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.

The transcription factor Sp1 regulates expression of numerous genes involved in many cellular processes. Different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, alternative splicing isoforms may also be the basis of its distinct functional activities. In this study, we identified a novel alternative splice isoform of Sp1 named Sp1c. This variant is generated by exclusion of a short domain, which we designate a, through alternative splice acceptor site usage in the exon 3. The existence of this new isoform was confirmed in vivo by Western blotting analysis. Although at very low levels, Sp1c is ubiquitously expressed, as seen in its fulllength Sp1. A preliminary characterization of Sp1c shows that: (a) Sp1c works as stronger activator of transcription than full-length Sp1; (b) percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than percentage of HEK293 Sp1-overexpressing cells.

Whey generated in cheese manufacturing poses serious environmental issues that limit process profitability. The innovation in the dairy sector recognizes the "bio-refinery" as a key to successful handling of whey disposal and economic rise. Cheese whey valorisation is a complex process involving multiple technologies that might lead to value-added products (biomass, fine or bulk chemicals). This work focuses on the optimization of a fermentation process using whey as growth medium and carbon source. Lactose, which is abundant in whey, is a valuable carbon source. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, is not a lactose-fermenting yeast. We set up an innovative biotechnological process for the production on large scale of a not-genetically modified yeast biomass that can be used in different contexts, such as bread making, production of probiotics, nutraceuticals, bio-active molecules. In order to use the cheese whey as raw material for the cultivation of S. cerevisiae and to overcome the limitations in the use of lactose we used and externally added the enzyme β-galactosidase. The careful optimization of the amount of added enzyme allowed the gradual release by hydrolysis and the simultaneous consumption of glucose and galactose with a consequent decrease of ethanol and an increase of the biomass produced.

Although the decrease in pyruvate secretion by brewer’s yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl a-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and 13C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer’s yeasts.

In yeast, pyruvate is placed at the crossroad of fermentation, oxidative metabolism and anabolic pathways. In this study we have characterized a previously developed pyruvate undersecreting sake yeast obtained by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, we investigated the mitochondrial activity of TCR7 by oxigraphy and 13C-metabolic flux analysis. The mutant strain (TCR7), displayed an higher mitochondrial pyruvate influx and oxidation, and a decreased glycerol production compared to the reference strain. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate extracellular secretion. Surprisingly mitochondrial activity resulted much higher in the sake yeast compared to CEN.PK 113-7D, the reference strain employied in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retained a branched mitochondrial structure for a longer time than laboratory strains. Further studies are needed to unveil the molecular mechanisms underlying these phenotypes.

The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called mitochondrial carriers (MCs) [1]. The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions with different tissuespecific expression patterns, and the identification of several diseases caused by alterations of their genes [2]. Until now, 22 MC subfamilies have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes [1]. In particular two well characterized MC subfamilies are known to play a crucial role in activating the mitochondrial apoptotic pathway, the first is the subfamily of the ADP/ATP carriers and the second is the subfamily of the citrate carrier. ADP/ATP carriers catalyze the efflux of ATP from the mitochondrial matrix in exchange for cytosolic ADP and their specific inhibition can lead the permeability transition pore opening in case of oxidative stress [3]. Citrate carrier catalyses the efflux of citrate from the mitochondrial matrix in exchange for cytosolic malate and plays a key role in inflammation [4,5]. Our data together with literature data let us suppose that these two MC subfamilies are promising molecular targets for cancer treatment. In particular basing on our knowledge of MC structure, translocation mechanism and substrate specificity [6] we are evaluating neuroendocrine cancer cell resistance to old MC inhibitors and we are screening chemical libraries to develop new specific drugs to be used for viability assays.

Saccharomyces cerevisiae is the preferred microorganism in the ethanol fermentation industry. However its use for the fermentation of renewable resources such as lignocellulosic biomass is impaired by several metabolic bottlenecks and toxic by-products formed in the pre-treatment process. Furfural is the most abundant by-product of hemicellulose hydrolysis which inhibits yeast growth [1]. At low concentrations, S. cerevisiae can overcome furfural toxicity by converting it to the corresponding alcohol (furfuryl alcohol) by NAD(P)H-dependent reactions [2]. Unfortunately these mechanisms of detoxification compete for key enzymes and cofactors needed to branch carbon flow to respiration and to ethanol production. Several studies have reported that furfural at high concentrations decreases yeast viability, specific growth rate and volumetric fermentation rate. Recently it has been proposed that furfural induces reactive oxygen species (ROS) generation and cellular damage in S. cerevisiae (3) ever the mechanism of furfural toxicity in yeast is not yet fully understood. We have carried out a flow cytometry analysis of yeast CEN-PK cells in the presence of different concentrations of furfural. We did not observe any significant increase of ROS production even at concentrations that abolished growth. However, we found that furfural induced a strong membrane depolarization during the lag phase, followed by hyperpolarization and cell death after the cells had started growing. Our results shed light on the mechanisms of toxicity of furfural in yeast and pave the way to a rational approach to improvement of tolerance of S. cerevisiae.

Saccharomyces cerevisiae is the preferred microorganism in the ethanol fermentation industry from renewable resources such as lignocellulosic biomass. In the process of obtaining monomeric sugars from lignocellulose trough a combination of physical, chemical and enzymatic treatments of biomass formation of inhibitory compounds occurs among wich furfural is the most abundant (Almeida et al, 2007). S. cerevisiae is relatively tolerant to furfural because it is capable of converting it to the corresponding alcohol with lower inhibitory capability by NAD(P)H dependent reactions (Taherzadeh et al, 1999). Unfortunately these mechanisms of detoxification compete for key enzymes and cofactors needed to bra nch carbon flow to respiration and to ethanol production. Several studies have reported that furfural decreased yeast viability, specific growth rate and volumetric fermentation rate. A recent work (Allen et al, 2010) has shown that furfural induced reactive oxygen species (ROS) accumulation and cellular damage in S. cerevisiae. However the toxicity mechanism of furfural on yeast cells is not yet fully understood. In our study we have conducted a flow cytometry analysis on yeast cells from CEN-PK family in response to treatment with different concentrations of furfural. Exposure to high levels of inhibitor did not caused ROS accumulation but resulted in increased intracellular depolarization and accumulation of dead cells. These results may shed light on the mechanism of toxicity of furfural in yeast and might be useful to improve the inhibitor resistance in S. cerevisiae.

La presente invenzione ha per oggetto un nuovo procedimento per la sintesi di alcoli chirali mediante l’impiego di Lactobacillus reuteri. L’invenzione ha anche per oggetto l’uso di tale microorganismo per la sintesi di detti alcoli chirali.

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated to respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated to reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.