Department of Chemical and Biomolecular Engineering
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Browsing Department of Chemical and Biomolecular Engineering by Author "Antoniewicz, Maciej Robert"
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Item Enzyme I facilitates reverse flux from pyruvate to phosphoenolpyruvate in Escherichia coli(Nature Publishing Group, 2017-01-27) Long, Christopher P; Au, Jennifer; Sandoval, Nicholas R; Gebreselassie, Nikodimos A; Antoniewicz, Maciek R; Christopher P. Long; Jennifer Au; Nicholas R. Sandoval; Nikodimos A. Gebreselassie; Maciek R. Antoniewicz; Antoniewicz, Maciej RobertThe bacterial phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) consists of cascading phosphotransferases that couple the simultaneous import and phosphorylation of a variety of sugars to the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. As the primary route of glucose uptake in E. coli, the PTS plays a key role in regulating central carbon metabolism and carbon catabolite repression, and is a frequent target of metabolic engineering interventions. Here we show that Enzyme I, the terminal phosphotransferase responsible for the conversion of PEP to pyruvate, is responsible for a significant in vivo flux in the reverse direction (pyruvate to PEP) during both gluconeogenic and glycolytic growth. We use 13C alanine tracers to quantify this back-flux in single and double knockouts of genes relating to PEP synthetase and PTS components. Our findings are relevant to metabolic engineering design and add to our understanding of gene-reaction connectivity in E. coli.Item Evolution of E. coli on [U-C-13] Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology(Public Library Science, 3/10/16) Sandberg,Troy E.; Long,Christopher P.; Gonzalez,Jacqueline E.; Feist,Adam M.; Antoniewicz,Maciek R.; Palsson,Bernhard O.; Troy E. Sandberg, Christopher P. Long, Jacqueline E. Gonzalez, Adam M. Feist, Maciek R. Antoniewicz, Bernhard O. Palsson; Antoniewicz, Maciej RobertC-13-Metabolic flux analysis (C-13-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for similar to 1000 generations on uniformly labeled C-13-glucose, a commonly used isotope for C-13-MFA. Phenotypic characterization of these evolved strains revealed similar to 40% increases in growth rate, with no significant difference in fitness when grown on either labeled (C-13) or unlabeled (C-12) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between C-12 and C-13-glucose as a carbon source for E. coli growth.