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A broad range of health benefits have been attributed to resveratrol (RSV) supplementation in mammalian systems, including the increases in longevity. Nonetheless, despite the growing number of studies performed with RSV, the molecular mechanism by which it acts still remains unknown. Recently, it has been proposed that inhibition of the oxidative phosphorylation activity is the principal mechanism of RSV action. This mechanism suggests that RSV might induce mitochondrial dysfunction resulting in oxidative damage to cells with a concomitant decrease of cell viability and cellular life span. To prove this hypothesis, the chronological life span (CLS) of Saccharomyces cerevisiae was studied as it is accepted as an important model of oxidative damage and aging. In addition, oxygen consumption, mitochondrial membrane potential, and hydrogen peroxide (H2O2) release were measured in order to determine the extent of mitochondrial dysfunction. The results demonstrated that the supplementation of S. cerevisiae cultures with 100 μM RSV decreased CLS in a glucose-dependent manner. At high-level glucose, RSV supplementation increased oxygen consumption during the exponential phase yeast cultures, but inhibited it in chronologically aged yeast cultures. However, at low-level glucose, oxygen consumption was inhibited in yeast cultures in the exponential phase as well as in chronologically aged cultures. Furthermore, RSV supplementation promoted the polarization of the mitochondrial membrane in both cultures. Finally, RSV decreased the release of H2O2 with high-level glucose and increased it at low-level glucose. Altogether, this data supports the hypothesis that RSV supplementation decreases CLS as a result of mitochondrial dysfunction and this phenotype occurs in a glucose-dependent manner.

Resveratrol is a phytochemical that may promote health. However, it has also been reported to be a toxic compound. The molecular mechanism by which resveratrol acts remains unclear. The inhibition of the oxidative phosphorylation (OXPHOS) pathway appears to be the molecular mechanism of resveratrol. Taking this into account, we propose that the cytotoxic properties of resveratrol depend on the energy (e.g., carbohydrates, lipids, and proteins) availability in the cells. In this regard, in a condition with low energy accessibility, resveratrol could enhance ATP starvation to lethal levels. In contrast, when cells are supplemented with high quantities of energy and resveratrol, the inhibition of OXPHOS might produce a low-energy environment, mimicking the beneficial effects of caloric restriction. This review suggests that investigating a possible complex relationship between caloric intake and the differential effects of resveratrol on OXPHOS may be justified. PRACTICAL APPLICATIONS: A low-calorie diet accompanied by significant levels of resveratrol might modify cellular bioenergetics, which could impact cellular viability and enhance the anti-cancer properties of resveratrol.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

The switch between mitochondrial respiration and fermentation as the main ATP production pathway through an increase glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response including glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 gene on the glycolytic flux and mitochondrial respiration through the glucose concentration variation to gain insights about its relationship with the Crabtree effect. Herein, we found that SNF1 deletion in Saccharomyces cerevisiae cells grown at 1% glucose, decreased glycolytic flux, increased NAD(P)H concentration, enhanced HXK2 gene transcription, and decreased mitochondrial respiration. Meanwhile, the same deletion increased the mitochondrial respiration of cells grown at 10% glucose. Altogether, these findings indicate that SNF1 is important to respond to glucose concentration variation and is involved in the switch between mitochondrial respiration and fermentation.

The switch of mitochondrial respiration to fermentation as the main pathway to produce ATP through the increase of glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response that includes glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 complex on the glycolytic flux and mitochondrial respiration, to gain insights about its relationship with the Crabtree effect. Herein, we found that inSaccharomyces cerevisiaecells grown at 1% glucose, mutation ofSNF1gene decreased glycolytic flux, increased NAD(P)H, enhancedHXK2gene transcription, and decreased mitochondrial respiration. Meanwhile, the same mutation increased the mitochondrial respiration of cells grown at 10% glucose. Moreover,SNF4gene deletion increased respiration and growth at 1% of glucose. In the case of theGAL83gene, we did not detect any change in mitochondrial respiration or growth. Altogether, these findings indicate thatSNF1is vital to switch from mitochondrial respiration to fermentation.

Nutritional homeostasis is fundamental for alcoholic fermentation in Saccharomyces cerevisiae. Carbon and nitrogen have been related to this metabolic process; nevertheless, little is known about their interactions with the media and the energetic metabolism. Rim15p kinase is a point of convergence among different nutrient-activated signaling pathways; this makes it a target to investigate the relationship between nutritional status and energetic metabolism. To improve the current knowledge of nutrient interactions and their association with RIM15, we validated the doubling time as an indicator of growth phenotype, confirming that this kinetic parameter can be related to the cellular bioenergetic status. This endorses the usefulness of a threshold in doubling time values as an indicator of fermentative (≤ 6.5 h) and respiratory growth (≥ 13.2 h). Using the doubling time as response variable, we find that (i) two second-order interactions between type and concentration of carbon and nitrogen sources significantly affected the growth phenotype of S. cerevisiae; (ii) these metabolic interactions changed when RIM15 was deleted, suggesting a dependence on this gene; (iii) high concentration of ammonium (5% w/v) is toxic for S. cerevisiae cells; (iv) proline prompted fermentative growth phenotype regardless presence or absence of RIM15; (v) RIM15 deletion reverted ammonium toxicity when cells were grown in glucose (10% w/v); and (vi) RIM15 deletion improves fermentative metabolism probably by a partial inhibition of the respiration capacity. This study reveals the existence of synergic and diverse roles of carbon and nitrogen sources that are affected by RIM15, influencing the fermentative and respiratory growth of S. cerevisiae.

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases lifespan whereas a high-glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences ROS generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the geneGSH1diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in thegsh1Δand WT strains. Interestingly, the chronological life span (CLS) was lowered in thegsh1Δstrain cultured with 10% glucose but not under dietary restriction. Thegsh1Δstrain also showed an inhibition of the mitochondrial respiration in 0.5 and 10% of glucose but only increased the H2O2levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease ingsh1Δstrain cultured with 0.5% glucose. Altogether these data indicate that glutathione has a major role in the function of electron transport chain (ETC) and is essential to maintain life span ofSaccharomyces cerevisiaein 10% glucose.

PodC es una enzima con actividad peroxidasa que puede tener diversas aplicaciones como la remoción de compuestos fenólicos de agua y su empleo en biosensores. No obstante, es necesario mejorar su proceso de producción para permitir que el proceso sea redituable, permitiendo aumentar su disponibilidad. Por lo que el objetivo del presente trabajo fue evaluar la producción de PodC bajo dos estrategias de cultivo. Además, se realizó la caracterización cinética de la cepa transformante, encontrando que sus parámetros cinéticos fueron inferiores (¿= 0.51±0.014 h-1 y td= 1.34±0.037 h) en comparación con los parámetros cinéticos de la cepa parental (¿= 0.62±0.011 h-1 y td= 1.10±0.020 h). Se eligió el inductor lactosa para su uso en los cultivos en biorreactor debido a que mejoró los parámetros cinéticos (¿= 0.092±0.004 h-1 y td= 7.50±0.326 h) de la cepa transformante en comparación con los obtenidos usando IPTG (¿= 0.057±0.002 h-1 y td= 12.16±0.498 h). Las estrategias de cultivo elegidas fueron cultivo por lote, empleado anteriormente para producir a PodC y el cultivo por auto-inducción, propuesto como una de las estrategias idóneas para la producción de proteínas recombinantes en cepas derivadas de E. coli BL21. Sin embargo, contrario a lo esperado el cultivo por lote tuvo un mejor desempeño que el cultivo por autoinducción. Lo cual pudo ser causado por el fenómeno de represión catabólica de carbono y a la inhabilidad del medio de cultivo auto-inductor para alcanzar altas densidades celulares. El empleo del cultivo por lote permitió alcanzar un rendimiento final del proceso de 84.84±0.84 mg/mL con una actividad específica de 911.63±8.009 U/mg. El cual fue dos veces mayor al reportado anteriormente (36 mg/mL con actividad específica de 1004 U/mg), si bien, el rendimiento final de PodC empleando cultivo por lote fue de solo del 0.30%, por lo que es necesario mejorar los procesos de solubilización y purificación de la proteína. PodC is an enzyme with peroxidase activity that can have different applications such as the removal of phenolic compounds from wastewater y its use in biosensors. However, it is necessary to improve its production process to allow it to be profitable y increase their availability. The aim of this study was to evaluate the production of PodC under two growth strategies. In addition, kinetic characterization of the transformant strain was performed, founding that its kinetic parameters (¿= 0.51±0.014 h-1 y td= 1.34±0.037 h) were inferiors compared with those of the parental strain (¿= 0.62±0.011 h-1 and td= 1.10±0.020 h). The lactose inducer was chosen for cultures in bioreactor due to the capacity of improved kinetic parameters of the transformant strain (¿= 0.092±0.004 h-1 and td= 7.50±0.326 h) were lower in comparison with those obtained using IPTG (¿= 0.057 ± 0.002 h-1 and td= 12.16 ± 0.498 h). The culture strategies chosen were batch cultivation, which has been previously used for produce PodC, and the autoinduction strategy, proposed as one of the best strategies for produced recombinant proteins, in strains derived from E. coli BL21. Nevertheless, we found that contrary our expectations the batch culture had outperformed the autoinduction culture. Which could be caused by the phenomenon called carbon catabolite repression y the inability of the autoinducer medium to achieve high cell density cultures. The use of batch culture allowed a final process yield of 84.84±0.84 mg/mL with a specific activity of 911.63±8.009 U/mg. Which was twice that reported previously (36 mg/mL with specific activity of 1004 U / mg), although the final yield of PodC was only 0.30%, so it is necessary to improve the processes of solubilization y protein purification.