<sup>13</sup>C NMR Flux Ratio Analysis of <i>Escherichia coli</i> Central Carbon Metabolism in Microaerobic Bioprocesses

Fiaux, Jocelyne; Andersson, Charlotte I.J.; Holmberg, Niklas; Bülow, Leif; Kallio, Pauli T.; Szyperski, Thomas; Bailey, James E.; Wüthrich, Kurt

doi:10.1021/ja983786y

Cited by 37 publications

(35 citation statements)

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“…The 13 C-labeling experiments revealed major differences in the central carbon metabolism when comparing VHb-and VHb-Red-expressing cells to FHPg-and FHP-expressing cells. VHb-and VHb-Red-positive cells yielded labeling patterns similar to those previously observed for anaerobically grown wild-type E. coli cells (43) and microaerobic MG1655 cells (11), whereas for FHPg-and FHP-expressing cells, a more aerobic fluxome was found (Fig. 3 and Table 4).…”

Section: Fig 2 Growth Trajectories Of the Four Hemoglobin-expressingsupporting

confidence: 81%

“…Higher glycolytic activity in VHb-expressing cells may then satisfy the ATP and NAD(P)H demands for growth and maintenance. To support this hypothesis, we included some previously published NMR data on hemoglobinnegative control cells in Table 4 (11). Comparison of the data on VHb-and VHb-Red-expressing cells with the VHb-negative control reveals similar flux ratios.…”

Section: Discussionmentioning

confidence: 97%

“…The ratios of intracellular metabolic fluxes in the central carbon metabolism of E. coli MG1655 cells grown under oxygen-limited conditions have previously been determined using biosynthetically directed fractional 13 C-labeling of amino acids and two-dimensional (2D) [ 13 C, 1 H]-correlation nuclear magnetic resonance (NMR) spectroscopy (11). This investigation, which serves as a reference for the presently studied hemoglobin-expressing cells, showed that, under microaerobic conditions, the topology of the active pathways is characteristic of anaerobic metabolism, as was primarily evidenced by the activity of the pyruvate-formate lyase and the suppression of 2-oxoglutarate dehydrogenase activity.…”

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Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Frey

Fiaux

Szyperski

et al. 2001

Appl Environ Microbiol

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Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and the A. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional 13 C-labeling of the proteinogenic amino acids and two-dimensional [ 13 C, 1 H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressing A. eutrophus FHP or the truncated heme domain (FHPg). E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb-and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressing A. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and D-lactate. The implications of these observations for biotechnological applications are discussed.Hemoglobins are a specific group of oxygen-binding proteins that can be found in mammals, plants, and microorganisms (15). The homodimeric hemoglobin of Vitreoscilla (VHb) is the best characterized bacterial hemoglobin. The expression of VHb is up-regulated by oxygen limitation (hypoxia) in Vitreoscilla (50), but its physiological functions have not yet been entirely elucidated. Nonetheless, heterologous expression of VHb has been used to alleviate physiologically unfavorable effects of oxygen limitation and to improve growth properties and productivity of various microorganisms, plants, and mammalian cells that yield industrially important metabolites (7, 18, 21, 28-30, 32, 35).Previous research on the effects arising from heterologous VHb expression has mainly focused on the operation of the respiratory chain. Expression of VHb in Escherichia coli cells lacking either cytochrome o (aerobic terminal oxidase) or cytochrome d complexes (microaerobic terminal oxidase) revealed a 5-fold increase in cytochrome o (in a cyd mutant, cyo ϩ strain) and a 1.5-fold increase in cytochrome d (in cyd ϩ , cyo mutant strain) complexes relative to wild-type cells (48). These results have led to the hypothesis that VHb is able to increase the effective intracellular oxygen concentration with concomitant increase of the amount of cytochrome o complexes (20): the proton translocation activity of cytochrome o complexes is characterized by a higher H/O ratio than cytochrome d complexes and is able to generate a larger proton gradient across the cell membrane (25,36,38). VHb-expressing cells are indeed able to generate a larger proton flux ...

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Section: Fig 2 Growth Trajectories Of the Four Hemoglobin-expressingsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 97%

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confidence: 99%

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Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Frey

Fiaux

Szyperski

et al. 2001

Appl Environ Microbiol

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Revisiting the ¹³C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

et al. 2005

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During the past decade, 13 C-labeling based metabolic flux analysis (MFA) has increasingly been used to understand the effect of genetic alterations [1,2], changes in external conditions [3,4] and different nutritional regimes [5,6] on the metabolism of micro-organisms. 13 C-labeling based MFA relies on the feeding of 13 C-labeled substrate to a biological system, allowing the labeled carbon atoms to distribute over the metabolic network, and subsequently measuring the 13 C-label distributions of intracellular and ⁄ or secreted The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C 2 ) and dihydroxyacetone moiety (C 3 ) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C 2 and C 3 fragment pools in the pentose phosphate pathway and can lead to different label distributions for 13 C-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published 13 C-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several microorganisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. 7It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the 13 C-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the halfreaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.Abbreviations C 2 , glycolaldehyde moiety; C 3 , dihydroxyacetone moiety; e4p, erythrose 4-phosphate; f6p, fructose 6-phosphate; fbp, fructose 1,6-bisphosphate 3;4;5 ; g1p, glucose 1-phosphate; g6p, glucose 6-phosphate; g3p, glyceraldehyde 3-phosphate; MFA, metabolic flux analysis; p5p, pentose pool consisting of ribulose 5-phosphate, ribose 5-phosphate and xylulose 5-phosphate; PPP, pentose phosphate pathway; r5p, ribose 5-phosphate; s7p, sedoheptulose 7-phosphate; SS res , sum of squared residuals; S 2 res , estimated error variance; TA, transaldolase; TK, transketolase; TPP, thiamine pyrophosphate; x5p, xylulose 5-phosphate.

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“…In this paper, we determine the metabolic history of monosaccharide units before their incorporation into starch by retrobiosynthetic NMR analysis (Eisenreich et al, 1993), a technique that is nonintrusive and nondestructive (Szyperski, 1995;Schmidt et al, 1998;Fiaux et al, 1999;Park et al, 1999;Eisenreich and Bacher, 2000;Glawischnig et al, 2001). The maize kernel was chosen as a model system to study starch biosynthesis because seeds of cereals are an important metabolic sink.…”

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Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance

et al. 2002

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The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) Plant metabolism is a complex network of many interconnected reactions and metabolites (Fien et al., 2000). For the analysis of metabolic networks, it is important to study metabolic pathways not only on the level of isolated genes or enzymes but also to quantify metabolite flux, which is involved in the formation of sink metabolites, such as starch.The biosynthesis of starch in the storage tissue of monocotyledonous plants has been studied in detail (for review, see Neuhaus and Emes, 2000). In maize (Zea mays), Suc from source leaves is imported into the developing cob tissue and converted into a mixture of Fru and UDP-Glc in the cytosol of endosperm cells (Chourey and Nelson, 1976;Chourey et al., 1998). UDP-Glc is converted into activated hexoses (i.e. Glc-1-P and Glc-6-P), which have been reported as starch precursors in various species. In maize, Glc-1-P is converted to the starch precursor ADP-Glc, which is transported into the plastid (Shannon et al., 1998). In contrast to the well-characterized import of activated hexoses into the amyloplasts as starch precursors, there is little evidence for the incorporation of trioses into starch (Neuhaus and Emes, 2000). It is therefore conceivable that starch is formed from intact C 6 units derived from cleaved Suc. However, in different systems, redistribution between C-1 and C-6 of Glc moieties of starch was observed, indicating metabolic cycling between trioses and hexoses in the cytosol (Hatzfeld and Stitt, 1990;Viola et al., 1991;Dieuaide-Noubhani et al., 1995;Krook et al., 1998). This phenomenon is also observed in starchaccumulating organs of cereals. In wheat (Triticum aestivum), 15% to 20% redistribution of 13 C-label between C-1 and C-6 of Glc recovered from starch was observed (Keeling et al., 1988). In maize, randomization of the carbon moieties of starch was detected using [1-14 C]Glc that was injected into developing kernels (Hatzfeld and Stitt, 1990).In this paper, we determine the metabolic history of monosaccharide units before their incorporation into starch by retrobiosynthetic NMR analysis (Eisenreich et al., 1993), a technique that is nonintrusive and nondestructive (Szyperski, 1995;Schmidt et al., 1998;Fiaux et al., 1999;Park et al., 1999;Eisenreich and Bacher, 2000;Glawischnig et al., 2001 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

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¹³C NMR Flux Ratio Analysis of Escherichia coli Central Carbon Metabolism in Microaerobic Bioprocesses

Cited by 37 publications

References 18 publications

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Revisiting the ¹³C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance

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13C NMR Flux Ratio Analysis of Escherichia coli Central Carbon Metabolism in Microaerobic Bioprocesses

Cited by 37 publications

References 18 publications

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by 13 C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by 13 C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Revisiting the 13C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance

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¹³C NMR Flux Ratio Analysis of Escherichia coli Central Carbon Metabolism in Microaerobic Bioprocesses

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by ¹³ C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

Revisiting the ¹³C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence