Taxus Chinensis Var Research Articles

Paclitaxel production using co-culture of Taxus suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.

Microsatellite loci from Taxus chinensis var. mairei (Taxaceae), an endangered and economically important tree species in China

Taxus chinensis var. mairei is an endangered and economically important tree species in China. Four polymorphic microsatellite markers were developed and characterized using the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) protocol. Polymorphism of each locus was quantified for five natural populations from Hubei Province, China. The number of alleles per locus varied from 6 to 24. The ranges of observed and expected heterozygosity were 0.493–1.000 and 0.440–0.845, respectively. These are the first microsatellites reported for the family of Taxaceae and will be helpful for the ongoing population and conservation genetics research of Taxus chinensis var. mairei.

Determination of paclitaxel and other six taxoids in Taxus species by high-performance liquid chromatography–tandem mass spectrometry

A method of high-performance liquid chromatography–tandem mass spectrometry (LC–MS-MS) has been developed for the trace analysis of paclitaxel and other six taxoids in three Taxus species including Taxus cuspidata, Taxus media and Taxus chinensis var. mairiei. Seven taxoids were separated using a gradient mode on an Eclipse XDB-C18 column (4.6 × 150 mm; i.d., 5 μm) at 20 °C. The compound separations were detected by an API 3000 mass spectrometer equipped with a TurboIonSpray™ interface. The compounds were detected using electrospray ionization (ESI) in positive-ion mode and quantified by multiple-reaction monitoring (MRM) mode using the transition mass of m/z 567.4 → 445.4, 609.3 → 549.5, 944.9 → 286.4, 812.6 → 286.1, 832.8 → 264.1, 854.4 → 286.1, and 812.6 → 286.1 for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. The ranges of limit of quantitation (LOQ) for taxoids were 32, 14, 26, 14, 20, 32, and 26 ng/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. Linearity was confirmed over the whole calibration range (0.07–45, 0.058–37.5, 0.058–37.5, 0.056–36.3, 0.053–33.8, 0.057–37.5, and 0.06–38.8 μg/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively) with coefficients higher than 0.9903. The inter- and intra-day precision of taxoids ranged from 2.86% to 6.31% for retention time and ranged from 3.91% to 7.33% for peak area. The recovery rates of this method were higher than 94.32% for 10-DAB III, 94.68% for baccatin III, 93.65% for 7-xyl-10-DAT, 93.29% for 10-DAT, 92.91% for cephalomannine, 93.41% for paclitaxel, and 93.06% for 7-epi-10-DAT, respectively.

Structural characterisation of a water-soluble polysaccharide with high branches from the leaves of Taxus chinensis var. mairei

A large number of polysaccharides present in the leaves of Taxus chinensis var. mairei were successively extracted with hot water after preliminary treatments. The ion-exchange and gel-permeation chromatography was used to isolate and purify the major polysaccharides to afford a complex water-soluble polysaccharide, named T1 with a molecular mass of 3.44 × 10 6 Da determined by HPGPC. T1 consisted of 2,4-di- O-methyl-mannose, rhamnose, arabinose, xylose, galactose, mannose, galacturonic acid and glucuronic acid in a molar ratio of 2:5:24:9:3:46:1:10, and the acetyl content was estimated to be 2.1%. On the basis of methylation analysis, periodate and chromium trioxide oxidations, Smith degradation, graded acid hydrolysis, and NMR and IR spectroscopy, T1 possessed a 1,3,6-linked β- d-Man p main chain, substituted by mannose, arabinose, galactose, xylose, rhamnose, 2,4-di- O-methyl-mannose, galacturonic acid, and glucuronic acid residues. These contained non-reducing end-units of mannose, arabinose, xylose, 2,4-di- O-methyl-mannose, and glucuronic acid. This is the first report on isolation of an acid heteropolysaccharide from Taxus species.

Salicylic acid‐induced taxol production and isopentenyl pyrophosphate biosynthesis in suspension cultures of Taxus chinensis var. mairei

The influences of salicylic acid (SA) on taxol production and isopentenyl pyrophosphate (IPP) biosynthesis pathways in suspension cultures of Taxus chinensis var. mairei were investigated by adding SA and mevastatin (MVS), a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase in the mevalonate pathway for IPP biosynthesis, into the culture systems. The cell death and taxol production were induced upon the introduction of SA, and 20mg/l was proved to be the optimal SA concentration in terms of the less damage to Taxus cells and marked activation of phenylalanine ammonia lyase (PAL). In the coexistence of SA (20mg/l) and MVS (100 nmol/l), the taxol content (1.626 mg/g dry wt) was higher than that (0.252 mg/g dry wt) of the MVS-treated system but almost equal to that (1.581 mg/g dry wt) of the SA-treated system. It is thus inferred that the activated non-mevalonate pathway should be responsible for the formation of IPP in taxol biosynthesis in the presence of SA.

Effect of Light Stress on the Content of Chloroplast Pigment and Taxol in the Leaves of Taxus chinensis var. mairei

The changes of the content of chloroplast pigment and taxol in the leaves of Taxus chinensis var.mairei were studied with light stress.The results comparing with the comparison showed that the contents of chlorophyll-a,chlorophyll-b and chlorophyll and the ratio of chlorophyll-a to chlorophyll-b were lower.However,the contents of chlorophyll-a,chlorophyll-b and chlorophyll tended to gradually rise as the stress prolonged,but the ratio of chlorophyll-a to chlorophyll-b first rose and then decreased.The content of carotenoid was lower than the comparison in the first two weeks and then surpassed the comparison.The ratio of carotenoid to chlorophyll was higher than the comparison and tended to first decreased and then rose as the stress prolonged.The contents of taxol was slightly lower than the comparison in the first week and then begun to gradually accumulate in the leaves of Taxus chinensis var.mairei at the second week,the content of taxol had increased to 1.5 times of the comparison at the third week,which is very significant to improve the content of taxol in the growed Taxus chinensis var.mairei.

Effects of Shading Treatment on Photosynthetic Response and Life Cycle form of Taxus chinensis var. mairei

The differences of photosynthetic response,photopigment and life cycle form of 5 years old Taxus chinensis var. mairei which live in the environment of 89% Shading treatment and change the light condition into 46.4% Shading treatment and full light for 50 days was studied.The result showed that the light compensation point of 89% Shading treatment,46.4% Shading treatment and full light was 18.88,30.52,65.34 μmol·m-2·s-1,and the light saturation point was 287.01,258.25,358.92 μmol·m-2·s-1,we can see that the shading treatment can reduce the light compensation point and light saturation point,so the plants can make the best of faint light,height the Photosynthesis rate,enhance the photosynthetic capacity,these changes of 89% is the biggest.Along with the degree increasing of shading treatment,the content of photopigment in the leaves of Taxus chinensis var. mairei which include chlorophyll a,chlorophyll b and carotenoids increased too,and that the ratio of the content of chlorophyll a,chlorophyll b decreased,the result showd that shading treatment can make the best of the light energy,especially blue-violet light,enhance the photosynthetic capacity.We also studied the effects of shading treatment on life cycle form of Taxus chinensis var. mairei,the life cycle form of 89% Shading treatment,46.4% Shading treatment and full light is V0.836C0.164,V0.625C0.375 and V0.772C0.228,the vegetative growth of 89% is the most exuberant.So we can conclude that 89% shading treatment can provide the most suitable condition for Taxus chinensis var. mairei in these three kinds of conditions.

Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.

Comparative lipidomics analysis of cellular development and apoptosis in two Taxus cell lines

A comparative lipidomics approach was employed to investigate the changes in membrane phospholipids during the procession of cellular development and apoptosis of two plant cell lines, Taxus cuspidata and Taxus chinensis var. mairei. Analysis of lipids by LC/ESI/MS n showed more than 90 phospholipid molecular species and indicated significant differences in the abundance throughout a 3-week period. Phosphatidic acid (PA), phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) were three important lipid groups that were responsible for the discrimination between the apoptotic T. chinensis var. mairei and living T. cuspidata cells. Continuous increase of phospholipase D (PLD) activity led to PA production in apoptotic T. chinensis var. mairei cells suggesting that the PLD activation and PA formation mediated the apoptosis. Comparison of the profiles of phosphatidylbutanol (PtdBut) with those of PC or phosphatidylethanolamine (PE) indicated that PC rather than PE was the major substrate of PLD in vivo. These results suggest that the alternation of membrane phospholipids may regulate apoptosis, triggering an increase in taxol production of T. chinensis var. mairei cells.

FRACTAL PROPERTIES OF DISTRIBUTION PATTERNS OF TAXUS CHINENSISVAR. MAIREI CLONAL POPULATIONS IN THE YUANBAOSHAN MOUNTAIN, CHINA

Aims Taxus chinensis var. mairei is the material from which taxol, an effective low-toxicity cancer-resistance medicine, is distilled. This species reproduces asexually, especially through sprouting from stems, which enables Taxus to expand its spatial occupation and maintain its population. Therefore, study on clonal properties of Taxus populations may provide a scientific basis for protecting and regenerating populations. This study addresses the following questions: 1) what are the fractal properties of population distribution patterns, 2) are clonal architecture and fractal properties correlated and 3) are fractal properties and aggregation correlated? Methods Based on a survey of Taxus, we established four 40 m × 15 m plots in its central distribution area in Yuanbaoshan Nature Reserve. For each plot, we determined the spatial coordinates of each Taxus individual. For Taxus 3 m tall, we measured height, crown size, height to branches and diameter at breast height; for shorter Taxus seedlings and saplings, only height was measured. We also estimated or measured cover and height of shrubs and herbs. Fractal properties of population patterns were analyzed by using the box-counting dimension and the information dimension. Important findings The box-counting dimension ranged from 0.993 1 to 1.353 1, which was far from the theoretical value 2, indicating low degree of spatial occupation. Significant correlation between the clonal architecture index and the box-counting dimension indicates that degree of spatial occupation was closely related to clonal architecture, i.e., populations tending to have phalanx clonal architecture had a stronger degree of spatial occupation than those tending to have guerrilla clonal architecture. The information dimension varied from 1.350 8 to 1.652 1 with community type. The Pearson correlation between information dimension and pattern index is significant, suggesting that differences of information dimension could reflect variation of the aggregation intensity. Information dimension was closely related to number and spatial distribution patterns of ramets.

An endophytic Taxol-producing fungus BT2 isolated from Taxus chinensis var. mairei

BT2, a newly isolated endophytic fungus from Taxus chinensis var. mairei, was observed to produce Taxol. Besides Taxol, a potent anticancer drug, BT2 could also yield taxane baccatin III, which was an important intermediate for Taxol and semi-synthesis of Taxol in industry. The isolation of such a fungus may provide a promising alterative approach to produce Taxol, and BT2 can serve as a potential material for fungus engineering to improve Taxol production. Key words: Baccatin III, endophytic fungus, Taxol, Taxus chinensis var. mairei.

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.

Inhibitor studies of isopentenyl pyrophosphate biosynthesis in suspension cultures of the yew Taxus chinensis var. mairei

Some metabolic inhibitors, mevastatin (MVS), chlorocholine chloride (CCC), sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), were respectively added into the suspension cultures of the yew Taxus chinensis var. mairei at the late phase of cell growth to study isopentenyl pyrophosphate (IPP) biosynthesis. The content of total taxanes decreased in the cases of MVS, NaPP and DLG addition, regardless of the inhibitor concentration, whereas it increased in the case of CCC addition. It was thus presumed that a mevalonate pathway might exist in IPP biosynthesis at the late growth phase of Taxus cells and that some IPP might transfer from the cytoplasm to the plastids. This presumption was also confirmed by analysing the effects of the inhibitors on taxane content. It was further demonstrated that IPP in the biosynthesis of taxol, baccatin III and 10-deacetylbaccatin III might transfer from the cytoplasm to the plastids, whereas the translocation of IPP might be not involved in cephalomannine biosynthesis. Furthermore, in combination with the results of previous studies, it is very probable that different IPP biosynthesis pathways exist during different growth phases in Taxus cells.

Biological responses of suspension cultures of Taxus chinensis var. mairei to shear stresses in the short term.

The biological responses of Taxus chinensis var. mairei suspension cultures to a range of shear rates were investigated in a Couette-type shear reactor. It was found that the shear rate below 458 s-1 enhanced the primary metabolism, increasing mitochondrial activity and protein expression and inhibiting the activity of phenylalanine ammonia lyase (PAL), an enzyme relevant to the secondary metabolism. However, the shear rate over 719 s-1 damaged taxus cells to some extent, decreasing the mitochondrial activity, increasing the membrane permeability, and even causing cell hypersensitive responses. As a result, PAL and intracellular peroxidase, an enzyme in cells relevant to reactive oxygen species scavenging, were activated and extracellular phenolics were accumulated. Additionally, shear-induced accumulation of free radicals and alkalization of the culture medium were observed. The results might shed light on a better understanding of the mechanism of plant cells responses to shear stress through the signal transduction pathway.

Translocation of isopentenyl pyrophosphate for Taxol biosynthesis in suspension cultures of Taxus chinensis var. mairei

Two inhibitors of metabolite translocators, sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), respectively, were added into suspension cultures of Taxus chinensis var. mairei at the early and late growth phases to investigate the translocation of isopentenyl pyrophosphate (IPP) from cytoplasm to plastids for Taxol biosynthesis. NaPP and DLG hardly affected cell biomass and viability regardless of the phases of their introduction. The Taxol content varied less when NaPP or DLG was added at day 7 but decreased obviously when NaPP or DLG was introduced at day 14. It is thus inferred that NaPP and DLG inhibited Taxol biosynthesis by blocking IPP translocation at the late growth phase, suggesting that the translocation of IPP be involved only at the late growth phase of Taxus cells.

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.

Fungal elicitor-induced cell apoptosis in suspension cultures of Taxuschinensis var. mairei for taxol production

A crude extract from Fusarium oxysprum caused significant cell apoptosis in suspension cultures of Taxus chinensis var. mairei as determined by transmission electron microscopy, DNA gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) analysis. Cells of T. chinensis var. mairei responded to the fungal elicitors by altering the general phenylpropanoid pathway and affecting the biosynthesis of taxol. The activity of l-phenylalanine ammonia lyase (PAL) increased appreciably within the first 36 h of elicitor treatment, and then decreased rapidly. A large quantity of phenolic compounds accumulated in the culture medium. The maximum concentration of taxol was three times higher than that of the control. Cell apoptosis and taxol production are closely relevant in elicitor-treated culture of T. chinensis var. mairei.

A model for signal transduction in suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum

A non-linear cascade model is proposed to describe the signal transduction pathway in suspension cultures of Taxuschinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum. The oxidative burst intensity, which was defined as the amount of the free radicals including superoxide anion (O2 ⋅), H2O2 and OH− and measured by ESR spectrometry, was used as the signal characteristic and the theory of electronic signal transduction cascade was then applied to derive the initial model. The model shows that the signal transduction cascade process is composed of three cascade nets, represented by the phosphorylation of a G-protein, activation of an ion channel and phospholipase C, and phosphorylation of protein kinase C. The three reactions were calculated as beginning at 30.5, 48.8 and 141.4 min after elicitation, and the maximum variation rates of the signal intensity in the three nets occurred at 43.2, 80.6 and 192.6 min, respectively. The validity of the model predictions was verified by experimental data.

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l−1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.