Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida

The T7 phage RNA polymerase (RNAP) is a widely used expression platform, but its implementation in non-model microbial hosts poses significant challenges due to cytotoxicity. We constructed an optimized phage phi15-based expression system as alternative to the T7 platform for a wide range of applications in Pseudomonas putida. The new system employs the small phi15 RNAP, driving expression from an orthogonal phi15 promoter. By finetuning expression levels of phi15rnap and introducing a phi15 lysozyme mutant that inhibits phi15 RNAP in uninduced conditions, a stringent system was created with 200-fold inducibility. Moreover, by successfully decoupling cell growth and protein production using phi15 gp16, a host RNAP inhibitor, expression levels could be enhanced further (20%). Apart from creating four optimized platform P. putida hosts and a set of Golden Gate-compatible vectors, we demonstrate the extensive flexibility of the phi15 system. A proof-of-concept expression for industrially relevant fluorinase resulted in 2.5- and 5-fold increased yield compared to other widely-adopted expression systems. The system functions well in combination with several inducer systems, and in a variety of vector-based and genomically integrated set-ups. In conclusion, the phi15 RNAP, promoter, lysozyme and growth-decoupler provide a valuable plug-and-play set of genetic parts for the P. putida toolbox.