Department of Bioengineering, Rice University, 6100 Main St., Houston, TX, 77005, USA.
Department of Biosciences, Rice University, 6100 Main St., Houston, TX, 77005, USA.
Nat Commun. 2019 Jul 15;10(1):3099. doi: 10.1038/s41467-019-10906-6.
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.
革兰氏阳性菌枯草芽孢杆菌表现出复杂的时空基因表达信号。尽管光遗传学工具非常适合研究此类过程，但尚未为该生物体设计出此类工具。在这里，我们将包含绿色/红色光可逆双组分系统 CcaSR、用于产生藻胆色素原（PCB）的两种代谢酶以及用于控制感兴趣基因转录的输出启动子的蓝细菌光传感器途径导入枯草芽孢杆菌。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来增强 PCB 的产生，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来增加动态范围。我们的最终设计表现出超过 70 倍的激活和快速响应动力学，非常适合研究广泛的基因调控过程。此外，我们开发的用于导入该途径的合成生物学方法应该使枯草芽孢杆菌在未来更容易进行工程改造。
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