Optogenetic control of Bacillus subtilis gene expression
Optogenetic control of Bacillus subtilis gene expression
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DOI：
10.1038/s41467-019-10906-6
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发表时间：
2019-07-15
影响因子：
16.6
通讯作者：
Tabor, Jeffrey J.
中科院分区：
文献类型：
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作者：
Castillo-Hair, Sebastian M.;Baerman, Elliot A.;Tabor, Jeffrey J.
革兰氏阳性菌枯草芽孢杆菌表现出复杂的空间和时间的基因表达信号。虽然光遗传学工具是研究这些过程的理想工具，但没有一个是针对这种生物体设计的。在这里，我们端口的蓝藻光传感器的途径，包括绿色/红色光可逆的双组分系统CcaSR，两个代谢酶的发色团藻蓝胆素（PCB）的生产，和输出启动子控制感兴趣的基因转录到B。枯草杆菌。在最初的非功能性设计之后，我们优化了途径基因的表达，通过生物合成酶的翻译融合增强PCB生产，设计了一个强大的嵌合输出启动子，并使用小型化的光传感器激酶增加动态范围。我们的最终设计展示了超过70倍的激活和快速响应动力学，使其非常适合研究广泛的基因调控过程。此外，我们开发的移植这一途径的合成生物学方法应该可以使B。枯草芽孢杆菌在未来更容易工程化。
The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.