Burkholderia thailandensis E264/pBMTL3-tdpR发酵生产抗癌药物ThailandepsinA的研究

Production of Thailandepsin A from Burkholderia thailandensis E264/p BMTL3-tdp R Fermentation

在伯克氏菌Bth264野生株产新型抗癌药物Thailandepsin A以及调节基因tdp R正向调控Thailandepsin A生物合成的基础上,利用基因工程菌Bth264/p BMTL3-tdp R发酵生产Thailandepsin A,以提高产量。以0.5%乳糖为诱导剂,确定最佳诱导条件:发酵15 h添加乳糖,诱导时间6 h;通过单因素实验,确定葡萄糖和胰蛋白胨作为碳氮源、装液量65/250 m L以及接种量1%;同时结合优化发酵培养基进行发酵,Thailandepsin A产量达到252.14 mg·L-1,比优化前的产量提高56%;另外在发酵过程中,添加大孔树脂HP-20原位吸附产物,Thailandepsin A产量可达283.75 mg·L-1,比不加树脂提高13.8%;最后,基于RT-PCR和比较Ct值法,基因工程菌和野生菌相比,Thailandepsin A生物合成基因tdp B、tdp C1的转录水平分别提高11.4倍和6.0倍,对应的产量增加4.6倍,从而在很大程度上说明调节基因tdp R的过表达促进生物合成基因转录水平的提高以及产量的增加。

The wild strain of Burkholderia thailandensis E264 can produce Thailandepsin A which is a promising anti-cancer drug. Based on positive regulation of tdpR, agenetic engineered strain of Burkholderia thailandensis E264/pBMTL3-tdpR was constructed to improve the yield of Thailandepsin A. An optimized culturing condition was obtained as： glucose and tryptone as carbon and nitrogen sources, respectively; medium volume 65/250 mL; inoculum ratio 1%; induction by 0.5% lactose for 6 h after culturing for 15 h. After further optimization of the fermentation media, the yield of Thailandepsin A was 252.14 mg.L^-1 which increased over 56% in comparison with the control. In addition, macroporous resin HP-20 was added to adsorb Thailandepsin A in the fermentation, and the yield of Thailandepsin A reached 283.75 mg.L^-1, which increased 13.8% comparing that without HP-20. RT-PCR and the Ct value method were used to compare the genetic engineered strain with the wild strain, and the results show that tdpB and tdpC1 of the genetic engineered strain were enhanced 11.4 and 6.0 times respectively, and the yield of Thailandepsin A was enhanced 4.6 times, which indicates that the over expression of regulatory gene tdpR promoted the transcription of biosynthetic genes and the production.