Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures

The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.     

Enhancement of paclitaxel production by abscisic acid in cell suspension cultures of Taxus chinensis

Addition of 5 mg abscisic acid l^–1 after 12 days' growth of Taxus chinensis suspension culture gave the greatest paclitaxel accumulation at 11 mg l^–1, which was almost 5 times that of the control culture. The highest paclitaxel production, 18 mg l^–1, was obtained using 5 mg abscisic acid l^–1 and 20 mg methyl jasmonate l^–1.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate

Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T‐DNA of A. rhizogenes 9,402 and TXS, carrying both the T‐DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two‐stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Paclitaxel and baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata

The effects of 100 and 200 μM methyl jasmonate (MJA) on cell proliferation and paclitaxel and baccatin III production were investigated in free and alginate immobilized cells of Taxus baccata growing in a selected product formation culture medium. The greatest accumulation of paclitaxel (13.20 mg dm⁻³) and baccatin III (4.62 mg dm⁻³) occurred when 100 μM MJA was added to the culture medium of cells entrapped using a 1.5 and 2.5 % alginate solution. The effects of different treatments on the viability of cultured cells and their capacity to excrete both taxanes into the surrounding medium were considered.     

Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis

Taxus chinensis suspension cells were cultured in the modified Gamborg's B5 medium. Addition of 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ resulted in the greatest paclitaxel production, at 25 mg l^–1 in the cultures, being almost 40 times higher than that of the control culture, 10 times higher than that of the culture exposed to Ag⁺, 6 times higher than that of the culture elicited by chitosan and almost double that of the culture elicited by methyl jasmonate.     

Improved paclitaxel production by in situ extraction and elicitation in cell suspension cultures of Taxus chinensis

Dibutyl phthalate, oleic acid and terpineol were used to extract paclitaxel in situ fromTaxus chinensis suspension cultures. Oleic acid/terpineol (1:1, v/v) added to the cultures gave a higher paclitaxel concentration, compared with either of them alone. Oleic acid/terpineol (1:1, v/v) incorporated into the cultures at 3:50 (v/v) 4 days after elicitation, which was carried out by adding 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ to 10-day-old cultures, resulted in the greatest paclitaxel production of 48 mg l^–1 at day 10 after elicitation. This was double that of the culture by elicitation, and 7-fold higher than that of the culture by in situ extraction.     

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75–160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 M on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.     

Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of taxol and baccatin III in cell cultures of Taxus, three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.     

Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: application to taxane production byTaxus sp.

TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol®) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.Abbreviations TLC Thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - HPLC high pressure liquid chromotography - UV ultraviolet - Rf retention factor     

Hairy root cultures of Taxus × media var. Hicksii Rehd. as a new source of paclitaxel and 10-deacetylbaccatin III

Hairy root cultures of Taxus × media var. Hicksii were established by infection of the plantlets with the Agrobacterium rhizogenes strain LBA 9402. The paclitaxel accumulation in hairy root cultures increased after 100 M methyl jasmonate treatment from 69 to 210 g g^–1 dry wt while the 10-deacetybaccatin III content was not affected by the elicitor.     

Elicitation kinetics of taxane production in suspension cultures of Taxus baccata Pendula

Methyl jasmonate increased taxane production in suspension cultures of Taxus baccata Pendula. Time course changes of taxane production after methyl jasmonate addition were different from normal kinetics without elicitation. Baccatin III and 10-deacetyl baccatin III were detected first and paclitaxel, 10-deacetyl taxol and cephalomanine followed each other in sequence. Paclitaxel was not a dead-end metabolite.     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L^?1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L^?1 and biomass productivity of 18.7 g L^?1 d^?1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Characterization of Coptis japonica cells with different alkaloid productivities

Several cell lines of Coptis japonica with different alkaloid productivities were characterized to obtain information on how a high metabolite production is established. High and low metabolite producing cells, except those from one cell line, showed similar growth kinetics and a similar pattern of nutrient uptake. Amino acid contents, especially that of tyrosine, differed between cell lines, but no correlation was found between the amino acid or tyrosine levels and alkaloid production. Since the addition of tyrosine did not increase the production of berberine, this primary substrate is apparently not the limiting factor for high production in cultured Coptis cells. The addition of berberine to the medium revealed that low-producing cells also have the ability to store alkaloid, and that low productivity is not due to decomposition of alkaloids which have been produced. The direct measurement of the biosynthesis of berberine using ¹⁴C-tyrosine clearly showed that high-producing cells had a higher biosynthetic activity of berberine from tyrosine than low-producing cells. The measurement of enzyme activities in berberine biosynthesis indicated that the early steps of berberine biosynthesis are important in the increased production of berberine.     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits