Taxol Production In Suspension Cultures Research Articles

A double oxidative burst for taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum

The effects of oligosaccharide from Fusarium oxysprum on secondary metabolisms in suspension cultures of Taxus chinensis var. mairei were investigated by detecting the oxidative burst (OB) signals using an Electron Paramagnetic Resonance (ESR) spectrometer. The oxidative burst signals induced by oligosaccharide gradually increased to a maximum at 4 h. Phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) activities were respectively 2 and 4 times higher than those of control, and large amount of phenolics were accumulated in culture medium. Two times of obvious increase in taxol production was observed, which was in accordance with the two times of elicitation of oxidative burst signals. The dependence of taxol production on the intensity of OB signals obeyed a modified Logistic curve. It was inferred that the syntheses of the side chain and nucleus of taxol were enhanced at low and high intensities of OB signals, respectively.

Acting paths of elicitors on Taxol biosynthesis pathway and their synergistic effect

Many elicitors have been reported to be favorable to Taxol production in suspension cultures of Taxus cells. However, their acting paths were proved to be different, which were identified based on a quasi-steady-state transfer model. It was found that silver nitrate, ammonium citrate and methyl jasmonic acid acted on the path between Baccatin III and Taxol, while arachidonic acid and salicylic acid acted on the path before 10-deactyl Baccatin III (10-DAB). The mixtures of elicitors with different acting paths could give a synergistic effect in Taxol production while those with the same acting paths could not. A synergistic coefficient was introduced to describe the synergistic effect of elicitors quantitatively, which was defined as the ratio of the maximum Taxol concentration in the coexistence of two elicitors to the summation of the maximum Taxol concentrations in the cases with only one of the elicitors. The larger the synergistic coefficient, the more significant the synergistic effect. The synergistic coefficients of silver nitrate and arachidonic acid, ammonium citrate and arachidonic acid, ammonium citrate and salicylic acid were calculated as 1.1, 1.44 and 1.25, respectively, while those of arachidonic acid and salicylic acid, ammonium citrate and methyl jasmonic acid were only around 0.5.

Signal transduction pathway for oxidative burst and taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum

Signal transduction pathway for oxidative burst and taxol production was studied in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum. Suspension cells exhibited an oxidative burst approximately 4h after challenge with oligosaccharide. The secondary metabolism including accumulation of extracellular phenolics and taxol production were enhanced, while the alkalization of the outer medium and activity of phenylalanine ammonia lyase (PAL) was promoted significantly. Pretreatment with the tyrosine (Tyr) kinase inhibitor herbimycin A abolished the oxidative burst and the subsequent induction of taxol production within 15 min after addition of elicitor. Suramin, a G-protein inhibitor, inhibited the oxidative burst if added up to 30 min after oligosaccharide treatment. The oxidative burst was also induced by the ion channel generator amphotericin B. In contrast, the ion channel blocker anthracene-9-carboxylate acid and Ca2+ channel blocker nifedipine inhibited the oxidative burst within 45 min after treatment. Oligosaccharide rapidly induced Tyr phosphrylation of receptor, which was inhibited by herbimycin A and anthracene-9-carboxylate acid. These responses were also inhibited by phospholipase C-neomycin. The response time of oligosaccharide to phospholipase C pathway was ca at 60 min. These data suggested that the activation of ion channels and phospholipase C follow Tyr kinase and G-protein in the signal pathway leading to the oxidative burst.

Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum

The trivalent ion of a rare earth element, lanthanum, was tested for elicitor-like effects on taxol production in suspension cultures of four different Taxus spp cells. In T. yunnanensis cell cultures, the lanthanum ion at concentrations from 1.15 to 23.0 μM stimulated taxol production. The lanthanum ion also promoted taxol excretion by the T. yunnanensis cells considerably. The maximum stimulation of taxol production was achieved by the addition of 5.8 μM La 3+ to the culture during mid-log growth phase, increasing the volumetric taxol yield by nearly threefold, from 2.61±0.37 to 9.89±1.92 mg l −1 over a 28 day culture period. At higher concentrations, i.e. 23.1 and 46.2 μM, however, the lanthanum ion caused significant growth inhibition. For the other three Taxus cell lines, namely an embryo and a leave cell of T. chinensis and a stem cell of T. chinensis marv, the addition of lanthanum ion to the culture only had a significant effect on taxol production by the T. chinensis marv stem cells, increasing the volumetric yield by about threefold to 4.69±0.76 mg l −1. These results suggest that lanthanum has elicitor-like effects on secondary metabolite synthesis of plant cell cultures.

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l−1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l−1) was 5 times higher than that in the case without any additives (3.4 mg l−1) and 1.4 times of that in the case with only thein situ extraction (12 mg l−1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.

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.

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.