Studies On The Interactions Of Taxus Cuspidata Suspension Cells And Endophytic Fungi During Paclitaxel Synthesis

Fusarium mairei was a paclitaxel-producing fungal endophyte derived from Taxus. The interactions of the endophytic fungi and Taxus cuspidata cells were studied in this work. F. mairei was cultured in B5 medium. Fungal endophyte culture supernatant (FECS) was obtained via removing the mycelia from the fungal cultures and concentrating the filtrates obtained to 3 times. The effects of F. mairei on the growth and paclitaxel accumulation of T. cuspidata cells was investigated by adding the FECS to T. cuspidata cultures, and the active chemicals produced by F. mairei in FECS were separated and identified in this work. Similarly, T. cuspidata suspension cultures were added to the paclitaxel-producing medium of F. mairei for examining the effects of T. cuspidata cells on the growth and the paclitaxel production of F. mairei. Furthermore, based on the principle of the nature endophye-plant symbiosis, F. mairei and T. cuspidata cells were co-cultured in a co-bioreactor for further studying their interactions and the applications in paclitaxel production.The effects of F. mairei on the growth and paclitaxel accumulation of T. cuspidata cells were systemically studied in this work. When FECS was added to the suspension cultures of T. cuspidata cell, it induced the defense responses of T. cuspidata cells, such as medium alkalization, activation of phenylalanine ammonium-lyase (PAL) and phenolics accumulation etc. Meanwhile, FECS increased the paclitaxel yield in T. cuspidata cell cultures. The optimal FECS addition amount was 6-8% (v/v). The palitaxel yield of T. cuspidata cultures treated with 6 mL FECS (6.47 mg/L) was 3.3-fold than control cells (1.97 mg/L). The culture conditions of F. mairei affected the active chemicals in FECS, and which would affect the eliciting efficiencies of FECS. When F. mairei was cultured under higher dissolved oxygen or with longer periods, the FECS obtained had higher eliciting efficiencies. FECS cultured for 9 days with middle dissolved oxygen had the highest eliciting efficiency and resulted in a maximum paclitaxel yield of 8.52 mg/L. The active chemicals in FECS produced by F. mairei were gibberllic acid (GA3) and an oligosaccharide of around 2 kD, they co-mediated the growth and paclitaxel synthesis of T. cuspidata cells.Compared to MJ, FECS treatment caused less negative effects on the growth and viability of T. cuspidata cells. MJ treatment decreased the biomass of T. cuspidata cells by 44%, but only 11% decrease in FECS treatment. Likewise, the viability of T. cuspidata cells treated by FECS was obviously higher than from MJ treatment. The differences of their effects on the cell growth and cell viability of T. cuspidata were likely contributed to the endogenous abscisic acid in T. cuspidata cells. Compared to MJ treatment (3.39μg/g), FECS treatment caused much lower the endogenous abscisic acid (1.28μg/g) of T. cuspidata cells. FECS treatment at day 5 resulted in the maximum paclitaxel yield (6.74 mg/L), while the cultures treated with MJ at day 15 produced the maximum paclitaxel yield (6.23 mg/L), but the paclitaxel productivity by FECS treatment (0.449 mg/L?d) was double that by MJ treatment (0.249 mg/L?d). According to the comparative studies between FECS treatment and MJ treatment, it may be inferred that the induced site by F. mairei to paclitaxel synthesis possibly located prior to 10-deacetylbaccatin III (10-DAB).The effects of T. cuspidata cells on the growth and paclitaxel formation of F. mairei were investigated also in this work. The results show that T. cuspidata culture suspensions of 0-50% (v/v) the addition amount enhanced the growth of F. mairei, but 50-100% (v/v) the addition amount inhibited the growth of F. mairei. The effects of T. cuspidata culture suspensions were different with its culture stages. T. cuspidata culture suspensions of days 10 and 15 enhanced the growth of F. mairei, but T. cuspidata culture suspensions of day 20 inhibited the growth of F. mairei. Furthermore, T. cuspidata culture suspensions had no significant effects on the paclitaxel accumulation in F. mairei, and F. mairei had no significant functions to converte the precursors of paclitaxel in T. cuspidata cultures to paclitaxel.The co-culture of T. cuspidata cells and F. mairei in a co-bioreactor was investigated and the co-culture parameters were optimized. The results show that the yield of paclitaxel by co-culture (25.63 mg/L) was 27-fold that by flask-culture (0.96 mg/L) during 15 days. The paclitaxel yield by co-culture using B5 medium was 2-fold higher than that using MS medium (12.8 mg/L), thus the B5 medium was more conformable in co-culture compared to MS medium. The separate membrane in the center of the co-bioreactor controls the exchange of chemicals between Taxus cultures and fungal cultures. The results indicate that hydrophilic pyroxylin filter was more suitable for the cell growth and the paclitaxel accumulation of Taxus cultures than lipophilic filter. In co-culture, the aeration of fungal cultures affected the production of its active chemicals, and the optimal ventilation ratio was 1:0.86. Additionally, the optimal inoculation time of fungus was at day 5 after culturing Taxus cells.