Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD⁺-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.The traditional use of baker''s yeast, Saccharomyces cerevisiae, for ethanol production has resulted in the accumulation of substantial information about its genetics, metabolism, and process development. Consequently, the collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites (1, 25, 28). Unlike ethanol, many of these products are not redox neutral relative to commonly used substrates such as glucose. Therefore, in addition to stoichiometry, redox constraints play an important role in the formation of the products. Additional reducing power has to be supplied to produce compounds whose degree of reduction is higher than that of the substrate. On the other hand, producing compounds with a degree of reduction lower than that of the substrate will force the synthesis of other compounds with higher degrees of reduction to compensate for excess reducing power generated from substrate oxidation. These constraints may decrease the product yield substantially.The catabolic currency that balances the degree of reduction between the substrate and the products is usually NADH. In S. cerevisiae, NADH is produced in the cytosol by mainly glyceraldehyde-3-phosphate dehydrogenase and other assimilatory reaction enzymes (35). In the mitochondria, NADH is formed in the tricarboxylic acid (TCA) cycle and the reaction of the pyruvate dehydrogenase complex. Cytosolic NADH is oxidized by the glycerol-3-phosphate shuttle or the external cytosolic NADH dehydrogenases, which are part of the electron transport chain (21). NADH can be transported across the outer mitochondrial membrane (18, 19) but not across the inner mitochondrial membrane (39). Therefore, a dedicated internal mitochondrial NADH dehydrogenase is required to oxidize mitochondrial NADH as part of the electron transport chain (22). The compartmental restriction of NADH oxidation has important ramifications for metabolism and electron transport. The electrons originating from cytosolic NADH are preferred over those originating from mitochondrial NADH (6) for entrance into the electron transport chain. The direct consequence of preferential utilization of cytosolic NADH is a higher redox potential (NADH/NAD⁺) in the mitochondria than in the cytosol. Consequently, during rapid NADH synthesis, as during exponential growth, the TCA cycle ceases to operate as a cycle and branches into oxidative and reducing pathways (12).Metabolic consequences of the compartmentalization of NADH homeostasis were evident from the difference in the product formation profile upon lowering of cytosolic or mitochondrial NADH. Lowering cytosolic NADH by overexpressing bacterial NADH oxidase lowered the production of glycerol and biomass by S. cerevisiae (14, 36). On the other hand, decreasing the mitochondrial NADH level decreased ethanol production and increased the biomass yield (36). These results are likely to be a combination of effects from alleviating the feedback inhibition of the TCA cycle by mitochondrial NADH and increasing respiratory capacity due to improved efficiency of oxidative phosphorylation, as quantified by the P/O ratio (15). There are no reports that describe the effect of increasing NADH in S. cerevisiae, although formate has been used previously as a source of additional reducing power in S. cerevisiae (2, 4, 11, 23, 24, 27). Formate (HCOO⁻) is efficiently oxidized to CO₂ by NAD⁺-dependent formate dehydrogenase (27) and, therefore, cannot be used as a carbon source for biomass synthesis. Thus, using formate as an auxiliary substrate for the generation of NADH to study the effect of increased NADH may be a feasible option. Given the compartment-dependent regulation of NADH homeostasis in S. cerevisiae (36), increasing the NADH level in the cytosol is likely to elicit a response different from that obtained by increasing the NADH level in the mitochondria.The aim of the present study is to differentiate between the metabolic consequences of increasing NADH in the cytosol and those of increasing NADH in the mitochondria of S. cerevisiae. Toward this aim, we either overexpressed the native Fdh1 (NAD⁺-dependent formate dehydrogenase) in the cytosol or directed it into the mitochondria in a strain background that is otherwise devoid of formate metabolism. We present our understanding of the physiological characteristics of the mutant strains under steady-state or dynamic conditions in the presence of different levels of formate.     

The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution

Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.     

Nocardiosis in Mediterranean bivalves: First detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy)

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.     

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.