High-Level Protein Expression in Pseudomonas putida Using the Phage-Based pPUT Expression System

High-yield expression systems, such as the phage T7 RNAP-based pET system in Escherichia coli have typically been restricted to a selected chassis organisms due to inherent toxicity. Indeed, emerging production hosts, such as Pseudomonas putida, show that traditional T7-based pET systems are detrimental to growth, limiting stable recombinant protein production in this organism. The recently developed phage phi15-based pPUT expression system addresses this, as it is fully adapted to this host, giving access to exceptional expression levels in P. putida. The pPUT system is highly modular, with elements to tweak stringency or decouple growth from production to further enhance yields. This chapter presents how to achieve this phi15-based expression system in P. putida, either for a strain of interest or dedicated strains for optimal expression. It outlines the workflow starting from initial selection of the modules and their use case. Once a system is selected, the workflow encompasses cloning gene(s) of interest into the expression vector pPUT-ST via SEVAtile assembly, transforming it to P. putida and subsequently introducing the selected modules. Once the strains are prepared, a lab scale expression of the gene of interest is achieved through a series of incubation and induction steps. The expression system has demonstrated up to fivefold increased protein yields compared other widely used systems and is both modular and tunable. It, thus, makes high-yield recombinant protein production available in P. putida for a wide range of applications and use cases.