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1

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C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum (1996)

Peters-Wendisch, Petra G. ; Wendisch, Volker F. ; de Graaf, Albert A. ; [et al.]

Springer

Archives of microbiology 165 (1996), S. 387-396

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ISSN: 1432-072X

Keywords: Key words Corynebacterium glutamicum ; Anaplerotic ; reactions ; Phosphoenolpyruvate carboxylase ; Isocitrate ; lyase ; Glyoxylate cycle

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H13CO3 –-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% 13C enrichment in C-4 and 3.5% 13C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C3-carboxylation reaction that also takes place in the absence of PEPCx.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002030050342

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2

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Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing (1996)

Marx, Achim ; de Graaf, Albert A. ; Wiechert, Wolfgang ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 49 (1996), S. 111-129

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ISSN: 0006-3592

Keywords: intracellular fluxes ; metabolite balance ; carbon labeling balance ; lysine ; anaplerotic reactions ; NMR spectroscopy ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of 1H-detected 13C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-13C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. © 1996 John Wiley & Sons, Inc.

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3

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Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis (1997)

Wiechert, Wolfgang ; Siefke, Claudia ; de Graaf, Albert A. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 55 (1997), S. 118-135

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ISSN: 0006-3592

Keywords: stationary flux estimation ; sensitivity analysis ; covariance analysis ; nonlinear statistics ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Metabolic carbon labelling experiments enable a large amount of extracellular fluxes and intracellular carbon isotope enrichments to be measured. Since the relation between the measured quantities and the unknown intracellular metabolic fluxes is given by bilinear balance equations, flux determination from this data set requires the numerical solution of a nonlinear inverse problem. To this end, a general algorithm for flux estimation from metabolic carbon labelling experiments based on the least squares approach is developed in this contribution and complemented by appropriate tools for statistical analysis. The linearization technique usually applied for the computation of nonlinear confidence regions is shown to be inappropriate in the case of large exchange fluxes. For this reason a sophisticated compactification transformation technique for nonlinear statistical analysis is developed. Statistical analysis is then performed by computing appropriate statistical quality measures like output sensitivities, parameter sensitivities and the parameter covariance matrix. This allows one to determine the order of magnitude of exchange fluxes in most practical situations. An application study with a large data set from lysine-producing Corynebacterium glutamicum demonstrates the power and limitations of the carbon-labelling technique. It is shown that all intracellular fluxes in central metabolism can be quantitated without assumptions on intracellular energy yields. At the same time several exchange fluxes are determined which is invaluable information for metabolic engineering. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 118-135, 1997.

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4

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Response of the central metabolism of Corynebacterium glutamicum to different flux burdens (1997)

Marx, Achim ; Striegel, Katharina ; de Graaf, Albert A. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 56 (1997), S. 168-180

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ISSN: 0006-3592

Keywords: metabolic fluxes ; metabolite balance ; NMR spectroscopy ; amino acid production ; bidirectional fluxes ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-13C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of 13C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate ↔ sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate ↔ fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.

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5

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Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments (1997)

Wiechert, Wolfgang ; de Graaf, Albert A.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 55 (1997), S. 101-117

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ISSN: 0006-3592

Keywords: stationary flux analysis ; metabolite flux balancing ; 13C isotope labeling experiments ; NMR ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: The extension of metabolite balancing with carbon labeling experiments, as described by Marx et al. (Biotechnol. Bioeng. 49: 11-29), results in a much more detailed stationary metabolic flux analysis. As opposed to basic metabolite flux balancing alone, this method enables both flux directions of bidirectional reaction steps to be quantitated. However, the mathematical treatment of carbon labeling systems is much more complicated, because it requires the solution of numerous balance equations that are bilinear with respect to fluxes and fractional labeling. In this study, a universal modeling framework is presented for describing the metabolite and carbon atom flux in a metabolic network. Bidirectional reaction steps are extensively treated and their impact on the system's labeling state is investigated. Various kinds of modeling assumptions, as usually made for metabolic fluxes, are expressed by linear constraint equations. A numerical algorithm for the solution of the resulting linear constrained set of nonlinear equations is developed. The numerical stability problems caused by large bidirectional fluxes are solved by a specially developed transformation method. Finally, the simulation of carbon labeling experiments is facilitated by a flexible software tool for network synthesis. An illustrative simulation study on flux identifiability from available flux and labeling measurements in the cyclic pentose phosphate pathway of a recombinant strain of Zymomonas mobilis concludes this contribution. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 101-117, 1997.

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6

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13C tracer experiments and metabolite balancing for metabolic flux analysis: Comparing two approaches (1998)

Schmidt, Karsten ; Marx, Achim ; de Graaf, Albert A. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 58 (1998), S. 254-257

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ISSN: 0006-3592

Keywords: metabolic flux analysis ; 13C tracer experiments ; fractional enrichment ; NADH ; NADPH ; pentose phosphate pathway ; Aspergillus oryzae ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for intracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends heavily on the correctness of the assumed stoichiometry including the presence of all reactions with a noticeable impact on the model metabolite balances. Determination of fluxes in complex metabolic networks often requires the inclusion of NADH and NADPH balances, which are subject to controversial debate. Transhydrogenation reactions that transfer reduction equivalents from NADH to NADPH or vice versa can usually not be included in the stoichiometric model, because they result in singularities in the stoichiometric matrix. However, it is the NADPH balance that, to a large extent, determines the calculated flux through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using 13C tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well established stoichiometric equations and measurements of the labeling state of intracellular metabolites. Neither NADH/NADPH balancing nor assumptions on energy yields need to be included to determine the intracellular fluxes. Because metabolite balancing methods and the use of 13C labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviations in the results. Flux analysis based entirely on metabolite balancing and flux analysis, including labeling information, have been performed independently for a wild-type strain of Aspergillus oryzae producing α-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on the intracellular flux distribution. The two different approaches to the calculation of fluxes are compared and deviations in the results are discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:254-257, 1998.

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Type of Medium: Electronic Resource

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