Suspension Cultures Of Taxus Chinensis Research Articles

Enhancing taxane biosynthesis in cell suspension culture of Taxus chinensis by overexpressing the neutral/alkaline invertase gene

Sucrose metabolism generates material and energy foundations and regulates the biosynthesis of secondary metabolites in plant cell cultures. However, this regulation mechanism is unclear. The neutral/alkaline invertase (NINV) gene is among the key enzymes in sucrose hydrolysis. Thus, the complete open reading frame (ORF) of this gene was obtained from Taxus chinensis cells. This gene was also successfully expressed in Escherichia coli. The expressed protein was verified through the function of sucrose cleavage. Furthermore, the transcriptional levels of the NINV gene can respond to the sucrose concentration changes and relate to taxane biosynthesis in T. chinensis cells. Subsequently, we developed the gene expression vector and introduced it into T. chinensis suspension cells to confirm the role of the NINV gene in taxane biosynthesis. Results indicated that this gene was overexpressed and that it significantly enhanced the expression of the taxadiene synthase (TAS) gene, as well as the biosynthesis of the seven detected individual taxanes. Thus, NINV-mediated sucrose metabolism can promote the biosynthesis of secondary metabolites. This study presents new concepts and approaches to increase the production of secondary metabolites in plant cell cultures.

Significantly improved taxuyunnanine C production in cell suspension cultures of Taxus chinensis by process intensification of repeated elicitation, sucrose feeding, and in situ adsorption

A highly efficient process intensification strategy was developed to enhance the taxuyunnanine C (Tc) production in Taxus chinensis (T. chinensis) by combining repeated elicitation with a synthesized jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA), sucrose feeding and in situ adsorption. Suspension cultures of a high Tc-producing cell line of T. chinensis were supplemented twice, on day 7 and day 12, with 100 μM DHPJA. The cultures were also supplemented by feeding 20 g sucrose/l and 100 g Ambelite XAD-7/l on day 7. This strategy had a synergistic effect on Tc synthesis, causing a significant increase in the Tc yield to 1,715.13 ± 124.12 mg/l on day 21. The yield was 3.28-fold above that produced by repeated DHPJA elicitation combined with sucrose feeding. When XAD-7 was replaced by Amberlite XAD-7 HP, the result was slightly different. The highest Tc yield of 1,728.84 ± 50.37 mg/l was achieved on day 27. The Tc yield achieved here is greater than twice the highest yield reported previously, the sole reported taxoid production yield above the gram per liter level. Given the pharmaceutical importance of taxoids, the results of this study will greatly contribute to the potential industrial production of Tc and other taxoids by plant cell cultures.

Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis

It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H(2)O(2) production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

Effect of initial ammonium concentration on taxoid production and biosynthesis genes expression profile in suspension cultures of Taxus chinensis cells

The effect of initial ammonium concentration in medium on production of taxuyunnanine C (Tc) in suspension cultures of Taxus chinensis was investigated. The results showed that the cell growth was reduced at an initial ammonium concentration of 2 mM when compared to the control (20 mM of initial ammonium in medium), but interestingly the Tc content was enhanced by two fold to 8.07 mg/g DW. The transcription of a couple of key genes in taxoid biosynthetic pathway was investigated by quantitative real-time PCR to understand why Tc biosynthesis was improved at the low initial medium ammonium (2 mM). It was found that the transcription level of genes of geranylgeranyl diphosphate synthase, taxadiene synthase, taxadiene 5α-hydroxylase, taxadiene 5α-ol O-acetyltransferase, taxane 10β-hydroxylase and taxadiene 13α-hydroxylase were all induced in the early stage of cultivation at 2 mM of initial ammonium compared to the control (20 mM of initial ammonium). The stimulation of secondary metabolite synthesis by a lower ammonium concentration may be resulted from the ammonium deficiency-induced transcription of Tc biosynthetic genes.

Expression profiling of key genes involved in taxuyunnanine C biosynthesis in suspension cultures of Taxus chinensis by sucrose feeding and double elicitation with 2,3-dihydroxylpropanyl jasmonate

Expression profiling of key genes involved in taxuyunnanine C biosynthesis in suspension cultures of Taxus chinensis by sucrose feeding and double elicitation with 2,3-dihydroxylpropanyl jasmonate

Novel synthetic 2,6-dichloroisonicotinate derivatives as effective elicitors for inducing the biosynthesis of plant secondary metabolites

Two novel 2,6-dichloroisonicotinic acid (INA) derivatives [trifluoroethyl 2,6-dichloroisonicotinate (TFINA) and 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate (DPCEJ)] were chemically synthesized and evaluated by bioassay as potential elicitors for inducing the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which stably produces a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. A significant increase in Tc accumulation was observed in the presence of TFINA or DPCEJ. For example, addition of 100 microM TFINA or DPCEJ on day 7 led to a high Tc content of 21.6 +/- 2.0 or 27.7 +/- 1.0 mg g(-1) (on day 21), while the Tc content was 13.7 +/- 1.0 and 17.1 +/- 0.9 mg g(-1) for the control and that with addition of 100 microM INA, respectively. To the best of our knowledge, this is the first report on the use of synthetic INA derivatives for inducing the biosynthesis of plant secondary metabolites. The results indicate that the newly synthesized INA analogues can act as promising elicitors for secondary metabolism induction in plant cell cultures.

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.

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis*

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.

Novel fluoro- and hydroxyl-containing jasmonate derivatives as highly efficient elicitors in suspension cultures of Taxus chinensis

To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T. chinensis cell cultures.

Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism.

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.

Glutathione levels control glucose-6-phosphate dehydrogenase activity during elicitor-induced oxidative stress in cell suspension cultures of Taxus chinensis

Fungal elicitor from Aspergillus niger simultaneously induced H 2O 2 accumulation and increased activity of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR) and glutathione peroxidase (GP) in cell suspension cultures of Taxus chinensis. H 2O 2 accumulation was abolished by the inactivation of G6PDH, which was similar to the NADPH oxidase inhibitors diphenylene iodonium, imidazole, and pyridine did. Exogenous H 2O 2 and generators of superoxide anions enhanced G6PDH activity; however, the enhanced G6PDH activity was suppressed by the glutathione (GSH) precursor N-acetylcysteine (NAC). Moreover, buthionine sulfoximine, an inhibitor of GSH synthase, had little effect on H 2O 2 production, but a significant increase in G6PDH activity and a decrease in GSH level compared with only fungal elicitor. These data showed that G6PDH activation was dependent on decreased GSH pool. Catalase inhibitor aminotriazole had little effect on fungal elicitor-induced GSH level and G6PDH activity, indicating decreased GSH pool was independent of catalase.

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.

Optimization of elicitors and precursors for paclitaxel production in cell suspension culture of Taxus chinensis in the presence of nutrient feeding

Elicitors and precursors were optimized for paclitaxel production in cell cultures of Taxus chinensis in the presence of nutrient feeding. The effects of nutrients feeding on cell growth and paclitaxel production were investigated initially and then response surface methodology (RSM) was used to optimize the concentrations of several precursors and elicitors for paclitaxel production in the presence of nutrient feeding . In the first step, the effect of silver nitrate, abscisic acid, chitosan, phenylalanine, methyl jasmonate, sodium benzoate and glycine was evaluated using a fractional factorial design. The major factors proved to be methyl jasmonate, chitosan, and abscisic acid. The effects of these main factors were further investigated by a central composite design. Paclitaxel production reached 54 mg l −1 when 10 mg l −1 silver nitrate, 6 mg l −1 abscisic acid, 23 mg l −1 chitosan, 15 mg l −1 phenylalanine, 31 mg l −1 methyl jasmonate, 30 mg l −1 sodium benzoate and 30 mg l −1 glycine were added on day 12 together with feeding solution containing 20 g l −1 sucrose added on day 16, which was 2 times higher than paclitaxel production without optimization.

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.

Cell death unlikely contributes to taxol production in fungal elicitor-induced cell suspension cultures of Taxus chinensis.

Cell suspension cultures of Taxus chinensis, with 20, 40 and 100 mg fungal elicitor l(-1) from Aspergillus niger, underwent rapid cell death after 24 h, which was about 2, 3.7 and 5-fold of that of the control. At the same time, Taxol production was increased, respectively, to about 5, 8 and 3-fold of that of the control. Inhibition of phenolics biosynthesis resulted in a 150% increase in cell death but a 54% decrease in Taxol production compared with 40 mg elicitor l(-1) alone. O2-free N2 inhibited cell death but had little effect on Taxol production as induced by 40 mg fungal elicitor l(-1).

Ethylene inhibitors enhance elicitor-induced paclitaxel production in suspension cultures of Taxus spp. cells

The effects of ethylene biosynthesis inhibitors including α-amino isobutyric acid, CoCl 2 and NiCl 2, and an inhibitor of ethylene action, Ag +, on fungal elicitor-induced paclitaxel production were assessed in cell suspension cultures of Taxus chinensis, T. chinensis var. mairei and T. yunnanensis to elucidate the role of ethylene in paclitaxel synthesis. All these ethylene inhibitors enhanced the paclitaxel yield, and most significantly (up to three-fold) by the combination of CoCl 2 (20 μM) and Ag + (30 μM). On the contrary, ethrel, an ethylene-releasing reagent, and acetylsalicylic acid (ASA), an inhibitor of both jasmonic acid (JA) and ethylene biosynthesis, depressed the elicitor-induced paclitaxel production. Importantly, the positive or negative effects of these ethylene inhibitors or generator on paclitaxel production were mainly observed when they were added to the culture before or simultaneously with the fungal elicitor, but became less significant or negligible when they were added 24 h after the elicitor. These findings suggest that the control of ethylene production is important for the elicitation of paclitaxel biosynthesis of the cultured Taxus spp. cells. Therefore, the application of ethylene inhibitors to Taxus cell cultures before elicitor treatment is an effective measure to enhance elicitor-induced paclitaxel production.

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.

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.

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.

Improved paclitaxel production by fed-batch suspension cultures of Taxus chinensis in bioreactors

Feeding sucrose at 20 g l -1 on day 16 gave maximum paclitaxel production at 10 mg l -1 when Taxus chinensis in 5 I bioreactors, Paclitaxel accumulation was doubled by the cultivation of cells initially with dissolved O 2 tension at 60% for 20 days followed by being at 20% for another 12 days in the bioreactor. Combination of these two strategies gave maximum paclitaxel production of 19 mg l -1 after 32 days.