期刊
METABOLIC ENGINEERING
卷 66, 期 -, 页码 283-295出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2021.04.002
关键词
CRISPR activation; Pseudomonas putida; Biopterin; Mevalonate
资金
- NSF [1817623, 1844152]
- DOE [DE-EE0008927]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1817623] Funding Source: National Science Foundation
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [1844152] Funding Source: National Science Foundation
This study successfully transferred the CRISPRa system to P. putida by optimizing the expression levels of CRISPRa system components and applying rules based on systematic characterization of promoter features, enabling regulation of biosynthesis in the biopterin and mevalonate pathways. Multiple genes can be activated simultaneously by targeting multiple promoters or a single promoter in a multi-gene operon, paving the way for new metabolic engineering strategies in P. putida and other bacterial species.
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.
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