phi15-based expression system for Pseudomonas putida

The T7 expression system is widely used in Synthetic Biology, but poses challenges in Pseudomonas putida due to cytotoxicity. In this study, we present an optimized phi15-based expression system as a valuable alternative to finally translate advanced T7-based applications in this emerging host. The phi15 system employs the small phi15 RNA polymerase (RNAP), driving expression from the orthogonal phi15 promoter. By finetuning expression levels of the phi15 RNAP in various ways and introducing the phi15 lysozyme (G3RQ) mutant to inhibit the phi15 RNAP in uninduced conditions, a stringent system was created with a inducibility of 200-fold. In addition, by successfully decoupling cell growth and protein production using phi15 gp16, a host RNAP inhibitor, expression levels were further enhanced by 20%. Apart from the creation of four optimized and well-characterized platform strains (P. putida P15, P15-L, P15-GD and P15-LGD) and a GoldenGate-compatible vector set, the obtained results also indicate the enormous flexibility of the phi15 system. It functions well in combination with several inducer systems in a high variety of set-ups, both vector-based or genomically integrated. As such, the phi15 RNAP, promoter, lysozyme and growth-decoupler constitute valuable tools for the P. putida toolbox, as a plug-and-play set of genetic parts.