important compounds. Nodule cultures, consisting of cohesive multicellular units displaying a high degree of differentiation, were initiated from cultured needles of seven Taxus cultivars (Taxus cuspidata, Taxus x media 'Hicksii', Taxus x hunnewelliana 'Richard Horsey', Taxus x media 'Dark Green Spreader', Taxus x media 'L. C. Bobbick', and Taxus brevifolia). Under normal semicontinuous perfusion culture conditions (bimonthly refreshments to yield 0.2% sucrose), only trace amounts of taxol were detected from Taxus nodule cultures. However, with an elevated sucrose level (0.5% or 1.0%), taxol production was enhanced in T. cuspidata nodules to approximately 12 micrograms taxol/g nodule dry weight (dw). Stimulation of taxol production by elevated sucrose levels occurred even in the absence of other nutrients. The effect of increased sucrose on taxol induction does not appear to be due to an osmotic effect in the medium, suggesting that the increase in taxol production may be correlated with a metabolic process within the nodules. Although sucrose had a significant effect on taxol production, taxane precursors or elicitors of terpenes, as well as other plant secondary metabolites, had no effect on the production of taxol from these cultures. In addition to taxol, the higher sucrose levels also induced the production of 7-epi-10-deacetyltaxol, cephalomannine, and 7-epi-10-deacetylcephalomannine, so that total content of these taxanes equaled approximately 39 micrograms taxane/g dw nodules.

Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures.

Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95). The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase. This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine. The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.

Taxol and taxane formation in plant cell culture

The production of taxol, cephalomannine and baccatin III from a suspension cell culture of Taxus brevifolia has been investigated using HPLC and tandem mass spectrometry (MS/MS). Interference from other products in the cell culture medium led to development of an MS/MS assay based on negative ion desorption chemical ionization using [2H3]-labelled internal standards for measurements of these compounds at the level of 2–8 ng cm–3 of medium. The use of parent scans allowed the detection of a number of new taxanes for further study.

Elicitation of Taxus sp. cell cultures for production of taxol

The addition of cell extracts and cultures filtrate of Pencillium minioluteum, Botrytis cinerea, Verticillium dahliae, and Gilocladium deliqucescens on the tenth day after transferring Taxus sp. (RO1-M28) cell suspensions into an induction medium, further improved the production of Taxol Taxol is a registered trademark of Bristol Myers Squibb for paclitaxel. and total taxanes. Arachidonic acid (1mg/L) addition at the time of inoculation increased Taxol production by 150%. Oxidative stress induction and copper sulphate or sodium orthovanadate addition had no effect on Taxol production. Three categories of elicitors; those specifically stimulating Taxol production, those specifically stimulating the producion of other taxanes, and those stimulating taxane production uniformly, could be identified. The biosynthetic site of action of these elicitors is currently not known.

Gas phase composition effects on suspension cultures of Taxus cuspidata

The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. (c) 1995 John Wiley & Sons, Inc.

Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.

Production of taxol and related taxanes in Taxus brevifolia cell cultures: Effect of sugar

Suspension cells of Taxus brevifolia were cultured as an alternative source of taxol and related taxanes. The effects of basal medium, sugar type, and sugar concentration on growth and taxane production were investigated. To induce taxol production, modification of medium composition was necessary. Fructose supported the best taxol yield and the use of high concentration of sugar was desirable. The maximum level of taxol obtained after 10 days of culture in an optimized condition was 1.43 mg/L, which was 0.013% as a specific content.

Continuous production of taxol by cell culture of taxus cuspidata.

Continuous production of taxol by cell culture of taxus cuspidata.

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.

Effects of partial bark removal on the growth of Pacific yew

Whole-tree harvest of Pacific yew (Taxusbrevifolia Nutt.) to provide bark for production of the new anticancer drug taxol may adversely affect stand structure where yews provide thermal cover, browse, or riparian benefits. Harvesting only a portion of the bark on standing trees would maintain existing stand structure if the affected trees continued to grow, but partial bark removal seldom has been applied because its long-term effects are unknown. We measured 121 yews that had been scarred by windthrow or logging damage 3–92 years ago and found that their growth did not differ significantly from the growth of nearby unscarred yews. Radial growth of the scarred trees was strongly correlated with growth before scarring; it was slightly associated with overstory canopy density, elevation, and the amount of bark removed. Partial bark removal from one side of the tree probably will not seriously affect the growth of Pacific yew if less than 50% of the bark is removed.

Large-scale insect and plant cell culture.

Currently, insect and plant cell cultures are not widely used to make products of commercial interest, largely because the development of large-scale cultivation methods is still in its infancy. With the advances made over the past year, some of the limitations associated with scale-up of these two types of expression system have been addressed. Increasing the oxygen supply and the concentration of various nutrients supplied to insect cells after infection has enabled high specific protein production to be maintained to higher cell densities than ever before, improving overall volumetric yields. Detailed work has focused on the capacity of insect cells to carry out complex post-translational modifications; however, as yet, evidence is conflicting as to the extent of protein processing and complex glycosylation possible in infected cells. In plant cell culture, the accepted axioms concerning large-scale culture have been re-examined. Recent studies have assessed culture at high cell densities and the constraints in reactor design resulting from the 'shear sensitivity' of plant cells. Results show that, as cell densities increase, alterations occur in the pathways of secondary metabolism, leading to decreases in specific productivity. The use of nutrient supplements and a medium cycling strategy shows promise for increasing and sustaining product formation. Furthermore, the importance of dissolved gas composition has been clearly demonstrated by use of a gas recirculation reactor. Reports of taxol and vindoline production in vitro demonstrate the potential and the necessity for further research in scale-up of plant cell culture.

Total Synthesis of Anticancer Agent Taxol Achieved by Two Different Routes

After years of ardent efforts by synthetic organic chemists to synthesize the anticancer drug taxol from simple starting materials, two groups have accomplished the feat within six weeks of each other. Taxol, originally isolated from the Pacific yew tree in the early 1960s, was recently approved by the Food & Drug Administration for use against ovarian cancer and has also shown activity against breast, lung, and other cancers. The drug was for a time a rare commodity, available only by extraction from yew bark. Bristol-Myers Squibb, which markets taxol, now intends to produce it by partial synthesis from 10-deacetylbaccatin III, a precursor obtained from yew needles and twigs. The company believes this semisynthetic process, developed at Florida State University, will eliminate the taxol shortage. Neither of the two new syntheses will be of practical use for commercial production of taxol. However, they may enable researchers to devise more ...

The stimulation of taxol production in Taxus brevifolia by various growth retardants

We have shown that chlorocholine chloride (CCC), succinic acid, 2,2-dimethylhydrazide (Alar ®) and tetramethylammonium bromide (TMAB) stimulate [ 14C]acetate incorporation into taxol in intact pieces of the inner bark of Taxus brevifolia (Pacific yew). Both CCC and Alar also stimulated taxol production in yew logs as measured by the incorporation of [1- 14C]acetate into [ 14C]taxol. An increase in taxol accumulation also occured in silica gel placed under flaps of yew trees in the forest treated with these two compounds. CCC also caused an increase, in an 8-week treatment period, of recoverable taxol from Pacific yew bark in a forest setting. CCC, alar and TMAB are all known as growth retardants, but may cause an effect on taxol biosynthesis via an inhibition of sterol biosynthesis.

Taxus callus cultures: Initiation, growth optimization, characterization and taxol production

Callus was induced from Taxus baccata cv. Repandens Parsons ex Rehd., T. brevifolia Nutt., T. cuspidata Sieb. & Zucc., and T. x media cvs. Hicksii and Densiformis Rehd. using different concentrations of 2,4-d-(2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), or NAA α-naphthalene acetic acid in combination with kinetin. All cultures grew slowly following the first subculture, and a majority turned brown and ceased growth within the next six to twelve months. The callus cultures which lived, continued to grow very slowly for one to two years before the growth rate improved. Initiation of roots and shoot primordia-like structures occurred on some cultures maintained in the dark, and 16 h light/8 h dark, respectively. A fast-growing, habituated callus line (CR-1) derived from T. x media Rehd. cv. Hicksii was established from callus initiated in 1986. Supplementing the medium with casein hydrolysate and both fructose and glucose enhanced the growth rate. A great deal of heterogeneity was found among and within the callus, with respect to the amount of taxol produced. The callus exhibited levels of taxol ranging from 0.1 to 13.1 mg kg-1 (0.0001 to 0.0131%) on a dry weight basis. Overall, the older brown-colored callus produced more taxol than the younger pale yellow-colored callus. The presence of taxol in callus samples was established by high performance liquid chromatography, its biological activity confirmed by a microtubule-stabilizing bioassay and its structure confirmed using one-and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy.

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.