Inducible and tunable gene expression systems for Pseudomonas putida KT2440

Area of Science:

• Synthetic Biology
• Microbial Biotechnology
• Metabolic Engineering

Background:

• Inducible and tunable expression systems are critical for efficient microbial production of valuable biochemicals.
• Pseudomonas putida KT2440 is a widely used bacterial chassis for synthetic biology applications.
• Development of novel promoter systems is needed to expand the toolbox for precise gene expression control.

Purpose of the Study:

• To develop and evaluate novel carbon source- and substrate-inducible promoter systems in Pseudomonas putida KT2440.
• To assess the performance of these systems using green fluorescent protein (GFP) as a reporter.
• To identify cost-effective and efficient inducers for gene expression in P. putida.

Main Methods:

• Construction and characterization of five distinct inducible promoter systems in P. putida KT2440.
• Analysis of green fluorescent protein (GFP) expression levels under various inducer conditions.
• Evaluation of inducer agents including glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose.

Main Results:

• Several promoter systems demonstrated efficient induction by low-cost compounds such as 3HP, LA, and xylose.
• The HpdR/P_hpdH system showed efficient induction by both 3HP and LA.
• LvaR/P_lvaA and XutR/P_xutA systems were highly sensitive, responding to low concentrations of LA and xylose, respectively. HexR/P_zwf1 showed weak GFP expression with glucose.
• The performance of these novel systems was comparable to conventional chemical-inducible systems.

Conclusions:

• The newly developed promoter systems offer tunable and inducible gene expression in P. putida KT2440.
• These systems utilize readily available and low-cost inducers, making them suitable for industrial biochemical production.
• The characterized promoter systems enhance the synthetic biology capabilities of P. putida for diverse applications.