Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida
Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida
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DOI：
10.1016/j.ymben.2021.04.002
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发表时间：
2021-05-17
影响因子：
8.4
通讯作者：
Zalatan, Jesse G.
中科院分区：
文献类型：
--
作者：
Kiattisewee, Cholpisit;Dong, Chen;Zalatan, Jesse G.
细菌中的CRISPR-Cas转录编程是一种新兴的工具，用于调节代谢途径工程的基因表达。在这里，我们使用先前在E. putida中开发的系统在P. putida中实现CRISPR-Cas转录激活（CRISPRa）。杆菌我们提供了一种将CRISPRa转移到新宿主的方法，首先优化CRISPRa系统组分的表达水平，然后基于启动子特征的系统表征应用有效CRISPRa的规则。使用这个优化的系统，我们调节生物蝶呤和甲羟戊酸途径的生物合成。我们证明了多个基因可以通过靶向多个启动子或靶向多基因操纵子中的单个启动子来同时激活。这项工作将在恶臭假单胞菌中实现新的代谢工程策略，并为其他细菌物种中的CRISPR-Cas转录编程铺平道路。
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.