Taxus Chinensis Cells Research Articles

Responses of defense signals, biosynthetic gene transcription and taxoid biosynthesis to elicitation by a novel synthetic jasmonate in cell cultures of Taxus chinensis

Recently novel synthetic jasmonate derivatives were shown to have very powerful stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells (Biotech Bioeng, 86:595; ibid. 86: 809, 2004). To provide an insight into the elicitation mechanism of the newly synthesized elicitors, by taking 2-hydroxyethyl jasmonate (HEJ, at 100 microM) as a typical example, in this work the defense signals were detected and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in cell cultures of T. chinensis. The oxidative burst (induced H2O2 production) was confirmed, and the induction of lipoxygenase (LOX) activity and intracellular jasmonate acid (JA) synthesis was found. The gene transcription of geranylgeranyl diphosphate synthase (GGPPs) and taxa-4(5),11(12)-diene synthase (TS) was up-regulated by HEJ elicitation compared to control. Inhibition of JA biosynthesis by a putative LOX inhibitor, ibuprofen (IBU), effectively depressed the HEJ-induced up-regulation of GGPPs and TS gene transcription levels. In contrast, the inhibition of H2O2 production by membrane NADPH oxidase inhibitor, diphenylene iodonium (DPI), did not affect the transcriptions of those genes. For the HEJ-induced Tc production, it was suppressed with addition of DPI or IBU. The results suggest that both H2O2 and JA signals were involved in HEJ-induced Tc biosynthesis, and JA mediated the induction of GGPPs and TS genes expression, but H2O2 was not essential to activate them. Finally, a signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

Nitric Oxide is Involved in Methyl Jasmonate-induced Defense Responses and Secondary Metabolism Activities of Taxus Cells

Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures such as the valuable anticancer diterpenoid taxol (paclitaxel) in Taxus spp. This work examines the involvement of nitric oxide (NO) in MeJA-induced plant defense responses and secondary metabolism in Taxus chinensis cell cultures. Exogenously supplied MeJA at 100 microM induced rapid production of NO in the Taxus cell cultures, reaching a maximum within 6 h of MeJA supply. Several other responses occurred concomitantly, including the production of hydrogen peroxide (H2O2), and the increases in intracellular malondialdehyde (MDA) content, lipoxygenase (LOX) and phenylalanine ammonium-lyase (PAL) activities. The MeJA-induced H2O2 production was suppressed by an NO donor, sodium nitroprusside (SNP), but enhanced by NO inhibitors, N (omega)-nitro-L-arginine (L-NNA) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO). In contrast, the MeJA-induced MDA, LOX and PAL were all enhanced by the NO donor but suppressed by the NO inhibitors. The NO inhibitors also suppressed MeJA-induced taxol accumulation. These results are suggestive of a role for NO as a signal element for activating the MeJA-induced defense responses and secondary metabolism activities of plant cells.

Purification, cloning, and functional expression of phenylalanine aminomutase: The first committed step in Taxol side-chain biosynthesis

The conversion of α-phenylalanine to β-phenylalanine is the first committed step in the biosynthesis of the C-13 side chain of Taxol. Thus, the novel enzyme responsible for this step, phenylalanine aminomutase (PAM), is of considerable interest for studies of Taxol biosynthesis and represents a potential target for genetic engineering. A method is described for purifying PAM from Taxus chinensis cell cultures. The purified enzyme has a K m of 1.1 mM, a V max of 110.1 μm/min/mg protein, a pH optimum of 7.5–8.0, and a denatured molecular weight of about 80 kDa. Peptide sequences derived from the purified protein were used to design and synthesize degenerate primers enabling the PCR synthesis of the PAM cDNA. The PAM cDNA encodes a protein of 687 amino acid residues with a deduced molecular weight of 75.3 kDa. The PAM cDNA was cloned and expressed in Escherichia coli, and PAM activity was demonstrated. As a gene symbol for the PAM enzyme, pam is proposed. Protein sequence alignments of PAM, phenylalanine ammonia-lyase (PAL), and histidine ammonia-lyase (HAL) sequences exhibit significant similarity providing insight into potential active site residues of PAM.

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.

Involvement of nitric oxide in elicitor-induced defense responses and secondary metabolism of Taxus chinensis cells

This work was to characterize the generation of nitric oxide (NO) in Taxus chinensis cells induced by a fungal elicitor extracted from Fusarium oxysporum mycelium and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. The fungal elicitor at 10-100 microg/ml (carbohydrate equivalent) 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 h and the second within 12 h of elicitor treatment. The NO donor sodium nitroprusside potentiated elicitor-induced H2O2 production and cell death but had little influence on elicitor-induced membrane K+ efflux and H+ influx (medium alkalinization). NO inhibitors Nomega-nitro-L-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide partially blocked the elicitor-induced H2O2 production and membrane ion fluxes. Moreover, the NO inhibitors suppressed elicitor-induced activation of phenylalanine ammonium-lyase and accumulation of diterpenoid taxanes (paclitaxel and baccatin III). These results suggest that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the Taxus cells.

Identification of a cDNA Clone Specific for the Taxol Synthesis Phase of Taxus Chinensis Cells by mRNA Differential Display*

In order to identify genes related to Taxol biosynthesis, the mRNA differential display method was employed to compare mRNA populations from suspension cultured Taxus chinensis cells before and after beginning to produce Taxol. From a total display of about 4000 PCR products, 104 were derived from cells during the Taxol synthesis phase but not the non-Taxol synthesis phase. These products were cloned, and one such cDNA clone, named TS1, was confirmed to be specifically expressed in the Taxol synthesis phase by northern blot analysis. The length of the transcript corresponding TS1 was approximately 2.1 kb. DNA sequencing and homology search showed the sequence of TS1 contains a partial open reading frame and has no homologies with other known genes. Hence, this report demonstrated the potential of mRNA differential display for the isolation of genes specific for the period of secondary metabolite production as well as the feasibility of the approach for the identification of genes potentially related to the synthesis of secondary metabolites in higher plants.

Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures

After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied. The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells. The H2O2 forming enzyme was also investigated. The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor. Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter. The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.

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.

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.

Oxidative Stress and Taxol Production Induced by Fungal Elicitor in Cell Suspension Cultures of Taxus Chinensis

Treatment of Taxus chinensis cell suspension cultures with fungal elicitor resulted in an oxidative stress characterized by H2O2 production, malondiadehyde (MDA) accumulation and cell death. This oxidative stress was dependent on the concentration of elicitor. Cells exposed to elicitor accumulated taxol, however, not proportional to elicitor concentration. High production of taxol occurred in cells treated with the suitable elicitor concentration. We concluded that oxidative stress had the deleterious effect on taxol production. Simultaneous treatment with elicitor and ascorbic acid (ASA) changed the oxidative stress and taxol production. Production of taxol in cells treated with 200 mg dm−3 elicitor and ASA was enhanced compared with that in cells treated with only 200 mg dm−3 elicitor, while production of taxol in cells treated with 100 and 50 mg dm−3 elicitor and ASA was decreased compared with that in cells treated with 100 and 50 mg dm−3 elicitor.

Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time ( t m) and volumetric oxygen transfer coefficient ( k La ) in the bioreactor were characterized at various cell densities and operating conditions. A constant t m of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 μM methyl jasmonate (MJA). The lower Tc production at the longer t m of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.

Enhanced taxane productivity in bioreactor cultivation of Taxus chinensis cells by combining elicitation, sucrose feeding and ethylene incorporation

In conventional suspension cultures of Taxus chinensis, the accumulation of physiologically active taxuyunnanine C (Tc) in 1 l (working volume) airlift bioreactors was lower than in shake flasks. Elicitation by methyl jasmonate (MJA) or sucrose feeding could improve the taxane production in the bioreactors. A strategy of combining MJA elicitation and sucrose feeding was applied to cell cultures in bioreactors. Both the Tc production and productivity were significantly enhanced by combined addition of 100 μM MJA and 20 g l −1 sucrose at day 7 and their maximum levels reached were 344±12 mg l −1 (day 18) and 18.6±0.5 mg l −1 day −1 (until day 15), respectively. A further combination of MJA elicitation (at 100 μM), sucrose feeding (at 20 g l −1) and ethylene incorporation (at 18 ppm) in the inlet air improved the Tc productivity to 20.2±0.4 mg l −1 day −1 in the bioreactors. The results suggest that the above combination approach is a useful strategy for large-scale production of taxane diterpenes by plant cell cultures.

Combination of conditioned medium and elicitation enhances taxoid production in bioreactor cultures of Taxus chinensis cells

Stimulating effects of conditioned medium (CM) addition and its combination with methyl jasmonate (MJA) elicitation on production of physiologically active taxuyunnanine C (TC) by cell cultures of Taxus chinensis were demonstrated in both a shake flask and a 1-l (working volume) concentric-tube airlift bioreactor. In control cell cultures, average cell growth rates were 0.117±0.003 per day in the flask and 0.086±0.001 per day in the airlift bioreactor. With addition of 50% (by volume) CM on day 0, an average growth rate of 0.153±0.006 per day in flask and 0.125±0.003 per day in bioreactor was obtained, and the maximum cell density (on day 11) was 15.0±0.1 g l −1 in flask and 15.5±0.8 g l −1 in bioreactor. TC production and productivity were 43.0±17.6 mg l −1 and 1.0±0.8 mg l −1 per day in bioreactor control cultures, and a much higher production of 78.9±20.6 mg l −1 and productivity of 4.5±1.2 mg l −1 per day were obtained in bioreactor cultures with CM addition. Combination of CM addition with MJA (at 100 μM) elicitation enhanced TC productivity in bioreactors by about 30% when compared with the cultures with only MJA elicitation, i.e., increased from 9.2±0.9 to 11.8±0.9 mg l −1 per day. The work is potentially beneficial to large-scale plant cell cultures for production of the bioactive secondary metabolite.

High stable production of taxol in elicited synchronous cultures of Taxus chinensis cells

Elicitation was an efficient method for enhancing taxol production in asynchronous cultures of Taxus chinensis cells but high production of taxol was unstable in 250 ml Erlenmeyer flask and a 10 l working volume stirred bioreactor. This situation could be remedied using two synchronization methods, double phosphate starvation and low temperature treatment. Low temperature treatment could not only maintain stable high taxol production, but also further enhance taxol production compared with elicited asynchronous cultures. This resulted in the greatest taxol production of 27 mg l −1, being about 2 and 11 times higher than in asynchronous cultures and in elicited asynchronous cultures, respectively. Taxol production in a stirred bioreactor was less than in flasks, but changes of taxol production in asynchronous cultures and synchronous cultures in a stirred bioreactor were similar to those in 250 ml flask. Therefore, high stable taxol production in elicited synchronous cultures of T. chinensis cells was amenable to scaleup. The possible relationship between stable production of taxol and cell cycle phase is discussed.

Improvement of cephalomanine production in Taxus chinensis cells by a combination of sucrose and methyl jasmonate

Cell suspension cultures of Taxus chinensis, supplemented with 25 g sucrose l−1, produced 11 mg cephalomanine l−1, 21 g biomass l−1 and 19 nkat geranylgeranyl diphosphate (GGPP) synthase activity g protein−1. Supplementation of the cultures with 100 μM methyl jasmonate (MJA) produced 17 mg cephalomanine l−1, 6 g biomass l−1 and 78 nkat GGPP synthase activity g protein−1. Addition of sucrose and MJA together produced 24 mg cephalomanine l−1, 18 g biomass l−1 and 55 nkat GGPP synthase activity g protein−1.

Effect of Mixing Time on Taxoid Production Using Suspension Cultures of Taxus chinensis in a Centrifugal Impeller Bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.

Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal.

An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.

Enhanced taxol production and release in Taxus chinensis cell suspension cultures with selected organic solvents and sucrose feeding.

Suspension culture of Taxus chinensis cells was carried out in aqueous-organic two-phase systems for the production and in situ solvent extraction of taxol (paclitaxel). Three organic solvents, hexadecane, decanol, and dibutylphthalate, were tested at 5-20% (v/v) in the culture liquid. All of these solvents stimulated taxol release and the yield per cell, though decanol and higher concentrations of the other two solvents depressed biomass growth significantly. Ten percent dibutylphthalate was the optimal solvent for improving taxol production and release with minimal cell growth inhibition. The time of solvent addition to the culture also affected taxol production, with the addition during the late-log growth phase being most favorable. By feeding sucrose to the culture near the stationary growth phase, the cell growth and taxol production period was extended from 27 to 42 days. The combining of the two-phase culture and sucrose feeding increased the taxol yield by about 6-fold compared with the single-phase batch culture, to 36.0 +/- 3.5 mg/L, with up to 63% taxol released. This study shows that in situ solvent extraction combined with nutrient feeding is an effective process strategy for production and recovery of secondary metabolites in plant cell suspension culture.

Improved paclitaxel accumulation in cell suspension cultures of Taxus chinensis by brassinolide

When brassinolide was added at 17 ng l−1 to Taxus chinensis cell suspension cultures, the paclitaxel content was increased by over 100% to 580 μg g−1 dry weight. Brassinolide may therefore be a novel regulator of paclitaxel biosynthesis.

Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene.

Suspension cells of Taxus chinensis were cultivated in both shake flasks and bioreactors. The production of taxuyunnanine C (TC) was greatly reduced when the cell cultures were transferred from shake flasks to bioreactors. Oxygen supply, shear stress and stripping-off of gaseous metabolites were considered as potential factors affecting the taxane accumulation in bioreactors. The effects of oxygen supply on the cell growth and metabolism were investigated in a stirred tank bioreactor by altering its oxygen transfer rate (OTR). It was found that both the pattern and amount of TC accumulation were not much changed within the range of OTR as investigated. Comparative studies on the cell cultivation in low shear and high shear generating bioreactors suggest that the decrease of TC formation in bioreactors was not due to the different shear environments in different cultivation vessels. An incorporation of 2% CO(2) in the inlet air was beneficial for the cell growth, but did not improve the TC production in bioreactors. Furthermore, the effects of different levels of ethylene addition into the inlet air on the cell growth and TC production were investigated in a bubble column reactor. The average cell growth rate increased from 0.146 to 0.204 d(-1) as the ethylene concentration was raised from 0 to 50 ppm, and both the content and production of TC were also greatly improved by ethylene addition. At an ethylene concentration of 18 ppm, the highest TC content and volumetric production in the reactor reached 13.28 mg/(g DW) and 163.7 mg/L, respectively, which were almost the same as those in shake flasks. Compared with the control reactor (bubble column without ethylene supplementation), the maximum TC content was increased by 82% and the total production of TC was doubled. The results indicate that ethylene is a key factor in scaling up the process of the suspension cultures of T. chinensis from a shake flask to a bioreactor.