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

ABSTRACT: 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 cre

SUBMITTER: Lammens EM 

PROVIDER: S-EPMC11794488 | biostudies-literature | 2025 Feb

REPOSITORIES: biostudies-literature