A novel high-cell density recombinant protein production system based on Ralstonia eutropha

Recombinant protein production has traditionally relied on organisms with well developed molecular biological tools, such as E. coli. However, E. coli is not suited for large industrial scale production due to its propensity to produce organic acids under oxygen-limiting conditions, inclusion body formation and proteolysis of the recombinant proteins. In this study, 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 E. coli based protein expression, in particular the propensity of inclusion body formation during high level protein expression. Using a proteomics approach we identified promoters that can be induced by manipulation of simple process parameters or media composition 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 organophospho hydrolase (OPH), a model enzyme prone to inclusion body formation in E. coli. In addition, we have developed strategies based on multiple copy integration and T7 RNA polymerase-based expression to further enhance the recombinant protein titers. Using the T7 RNA polymerase approach, we have been able to produce approximately 9.5 g/liter of soluble active OPH. This represents the highest titer reported to date for this enzyme and is approximately 90 times greater than expression levels reported in E. coli.