saccharomyces-cerevisiae-exponential-growth-kinetics-batch-culture-to

<em>Saccharomyces cerevisiae</em> Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

mitochondrial-respiration-quantification-in-yeast-whole-cells

Mitochondrial Respiration Quantification in Yeast Whole Cells

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to ...

bergmeyer-glucose-quantification-for-microbiological-samples

Bergmeyer Glucose Quantification for Microbiological Samples

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, ...

Instituto Tecnológico Superior de Ciudad Hidalgo

Tecnológico Nacional de México/Instituto Tecnológico Superior de Ciudad Hidalgo

Resveratrol (3,4',5-trihydroxy-trans-stilbene, RSV) has emerged as an important molecule in the biomedical area. This is due to its antioxidant and health benefits exerted in mammals. Nonetheless, early studies have also demonstrated its toxic properties toward plant-pathogenic fungi of this phytochemical. Both effects appear to be opposed and caused by different molecular mechanisms. However, the inhibition of cellular respiration is a hypothesis that might explain both toxic and beneficial properties of resveratrol, since this phytochemical: (1) decreases the production of energy of plant-pathogenic organisms, which prevents their proliferation; (2) increases adenosine monophosphate/adenosine diphosphate (AMP/ADP) ratio that can lead to AMP protein kinase (AMPK) activation, which is related to its health effects, and (3) increases the reactive oxygen species generation by the inhibition of electron transport. This pro-oxidant effect induces expression of antioxidant enzymes as a mechanism to counteract oxidative stress. In this review, evidence is discussed that supports the hypothesis that cellular respiration is the main target of resveratrol. 

Resveratrol Inhibition of Cellular Respiration: New Paradigm for an Old Mechanism.

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 (HO) 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 HO 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 induces mitochondrial dysfunction and decreases chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner.

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases life span 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 reactive oxygen species generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in Saccharomyces cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and wild-type strains. Interestingly, the chronological life span was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed inhibition of the mitochondrial respiration in 0.5 and 10% glucose but only increased the H O levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the chronological life span of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H O levels.

Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner.

The antioxidant phenotype caused by resveratrol has been recognized as a key piece in the health benefits exerted by this phytochemical in diseases related to aging. It has recently been proposed that a mitochondrial pro-oxidant mechanism could be the cause of resveratrol antioxidant properties. In this regard, the hypothesis that resveratrol impedes electron transport to complex III of the electron transport chain as its main target suggests that resveratrol could increase reactive oxygen species (ROS) generation through reverse electron transport or by the semiquinones formation. This idea also explains that cells respond to resveratrol oxidative damage, inducing their antioxidant systems. Moreover, resveratrol pro-oxidant properties could accelerate the aging process, according to the free radical theory of aging, which postulates that organism's age due to the accumulation of the harmful effects of ROS in cells. Nonetheless, there is no evidence linking the chronological lifespan (CLS) shorten occasioned by resveratrol with a pro-oxidant mechanism. Hence, this study aimed to evaluate whether resveratrol shortens the CLS of Saccharomyces cerevisiae due to a pro-oxidant activity. Herein, we provide evidence that supplementation with 100 μM of resveratrol at 5% glucose: (1) shortened the CLS of ctt1Δ and yap1Δ strains; (2) decreased ROS levels and increased the catalase activity in WT strain; (3) maintained unaffected the ROS levels and did not change the catalase activity in ctt1Δ strain; and (4) lessened the exponential growth of ctt1Δ strain, which was restored with the adding of reduced glutathione. These results indicate that resveratrol decreases CLS by a pro-oxidant mechanism.

Resveratrol shortens the chronological lifespan of Saccharomyces cerevisiae by a pro-oxidant mechanism.

Adaptations of eukaryotic cells to environmental changes are important for their survival. However, under some circumstances, microenvironmental changes promote that eukaryotic cells utilize a metabolic signature resembling a unicellular organism named the Warburg effect. Most cancer cells share the Warburg effect displaying lactic fermentation and high glucose uptake. The Warburg effect also induces a metabolic rewiring stimulating glutamine consumption and lipid synthesis, also considered cancer hallmarks. Amino acid metabolism alteration due to the Warburg effect increases plasma levels of proline and branched-chain amino acids in several cancer types. Proline and lipids are probably used as electron transfer molecules in carcinogenic cells. In addition, branched-chain amino acids fuel the Krebs cycle, protein synthesis, and signaling in cancer cells.

Metabolic profile of the Warburg effect as a tool for molecular prognosis and diagnosis of cancer.

The Crabtree effect molecular regulation comprehension could help to improve ethanol production with biotechnological purposes and a better understanding of cancer etiology due to its similarity with the Warburg effect. Snf1p/Hxk2p/Mig1p pathway has been linked with the transcriptional regulation of the hexose transporters and phenotypes associated with the Crabtree effect. Nevertheless, direct evidence linking the genetic control of the hexose transporters with modulation of the Crabtree effect phenotypes by the Snf1p/Hxk2p/Mig1p pathway is still lacking. In this sense, we provide evidence that SNF1 and HXK2 genes deletion affects exponential growth, mitochondrial respiration, and transcript levels of hexose transporters in a glucose-dependent manner. The Vmax of the hexose transporters with the high transcript levels was correlated positively with the exponential growth and negatively with the mitochondrial respiration. HXT2 gene transcript levels were the most affected by the deletion of the SNF1/HXK2/MIG1 pathway. Deleting the orthologous genes SNF1 and HXK2 in Kluyveromyces marxianus (Crabtree negative yeast) has an opposite effect compared to Saccharomyces cerevisiae in growth and mitochondrial respiration. Overall, these results indicate that the SNF1/HXK2/MIG1 pathway regulates transcript levels of the hexose transporters, which shows an association with the exponential growth and mitochondrial respiration in a glucose-dependent manner.

Snf1p/Hxk2p/Mig1p pathway regulates hexose transporters transcript levels, affecting the exponential growth and mitochondrial respiration of Saccharomyces cerevisiae.

Quercetin is a flavonol ubiquitously present in fruits and vegetables that shows a potential therapeutic use in non-transmissible chronic diseases, such as cancer and diabetes. Although this phytochemical has shown promising health benefits, the molecular mechanism behind this compound is still unclear. Interestingly, quercetin displays toxic properties against phylogenetically distant organisms such as bacteria and eukaryotic cells, suggesting that its molecular target resides on a highly conserved pathway. The cytotoxicity of quercetin could be explained by energy depletion occasioned by mitochondrial respiration impairment and its concomitant pleiotropic effect. Thereby, the molecular basis of quercetin cytotoxicity could shed light on potential molecular mechanisms associated with its health benefits. Nonetheless, the evidence supporting this hypothesis is still lacking. Thus, this study aimed to evaluate whether quercetin supplementation affects mitochondrial respiration and whether this is related to quercetin cytotoxicity. Saccharomyces cerevisiae was used as a study model to assess the effect of quercetin on energetic metabolism. Herein, we provide evidence that quercetin supplementation: (1) decreased the exponential growth of S. cerevisiae in a glucose-dependent manner; (2) affected diauxic growth in a similar way to antimycin A (complex III inhibitor of electron transport chain); (3) suppressed the growth of S. cerevisiae cultures supplemented with non-fermentable carbon sources (glycerol and lactate); (4) promoted a glucose-dependent inhibition of the basal, maximal, and ATP-linked respiration; (5) diminished complex II and IV activities. Altogether, these data indicate that quercetin disturbs mitochondrial respiration between the ubiquinone pool and cytochrome c, and this phenotype is associated with its cytotoxic properties.

Cytotoxicity of quercetin is related to mitochondrial respiration impairment in Saccharomyces cerevisiae.

Cancer is a leading cause of death worldwide, reporting nearly 10 million deaths in 2020. One of the hallmarks of cancer cells is their capability to evade growth suppressors and sustain proliferative signaling resulting in uncontrolled growth. The AMPK pathway, a catabolic via to economize ATP, has been associated with cancer. AMPK activation is related to cancer progression in advanced stages, while its activation by metformin or phenformin is associated with cancer chemoprevention. Thus, the role of the AMPK pathway in cancer growth modulation is not clear. Saccharomyces cerevisiae might be a useful model to elucidate AMPK participation in growth regulation since it shares a highly conserved AMPK pathway. Therefore, this work is aimed at evaluating the role of the AMPK pathway on S. cerevisiae growth under different nutritional conditions. Herein, we provide evidence that the SNF1 gene is necessary to maintain S. cerevisiae growth with glucose as a sole carbon source at every concentration tested. Resveratrol supplementation inhibited the exponential growth of snf1∆ strain at low glucose levels and decreased it at high glucose levels. SNF1 gene deletion impaired exponential growth in a carbohydrate concentration-dependent manner independently of nitrogen source or concentration. Interestingly, deletion of genes encoding for upstream kinases (SAK1, ELM1, and TOS3) also had a glucose dose-dependent effect upon exponential growth. Furthermore, gene deletion of regulatory subunits of the AMPK complex impacted exponential growth in a glucose-dependent manner. Altogether, these results suggest that the SNF1 pathway affects the exponential growth of S. cerevisiae in a glucose-dependent manner.

The role of the SNF1 signaling pathway in the growth of Saccharomyces cerevisiae in different carbon and nitrogen sources.

Quercetin is a flavonoid with promising therapeutic applications; nonetheless, the phenotype exerted in some diseases is contradictory. For instance, anticancer properties may be explained by a cytotoxic mechanism, whereas antioxidant-related neuroprotection is a pro-survival process. According to the available literature, quercetin exerts a redox interaction with the electron transport chain (ETC) in the mitochondrion, affecting its membrane potential. It also affects ATP generation by oxidative phosphorylation, where ATP deprivation could partly explain its cytotoxic effect. Moreover, quercetin may support the generation of free radicals through redox reactions, causing a prooxidant effect. The nutrimental stress and prooxidant effect induced by quercetin might promote pro-survival properties such as antioxidant processes. Thus, in this review, we discuss the evidence supporting that quercetin redox interaction with the ETC could explain its beneficial and toxic properties.

The multifaceted role of quercetin derived from its mitochondrial mechanism.

Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 2 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 2 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.

ArticleTotal : 3	Year
Publication title Cited by 45	2018
Publication title Cited by 1	2024
Publication title Cited by 3	2025

Article	Year
Resveratrol Inhibition of Cellular Respiration: New Paradigm for an Old Mechanism. International journal of molecular sciences\| PubMed ID: 26999118	2016
Resveratrol induces mitochondrial dysfunction and decreases chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner. Journal of bioenergetics and biomembranes\| PubMed ID: 28401438	2017
Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner. Yeast (Chichester, England)\| PubMed ID: 29277922	2018
Resveratrol shortens the chronological lifespan of Saccharomyces cerevisiae by a pro-oxidant mechanism. Yeast (Chichester, England)\| PubMed ID: 34693568	2022
Metabolic profile of the Warburg effect as a tool for molecular prognosis and diagnosis of cancer. Expert review of molecular diagnostics\| PubMed ID: 35395916	2022
Snf1p/Hxk2p/Mig1p pathway regulates hexose transporters transcript levels, affecting the exponential growth and mitochondrial respiration of Saccharomyces cerevisiae. Fungal genetics and biology : FG & B\| PubMed ID: 35526810	2022
Cytotoxicity of quercetin is related to mitochondrial respiration impairment in Saccharomyces cerevisiae. Yeast (Chichester, England)\| PubMed ID: 36285422	2022
The role of the SNF1 signaling pathway in the growth of Saccharomyces cerevisiae in different carbon and nitrogen sources. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]\| PubMed ID: 36972016	2023
The multifaceted role of quercetin derived from its mitochondrial mechanism. Molecular and cellular biochemistry\| PubMed ID: 37656383	2024
Lipase activity of recombinant KmYJR107Wp and KmLIP3p enzymes expressed in Saccharomyces cerevisiae BY4742 from Kluyveromyces marxianus L2029. Journal, genetic engineering & biotechnology\| PubMed ID: 39179325	2024

Luis Alberto Madrigal-Perez

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