The Co-cultivation Of Taxus Cuspidata And Fusarium Mairei By Immobilization

Paclitaxel, a highly derivatized diterpenoid, was firstly found from some species of Taxus. Due to its unique anticancer mechanism and the remarkable curative effect for various malignant tumors, it has been the focus to which people pay attention all the time. Paclitaxel production by endophytic fungal fermentation, or plant cell cultivation had been the research hotspots in recent years. But the key bottleneck to realize the industrialization might be difficult to break; the output character can not meet the production requirement.In the long period symbiotic coordination, the endophytic fungi and the host plant had formed the mutually beneficial interaction. The related studies demonstrated that the endophytic fungi were able to participate in the synthesis or the transformation of the active constituents in their host plant. The inductor which produced by the endophytic fungi can limit the Taxus cells growing and promote the synthesis and secretion of paclitaxel. The inductor produced by the Taxus cells may also promote the synthesis and secretion of paclitaxel in the endophytic fungi. Therefore, studies on the simulation of the symbiotic environment of them were possibly helpful for the researches on their relation, and the synthesis mechanism of secondary metabolites and so on. But the cultivation of endophytic fungi and the host plant in vitro, not only in the environmental conditions(pH, dissolvable oxygen, shearing force, medium ingredients), but also in the growth metabolism (physiological condition, growth cycle, metabolites, pathways) and so on, did have a lot of differences.The interaction mechanism between Taxus cuspidate and Fusarium mairei K178 was studied preliminarily in this research for improving their respective paclitaxel productivity by immobilized cultivation, which imitated their symbiotic environment under the condition of maintaining their biological activity. The main methods and results were as follows:1. The choice and the cultivation of immobilized cells were explored. There were three immobilization methods using calcium alginate, two methods using polyvinyl alcohol for F. mairei, one immobilization method using calcium alginate for T. cuspidate cells had been explored. A chosen kind of cells would be taken to carry on a chosen immobilization method by comparing the culture states of immobilized T. cuspidate with that of F. mairei. The detailed researches were focused on the conditions of immobilization, including the culture medium, the concentration of sodium alginate solution, the concentration of CaCl2 solution, the embedding density of cells, the diameter of gelatin bead and so on. And how about these kinds of factors might make influences on the bioactivity and paclitaxel productivity of the immobilized cells. The result showed that the immobilized F. mairei did not have the usability. T. cuspidate cells were carried on the most temperate immobilization method by using calcium alginate. The optimum condition for the concentration of sodium alginate solution 20 g/L, for the embedding density of T. cuspidate cells 0.15~0.2 g/mL, for the diameter of gelatin bead 3~4 mm. Under these conditions immobilized T. cuspidate cells consumed sucrose quickly, growed stably with no leaking phenomenon, showed the low phenol output. 2. The co-cultivation conditions were optimized. Detailed researches focused on the inoculum volume of T. cuspidate and F. mairei, the inoculum time, the culture period and so on, and how about these kinds of factors might make influences on the bioactivity and paclitaxel productivity of the co-cultivation. The control experiments were carried on for removing the disturbances caused by the remaining intra-cellular paclitaxel, the secretion or the leakage of T. cuspidate plant cell fragments, and the materials in the operations. The co-cultivation conditions were optimized through the orthogonal experiment as the concentration of sodium alginate solution 2%(M/V), the density of entrapped suspension plant cells 0.15 g/mL, the concentration of CaCl2 solution 4%(M/V), the diameter of the beads 3～3.5 mm, 0.5h calcification curing, the inoculum volume of immobilized T. cuspidate cells 40 mL, amended MS medium 70 mL /250mL flask, the inoculum volume of F. mairei in log phase 10% (V/V) to 9th day cultured T. cuspidate cells, cultured in dark and 160 r/min at 25℃for another 10 days. The results showed that F. mairei could induce strong growth inhibition on T. cuspidate, and the extra-cellular secretion of paclitaxel and other secondary metabolites. The secretions of T. cuspidate could also influence the paclitaxel production of F. mairei significantly.3. The fermentation medium and conditions were investigated for promoting paclitaxel output of the co-cultivation. Detailed researches focused on the influences of the amended MS medium ingredients including the main nutritive ingredients, the chemical additives for bioactivity, for paclitaxel output and for secretion on the mass-transfering effects, on the paclitaxel output and the mycelium multiplication of F. mairei, on the the paclitaxel output and the bioactivity of the immobilized T. cuspidate cells in the co-cultivation system. Effects of medium volume content and adding sucrose content on the ultimate paclitaxel output were studied. The result showed that the elevated content of CA was advantageous to the multiplication of F. mairei, in view of the contradiction between the paclitaxel output and the mycelium multiplication, the best balance point was the original content. Archusia as 2,4-D and NAA, cytokinin as 6-BA, had little influence on the paclitaxel output and the multiplication of F. mairei, but KT had the inhibitory action when its content elevated. It could promote paclitaxel secretion and output under the condition of adding sucrose in the 6th day approximately 30 g/L.The differences in dissolvable oxygen owning to volume content could influence the paclitaxel output significantly. The mineral ingredients in the amended MS medium had different functions. When the content of KH2PO4 were elevated there was no benefit for the paclitaxel production of F. mairei, so did CaCl2, (NH4)2SO4 and FeSO4-Na2·EDTA.CaCl2, (NH4)2SO4 and FeSO4-Na2·EDTA even had the inhibitory action when their contents were elevated, possibly because the metabolism differences caused by the acid pH. Different densities of H3BO3 had little influence on the paclitaxel output and the multiplication of F. mairei, suggested that the major factor which inhibited the mycelium multiplication of F. mairei using PVA- H3BO3 immobilization method might be the other immobilization operations or the acidic pH. The chemical additives for paclitaxel secretion had no benefit for the paclitaxel output or the mycelium multiplication of F. mairei. To improve the secretion of immobilized T. cuspidate cells, the induction of F. mairei is the most remarkable.