便携式细菌 CRISPR 转录激活可实现恶臭假单胞菌的代谢工程。
Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida.
机构信息
Molecular Engineering & Sciences Institute and Center for Synthetic Biology,University of Washington,Seattle, WA, 98195, USA.
Molecular Engineering & Sciences Institute and Center for Synthetic Biology,University of Washington,Seattle, WA, 98195, USA; Department of Chemistry,University of Washington,Seattle, WA, 98195, USA.
出版信息
Metab Eng. 2021 Jul;66:283-295. doi: 10.1016/j.ymben.2021.04.002. Epub 2021 Apr 28.
CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for metabolic pathway engineering. Here we implement CRISPR-Cas transcriptional activation (CRISPRa) in P. putida using a system previously developed in E. coli. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of promoter features. Using this optimized system, we regulate biosynthesis in the biopterin and mevalonate pathways. We demonstrate that multiple genes can be activated simultaneously by targeting multiple promoters or by targeting a single promoter in a multi-gene operon. This work will enable new metabolic engineering strategies in P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species.
CRISPR-Cas 转录调控在细菌中是一种新兴的工具，可以用于代谢途径工程中基因表达的调控。在这里，我们使用先前在大肠杆菌中开发的系统，在 P. putida 中实现了 CRISPR-Cas 转录激活（CRISPRa）。我们提供了一种通过首先优化 CRISPRa 系统组件的表达水平，然后根据启动子特征的系统表征应用有效的 CRISPRa 规则，将 CRISPRa 转移到新宿主的方法。使用这个优化的系统，我们调节了生物蝶呤和甲羟戊酸途径中的生物合成。我们证明了通过靶向多个启动子或通过靶向多基因操纵子中的单个启动子，可以同时激活多个基因。这项工作将在 P. putida 中为新的代谢工程策略铺平道路，并为其他细菌物种中的 CRISPR-Cas 转录调控奠定基础。
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