Untersuchungen zum Überflussmetabolismus in Escherichia coli

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 Ptet
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.