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General Information info

Manuscript title A comparative transcriptomic, fluxomic and metabolomic analysis of the response of Saccharomyces cerevisiae to increases in NADPH oxidation.
PubMed ID 22805527
Journal BMC Genomics
Year 2012
Authors Magalie Celton, Isabelle Sanchez, Anne Goelzer, Vincent Fromion, Carole Camarasa and Sylvie Dequin
Affiliations INRA, UMR1083 SPO, 2 place Viala, F-34060 Montpellier, France
Keywords NADPH, Pentose Phosphate Pathway, Acetoin, Intracellular Metabolite, NADPH Oxidation
Full text article Downloadarticle Celton_2012.pdf
Project name not specified

Experiment Description info

Organism Saccharomyces cerevisiae
Strain 59A NADPH-Bdh
Data type flux measurements
Data units mmol/100 mmol glucose
Execution date not specified

Experimental Details info

Temperature (0C) 28
pH 5.0
Carbon source glucose,
Culture mode batch
Process condition anaerobic
Dilution rate (h-1)
Working volume (L) 0.01
Biomass concentration (g/L) for acetoin 0 mM = 2.9g, for acetoin 100 mM = 2.55g, for acetoin 200 mM = 2.65g and for acetoin 300 mM = 2.17g
Medium composition

2X SD minimal medium (13.4% yeast nitrogen base without amino acids, 100% glucose). The SD minimal medium was supplemented with 1.25 mg/l ergosterol, 0.164 g/l Tween 80, and 0.35 mg/l oleic acid, to satisfy the lipid requirements of the yeast cells during anaerobic growth. Acetoin was added at various concentrations (from 0 to 300 mM) at the start of fermentation, as previously described by Celton et al. [1].

General protocol information Flux analysis method: 13C constrained MFA

Platform: GC-MS

Methods description - Notes

At an OD600 of 3.0, corresponding to mid-exponential growth phase, the concentration of extracellular metabolites (glucose, glycerol, organic acids) was measured in the supernatant (these data will be used to constrain the stoichiometric model, as described below). Cells were harvested and hydrolyzed overnight with 6 M HCl for the determination of amino acids labeling patterns. One glucose derivative (glucose pentacetate) and two amino-acid derivatives (ethyl chloroformate (ECF) and dimethyl formamide dimethyl acetal (DMFDMA)) were analyzed by GC-MS [2], as previously described by Gombert et al. [3]. From the raw GC-MS data, the summed fractional labeling (SFL) of each fragment was calculated as follow: SFL = 100 × [(1.m 1 + 2.m 2 + …. + n.m n) × (m 0 + m 1 + m 2 + … + m n)-1, with m o the fractional abundance of the lowest corrected mass and mi > 0 the abundance of molecules with higher corrected masses. The labelling data are available in request.
The data set used for 13 C-flux analysis included the 25 SFLs calculated from labeling experiments and 22 measured fluxes, including the drain of metabolic intermediates to biomass and the formation of seven metabolites, giving a total of 47 items of experimental data. The distribution of carbon in central carbon metabolism (CCM) was estimated with the metabolic model described by Gombert et al.[3], modified to take into account features specific to fermentation [4]. The network used is described in additional file 2. Flux calculations (60 reactions) were carried out with Matlab 7, as previously described [5,3]. Differences between experimental and simulated SFLs and between experimental and simulated fluxes were minimized by an iterative procedure.
-----------------------------------References---------------------------------
[1] Celton M, Goelzer A, Camarasa C, Fromion V, Dequin S. Metab Eng. 2012, 14: 366-79. http://doi.org/f32tf6
[2] Christensen B, Nielsen J. Met Eng. 1999, 1: 282-290. http://doi.org/bjrp5z
[3] Gombert AK, dos Moreira Santos M, Christensen B, Nielsen J. J Bacteriol. 2001, 183: 1441-10. http://doi.org/crqbjx
[4] Heux S: Ingénierie métabolique et analyse13C-flux du métabolisme central des levuresSaccharomyces cerevisiaeœnologiques.PhD Thesis. 2006, University of Burgundy, Sciences de la vie et de la santé.
[5] Christensen B, Nielsen J. Met Eng. 1999, 1: 282-290. http://doi.org/bjrp5z

Data file
Downloadfluxes KIMODATAID115_v0.xlsx
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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-08-19 19:03:08 UTC

Updated 2018-08-19 19:03:08 UTC

Version 0

Status (reviewed) 2018-08-19 19:03:43 UTC




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