PHYSIOLOGICAL CHARACTERISATION OF YEAST STRAINS DISPLAYING ALTERED MITOCHONDRIAL NADH/NAD+ RATIOS

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 tool for both studying and engineering metabolism. In Saccharomyces cerevisiae pyridine coenzymes are involved in > 200 reactions. Manipulation of their cellular content as well as of their intracellular compartimentalization is expected to deeply affects overall metabolism determining modifications of the intracellular redox potential (NADH/NAD+ ratio), a fundamental requirement for sustained metabolism and growth in all biological systems. Redox balance and NAD+/NADH concentrations, together with mitochondrial activity, determine the extent of respiration, as well as the fermentative production of ethanol and glycerol. The dramatic difference in ATP production sustained by respirative or fermentative metabolism results in great difference in biomass yield, that in turn heavily affects the profitability of a biotechnological process. In S. cerevisiae biosynthesis of NAD+ takes place in the cytoplasm. Since mitochondria are impermeable to NAD+, a transporter is necessary to fuel these organelles with the requested amounts of the cofactor. Two mitochondrial transporters, named Ndt1p e Ndt2p, were identified as major responsible of transferring NAD+ from cytoplasm to the mitochondrial matrix. In the present paper we investigated the physiological effects of a double ndt1ndt2 deletion and of the overespression of NDT1 ( ndt1-over strain) in batch and chemostat cultures. Deletion of both genes was reflected by a relevant drop in the NAD+ mitochondrial level, while the concentration of the coenzyme was sensibly increased in NDT1 overespressing strain. Consequently, also the NAD+/NADH mitochondrial ratio was affected in the mutant strains, being increased in ndt1ndt2 and decreased in the ndt1-over strain. While the increase of NDT1 expression didn’t affect the growth features in any carbon source, the double deleted strain showed relevant defects when grown in minimal medium. Moreover, these defects were dependent on catabolite repression of the carbon source used, being light on glucose, and more marked on ethanol. Strikingly, we measured very high respiratory activity for ndt1ndt2 strain, together with a very high Respiratory State Value, that indicate an oxygen consumption close to the upper limit for the cell. Oxygen consumption measurements on isolated mitochondria revealed that both lowered and elevated mitochondrial pyridine nucleotide contents, characteristic of ndt1ndt2 and ndt1-over strain respectively, determined an impairment in the oxidation of substrates that generates NADH into the mitochondrial matrix. Chemostat analysis confirmed respiratory levels for both mutant higher than for the wild type. In this cultural condition metabolic alterations for NDT1 overespressing strain became evident, and a comparison with double deleted and control strains became possible. Despite the high oxygen consumption, both strains showed low biomass yield values, indicating an impairment in mitochondrial functions. Since glucose consumption rates were higher than for the wild type, ethanol was accumulated as a by product, and Crabtree effect was triggered at lower Dilution rates. To our knowledge, this is the first study in which the effect of mitochondrial NAD+ content on yeast cell physiology has been investigated. This study demostrates the importance of coenzyme redistribution between mitochondria and cytoplasm on metabolic fluxes, and could in perspective be very useful in the optimisation of biotechnological process based on enzymatic reactions involving pyridine cofactors.