Research papersEnhancement of paclitaxel production by temperature shift in suspension culture of Taxus chinensis
Introduction
Paclitaxel is a diterpene alkaloid compound derived from yew tree (Taxus sp.) and it has been used as a substance for anticancer drug since 1992 [1]. In the early developmental stage of paclitaxel, the drug was extracted from bark of slow growing yew tree, which caused severe environmental problems. Plant cell culture has been suggested as an attractive alternative that could overcome the limitations of extracting useful metabolites from natural resources. In spite of many reports from various groups [2], [3], [4], [5], [6], [7], [8], commercial paclitaxel production by plant cell culture has not been successful yet. The major obstacle to commercialization has been the low yield of paclitaxel from plant cell culture. To improve the production yield of paclitaxel, selection of high producing cell line and development of efficient processes are of importance.
It is known that many factors could affect cell growth and metabolite production in plant cell cultures. Those include components of basal medium, source of carbon, phytohormones, anti-metabolites, O2, pH, elicitors, temperature, stirring frequency, and light condition [9]. Among those conditions, culture temperature has been shown to influence the cell growth and the product yields in plant cells but no general trends were evident [10], [11], [12], [13], [14], [15], [16]. Most of Taxus species cell cultures have been performed at 25°C [2], [3], [4], [5], [6], [7], [8], and effect of temperature on the paclitaxel production has not been investigated.
The aim of this study was to enhance the yield of paclitaxel in suspension culture of Taxus chinensis cells by optimization of temperature condition. Effect of temperature on cell growth and paclitaxel production was investigated and it was followed by the optimization of the paclitaxel production by temperature change timing. Furthermore, effect of temperature shift on paclitaxel synthetic pathway, was studied by analyzing the change of taxane related compounds, and treating inhibitors of protein synthesis during cultivation.
Section snippets
Plant cell line and media
Taxus chinensis cells were grown under darkness at 24 ± 1°C on a gyratory shaker (New Brunswick Scientific Co. Inc., G-10, USA) at 150 rpm. The liquid medium contained inorganic salt formulation of Gamborg [17], 30 g/L sucrose, 10 μM naphthalene acetic acid (NAA), 0.2 μM 6-benzylamino purine (BA), 1 g/L casein hydrolysate, and 1 g/L 2-[N-morpholino]ethanesulfonic acid (MES). The cells were sub-cultured every two weeks by adding 50 ml of inoculum into 150 ml of fresh medium in 500-ml long neck
Effect of temperature condition on cell growth and paclitaxel production
Effect of culture temperature on cell growth was studied by incubating cells in temperature controlled shaking incubators at 20, 24, 29, 32, 36°C, respectively. As shown in the Table 1, specific growth rate reached to the maximum of 0.0856 at 24°C, and decreased severely when the temperature increased. Specific growth rate at 20°C was 0.0698, which was higher than that of 0.0453 at 29°C condition. That implied cell growth was more affected at higher temperature than lower temperature condition.
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