NMR isotopic filiation of 13 C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in Saccharomyces cerevisiae during anaerobic glucose fermentation. The assimilation of [3-13 C]aspartate led to the formation of [2,3-13 C]malate and [2,3-13 C]succinate, with equal levels of 13 C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [3-13 C]glutamate. The non-random distribution of 13 C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed 13 C 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.

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.