Production of recombinant proteins using multiple‐copy gene integration in high‐cell‐density fermentations of Ralstonia eutropha

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Abstract
We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid‐based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion‐body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple‐copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high‐cell‐density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 114–120, 2003.