A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

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Abstract
We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus ) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli -based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli .