Developing cumate-inducible gene expression systems for application in Pseudomonas putida KT2440.

AIMS: Pseudomonas putida KT2440 possesses a versatile metabolic network and broad substrate utilization capacity, making it as an attractive chassis for synthetic biology applications. However, the limited availability of efficient gene expression tools for precise metabolic regulation has constrained its engineering potential. To address this limitation, we developed two novel cumate-inducible expression systems. METHODS AND RESULTS: In this study, we developed two novel cumate-inducible expression systems by engineering distinct cumate-responsive operators, CuO1 and CuO2, respectively derived from the cym and cmt operons in P. putida KT2440. We assessed the expression strength of the constructed systems and optimized inducer concentrations using eGFP fluorescence intensity as a reporter. Through systematic promoter optimization, operator architecture optimization, and quantitative benchmarking, we achieved substantial improvements in system performance. Finally, we applied the optimized system to bio-transform limonene into perillyl alcohol via a heterologous cytochrome P450 to achieve an increase in the production. CONCLUSION: Cumate-inducible expression system R2CuO1 demonstrated superior gene expression strength than the IPTG-inducible system Ilac. We successfully implemented our optimized cumate-responsive platform for perillyl alcohol bio-production from limonene, achieving a titer of 40.93 mg·L⁻¹, 2.1 times higher than that of the conventional lac expression system.