Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis.
The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities.
Hoffmann
Frank
F
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Weber
Jan
J
Rinas
Ursula
U
D002952:Citric Acid Cycle / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D008291:Malate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D010100:Oxygen