Verlag des Forschungszentrums Jülich

JUEL-4199
Veit, Andrea
Untersuchungen zum Überflussmetabolismus in Escherichia coli
112 S., 2006



Characterization of overflow metabolism in Escherichia coli
In biotechnology, recombinant E. coli strains are used for the production of fine chemicals and heterologous proteins. During aerobic growth on glucose, E. coli produces acetate, a phenomenon referred to as overflow metabolism. The accumulation of acetate perturbs growth and recombinant protein production. Until now the molecular cause of aerobic acetate excretion is not fully understood. Previous work has revealed gene expression changes correlating with acetate formation. Genes coding for enzymes of both the tricarboxylic acid cycle and the respiratory chain as well as genes of unknown function (i.e. gadE, actP, yjcH and yjeJ) showed differential expression. In this work the following results were obtained:

  • • Deregulation of the sdhCDAB- b0725- sucABCD-operon in E. coli MG1655 was achieved by chromosomal exchange of the Psdh-promoter with the strong and constitutive P tet promoter and brought about a fourfold increase in the specific activities of both succinate dehydrogenase and succinate thiokinase. The specific activity of 2- oxoglutarate dehydrogenase was enhanced 1.7 fold.
    The constructed strain excreted significantly less acetate than the wild type while maintaining high glucose uptake rates. The reduced acetate formation was not accompanied by the formation of other incomplete oxidation products. Instead the formation of carbon dioxide increased as a likely consequence of elevated TCA cycle activity which accounts for the diminished acetate overflow. So aerobic acetate formation is primarily caused by transcriptional control of the sdhCDAB- b0725- sucABCD operon.
  • • The deletion of the genes gadE (yhiE), actP (yjcG), yjcH or yjeJ in E.coli MG1655 did not influence aerobic acetate excretion significantly. This shows that those genes are not essential for glucose overflow metabolism.
  • • Another regulator of the sdhCDAB- b0725- sucABCD-operon in E. coli MG1655 could be identified by DNA-affinity chromatography: YdcI. This regulator which belongs to the lysR family was isolated from cells grown in glucose minimal medium. YdcI is supposed to take part in the regulation of aerobic acetate formation.

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Letzte Änderung: 07.06.2022