Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase complex (PDHC)-deficient derivative reveals the role of PDHC in the fermentative metabolism of glucose.
Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of (13)C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional (13)C,(1)H NMR spectroscopy. Metabolite balancing, biosynthetic (13)C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO(2) and the calculated CO(2) evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO(2) for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO(2), thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO(2) and H(2)) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO(2) generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.
Murarka
Abhishek
A
Department of Chemical and Biomolecular Engineering, RiceUniversity, Houston, Texas 77005, USA.
Clomburg
James M
JM
Moran
Sean
S
Shanks
Jacqueline V
JV
Gonzalez
Ramon
R
D002245:Carbon Dioxide / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology*; D005560:Formate Dehydrogenases; D005947:Glucose / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase; D008190:Lyases / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D011768:Pyruvate Dehydrogenase Complex
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D002245:Carbon Dioxide; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D008190:Lyases; D005947:Glucose