To determine whether intracellular ATP levels can be affected, Escherichia coli overexpressing phosphoenolpyruvate carboxykinase (pck) or phosphoenolpyruvate carboxylase (ppc) were grown in glucose minimal medium. The Pck-overexpressing cells showed approximately twice the intracellular ATP concentration, with 22% slower growth than the Ppc-overexpressing strain. This unexpected result of higher ATP coupled with slower growth is discussed based on transcriptome analysis.