A modular T7-based gene expression platform in Pseudomonas putida: Construction and in silico analysis

Abstract

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 biotechnological hosts is still very scarce. To this intent, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in Pseudomonas putida, using the model protein eGFP as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic set-up of the system, such as slow growth and low protein production rates. By replacing the T7-phage inherent T{Phi} 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 could show that the system is saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. The saturation points to 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/494349v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@2f5a0corg.highwire.dtl.DTLVardef@c0e7aborg.highwire.dtl.DTLVardef@a9ca83org.highwire.dtl.DTLVardef@12e65fa_HPS_FORMAT_FIGEXP M_FIG C_FIG