枯草芽孢杆菌 bsrE /SR5转录后调控系统构建与优化

Construction and optimization of bsrE/SR5 posttranscriptional regulation system in Bacillus subtilis

枯草芽孢杆菌被认为是安全的菌株,其作为底盘细胞在发酵领域具有独特的优势。然而,与使用最广泛的原核生物宿主大肠杆菌和真核生物宿主酿酒酵母相比,枯草芽孢杆菌由于缺乏基因调控元件在生物技术领域受到限制。该研究通过与bsrG/SR4的比较,成功构建了1套以I型毒素-抗毒素系统bsrE/SR5为基础的基于小RNA的新型调控系统,抑制效率达到88.8%。该系统的建立为枯草芽孢杆菌基因调控提供了新的转录后水平的调控工具。其次,我们对SR5进行了截短表达。仅保留终止子序列的SR5截短突变体也能保持较高的抑制效率(83.8%)。截短后的SR5不仅能进一步减小菌体的代谢压力;同时因为片段较小(仅37 nt)能够直接通过引物添加至载体,为其应用提供了便利。bsrE/SR5转录后调控系统的构建丰富了MS-DOS[Modulation via the small RNA(sRNA)-dependent operation system]调控系统库,为枯草芽孢杆菌基因调控提供更多工具,对枯草芽孢杆菌在基础和应用研究中的发展起到了推动作用。

Bacillus subtilis is a generally recognized as safe(GRAS)organism,which has unique advantages in the fermentation field as a chassis cell.However,compared with the most widely used prokaryotic production host Escherichia coli and eukaryotic host Saccharomyces cerevisiae,B.subtilis is limited in the biotechnological research owing to the lack of genetic regulatory elements.In the present study,through comparison with bsrG/SR4,a new modulation via the small RNA(sRNA)-dependent operation system(MS-DOS)based on another type I toxin-antitoxin system bsrE/SR5 was constructed successfully,whose inhibition rate was 88.8%.This system provided a new post-transcriptional regulatory tool for B.subtilis gene regulation.Then,the truncation of SR5 was investigated.The SR5 mutant maintained high activity(83.8%inhibition rate)in the case of retaining only the terminator sequence.The truncated SR5 could further reduce the metabolic pressure of the bacteria,and thus could be directly added to the plasmid by designing primers since its fragment is short(only 37 nt),providing convenience for its application.The construction of the bsrE/SR5 post-transcriptional regulatory system enriches the MS-DOS regulatory system library which will provide more gene regulation tools for B.subtilis and promote the development of B.subtilis in basic and applied research.