Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Abstract

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

Fig. 1.

Specific activities (in units/mg protein) of NADH-dependent glycerol-3-phosphate dehydrogenase (G3P DH), NADH-dependent alcohol dehydrogenase (ADH), and NAD-dependent isocitrate dehydrogenase (IDH) in CON, NOX, and AOX during carbon-limited chemostats grown at a dilution rate of 0.1 h⁻¹ (yellow bars) and at respective D_crit (brown bars).

Fig. 2.

Measurement of indicators of the redox metabolism during chemostat cultivations under carbon or nitrogen limitation in CON, NOX, and AOX. (A) Total specific NADH oxidation activity. (B) Ratio of intracellular NADH/NAD. Carbon-limited chemostats were operated at a dilution of 0.1 h⁻¹ (white bars) or at the respective D_crit (gray bars), and the nitrogen-limited chemostat (black bars) was operated at a dilution rate of 0.1 h⁻¹.

Fig. 3.

Analysis of transcription responses of NOX vs. CON superimposed on the metabolic network, as described previously (25). Shown are the key enzymes that were identified in the central metabolism as part of the subnetwork. Genes that were up-regulated are indicated in red; those that were down-regulated are indicated in green.

Fig. 4.

Analysis of transcription responses of AOX vs. CON superimposed on the metabolic network, as described previously (25). Shown are the key enzymes that were identified in the central metabolism as part of the subnetwork. Genes that were up-regulated are indicated in red; those that were down-regulated are indicated in green.