Quantitative Analysis of Metabolic and Signaling Pathways in Saccharomyces cerevisiae

Mauch, Klaus; Vaseghi, Sam; Reuß, Matthias

doi:10.1007/978-3-642-59735-0_15

Cited by 11 publications

(6 citation statements)

References 56 publications

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“… † All these compounds, except for nuclear DNA, trehalose and glycogen, were assumed to maintain constant ratios among themselves during the whole cell cycle. Intracellular concentrations were calculated for each phase on the basis of concentration data for cell number and biomass dry weight in combination with mean cell volumes for the respective cell cycle phase (Mauch et al , 2000; Müller, 2006). …”

Section: Methodsmentioning

confidence: 99%

¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

Costenoble

Müller

Barl

et al. 2007

FEMS Yeast Research

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This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.

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Section: Methodsmentioning

confidence: 99%

¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

Costenoble

Müller

Barl

et al. 2007

FEMS Yeast Research

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“…(Or, can we simply sum up every enzyme reaction to understand the system quantitatively?). By comparison of in vitro, in situ, and in vivo results for enzyme kinetics of the phosphofructokinase I system in Saccharomyces cerevisiae, it has been demonstrated that remarkable dierences in structure of the kinetic expressions, as well as in parameter values, can be observed in the case of such complex enzyme systems (Mauch et al, 2000). Consequently, we recommend using the measurements of intracellular metabolites for identi®cation of in vivo enzyme kinetics in a similar way as measurements are applied to identi®cation of in vitro kinetics within test tubes.…”

Section: Introductionmentioning

confidence: 96%

Dynamic modeling of the central carbon metabolism of Escherichia coli

Chassagnole

Noisommit-Rizzi

Schmid

et al. 2002

Biotech & Bioengineering

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603

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Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.

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“…As shown, sudden changes of glucose availability caused intracellular metabolism dynamics, which has been the basis for identifying in vivo enzyme kinetics by a sequential procedure of glucose pulsing, rapid cell sampling, metabolism inactivation, intracellular metabolite analysis, and detailed modeling using structured, mechanistic models. In the following, similar (experimental) studies have been performed analyzing S. cerevisiae (Vaseghi et al, 1999; Mauch et al, 2000; Lange et al, 2001, Ostergaard et al, 2001), Zymomonas mobilis (Weuster‐Botz and de Graaf, 1996; Weuster‐Botz D., 1997;) and Escherichia coli (Schäfer et al, 1999; Sauter et al, 2002; Chassagnole et al, 2002; Hurlebaus et al, 2002; Degenring et al 2003).…”

Section: Introductionmentioning

confidence: 97%

Stimulation, Monitoring, and Analysis of Pathway Dynamics by Metabolic Profiling in the Aromatic Amino Acid Pathway

Oldiges¹,

Kunze²,

Degenring³

et al. 2004

Biotechnol. Prog.

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Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains. During the observation window from -4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy-d-arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P). Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed. On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion--called "pool efflux capacity" (PEC)--is derived. Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E. coli strain A (genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in strain B, genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent L-Phe producing strains. Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled. The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways.

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Quantitative Analysis of Metabolic and Signaling Pathways in Saccharomyces cerevisiae

Cited by 11 publications

References 56 publications

¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

Dynamic modeling of the central carbon metabolism of Escherichia coli

Stimulation, Monitoring, and Analysis of Pathway Dynamics by Metabolic Profiling in the Aromatic Amino Acid Pathway

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Quantitative Analysis of Metabolic and Signaling Pathways in Saccharomyces cerevisiae

Cited by 11 publications

References 56 publications

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

Dynamic modeling of the central carbon metabolism of Escherichia coli

Stimulation, Monitoring, and Analysis of Pathway Dynamics by Metabolic Profiling in the Aromatic Amino Acid Pathway

Contact Info

Product

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¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

¹³C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae