Abstract
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.
Highlights
BES-0331388/BES-0601549 and EEC-0813570. □S The on-line version of this article contains supplemental text, figures, tables, and references. 1 To whom correspondence should be addressed: 6100 Main St., MS-362, The fermentative metabolism of glucose has been extensively studied in many organisms, especially in the model bacterium Escherichia coli [1]
Kinetics of Glucose Fermentation in Wild-type MG1655 and a pyruvate dehydrogenase complex (PDHC)-deficient Strain and Analysis via Metabolite Balancing— We found that a PDHC-deficient strain (Pdh) grew and metabolized glucose slower than its wild-type MG1655 (Fig. 2)
Similar findings were made when another wild-type E. coli, K12 strain W3110, and its PDHC-deficient derivative were evaluated. These results indicate that PDHC is active during fermentative metabolism of glucose and that this enzyme performs a beneficial function
Summary
BES-0331388/BES-0601549 and EEC-0813570. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental text, figures, tables, and references. 1 To whom correspondence should be addressed: 6100 Main St., MS-362, The fermentative metabolism of glucose has been extensively studied in many organisms, especially in the model bacterium Escherichia coli [1]. In the absence of external electron acceptors (i.e. under fermentative conditions), E. coli converts most of the glucose to a mixture of organic acids (acetate, formate, lactate, and succinate), ethanol, carbon dioxide, and hydrogen (Fig. 1) [1]. Most of these fermentation products are generated from pyruvate (Fig. 1). Pyruvate is located at a major metabolic node linking carbohydrate catabolism to energy generation and biosynthesis and represents a major switch point between respiratory and fermentative metabolism In this context, pyruvate formatelyase (PFL) and the pyruvate dehydrogenase complex (PDHC) are considered the major routes for pyruvate dissimilation in E. coli (Fig. 1) [1].
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