Document Detail


Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data.
MedLine Citation:
PMID:  17132142     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.
Authors:
Roelco J Kleijn; Jan-Maarten A Geertman; Beckley K Nfor; Cor Ras; Dick Schipper; Jack T Pronk; Joseph J Heijnen; Antonius J A van Maris; Wouter A van Winden
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-11-21
Journal Detail:
Title:  FEMS yeast research     Volume:  7     ISSN:  1567-1356     ISO Abbreviation:  FEMS Yeast Res.     Publication Date:  2007 Mar 
Date Detail:
Created Date:  2007-02-22     Completed Date:  2007-05-08     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101085384     Medline TA:  FEMS Yeast Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  216-31     Citation Subset:  IM    
Affiliation:
Department of Biotechnology, Delft University of Technology, Delft, the Netherlands.
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MeSH Terms
Descriptor/Qualifier:
Carbon / metabolism
Carbon Isotopes / metabolism
Gas Chromatography-Mass Spectrometry
Gene Deletion
Glycerol / metabolism*
Glycerolphosphate Dehydrogenase / genetics
Magnetic Resonance Spectroscopy
Metabolic Networks and Pathways*
NADH Dehydrogenase / genetics
Oxidation-Reduction
Saccharomyces cerevisiae / enzymology,  genetics*,  metabolism*
Triose-Phosphate Isomerase / genetics
Up-Regulation*
Chemical
Reg. No./Substance:
0/Carbon Isotopes; 56-81-5/Glycerol; 7440-44-0/Carbon; EC 1.1.-/Glycerolphosphate Dehydrogenase; EC 1.6.99.3/NADH Dehydrogenase; EC 5.3.1.1/Triose-Phosphate Isomerase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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