Fabrication and characterization of a robust and strong bacterial promoter from a semi-rationally engineered promoter library in Bacillus subtilis
Fabrication and characterization of a robust and strong bacterial promoter from a semi-rationally engineered promoter library in Bacillus subtilis
复制标题
从枯草芽孢杆菌半合理工程启动子库中构建和表征强大的细菌启动子
DOI：
10.1016/j.procbio.2017.06.024
复制
发表时间：
2017-10-01
影响因子：
4.4
通讯作者：
Zhou, Zhemin
中科院分区：
文献类型：
--
作者：
Han, Laichuang;Suo, Feiya;Zhou, Zhemin
稳健启动子在合成生物学中具有重要用途。然而，这些启动子的重要生物组分在枯草芽孢杆菌中仍然有限。本研究从随机突变构建的启动子文库中获得了一系列P-srfA基因的变异启动子。通过对文库的筛选，发现变异启动子Pv 1的转录活性最高，约为天然启动子P-srfA的1.56倍。此外，Pv 1能够在培养期间以不断增加的水平触发GFP表达。Pv 1的耐用性通过过量生产天冬氨酸酶（aspA）得到证实。B。过量生产aspA的枯草芽孢杆菌在培养期间表现出正常的细胞生长和不断增加的产量。最后，由Pv 1变体触发的aspA表达在B中显示出较高的产量和aspA表达水平。枯草芽孢杆菌的P-srfA。这些结果表明，细菌启动子-10区域附近序列的随机突变显著影响启动子的转录活性。Pv 1具有较高的活性和鲁棒性，在B基因表达系统和遗传电路中具有潜在的应用价值。枯草杆菌。
Robust promoters have substantial usage in synthetic biology. The important biological components of these promoters, however, remain limited in Bacillus subtilis. In this study, an array of variant promoters derived from P-srfA was from a promoter library constructed by randomized mutation. By screening the library, the variant promoter Pv1 displayed the highest transcriptional activity, which was approximately 1.56-fold higher than that of native P-srfA. Moreover, Pv1 was able to trigger GFP expression at constantly increased levels over the culture period. The robustness of Pv1 was confirmed by the over-production of aspartase (aspA). B. subtilis that overproduced aspA exhibited normal cell growth and a constantly increased yield over the cultural period. Finally, aspA expression triggered by the Pv1 variant displayed a higher yield and expression levels of aspA in B. subtilis compared to those of native P-srfA. These results suggest that randomized mutation of sequences adjacent to the -10 region of the bacterial promoter significantly influenced the transcription activity of the promoter. Pv1, which has high activity and robustness, has potential applications in gene expression systems and genetic circuits in B. subtilis.