Deletion or Overexpression of Mitochondrial NAD ⁺ Carriers in Saccharomyces cerevisiae Alters Cellular NAD and ATP Contents and Affects Mitochondrial Metabolism and the Rate of Glycolysis

Abstract

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.Many metabolic engineering strategies rely on the manipulation of enzyme levels to achieve the amplification, interruption, or addition of a metabolic pathway. Modification of enzyme cofactor concentration is another, relatively unexplored tool for both studying and engineering the metabolism of a cell. In Saccharomyces cerevisiae, second only to ATP, pyridine coenzymes are involved in hundreds of reactions (10). Manipulation of their cellular contents as well as their intracellular compartmentalization would be expected to deeply affect the overall cellular metabolism by determining modifications in the redox potential of subcellular compartments. Mitochondrial NAD-dependent dehydrogenase reactions can affect the rate of respiration and the overflow metabolism, thus influencing the production of ethanol, glycerol, and biomass (18, 32). Mitochondrial NAD is also crucial for anabolism; for example, the synthesis of many intermediates of the amino acid pathway(s) generated by the Krebs cycle is strongly dependent on the mitochondrial NAD ϩ /NADH ratio (21, 34). The regulation of catabolism and anabolism by mitochondrial NAD has been extensively studied in plants, where the physiological modulation of mitochondrial NAD ϩ content has long been postulated and the proteins responsible for it have been recentl...

The dynamic regulation of NAD metabolism in mitochondria

“…NAD transport by Ndt1 is highly specific to NAD, with an apparent K m of 0.38 mM [44]. Deletion of these transporters in yeast, reduces mitochondrial NAD levels (>4 fold), impairs the activity of mitochondrial NAD-requiring enzymes, and delays growth on nonfermentable carbon (respiratory conditions) [44, 45]. Conversely, overexpression of Ndt1 doubles NAD levels [44, 45].…”

“…Deletion of these transporters in yeast, reduces mitochondrial NAD levels (>4 fold), impairs the activity of mitochondrial NAD-requiring enzymes, and delays growth on nonfermentable carbon (respiratory conditions) [44, 45]. Conversely, overexpression of Ndt1 doubles NAD levels [44, 45]. A mitochondrial NAD transporter, AtNDT2, has also been found in Arabidopsis , which transports NAD in a counter-exchange mode for ADP or AMP [46].…”

NAD Biosynthesis in Humans - Enzymes, Metabolites and Therapeutic Aspects

“…Though it was surprising that seemingly opposite mitochondrial NAD + modulations resulted in similar metabolic profiles, comparable observations have been reported in yeast: overexpression as well as deletion of mitochondrial NAD + carriers lowers ATP production, and upon complete respiratory growth conditions both mutants exhibited a low biomass yield indicating impaired respiratory functions [165].…”

Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the CAT8 Gene

“…Ϫ1 for TET and CCCP, respectively (18,19). The DO slope as a function of time was estimated after adding TET at 15 g mg (DCW)…”