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Investigation by ¹³C-NMR and tricarboxylic acid (TCA) deletion mutant analysis of pathways for succinate formation in Saccharomyces cerevisiae during anaerobic fermentation

¹ UMR-Sciences pour l'nologie, Microbiologie et Technologie des Fermentations, Institut National de la Recherche Agronomique, 2 Place Viala, 34060 Montpellier, France
² Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique et Université d'Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France

Correspondence
Carole Camarasa
camarasa@ensam.inra.fr Sylvie Dequin
dequin{at}ensam.inra.fr

NMR isotopic filiation of ¹³C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in Saccharomyces cerevisiae during anaerobic glucose fermentation. The assimilation of [3-¹³C]aspartate led to the formation of [2,3-¹³C]malate and [2,3-¹³C]succinate, with equal levels of ¹³C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [3-¹³C]glutamate. The non-random distribution of ¹³C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed ¹³C distribution suggests that the succinate dehydrogenase (SDH) complex is not active during glucose fermentation. This hypothesis was tested by deleting the SDH1 gene encoding the flavoprotein subunit of the SDH complex. The growth, fermentation rate and metabolite profile of the sdh1 mutant were similar to those of the parental strain, demonstrating that SDH was indeed not active. Filiation experiments indicated the reductive branch of the TCA pathway was the main pathway for succinate production if aspartate was used as the nitrogen source, and that a surplus of succinate was produced by oxidative decarboxylation of 2-oxoglutarate if glutamate was the sole nitrogen source. Consistent with this finding, a kgd1 mutant displayed lower levels of succinate production on glutamate than on other nitrogen sources, and higher levels of oxoglutarate dehydrogenase activity were observed on glutamate. Thus, the reductive branch generating succinate via fumarate reductase operates independently of the nitrogen source. This pathway is the main source of succinate during fermentation, unless glutamate is the sole nitrogen source, in which case the oxidative decarboxylation of 2-oxoglutarate generates additional succinate.

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