Overexpression of metabolic enzymes at the junction of glycolysis and the TCA cycle in Escherichia coli: physiological effects and application

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references (leaves 58-65).Issued also on microfiche from Lange Micrographics.The metabolism of Escherichia coli has central importance in biochemical engineering, metabolic engineering, and molecular biology. This study relates to all three disciplines. In biochemical engineering, previous studies show that overexpressing the enzyme phosphoenolpyruvate synthase (Pps) has increased the yield of the aromatic precursor 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) from glucose by two- fold compared to previous methods. In this study, Pps overexpression is again applied to DAHP production, but this time, independent modulation of three different overexpression enzymes (DAHP synthase (AroG), transketolase (Tkt), and Pps) was allowed to attempt to optimize DAHP production. In addition to using glucose for this optimization, xylose was also used as a carbon source. With glucose, pathway analysis shows the potential for carbon flux recycle due to Pps because phosphoenolpyruvate (PEP, a precursor for DAHP production) is converted to private during glucose uptake by the phosphotransferase (PTS) system. However, with xylose, pathway analysis showed no theoretical need for Pps overexpression because xylose does not use the PTS system. With xylose, DAHP production is optimized during AroG overexpression alone; Pps helped produce DAHP only slightly when AroG was not overexpressed. The implications of this work to metabolic engineering and molecular biology are the physiological effects of enzyme overexpression on central metabolism. Overexpression of glycolytic phosphoenolpyruvate carboxylase (Ppc) and gluconeogenic phosphoenolpyruvate carboxykinase (Pck) show physiological effects of increased glucose conscription, along with other effects previously observed. When both enzymes are overexpressed together in a futile cycle, more pronounced effects occur including private excretion. Analysis of internal metabolite concentrations shows that fructose 1,6-diphosphate (FDP) concentrations increased for cases increased glucose consumption. This provides some insight into the types of feedback mechanisms that occur within the bacteria. The effects decreased somewhat when RelA mutants overexpressed the futile cycle enzymes. This difference is due to differences in protein concentrations of overexpressed enzymes. The results show the benefits and ramifications of enzyme overexpression