Physical decoupling of XylS/Pm regulatory elements and conditional proteolysis enable precise control of gene expression in Pseudomonas putida

Most of the gene expression systems available for Gram‐negative bacteria are afflicted by relatively high levels of basal (i.e. leaky) expression of the target gene(s). This occurrence affects the system dynamics, ultimately reducing the output and productivity of engineered pathways and synthetic circuits. In order to circumvent this problem, we have designed a novel expression system based on the well‐known XylS/Pm transcriptional regulator/promoter pair from the soil bacterium Pseudomonas putida mt‐2, in which the key functional elements are physically decoupled. By integrating the xylS gene into the chromosome of the platform strain KT2440, while placing the Pm promoter into a set of standard plasmid vectors, the inducibility of the system (i.e. the output difference between the induced and uninduced state) improved up to 170‐fold. We further combined this modular system with an extra layer of post‐translational control by means of conditional proteolysis. In this setup, the target gene is tagged with a synthetic motif dictating protein degradation. When the system features were characterized using the monomeric superfolder GFP as a model protein, the basal levels of fluorescence were brought down to zero (i.e. below the limit of detection). In all, these novel expression systems constitute an alternative tool to altogether suppress leaky gene expression, and they can be easily adapted to other vector formats and plugged‐in into different Gram‐negative bacterial species at the user's will.