Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using C-13-NMR/MS

Acetate accumulation is a common problem observed in aerobic high cell dens ity Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and ina ctive in a high acetate producer, JM109. To further investigate this hypoth esis, we now develop a model for the incorporation of C-13 from uniformly l abeled glucose into key TCA cycle intermediates. The C-13 isotopomer distri butions of oxaloacetate and acetyl-CoA are first determined using NMR and M S techniques. These distributions are next validated by predicting the NMR spectrum of glutamate, Under steady state isotopic conditions, and with kno wledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtain ed with respect to the flux through the PPC anaplerotic shunt. We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109. Further, in BL21, the flux through th e TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA c ycle flux is only third of the flux through the PPC shunt. (C) 2000 John Wi ley & Sons, Inc.