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

Abstract

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.

Metabolic flux analysis using 13C peptide label measurements

“…Recent technological advances have made it possible to follow the metabolism of a cell culture over time (Antoniewicz et al, 2007a), but cell cycle-resolved MFA (Costenoble et al, 2007) of unsynchronized cells is not yet possible. We introduce technology with the potential to enable such investigations.…”

Metabolic flux analysis in eukaryotes

“…Presently, the only way to address this question is the application of synchronized cultures. In partly synchronized S. cerevisiae cells the labeling patterns of intracellular metabolites determined using LC-MS changed in different phases of the cell cycle, especially the PPP activity changed by a factor of two [18]. S. cerevisiae represents the most extensively studied fungal organism concerning MFA.…”

Integration of Metabolic Reactions and Gene Regulation

“…Current datasets of metabolic fluxes are concentrated on microbes such as E. coli (e.g., [14][15][16]) and S. cerevisiae (e.g., [17][18][19]). Most datasets probe the well-known glucose metabolism network (glycolysis, TCA cycle, pentose phosphorylation).…”