Metabolism of recombinant E. coli

Diabetes, Digestive, Kidney Diseases

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Abstract

In our previous work using indirect analysis we established that the glyoxylate shunt, the TCA cycle and acetate uptake by acetyl-CoA synthetase, are more active in E.coli B than in E.coli K. By implementing the system biology approach we showed that in addition to what we knew, other metabolic pathways are active differently in the two strains. These are glucoenogensis, sfcA shunt, ppc shunt, glycogen biosynthesis and fatty acid degradation. It was found that in E.coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase -acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconegensis enzyme phosphoenolpyruvate synthase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF), cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations, these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis (fbp, pckA, and ppsA) lead to glycogen accumulation and are constitutively active in E. coli BL21 regardless of glucose feeding strategy.[unreadable] Resulting from this work is the development of a statistical analysis method which implements a semiparametric algorithm, based on a density ratio model. By using this method it was possible to compare and quantify the expression patterns of groups of genes involved in several central carbon metabolic pathways.