Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium

Abstract

Isotope effects on the central carbon metabolism due to the addition of variable amounts of D₂O (0 to 70%) were investigated with biosynthetically directed fractional ¹³C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly ¹³C-labeled glucose as the sole carbon source. The resulting ¹³C-labeling patterns in the amino acids were analysed by two-dimensional [¹³C,¹H]-correlation spectroscopy. With the aforementioned growth conditions, higher D₂O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA. Furthermore, the addition of D₂O affected the C₁ metabolic pathways that involve Ser and Gly. Otherwise the E. coli cells showed identical topologies of the active biosynthetic pathways in H₂O and at elevated D₂O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D₂O. Cells that had been adapted for growth in D₂O exhibited the same response to the presence of D₂O in the nutrient medium as non-adapted cells. Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed.