CRISPR interference-mediated gene regulation in Pseudomonas putida KT2440

Area of Science:

• Synthetic Biology
• Microbial Engineering

Background:

• Targeted gene regulation is crucial for modifying microbial phenotypes.
• The CRISPR interference (CRISPRi) system offers a powerful tool for gene regulation.

Purpose of the Study:

• To establish and validate a CRISPRi system for gene regulation in Pseudomonas putida KT2440.
• To demonstrate the system's efficacy in controlling both exogenous and endogenous genes.
• To enhance microbial production of valuable compounds through targeted gene repression.

Main Methods:

• Constructed a single CRISPRi plasmid expressing nuclease-deficient Cas9 and single guide RNA.
• Utilized inducible (P_{rha BAD}) and constitutive (J23119) promoters for gene expression control.
• Targeted exogenous green fluorescent protein and endogenous glpR genes in P. putida KT2440.

Main Results:

• Successfully repressed target genes, confirmed by reduced fluorescence and growth lag.
• CRISPRi-mediated repression of glpR improved cell growth and glycerol utilization.
• Enhanced mevalonate production in engineered P. putida by repressing glpR.

Conclusions:

• The developed CRISPRi system provides simple and efficient gene regulation in P. putida KT2440.
• This system is a robust tool for reprogramming P. putida for microbial cell factory applications.
• CRISPRi facilitates the development of microbial platforms for producing industrially valuable products.