Metabolism of recombinant E. coli

National Institute Of Diabetes And Digestive And 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 genes (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.[unreadable] To further understand this phenomenon, several key genes producing enzymes involved in acetate production and consumption were knocked out in E. coli B. Both bacterial growth and metabolite production for these knockout strains were analyzed. [unreadable] Deletions of genes involved in both acetate utilization such as acs and aceA, and acetate production such as poxB did not have a significant effect on the acetate production pattern of the E. coli B. Only the following deletions reduced acetate production: ackA, pta and ackA, pta ackA and poxB. Metabolite analysis of the strains without ackA and pta or ack, pta and poxB showed no significant effect in succinate, citrate, malate, and lactate accumulation, but these strains did show an effect on acetate, pyruvate and formate. Acetate production of 1.67 g/L in the mutant strain lacking ackA, pta and poxB may be an indication of an alternative acetate producing pathway. Additional outcome of this study is the observation that deletion of poxB is followed by activation of the TCA cycle, gluconeogenesis, and anaplerotic pathways, thus contributing to a better protein producing host cell.