Crabtree Effect Research Articles

Novel Strategy for the Calorimetry-Based Control of Fed-Batch Cultivations of Saccharomyces cerevisiae

Typical controllers for fed-batch cultivations are based on the estimation and control of the specific growth rate in real time. Biocalorimetry allows one to measure a heat signal proportional to the substrate consumed by cells. The derivative of this heat signal is usually used to evaluate the specific growth rate, introducing noise to the resulting estimate. To avoid this, this study investigated a novel controller based directly on the heat signal. Time trajectories of the heat signal setpoint were modelled for different specific growth rates, and the controller was set to follow this dynamic setpoint. The developed controller successfully followed the setpoint during aerobic cultivations of Saccharomyces cerevisiae, preventing the Crabtree effect by maintaining low glucose concentrations. With this new method, fed-batch cultivations of S. cerevisiae could be reliably controlled at specific growth rates between 0.075 h−1 and 0.20 h−1, with average root mean square errors of 15 ± 3%.

A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism.

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer, and neurological disorders. Therefore, unraveling the role of nutrients as signaling molecules and metabolites together with their interconnectivity may provide a deeper understanding of how these conditions occur. Both signaling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition, current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism in yeast cells, we developed a hybrid model, combining a Boolean module, describing the main pathways of glucose and nitrogen signaling, and an enzyme-constrained model accounting for the central carbon metabolism of Saccharomyces cerevisiae, using a regulatory network as a link. The resulting hybrid model was able to capture a diverse utalization of isoenzymes and to our knowledge outperforms constraint-based models in the prediction of individual enzymes for both respiratory and mixed metabolism. The model showed that during fermentation, enzyme utilization has a major contribution in governing protein allocation, while in low glucose conditions robustness and control are prioritized. In addition, the model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression, as well as regulatory effects associated with lifespan increase during caloric restriction. Overall, we show that our hybrid model provides a comprehensive framework for the study of the non-trivial effects of the interplay between signaling and metabolism, suggesting connections between the Snf1 signaling pathways and processes that have been related to chronological lifespan of yeast cells.

Saccharomyces cerevisiae as a model to study synthetic cannabinoids: the impact of using different carbon sources

Introduction Synthetic cannabinoids (SC) are potent agonists of cannabinoid receptors, that mime the psychoactive effect of Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive component of cannabis [1]. JWH-018 was the first novel psychoactive SC found in the recreational drug marketplace in 2008. A few years later, JWH-018 started to be controlled by authorities, so alternative molecules started to emerge. THJ-018 was designed to replace JWH-018, having a similar structural skeleton and also a naphthalene and pentyl chain connected via a middle core substructure. Eventually, THJ-018 was scheduled and alternatives emerged, such as EG-018 [2]. This practice makes almost impossible to characterise SC toxicological profiles on an acceptable time scale, mostly due to the time-consuming experiments that must be held in animal models or human cells by standard methods. The yeast Saccharomyces cerevisiae shares highly conserved molecular and cellular mechanisms with human cells and has been used before for synthetic cathinones [3]. The present work has studied the best carbon source (glucose or galactose) to measure the impact of synthetic cannabinoids on S. cerevisiae growth, aiming to develop a method able to profile synthetic cannabinoids toxicity in a short time scale. The difference between carbon sources is that in Crabtree-positive yeast strains, glucose induce a strong inhibition of mitochondrial oxidative phosphorylation [4]. Materials and methods The effect of EG-018 was evaluated by its impact on S. cerevisiae BY4741 (WT) growth on minimal medium with 2% Glucose or 1% Galactose as sole carbon sources, in the presence and absence of the SC, followed at OD600nm. Growth rates at each condition was fitted to a logistic equation. Results Figure 1 shows OD600max obtained from the non-linear regression to the logistic equation in the presence of different concentrations of EG-018. While in galactose there is no effect, in 2% Glucose the results points to a growth decrease with EG-018. Discussion and conclusions: Comparing the two carbon sources, yeast growth on glucose is more susceptible to EG-018. Recent studies points that JWH-018, a similar SC, exerts an effect on glycolytic and pentose phosphate pathway at high concentrations of glucose (personal communication). As yeast growth in galactose proceeds simultaneously via respiration and fermentation due to the “Crabtree effect” [4] an impact of EG-018 on glycolysis might be less effective. These results, suggest that glucose is the best carbon source to develop a yeast based toxicity sensor for SC. Figure 1. OD600max results in S. cerevisiae BY4741 in the presence of 0, 25 and 50 µM of EG-018 in glucose and galactose.

Impact of the Whole Genome Duplication Event on PYK Activity and Effects of a PYK1 Mutation on Metabolism in S. cerevisiae.

Background: Evolution of aerobic fermentation (crabtree effect) in yeast is associated with the whole genome duplication (WGD) event, suggesting that duplication of certain genes may have altered yeast metabolism. The pyruvate kinase (PYK) gene is associated with alterations in cell metabolism, and duplicated during the WGD, generating PYK1 and PYK2. Thus, the impact of WGD on PYK activity and role of PYK in yeast metabolism were explored.Methods: PYK activity in the presence or absence of fructose-1,6-bisphosphate (FBP) was compared between pre- and post-WGD yeast. Glucose consumption, ethanol production, and oxygen consumption were measured in wildtype yeast and yeast with a T403E point mutation, which alters FBP binding affinity.Results: FBP stimulated increased PYK activity in pre-WGD yeast and in the PYK1 isoforms of post-WGD yeast, but not in the PYK2 isoforms of post-WGD yeast. Compared to wildtype, T403E mutant yeast displayed reduced glucose consumption, reduced ethanol production, and increased mitochondrial metabolism.Conclusion: The WGD event impacted the sensitivity of PYK activity to FBP. Mutations in the FBP binding domain of PYK induce metabolic shifts that favor respiration and suppress fermentation.

Gene expression regulates metabolite homeostasis during the Crabtree effect: Implications for the adaptation and evolution of Metabolism

A key issue in both molecular and evolutionary biology has been to define the roles of genes and phenotypes in the adaptation of organisms to environmental changes. The dominant view has been that an organism's metabolic adaptations are driven by gene expression and that gene mutations, independent of the starting phenotype, are responsible for the evolution of new metabolic phenotypes. We propose an alternate hypothesis, in which the phenotype and genotype together determine metabolic adaptation both in the lifetime of the organism and in the evolutionary selection of adaptive metabolic traits. We tested this hypothesis by flux-balance and metabolic-control analysis of the relative roles of the starting phenotype and gene expression in regulating the metabolic adaptations during the Crabtree effect in yeast, when they are switched from a low- to high-glucose environment. Critical for successful short-term adaptation was the ability of the glycogen/trehalose shunt to balance the glycolytic pathway. The role of later gene expression of new isoforms of glycolytic enzymes, rather than flux control, was to provide additional homeostatic mechanisms allowing an increase in the amount and efficiency of adenosine triphosphate and product formation while maintaining glycolytic balance. We further showed that homeostatic mechanisms, by allowing increased phenotypic plasticity, could have played an important role in guiding the evolution of the Crabtree effect. Although our findings are specific to Crabtree yeast, they are likely to be broadly found because of the well-recognized similarities in glucose metabolism across kingdoms and phyla from yeast to humans.

Creation of a Low-Alcohol-Production Yeast by a Mutated SPT15 Transcription Regulator Triggers Transcriptional and Metabolic Changes During Wine Fermentation.

There is significant interest in the wine industry to develop methods to reduce the ethanol content of wine. Here the global transcription machinery engineering (gTME) technology was used to engineer a yeast strain with decreased ethanol yield, based on the mutation of the SPT15 gene. We created a strain of Saccharomyces cerevisiae (YS59-409), which possessed ethanol yield reduced by 34.9%; this was accompanied by the increase in CO2, biomass, and glycerol formation. Five mutation sites were identified in the mutated SPT15 gene of YS59-409. RNA-Seq and metabolome analysis of YS59-409 were conducted compared with control strain, suggesting that ribosome biogenesis, nucleotide metabolism, glycolysis flux, Crabtree effect, NAD+/NADH homeostasis and energy metabolism might be regulated by the mutagenesis of SPT15 gene. Furthermore, two genes related to energy metabolism, RGI1 and RGI2, were found to be associated with the weakened ethanol production capacity, although the precise mechanisms involved need to be further elucidated. This study highlighted the importance of applying gTME technology when attempting to reduce ethanol production by yeast, possibly reprogramming yeast’s metabolism at the global level.

High Cell Density Cultivation of Saccharomyces cerevisiae with Intensive Multiple Sequential Batches Together with a Novel Technique of Fed-Batch at Cell Level (FBC)

High cell density cultivation (HCDC) is developed for the production of microbial biomasses and their products. They must be produced from high concentrations of substrate, e.g., glucose or sucrose. In batch culture, a high concentration of those sugars >40–50% (w/v) cannot efficiently be utilized because of a dissolved O2 limitation causing the Crabtree effect that produces toxic by-products, i.e., ethanol and/or acetate, that inhibit cell growth. To prevent this effect, the HCDC is conducted with the fed-batch strategies. However, it has many disadvantages, i.e., complicated operations. To overcome those problems, this study was designed to use a new, efficient C-source (carbon source) substrate, namely dextrin, an oligomer of glucose. It can be utilized by yeast at a very high concentration of ~100 g/L although using just batch cultivation. As it is gradually hydrolyzed to release glucose molecules and gradually assimilated into the cells as “fed-batch at the cell level” (FBC), it prevents the yeast cell system from undergoing the Crabtree effect. In this research, the types of medium, the types of sugar compared with dextrin, and the concentrations of yeast extract (YE) were studied. The batch production medium (BPM) with dextrin and YE performed very good results. The concentrations of dextrin for yeast cultivation were studied in the aerobic batch 5-L bioreactors. Its optimum concentration was at 90 g/L with 9 g/L of YE in 3× BPM. It was operated at 3 W/kg energy dissipation rate per unit mass (ε¯T) and 3 vvm airflow rate. Further, the intensive multiple sequential batch (IMSB) technique of high intensities of agitation speed and airflow was developed to achieve higher yield and productivity. The maximum values of cell biomass, specific growth rate, yield coefficient, productivity, and efficiency were at 55.17 g/L, 0.21 h−1, 0.54 g/g, 2.30 g/L/h, and 98.18%, respectively. The studies of cell growth kinetics, biochemical engineering mass balances, and fluid dynamics for the design of impeller speeds of the 5-L bioreactors during the cultivations of yeast using dextrin at the high concentrations were successful. The results can be used for the scale-up of bioreactor for the industrial production of yeast cell biomass at high concentrations.

Dissolved-oxygen feedback control fermentation for enhancing \u03b2-carotene in engineered Yarrowia lipolytica

The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of β-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L β-carotene. Both biomass and β-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP+/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, μm′, Yx/s′, and Yp/s′ was 0.527, 0.353, and 0.158, respectively. The μm′ was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Yx/s′ and Yp/s′ were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of β-carotene.

Reconstruction and analysis of genome-scale metabolic model of weak Crabtree positive yeast Lachancea kluyveri

Lachancea kluyveri, a weak Crabtree positive yeast, has been extensively studied for its unique URC pyrimidine catabolism pathway. It produces more biomass than Saccharomyces cerevisiae due to the underlying weak Crabtree effect and resorts to fermentation only in oxygen limiting conditions that renders it as a suitable industrial host. The yeast also produces ethyl acetate as a major overflow metabolite in aerobic conditions. Here, we report the first genome-scale metabolic model, iPN730, of L. kluyveri comprising of 1235 reactions, 1179 metabolites, and 730 genes distributed in 8 compartments. The in silico viability in different media conditions and the growth characteristics in various carbon sources show good agreement with experimental data. Dynamic flux balance analysis describes the growth dynamics, substrate utilization and product formation kinetics in various oxygen-limited conditions. We have also demonstrated the effect of switching carbon sources on the production of ethyl acetate under varying oxygen uptake rates. A phenotypic phase plane analysis described the energetic cost penalty of ethyl acetate and ethanol production on the specific growth rate of L. kluyveri. We generated the context specific models of L. kluyveri growing on uracil or ammonium salts as the sole nitrogen source. Differential flux calculated using flux variability analysis helped us in highlighting pathways like purine, histidine, riboflavin and pyrimidine metabolism associated with uracil degradation. The genome-scale metabolic construction of L. kluyveri will provide a better understanding of metabolism behind ethyl acetate production as well as uracil catabolism (pyrimidine degradation) pathway. iPN730 is an addition to genome-scale metabolic models of non-conventional yeasts that will facilitate system-wide omics analysis to understand fungal metabolic diversity.

Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity of HepG2 cells to the purine derivative kinetin riboside

Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucose-induced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosis-inducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress.

Performance, genomic rearrangements, and signatures of adaptive evolution: Lessons from fermentative yeasts.

The capacity of some yeasts to extract energy from single sugars, generating CO2 and ethanol (=fermentation), even in the presence of oxygen, is known as the Crabtree effect. This phenomenon represents an important adaptation as it allowed the utilization of the ecological niche given by modern fruits, an abundant source of food that emerged in the terrestrial environment in the Cretaceous. However, identifying the evolutionary events that triggered fermentative capacity in Crabtree‐positive species is challenging, as microorganisms do not leave fossil evidence. Thus, key innovations should be inferred based only on traits measured under culture conditions. Here, we reanalyzed data from a common garden experiment where several proxies of fermentative capacity were recorded in Crabtree‐positive and Crabtree‐negative species, representing yeast phylogenetic diversity. In particular, we applied the “lasso‐OU” algorithm which detects points of adaptive shifts, using traits that are proxies of fermentative performance. We tested whether multiple events or a single event explains the actual fermentative capacity of yeasts. According to the lasso‐OU procedure, evolutionary changes in the three proxies of fermentative capacity that we considered (i.e., glycerol production, ethanol yield, and respiratory quotient) are consistent with a single evolutionary episode (a whole‐genomic duplication, WGD), instead of a series of small genomic rearrangements. Thus, the WGD appears as the key event behind the diversification of fermentative yeasts, which by increasing gene dosage, and maximized their capacity of energy extraction for exploiting the new ecological niche provided by single sugars.

The role of hexokinase in cancer

A thorough understanding of the processes occurring in cancer cells is necessary to make cancer treatment as effective as possible. Changes in cellular metabolism in relation to normal cells are considered particularly important. One of the most interesting and promising areas is glucose metabolism and the factors affecting this process, with special emphasis on the potential role of hexokinases, especially the isoform II of this enzyme. Hexokinases (HK) are transferase enzymes involved in the process of glycolysis. Hexokinase II (HK II) plays an important role in initiating and maintaining the glycolysis process at a high level of efficiency, which is crucial for the growth and proliferation of cancer cells. An increase in the number of copies of the HK II gene and increased transcription of this enzyme resulting in the suppression of apoptosis and the enhancement of cell proliferation have been found in tumor cells. Hexokinase II also participates in the Crabtree effect by affecting the amount of ATP and thus the efficiency of the Ca2+ removal process outside the cell membrane by Ca2+ ATPase. Overexpression of HK II has thus far been found in pancreatic cancer, gastric cancer, breast cancer, squamous cell carcinoma of the larynx, glioblastoma multiforme, ovarian cancer and biliary tract cancer, indicating the possible key role of this enzyme in their formation and progression and providing the basis for seeking potential benefits of cancer treatment using HK II as a target of new drugs.

Fructose-Induced Crabtree Effect: Inhibition of Respiration and Stimulation of Glycolysis in HepG2 Cells

Background: Non-alcoholic fatty liver disease (NAFLD) is approaching epidemic proportions in the US. Increased consumption of fructose (in the form of sucrose or high-fructose corn syrup) has been implicated in the development of NAFLD. The acute effects of fructose on energy metabolism of the liver have not been explored yet. Glucose is known to shift the energy source of ATP generation from respiration to glycolysis (the Crabtree effect). The liver is unique since glucose metabolism is very low in normoglycemia, however, fructose is readily metabolized and is not subject to enzyme regulation that limits the metabolism of glucose in liver. We hypothesized that fructose could induce the Crabtree effect in liver cells. Methods: The hepatoma cell line (HepG2) was cultured in MEM medium. Energy metabolism was investigated using the Agilent Seahorse XFe24 Analyzer, which simultaneously measures respiration (as oxygen consumption rate, OCR) and glycolysis (related to extracellular acidification rate, ECAR). These rates were used to estimate ATP production rates (JATP; see Mookerjee et al., 2018, J.Biol..Chem. 292:7189). Results: Glucose (10 mM) inhibited mitochondrial (oxidative) ATP production by 67% and, at the same time, increased glycolytic ATP production 6.2-fold. The effects of fructose were similar albeit somewhat smaller: fructose (10 mM) inhibited mitochondrial ATP production by 48% and increased glycolytic ATP production 3.6-fold. The effects of glucose and fructose were not additive. Conclusions: This preliminary study shows that fructose alters energy metabolism in a model system of the liver. A similar effect in may contribute to the development of NAFLD.

Fumarate production with Rhizopus oryzae: utilising the Crabtree effect to minimise ethanol by-product formation

BackgroundThe four-carbon dicarboxylic acids of the tricarboxylic acid cycle (malate, fumarate and succinate) remain promising bio-based alternatives to various precursor chemicals derived from fossil-based feed stocks. The double carbon bond in fumarate, in addition to the two terminal carboxylic groups, opens up an array of downstream reaction possibilities, where replacement options for petrochemical derived maleic anhydride are worth mentioning. To date the most promising organism for producing fumarate is Rhizopus oryzae (ATCC 20344, also referred to as Rhizopus delemar) that naturally excretes fumarate under nitrogen-limited conditions. Fumarate excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. Attempts to eliminate ethanol production classically focus on enhanced oxygen availability within the mycelium matrix. In this study our immobilised R. oryzae process was employed to investigate and utilise the Crabtree characteristics of the organism in order to establish the limits of ethanol by-product formation under growth and non-growth conditions.ResultsAll fermentations were performed with either nitrogen excess (growth phase) or nitrogen limitation (production phase) where medium replacements were done between the growth and the production phase. Initial experiments employed excess glucose for both growth and production, while the oxygen partial pressure was varied between a dissolved oxygen of 18.4% and 85%. Ethanol was formed during both growth and production phases and the oxygen partial pressure had zero influence on the response. Results clearly indicated that possible anaerobic zones within the mycelium were not responsible for ethanol formation, hinting that ethanol is formed under fully aerobic conditions as a metabolic overflow product. For Crabtree-positive organisms like Saccharomyces cerevisiae ethanol overflow is manipulated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae for both growth and production fermentations. It was shown that all ethanol can be eliminated during growth for a glucose addition rate of 0.07,hbox {g},hbox {L}^{-1},hbox {h}^{-1}. The production phase behaved in a similar manner, where glucose addition of 0.197,hbox {g},hbox {L}^{-1},hbox {h}^{-1} resulted in fumarate production of 0.150,hbox {g},hbox {L}^{-1},hbox {h}^{-1} and a yield of 0.802,hbox {g},hbox {g}^{-1} fumarate on glucose. Further investigation into the effect of glucose addition revealed that ethanol overflow commences at a glucose addition rate of 0.395,hbox {g},hbox {g}^{-1},hbox {h}^{-1} on biomass, while the maximum glucose uptake rate was established to be between 0.426 and 0.533,hbox {g},hbox {g}^{-1},hbox {h}^{-1}.ConclusionsThe results conclusively prove that R. oryzae is a Crabtree-positive organism and that the characteristic can be utilised to completely discard ethanol by-product formation. A state referred to as “homofumarate production” was illustrated, where all carbon input exits the cell as either fumarate or respiratory hbox {CO}_{2}. The highest biomass-based “homofumarate production”: rate of 0.243,hbox {g},hbox {g}^{-1},hbox {h}^{-1} achieved a yield of 0.802,hbox {g},hbox {g}^{-1} on glucose, indicating the bounds for developing an ethanol free process. The control strategy employed in this study in conjunction with the uncomplicated scalability of the immobilised process provides new direction for further developing bio-fumarate production.

Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur.

Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and in vivo, acquired with genetically-encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K+ coincides with a Warburg effect mediated by NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4+. The compounded outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na+ pump. We conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.

Tracking Yeast Metabolism and the Crabtree Effect in Real Time via CO2 Production using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS)

In this study, a new approach to measure metabolic activity of yeast via the Crabtree effect is described. BARDS is an analytical technique developed to aid powder and tablet characterisation by monitoring changes in the compressibility of a solvent during solute dissolution. It is a rapid and simple method which utilises a magnetic stir bar to mix added solute and induce the acoustic resonance of a vessel containing a fixed volume of solvent. In this study it is shown that initiation of fermentation in a yeast suspension, in aqueous buffer, is accompanied by reproducible changes in the frequency of induced acoustic resonance. These changes signify increased compressibility of the suspension due to CO2 release by the yeast. A simple standardised BARDS protocol reveals yeast carbon source preferences and can generate quantitative kinetic data on carbon source metabolism which are characteristic of each yeast strain. The Crawford-Woods equation can be used to quantify total gaseous CO2 produced by a given number of viable yeast when supplied with a fixed amount of carbon source. This allows for a value to be calculated for the amount of gaseous CO2 produced by each yeast cell. The approach has the potential to transform the way in which yeast metabolism is tracked and potentially provide an orthogonal or surrogate method to determining viability, vitality and attenuation measurements in the future.

Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect.

Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.

Comparison of metabolic profiles of yeasts based on the difference of the Crabtree positive and negative

The Crabtree effect involves energy management in which yeasts utilize glycolysis as the terminal electron acceptor instead of oxygen, despite the presence of sufficient dissolved oxygen, when oxygen concentrations exceed a certain limit. The Crabtree effect is detrimental to bakery yeast production, because it results in lower cellular glucose yields. Batch culture of Saccharomyces cerevisiae, a Crabtree positive yeast, decreased the cell yield of glucose and produced large amounts of ethanol despite a high specific glucose consumption rate compared to Candida utilis, a Crabtree negative yeast. This study investigated the effect of these characteristics on metabolite levels. We performed metabolome analysis of both yeasts during each growth phase of batch culture using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Principle component analysis of metabolome data indicated that the Crabtree effect affected metabolites related to NADH synthesis in central metabolism. The amount of these metabolites in S.cerevisiae was lower than that in C.utilis. However, to maintain the specific glucose consumption rate at high levels, yeasts must avoid depletion of NAD+, which is essential for glucose utilization. Our results indicated that NADH was oxidized by converting acetaldehyde to ethanol in S.cerevisiae, which is in accordance with previous reports. Therefore, the specific NADH production rates of S.cerevisiae and C.utilis did not show a difference. This study suggested that NAD+/NADH ratio is disrupted by the Crabtree effect, which in turn influenced central metabolism and that S.cerevisiae maintained the NAD+/NADH ratio by producing ethanol.

Energy metabolism controls phenotypes by protein efficiency and allocation

Cells require energy for growth and maintenance and have evolved to have multiple pathways to produce energy in response to varying conditions. A basic question in this context is how cells organize energy metabolism, which is, however, challenging to elucidate due to its complexity, i.e., the energy-producing pathways overlap with each other and even intertwine with biomass formation pathways. Here, we propose a modeling concept that decomposes energy metabolism into biomass formation and ATP-producing pathways. The latter can be further decomposed into a high-yield and a low-yield pathway. This enables independent estimation of protein efficiency for each pathway. With this concept, we modeled energy metabolism for Escherichia coli and Saccharomyces cerevisiae and found that the high-yield pathway shows lower protein efficiency than the low-yield pathway. Taken together with a fixed protein constraint, we predict overflow metabolism in E. coli and the Crabtree effect in S. cerevisiae, meaning that energy metabolism is sufficient to explain the metabolic switches. The static protein constraint is supported by the findings that protein mass of energy metabolism is conserved across conditions based on absolute proteomics data. This also suggests that enzymes may have decreased saturation or activity at low glucose uptake rates. Finally, our analyses point out three ways to improve growth, i.e., increasing protein allocation to energy metabolism, decreasing ATP demand, or increasing activity for key enzymes.

SNF1 controls the glycolytic flux and mitochondrial respiration.

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.