红豆杉内生真菌中巴卡亭Ⅲ产生菌的筛选及培养基的初步优化

Screening of Endophytic Fungi Producing BaccatinⅢfrom Taxus Yunnanensis and Preliminary Optimization of the Culture Media

目的:筛选出产巴卡亭Ⅲ的红豆杉内生真菌,为抗癌药物紫杉醇提供一个新的来源。方法:从云南红豆杉树皮中分离纯化出内生真菌,对其进行液体发酵培养,过滤,收集菌丝体并破碎,用二氯甲烷萃取,萃取液进行薄层层析,与巴卡亭Ⅲ标准品有相同比移值的样品再利用高效液型色谱与标样进行比照,进而筛选出产巴卡亭Ⅲ的内生真菌。同时以发酵培养基基础,对筛选出的真菌进行碳源和氮源的优化。并在此基础上探讨几种常见的紫杉烷类化合物代谢诱导剂乙酸钠、苯甲酸钠、苯丙氨酸和亮氨酸的浓度对巴卡亭Ⅲ积累的影响。结果:得到一株产巴卡亭Ⅲ的内生真菌NSZJ043,该菌株易培养,生长迅速,经鉴定属于曲霉属。NsZJ043产巴卡亭Ⅲ的培养条件的初步优化结果表明:最适碳源、氮源分别是蔗糖和蛋白胨;在含20g/L蔗糖、1．5g/L蛋白胨、0．1 mmol/L乙酸钠、0．01mmol/L苯甲酸钠的优化培养基中培养8d,巴卡亭Ⅲ含量为34．6μg/L。结论:筛选出一株产巴卡亭Ⅲ的内生真菌,其具有优良的发酵特性,巴卡亭Ⅲ前体物质苯甲酸钠和乙酸钠对巴卡亭Ⅲ的合成有促进作用;苯丙氨酸和亮氨酸对其含量影响不显著,优化后NSZJ043产巴卡亭Ⅲ含量有较大的提高。

Objective： Baccatin Ⅲ is the precursor of the anticancer drug taxol （Paclitaxel）. The semi-synthesis of taxol is the important method to afford the consumption of the cancer patients. The purpose of this work was to isolate and identify baccatin Ⅲ-producing endophyfic fungi from the bark of the Taxus Yurmanensis and optimize the culture conditions. Methods ： Potato dextrose agar （PDA） culture medium was used to isolate endophyte from the bark of the Taxus Yunnanensis. The isolated fungi were incubated in 250 mL conical flasks containing 100 mL of semi-synthesis medium at 28℃, and the mycelium was obtained by filtering. Methylene chloride was used to extract the organic compounds from the disrupted mycelium. The baccatin Ⅲ -producing ability of the isolates was identified by the technology of thin layer chromatography （TLC） and high performance liquid chromatography （HPLC）. The optimal carbon and nitrogen source were tested for mycelium fermentation. Several compounds, known previously as activators of taxane synthesizing, including sodium benzoate, sodium acetate, 1-phenylalanine, and leucine, were tested for their effects on accumulation of baccatin Ⅲ. Results： More than 200 strains of fungi were isolated and an baccatin HI-producing endophytic fungus NSZJ043 was isolated from the bark of the Taxus Yunnanensis. The strain NSZJ043 belongs to Aspergillus sp. according to the morphology of colony and characteristics of spores. The optimal carbon and nitrogen source for mycelium fermentation was saccharose and peptone, respectively. The yielding of baccafin Ⅲ was up to 34.6μg/L after incubated in the optimal medium containing 20g/L saccharose, 1.5g/L peptone, 0.1mmol/L sodium acetate and 0.01mmol/L sodium benzoate for 8 days. Conclusions：An baccatin Ⅲ-producing endophyfic fungus NSZJ043 was isolated from the bark of the Taxus Yunnanensis. Sodium benzoate and sodium acetate could promote the accumulation of baccatin Ⅲ as the precursor, but phenylalanine or leucine has litde effect on the biosynthesis of baccafin Ⅲ. Isolation of the baccatin Ⅲ-producing fungi provides a new approach to taxol and taxane extraction.