【作者】 李永成；

【导师】 陶文沂；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 为了研究产紫杉醇内生真菌—美丽镰刀菌(Fusarium mairei)与东北红豆杉(Taxus cuspidata)细胞在合成紫杉醇中的关系。首先,将Fusarium mairei培养在B5培养基中,去菌丝,浓缩滤液至3倍,获得内生真菌培养液(Fungal endophyte culture supernatants, FECS)。将FECS添加到植物细胞悬浮培养物中,以考察内生真菌代谢物对植物细胞生长与紫杉醇合成的影响,并对其活性成分进行了分离鉴定。同样也将植物细胞悬浮培养液加入到内生真菌产紫杉醇培养基中,以考察植物细胞对美丽镰刀菌生长与紫杉醇合成的影响。此外,利用内生真菌与寄主植物在自然界的共生机理,将内生真菌与植物细胞耦合培养在耦合生物反应器中,进一步考察它们的关系以及在紫杉醇生产中的应用。系统研究了美丽镰刀菌对东北红豆杉细胞生长与紫杉醇合成的影响。当FECS加入到东北红豆杉悬浮细胞培养物中时,可引起东北红豆杉细胞的防御反应,如:培养基碱化、PAL激活、和酚的积累,并促进紫杉醇的合成。FECS的最佳添加量在6-8% (v/v)。6 mLFECS处理,其紫杉醇含量(6.47 mg/L)是对照(1.97 mg/L)的3.3倍。美丽镰刀菌的培养条件影响其活性成分的产生,从而影响FECS的诱导效益,长时间培养与高溶氧培养条件下的FECS有较好的诱导效应。其中以9天中等溶氧水平培养的FECS效果最好,其紫杉醇含量达8.52 mg/L,是对照的4.3倍。美丽镰刀菌产生的活性物质是赤霉素GA3与一分子量为～2 kD的寡糖。它们协同调节东北红豆杉细胞的生长与紫杉醇合成。FECS与茉莉酸甲酯(Methyl jasmonate, MJ)比较,FECS对红豆杉细胞生长与活性的影响小,MJ处理可使东北红豆杉细胞最终的生物量下降44%,而FECS处理仅使东北红豆杉细胞最终生物量下降11%。同时MJ处理对细胞活性的影响也明显高于FECS处理。MJ处理与FECS处理对红东北豆杉细胞生长与活性的差异可能与东北红豆杉细胞内内源脱落酸有关,MJ处理可诱导东北红豆杉细胞内产生较高浓度的脱落酸(3.39μg/g),而FECS处理的脱落酸相对较低(1.28μg/g)。FECS在红豆杉细胞培养早期(5天)添加可获得最佳紫杉醇产量(6.74 mg/L),而MJ处理在红豆杉细胞培养中后期(15天)添加可获得最佳紫杉醇产量(6.23 mg/L),但FECS处理所获得的紫杉醇生产力(0.449 mg/L?d)是MJ处理(0.249 mg/L?d)的双倍。通过与MJ的比较研究发现,美丽镰刀菌对紫杉醇合成的诱导作用的位点可能在10-去乙酰巴卡亭III(10-deacetylbaccatin III ,10-DAB)代谢途径前。系统研究了东北红豆杉细胞对美丽镰刀菌生长与紫杉醇合成的影响。实验结果表明,东北红豆杉细胞悬浮培养液添加量在0-50%(v/v)时,对美丽镰刀菌生长有促进作用,而在50-100%(v/v)时,则抑制美丽镰刀菌生长。不同培养阶段的植物细胞悬浮培养液对美丽镰刀菌生长有不一样的影响。10天与15天的植物细胞悬浮培养液对内生真菌的生长有促进作用,而20天的植物细胞悬浮培养液对内生真菌的生长有抑制作用。但植物细胞悬浮培养液对美丽镰刀菌合成紫杉醇没有明显影响。美丽镰刀菌对植物细胞悬浮培养液中的紫杉醇前体物无明显转化作用。利用生物耦合反应器,对东北红豆杉细胞与美丽镰刀菌的耦合培养进行了系统研究,并对耦合培养的相关参数进行了优化。结果表明,15天耦合培养中植物细胞紫杉醇的含量(25.63 mg/L)是三角瓶培养(0.96 mg/L)的27倍。B5培养基与MS培养基相比更适合于耦合培养,前者在耦合培养中所得的紫杉醇含量比后者(12.8 mg/L)要高一倍。耦合培养罐的中间隔膜控制植物细胞与内生真菌间的物质交换。亲水膜比油膜更适合于植物细胞的生长与紫杉醇的合成。在耦合培养中,内生真菌培养物的通气条件影响内生真菌活性物质的产生,最佳的通气比为1:0.86。内生真菌接入耦合罐的最佳时间是在植物细胞培养后的第五天。更多还原

【Abstract】 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.更多还原