Metabolic flux analysis of a glycerol‐overproducing Saccharomyces cerevisiae strain based on GC‐MS, LC‐MS and NMR‐derived 13C‐labelling data

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 13C-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 13C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and 13C-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 13C-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.