Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS.
Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and inactive in a high acetate producer, JM109. To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates. The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques. These distributions are next validated by predicting the NMR spectrum of glutamate. Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained 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 the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt.
Noronha
S B
SB
Biotechnology Unit, LCDB, NIDDK, NIH, Bethesda, Maryland 20892, USA.
Yeh
H J
HJ
Spande
T F
TF
Shiloach
J
J
D036002:ADP Ribose Transferases; D000105:Acetyl Coenzyme A / Q000378:metabolism; D001427:Bacterial Toxins; D002247:Carbon Isotopes; D002952:Citric Acid Cycle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000493:pharmacokinetics; D006038:Glyoxylates / Q000378:metabolism*; D016018:Least-Squares Analysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D062907:Oxaloacetic Acid / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000105:Acetyl Coenzyme A; D001427:Bacterial Toxins; D002247:Carbon Isotopes; D005098:Exotoxins; D005947:Glucose; D006038:Glyoxylates; D062907:Oxaloacetic Acid; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C031150:glyoxylic acid