基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用  

Construction and Application of Plasmid‑based and Xylose‑induced Transformation System of Bacillus subtilis

作　　者：田辉 王帅 刘波[1] TIAN Hui;WANG Shuai;LIU Bo(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《生物技术进展》2021年第6期741-748,共8页Current Biotechnology

基　　金：国家自然科学基金项目(31671802)。

摘　　要：枯草芽孢杆菌(Bacillus subtilis)作为食品级安全菌株,因其具有理化特征清晰、培养发酵方便等特点,广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B.subtilis遗传转化方法存在操作流程繁琐、效率低等缺点,因此,开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B.subtilis感受态的形成,并在B.subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_(xyl)调控comK表达的穿梭质粒pUBC01⁃P_(xyl)⁃comK的菌株B.subtilis K1,经木糖诱导条件优化后,质粒pHY300⁃p43⁃egfp的转化效率达到4.8×10^(3) CFU·μg^(-1)。此外,质粒pUBC01⁃P_(xyl)⁃comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性,同时增强了菌株尤其是生产菌株的性状稳定性。Bacillus subtilis is a kind of food⁃grade safe strain.Due to clear physicochemical characteristics and simple fermenta⁃tion,it is widely used in the high⁃efficiency expression of heterologous proteins and the synthesis of high value⁃added compounds.The traditional genetic transformation methods for B.subtilis have some demerits,such as complicated operation procedures and low efficiency.It is significant important to develop a convenient and highly efficient genetic transformation system.The transcription factor ComK was proved to regulate the formation of competent cells and play an important role in highly efficient transformation of B.subtilis.A shuttle plasmid pUBC01⁃P_(xyl)⁃comK containing a xylose⁃induced P_(xyl)⁃comK expression cassette was constructed and a recombinant strain B.subtilis K1 containing this plasmid was developed.The transfor⁃mation efficiency of plasmid pHY300⁃p43⁃egfp into B.subtilis K1 could reach 4.8×10^(3) CFU·μg^(-1) after optimized xylose induction condition.In addition,pUBC01⁃P_(xyl)⁃comK could be cured via serial subculture under nonselective condition.The system of xylose⁃induced competent cells and plasmid curing greatly increased the convenience of gene editing and strain improvement of B.subtilis and enhanced character stability of the transformed strains,especially production strains.

关 键 词：枯草芽孢杆菌 ComK 感受态 木糖诱导 质粒消除 

分 类 号：Q78[生物学—分子生物学] Q93