Metabolic engineering of Escherichia Coli central metabolism : physiological effects of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase overproduction

Abstract

An ultimate goal of metabolic engineering is to achieve the maximum product yield. Much effort to date has been made to remove bottlenecks in the terminal pathways. As a result, the rate-controlling steps have been shifted to central pathways. However, manipulations of the central metabolism may face an inherent resistance, which in turn may result in a significant change in cell physiology and restrict the practical application. To approach a rational design of metabolic pathways, it is important to understand how bacteria respond to alterations in central metabolite pools. In an attempt to investigate this problem, a perturbation in phosphoenolpyruvate (PEP) and oxaloacetate (OAA) levels has been applied. PEP and OAA are precursors for the synthesis of amino acids and key metabolites at the junction between glycolysis and the tricarboxylic acid cycle. The flux between PEP and OAA is mediated by the reactions of PEP carboxylase (Ppc) and PEP carboxykinase (Pck). In this study, we attempted to disturb the flux distribution between PEP and OAA by overexpression of Ppc, Pck, or both. By doing so, we found that (1) overexpression of Ppc reduces glucose consumption but without affecting cell growth, which improves the growth yield on glucose as a result. (2) overexpression of Pck increases glucose uptake rate relative to growth rate. In particular, the expression level of glnA (encoding glutamine synthetase) is under the effects of Pck activity. (3) the growth rate on succinate is not optimized. Increased levels of Pck can increase the growth rate on succinate, indicating the level of Pck in E. coli is suboptimal under this condition. (4) simultaneous expression of Ppc and Pck forms futile cycling which stimulates metabolic activities. In responses to futile cycling, E. coli is forced to raise oxygen and glucose consumption rates. As a consequence, the surplus of glucose uptake results in increased fermentation products.