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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*

Open AccessPublished:July 28, 2010DOI:https://doi.org/10.1074/jbc.M110.121095
      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 13C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional 13C,1H NMR spectroscopy. Metabolite balancing, biosynthetic 13C 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 CO2 and the calculated CO2 evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO2 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 CO2, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO2 and H2) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO2 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.

      Introduction

      The fermentative metabolism of glucose has been extensively studied in many organisms, especially in the model bacterium Escherichia coli (
      • Sawers R.G.
      • Clark D.P.
      ). As shown in Fig. 1, glucose is concomitantly transported and phosphorylated by the phosphoenolpyruvate-dependent phosphotransferase system (
      • Deutscher J.
      • Francke C.
      • Postma P.W.
      ,
      • Mayer C.
      • Boos W.
      ). The resulting glucose 6-phosphate is processed via the Embden-Meyerhof-Parnas (EMP)
      The abbreviations used are: EMP
      Embden-Meyerhof-Parnas
      FHL
      formate hydrogen-lyase
      AcCoA
      acetyl coenzyme A
      HSQC
      heteronuclear single-quantum coherence
      MFA
      metabolic flux analysis
      non-ox-PPP
      nonoxidative branch of the pentose phosphate pathway
      ox-PPP
      oxidative branch of the pentose phosphate pathway
      PDHC
      pyruvate dehydrogenase complex
      PFL
      pyruvate formate-lyase
      PPP
      pentose phosphate pathway.
      pathway and the pentose phosphate pathway (PPP) (
      • Romeo T.
      • Snoep J.L.
      ) to provide ATP, reducing power and carbon skeletons for biosynthesis. 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) (
      • Sawers R.G.
      • Clark D.P.
      ). Most of these fermentation products are generated from pyruvate (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Pathways involved in the synthesis of fermentation products, precursor metabolites, ATP, and reducing equivalents during the fermentative utilization of glucose by E. coli. Enzyme(s) catalyzing shown reaction(s) are as follows. Glucose transport and phosphorylation: (1), phosphoenolpyruvate-dependent phosphotransferase system (PTS); EMP: (2), phosphoglucose isomerase; (3), 6-phosphofructokinase; (4), fructose bisphosphate aldolase; (5), triose-phosphate isomerase; (6), glyceraldehyde-3-phosphate dehydrogenase; (7), phosphoglycerate kinase; (8), phosphoglycerate mutases and enolase; (9), pyruvate kinase. Ox-PPP: (10), glucose-6-phosphate dehydrogenase; (11), 6-phosphogluconolactonase; and (12), 6-phosphogluconate dehydrogenase. Non-ox-PPP: (13), ribulose phosphate 3-epimerase; (14), ribose-5-phosphate isomerases; (15), transketolases; and (16), transaldolases. Oxidative and reductive branches of the TCA cycle: (17), citrate synthase and acomitases; (18), isocitrate dehydrogenase; (19), malate dehydrogenase and fumarases; and (20), fumarate reductase. Anaplerotic reaction: (21), phosphoenolpyruvate carboxylase. Pyruvate dissimilation: (22), pyruvate formate-lyase; (23), pyruvate dehydrogenase complex. Fermentation: (24), lactate dehydrogenase; (25), formate hydrogen-lyase; (26), phosphate acetyltransferase; (27), acetate kinase; (28), alcohol/acetaldehyde dehydrogenase. Transhydrogenases: (29), soluble and membrane-bound pyridine nucleotide transhydrogenases. Cell growth: (30), synthesis of cell mass from precursor metabolites (★), ATP, and reducing equivalents. Broken lines indicate multiple steps. The following abbreviation are used: ACK, acetate kinase; ADH, alcohol dehydrogenase; AKG, α-ketoglutarate; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; FRU16P, fructose 1,6-bisphosphate; FRU6P, fructose 6-phosphate; FUM, fumarate; GALP, glyceraldehyde 3-phosphate; GLC6P, glucose 6-phosphate; ICIT, isocitrate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PGLU, 6-phospho-d-gluconate; PGLUL, d-glucono-δ-lactone-6-phosphate; POX, pyruvate oxidase; PP, pentose phosphate; PPP, pentose phosphate pathway; PTA, phosphate acetyltransferase; PYK, pyruvate kinase; PYR, pyruvate; RL5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7-phosphate; T3P, combined pool of GALP and DHAP; X5P, xylose 5-phosphate; 13PG, 1,3-diphosphate glycerate; 3PG, 3-phosphoglycerate.
      Pyruvate is located at a major metabolic node linking carbohydrate catabolism to energy generation and biosynthesis and also represents a major switch point between respiratory and fermentative metabolism. In this context, pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHC) are considered the major routes for pyruvate dissimilation in E. coli (Fig. 1) (
      • Sawers R.G.
      • Clark D.P.
      ). During aerobic growth, pyruvate is converted to acetyl coenzyme A (AcCoA), carbon dioxide, and NADH by PDHC. AcCoA enters the tricarboxylic acid cycle, where it is further oxidized to carbon dioxide, generating reducing equivalents that support ATP production through coupling to the electron transport systems and oxidative phosphorylation. During anaerobic fermentation, PFL replaces PDHC converting pyruvate to AcCoA and formate. AcCoA leads to the synthesis of fermentation products acetate and ethanol, which contribute to ATP generation and maintenance of redox balance, respectively (Fig. 1). The in vivo activity of PDHC during glucose fermentation has been reported as either very low or undetectable (
      • Sawers R.G.
      • Clark D.P.
      ,
      • Smith M.W.
      • Neidhardt F.C.
      ,
      • de Graef M.R.
      • Alexeeva S.
      • Snoep J.L.
      • Teixeira de Mattos M.J.
      ,
      • Snoep J.L.
      • de Graef M.R.
      • Westphal A.H.
      • de Kok A.
      • Teixeira de Mattos M.J.
      • Neijssel O.M.
      ). Even when active to some extent, PDHC cannot support fermentative growth on glucose in the absence of PFL unless the medium is supplemented with acetate (
      • Sawers R.G.
      • Clark D.P.
      ,
      • de Graef M.R.
      • Alexeeva S.
      • Snoep J.L.
      • Teixeira de Mattos M.J.
      ,
      • Varenne S.
      • Casse F.
      • Chippaux M.
      • Pascal M.C.
      ). The role of PDHC in the fermentative metabolism of glucose by E. coli remained unknown prior to the work reported here.
      This study focuses on a comprehensive metabolic analysis of wild-type E. coli and a PDHC-deficient derivative during the fermentative metabolism of glucose. Significant differences between the two strains were identified with the use of metabolite balancing, biosynthetic 13C labeling of proteinogenic amino acids, and isotopomer balancing. This analysis led to the hypothesis that PDHC is responsible for the generation of CO2 during glucose fermentation. The proposed role of PDHC was verified experimentally.

      DISCUSSION

      The metabolic characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) reported here led to identification of the role of PDHC in the fermentative metabolism of glucose. An exhaustive examination of these strains was conducted by using a mixture of 13C-labeled and -unlabeled glucose followed by analysis of protein-bound amino acids via two-dimensional 13C,1H HSQC NMR spectroscopy. Metabolite balancing (
      • Stephanopoulos G.
      • Aristidou A.A.
      • Nielsen J.
      ), biosynthetic fractional 13C labeling of proteinogenic amino acids (
      • Szyperski T.
      ), and isotopomer balancing (
      • Klapa M.I.
      • Park S.M.
      • Sinskey A.J.
      • Stephanopoulos G.N.
      ) were employed as a means to evaluate the metabolic differences between MG1655 and Pdh. Metabolite balancing and the analysis of intact and broken carbon bonds in key amino acids provided initial evidence of a potential redistribution of fluxes in response to the PDHC deficiency. The intracellular fluxes obtained via both metabolite and isotopomer balancing revealed a significant flux through PDHC in MG1655 and dramatic changes in the flux and operation of the PPP in response to the PDHC deficiency. Specifically, a 5-fold increase in the flux of the oxidative branch of the PPP was observed in strain Pdh compared with MG1655, and most of this carbon was cycled back to the EMP pathway. Because the ox-PPP, as PDHC, generates CO2, the aforementioned findings led to the hypothesis that the role of PDHC during the anaerobic fermentation of glucose is to generate CO2 for cell growth.
      E. coli is known to require a supply of CO2 as metabolic substrate during normal growth for the biosynthesis of small molecules (e.g. arginine, pyrimidines, and purines), fatty acids, and precursor metabolites (e.g. oxaloacetate). For example, anaerobic growth without a lag period can be achieved if a suitable concentration of CO2 is provided, and the subsequent exponential growth rate can be controlled by the concentration of this species (
      • Lacoursiere A.
      • Thompson B.G.
      • Kole M.N.
      • Ward D.
      • Gerson D.F.
      ,
      • Repaske R.
      • Clayton M.A.
      ). When not externally provided, CO2 can be endogenously generated through several metabolic pathways that involve decarboxylation reactions such as ox-PPP (6-phosphogluconate dehydrogenase) (
      • Romeo T.
      • Snoep J.L.
      ), TCA cycle (2-oxoglutarate dehydrogenase, isocitrate dehydrogenase) (
      • Cronan J.E.
      • LaPorte D.
      ), fatty acids biosynthesis (β-ketoacyl acyl carrier protein synthase) (
      • Cronan J.E.
      • Rock C.O.
      ), and enzymatic complexes PDHC and FHL (
      • Sawers R.G.
      • Blokesch M.
      • Bock A.
      ). The low activity of the TCA cycle under fermentative conditions limits the contribution of 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase. Fatty acids biosynthesis, on the other hand, generates CO2 (β-ketoacyl acyl carrier protein synthase) but also consumes it (acetyl-CoA carboxylase), making its overall contribution to CO2 generation very low. The ox-PPP (6-phosphogluconate dehydrogenase), PDHC, and FHL then become the three main sources of endogenous CO2 during fermentative metabolism. However, under neutral to alkaline conditions and early phases of growth, FHL provides very little endogenous CO2 (
      • Merlin C.
      • Masters M.
      • McAteer S.
      • Coulson A.
      ), as this enzyme is triggered by acidic pH and accumulation of formate (
      • Sawers R.G.
      • Blokesch M.
      • Bock A.
      ). It then follows that PDHC and ox-PPP represent the main sources of endogenous CO2 during the early stages of anaerobic cultures conducted at neutral to alkaline pH. Several lines of evidence presented in this study clearly support the role of PDHC and ox-PPP as the primary source of CO2. First, single mutants Pdh and Zwf exhibited less efficient growth than their wild-type MG1655, an effect that was even more pronounced in double mutant Pdh-Zwf (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6A). Growth deficiencies in these three strains were completely eliminated by supplementing the growth medium with bicarbonate (Fig. 6A), a well known source of CO2 (
      • Lacoursiere A.
      • Thompson B.G.
      • Kole M.N.
      • Ward D.
      • Gerson D.F.
      ,
      • Repaske R.
      • Clayton M.A.
      ). Activation of FHL by maintaining an acidic extracellular pH rendered the PDHC deficiency silent (Fig. 6B), which demonstrates that, when active, FHL is an alternative source of CO2. This proposal was further supported by the observation that the negative impact of the PDHC deficiency reappeared at acidic conditions upon elimination of the FHL activity (Fig. 6B).
      The role proposed here for PDHC in the anaerobic fermentation of glucose is to fulfill the metabolic requirements of CO2. The efficiency of CO2 generation by PDHC can be examined by comparing this pathway to the ox-PPP, the alternative pathway the cells use to generate CO2 under the conditions investigated in this study. Equations 1 and 2 compare the overall stoichiometry of each pathway assuming equal CO2 yields and interconversion of NADH and NADPH via transhydrogenases (see also supplemental Fig. 4).
      PDHC,3glucose6AcCoA+3CO2+3formate+9NAD(P)H+9ATP
      (Eq. 1)


      ox-PPP,3glucose5AcCoA+3CO2+5formate+11NAD(P)H+8ATP
      (Eq. 2)


      As can be seen, for the same amount of CO2 generated upon consumption of the same amount of glucose, PDHC generates more ATP (energy) and AcCoA (carbon) and less formate and reduced cofactors. Cells utilize both energy and carbon to generate the building blocks of biomass, and formate is a toxic metabolite detrimental for cell growth. Also, during fermentative metabolism of glucose, cells operate in a very reduced environment (i.e. high NADH/NAD+ ratio) (
      • de Graef M.R.
      • Alexeeva S.
      • Snoep J.L.
      • Teixeira de Mattos M.J.
      ), and further generation of reducing equivalents is clearly undesirable. Thus, overall, PDHC is more energy- and carbon-efficient than ox-PPP and also generates less toxic by-products and reduced cofactors. We speculate that this could be the reason why strain Pdh grows slower even when ox-PPP is present to substitute for this function and why wild-type MG1655 prefers the PDHC pathway to fulfill its metabolic requirements of CO2.

      Acknowledgments

      We thank F. R. Blattner and H. Mori for providing research materials, Y. Dharmadi for assistance with genetic methods, and B. Fulton for assistance with NMR.

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