短小芽孢杆菌基因无痕修饰系统的建立及应用

Establishment and application of a markerless genetic modification system in Bacillus pumilus

为了实现对短小芽孢杆菌(Bacillus pumilus)SCU11基因组连续地无痕改造,提高碱性蛋白酶产量,本文建立了基于Upp基因反向筛选的短小芽孢杆菌基因无痕修饰系统,将碱性蛋白酶基因AprE的1份拷贝无痕插入至短小芽孢杆菌染色体16S rDNA区.摇瓶发酵实验显示,突变菌株碱性蛋白酶活最高达到7125 U/mL,与出发菌株相比提高了33.1%.结果表明利用该系统可实现对短小芽孢杆菌基因组的无痕修饰,对AprE插入突变菌株进行双交换筛选的最终效率约为23.07%.

In order to achieve consecutive and markerless modification in B.pumilus SCU11 genome and increase the production of alkaline protease, we established a markerless genetic modification system for B.pumilus based on counter-selection of Upp gene, and a copy of the alkaline protease gene AprE was inserted into the 16S rDNA region in the genome of B.pumilus. The shaking flask fermentation experiment showed that the alkaline protease activity of mutant strain reached 7125 U/mL, which was 33.1% higher than that of the parental strain. The results showed that the system can achieve markerless genetic modification to the Bacillus pumilus genome, and the final screening efficiency in double-crossover of AprE insertion strains was about 23.07%.