Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida

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.

Automated summary

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.