Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida

The T7 RNA polymerase is considered one of the most popular tools for heterologous gene expression in the gold standard biotechnological host Escherichia coli. However, the exploitation of this tool in other prospective hosts, such as the biotechnologically relevant bacterium Pseudomonas putida, is still very scarce. The majority of the existing T7-based systems in P. putida show low expression strengths and possess only weak controllability. A fundamental understanding of these systems is necessary in order to design robust and predictable biotechnological processes. To fill this gap, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in P. putida, using the enhanced Green Fluorescent Protein (eGFP) as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic setup of the system, such as slow growth and low protein production rates. By replacing the T7 phage-inherent TΦ terminator downstream of the heterologous gene with the synthetic tZ terminator, growth and protein production rates improved drastically, and the T7 RNA polymerase system reached a productivity level comparable to that of an intrinsic RNA polymerase-based system. Furthermore, we were able to show that the system was saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. This saturation indicates an essential role for the ribosome binding sites of the T7 RNA polymerase since, in an oversaturated system, cellular resources are lost to the synthesis of unnecessary T7 RNA polymerase. Eventually, we combined the experimental data into a model that can predict the eGFP production rate with respect to the relative strength of the ribosome binding sites upstream of the T7 gene.