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Cat8 is a C6 zinc cluster transcription activator in yeast. It is generally recognized that the transcription of CAT8 is inhibited and that Cat8 is inactive in the presence of high concentrations of glucose. However, our recent study found that constitutively overexpressed Cat8 played a regulatory role in Saccharomyces cerevisiae in the presence of 20 g/L glucose. To explore the regulatory network of Cat8 at high glucose concentrations, CAT8 was both overexpressed and deleted in this study. Cell growth and glucose consumption in different media were significantly accelerated by the deletion of CAT8, while the lag period was greatly shortened. RNA-seq and genetic modification showed that the deletion of CAT8 changed the type of energy metabolism in yeast cells. Many genes related to the mitochondrial respiratory chain were downregulated, resulting in a reduction in aerobic respiration and the tricarboxylic acid cycle. Meanwhile, both the energy supply of anaerobic ethanol fermentation and the Crabtree effect of S. cerevisiae were enhanced by the deletion of CAT8. CAT8 knockout cells show a higher sugar uptake rate, a higher cell growth rate, and higher tolerance to glucose than the wild-type strain YS58. This study expands the understanding of the regulatory network of Cat8 and provides guidance for modulating yeast cell growth. Key points: • The deletion of CAT8 promoted cell growth of S. cerevisiae. • Transcriptome analysis revealed the regulation network of Cat8 under 1% glucose condition. • CAT8 deletion increases the glucose tolerance of cells by enhancing the Crabtree effect. [ABSTRACT FROM AUTHOR]

Supply of Gibbs free energy and precursors are vital for cellular function and cell metabolism have evolved to be tightly regulated to balance their supply and consumption. Precursors and Gibbs free energy are generated in the central carbon metabolism (CCM), and fluxes through these pathways are precisely regulated. However, how fluxes through CCM pathways are affected by posttranslational modification and allosteric regulation remains poorly understood. Here, we integrated multi-omics data collected under nine different chemostat conditions to explore how fluxes in the CCM are regulated in the yeast Saccharomyces cerevisiae. We deduced a pathway- and metabolism-specific CCM flux regulation mechanism using hierarchical analysis combined with mathematical modeling. We found that increased glycolytic flux associated with an increased specific growth rate was accompanied by a decrease in flux regulation by metabolite concentrations, including the concentration of allosteric effectors, and a decrease in the phosphorylation level of glycolytic enzymes. [ABSTRACT FROM AUTHOR]

Background: Owing to the Crabtree effect, Saccharomyces cerevisiae produces a large amount of ethanol in the presence of oxygen and excess glucose, leading to a loss of carbon for the biosynthesis of non-ethanol chemicals. In the present study, the potential of a newly constructed Crabtree negative S. cerevisiae, as a chassis cell, was explored for the biosynthesis of various non-ethanol compounds. Results: To understand the metabolic characteristics of Crabtree negative S. cerevisiae sZJD-28, its transcriptional profile was compared with that of Crabtree positive S. cerevisiae CEN.PK113-11C. The reporter GO term analysis showed that, in sZJD-28, genes associated with translational processes were down-regulated, while those related to carbon metabolism were significantly up-regulated. To verify a potential increase in carbon metabolism for the Crabtree negative strain, the production of non-ethanol chemicals, derived from different metabolic nodes, was then undertaken for both sZJD-28 and CEN.PK113-11C. At the pyruvate node, production of 2,3-butanediol and lactate in sZJD-28-based strains was remarkably higher than that of CEN.PK113-11C-based ones, representing 16.8- and 1.65-fold increase in titer, as well as 4.5-fold and 0.65-fold increase in specific titer (mg/L/OD), respectively. Similarly, for shikimate derived p-coumaric acid, the titer of sZJD-28-based strain was 0.68-fold higher than for CEN.PK113-11C-based one, with a 0.98-fold increase in specific titer. While farnesene and lycopene, two acetoacetyl-CoA derivatives, showed 0.21- and 1.88-fold increases in titer, respectively. From malonyl-CoA, the titer of 3-hydroxypropionate and fatty acids in sZJD-28-based strains were 0.19- and 0.76-fold higher than that of CEN.PK113-11C-based ones, respectively. In fact, yields of products also improved by the same fold due to the absence of residual glucose. Fed-batch fermentation further showed that the titer of free fatty acids in sZJD-28-based strain 28-FFA-E reached 6295.6 mg/L with a highest reported specific titer of 247.7 mg/L/OD in S. cerevisiae. Conclusions: Compared with CEN.PK113-11C, the Crabtree negative sZJD-28 strain displayed a significantly different transcriptional profile and obvious advantages in the biosynthesis of non-ethanol chemicals due to redirected carbon and energy sources towards metabolite biosynthesis. The findings, therefore, suggest that a Crabtree negative S. cerevisiae strain could be a promising chassis cell for the biosynthesis of various chemicals. [ABSTRACT FROM AUTHOR]

The respiro-fermentative metabolism in the yeast Saccharomyces cerevisiae, also called the Crabtree effect, results in lower energy efficiency and biomass yield which can impact yields of chemicals to be produced using this cell factory. Although it can be engineered to become Crabtree negative, the slow growth and glucose consumption rate limit its industrial application. Here the Crabtree effect in yeast can be alleviated by engineering the transcription factor Mth1 involved in glucose signaling and a subunit of the RNA polymerase II mediator complex Med2. It was found that the mutant with the MTH1A81D&MED2*432Y allele could grow in glucose rich medium with a specific growth rate of 0.30 h−1, an ethanol yield of 0.10 g g−1, and a biomass yield of 0.21 g g−1, compared with a specific growth rate of 0.40 h−1, an ethanol yield of 0.46 g g−1, and a biomass yield of 0.11 g g−1 in the wild-type strain CEN.PK 113-5D. Transcriptome analysis revealed significant downregulation of the glycolytic process, as well as the upregulation of the TCA cycle and the electron transfer chain. Significant expression changes of several reporter transcription factors were also identified, which might explain the higher energy efficiencies in the engineered strain. We further demonstrated the potential of the engineered strain with the production of 3-hydroxypropionic acid at a titer of 2.04 g L−1, i.e., 5.4-fold higher than that of a reference strain, indicating that the alleviated glucose repression could enhance the supply of mitochondrial acetyl-CoA. These results suggested that the engineered strain could be used as an efficient cell factory for mitochondrial production of acetyl-CoA derived chemicals. [ABSTRACT FROM AUTHOR]

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast. [Display omitted] • D. bruxellensis directed most of carbon to fermentation even under aerobic conditions. • Disulfiram decreased growth and stimulated ethanol production in D. bruxellensis. • Acdh activity might take control of the carbon flowing in the central metabolism. • Nitrate impairs the production of ethanol in glucose. • Sucrose improved the metabolism of D. bruxellensis regardless the nitrogen source. [ABSTRACT FROM AUTHOR]

In view of unified cell bioenergetics, cell bioenergetic problems related to cell overenergization can cause excessive disturbances in current cell fate and, as a result, lead to a change of cell-fate. At the onset of the problem, cell overenergization of multicellular organisms (especially overenergization of mitochondria) is solved inter alia by activation and then stimulation of the reversible Crabtree effect by cells. Unfortunately, this apparently good solution can also lead to a much bigger problem when, despite the activation of the Crabtree effect, cell overenergization persists for a long time. In such a case, cancer transformation, along with the Warburg effect, may occur to further reduce or stop the charging of mitochondria by high-energy molecules. Understanding the phenomena of cancer transformation and cancer development has become a real challenge for humanity. To date, many models have been developed to understand cancer-related mechanisms. Nowadays, combining all these models into one coherent universal model of cancer transformation and development can be considered a new challenge. In this light, the aim of this article is to present such a potentially universal model supported by a proposed new model of cellular functionality evolution. The methods of fighting cancer resulting from unified cell bioenergetics and the two presented models are also considered. [ABSTRACT FROM AUTHOR]

Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae , a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism. • S. cerevisiae NAD+/NADH ratios vary significantly between aerobic and anaerobic conditions. • B3 salvage is the preferred pathway to convert nicotinamide riboside to nicotinamide during anaerobiosis. • Total NADP(H) levels are higher during aerobiosis with no difference in the ratio. • Anaerobiosis increases the expression of genes in glycolysis and lipid biosynthetic pathways. • Increased expression of thiamine regulation genes during aerobiosis suggests an increased thiamine requirement. [ABSTRACT FROM AUTHOR]

The Crabtree effect in the yeast, Saccharomyces cerevisiae , has been extensively studied, but only few studies have analyzed the dynamic conditions across the critical specific growth rate where the Crabtree effect sets in. Here, we carried out a multi-omics analysis of S. cerevisiae undergoing a specific growth rate transition from 0.2 h−1 to 0.35 h−1. The extracellular metabolome, the transcriptome and the proteome were analyzed in an 8-h transition period after the specific growth rate shifted from 0.2 h−1 to 0.35 h−1. The changing trends of both the transcriptome and proteome were analyzed using principal component analysis, which showed that the transcriptome clustered together after 60 min, while the proteome reached steady-state much later. Focusing on central carbon metabolism, we analyzed both the changes in the transcriptome and proteome, and observed an interesting changing pattern in the tricarboxylic acid (TCA) pathway, which indicates an important role for citric acid shuttling across the mitochondrial membrane for α-ketoglutarate accumulation during the transition from respiratory to respiro-fermentative metabolism. This was supported by a change in the oxaloacetate and malate shuttle. Together, our findings shed new light into the onset of the Crabtree effect in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Aerobic fermentation, also referred to as the Crabtree effect in yeast, is a well-studied phenomenon that allows many eukaryal cells to attain higher growth rates at high glucose availability. Not all yeasts exhibit the Crabtree effect, and it is not known why Crabtree-negative yeasts can grow at rates comparable to Crabtree-positive yeasts. Here, we quantitatively compared two Crabtree-positive yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two Crabtree-negative yeasts, Kluyveromyces marxianus and Scheffersomyces stipitis, cultivated under glucose excess conditions. Combining physiological and proteome quantification with genome-scale metabolic modeling, we found that the two groups differ in energy metabolism and translation efficiency. In Crabtree-positive yeasts, the central carbon metabolism flux and proteome allocation favor a glucose utilization strategy minimizing proteome cost as proteins translation parameters, including ribosomal content and/or efficiency, are lower. Crabtree-negative yeasts, however, use a strategy of maximizing ATP yield, accompanied by higher protein translation parameters. Our analyses provide insight into the underlying reasons for the Crabtree effect, demonstrating a coupling to adaptations in both metabolism and protein translation. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from oxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect. [ABSTRACT FROM AUTHOR]

The ability of the oleaginous yeast Trichosporon fermentans to efficiently produce lipids when cultivated in dried sweet sorghum was evaluated. First, lipid production was evaluated in synthetic media mimicking the composition of sweet sorghum stalks and optimized based on the nitrogen source and C: N ratio. Under optimum conditions, the lipid production reached 3.66 g/L with 21.91% w/w lipid content by using a mixture of sucrose, glucose and fructose and peptone at C: N ratio 160. Cultivation on pre-saccharified sweet sorghum stalks offered 1.97 g/L, while it was found that sweet sorghum stalks can support yeast growth and lipid production without the need for external nitrogen source addition. At an attempt to increase the carbon source concentration for optimizing lipid production, the Crabtree effect was observed in T. fermentans. To this end, the yeast was evaluated for its potential to produce ethanol under anaerobic conditions in synthetic media and sweet sorghum. The ethanol concentration at 100 g/L glucose was 40.31 g/L, while utilizing sweet sorghum by adding a distinct saccharification step and external nitrogen source offered ethanol concentration equal to 23.5 g/L. To the authors' knowledge, this is the first time that the Crabtree effect is observed in T. fermentans. • Lipid production was evaluated in synthetic media and sweet sorghum. • Lipid production in sweet sorghum was 1.97 g/L adding a saccharification step and no nitrogen source. • Crabtree effect was observed in Trichosporon fermentans. • Ethanol production in 20% w/v sweet sorghum was 23.5 g/L. [ABSTRACT FROM AUTHOR]

Background: The 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. Results: All 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 g L - 1 h - 1 . The production phase behaved in a similar manner, where glucose addition of 0.197 g L - 1 h - 1 resulted in fumarate production of 0.150 g L - 1 h - 1 and a yield of 0.802 g 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 g g - 1 h - 1 on biomass, while the maximum glucose uptake rate was established to be between 0.426 and 0.533 g g - 1 h - 1 . Conclusions: The 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 CO 2 . The highest biomass-based "homofumarate production": rate of 0.243 g g - 1 h - 1 achieved a yield of 0.802 g 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. [ABSTRACT FROM AUTHOR]

Summary: Yeast activity in solution can be quantified by its production of Carbon Dioxide which affects the compressibility of the solution which can be tracked acoustically with a microphone. Yeast is found to respond predictably to a fixed amount of glusose. • BARDS is a rapid method for real-time analysis of CO 2 production by yeast cells via the Crabtree Effect. • Yeast strains can be differentiated by real-time CO 2 production profiling. • BARDS detects intracellular v extra-cellular timelines for CO 2 production. • BARDS can determine cellular CO 2 gaseous production using the Crawford Equation. • BARDS can detect metabolic pathway switching depening on the carbon source bolus used. 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 CO 2 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 CO 2 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 CO 2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Accumulation of malic acid due to overflow carbon metabolism. • Influence of ambient pH on sclerotiorin biosynthesis. • Repression of sclerotiorin biosynthesis by Crabtree effect. • Efficient biosynthesis of sclerotiorin via carbon metabolite repression. Sclerotiorin is the secondary metabolite of Penicillium sclerotiorum , which is usually produced by solid state fermentation or liquid culture without stirring (formation of mycelial mat on air-liquid surface). In the present work, it was confirmed that malic acid was produced during cell suspension culture in a liquid medium (mycelia acting as planktonic cells) using resting cells as whole cell biocatalyst. The accumulation of malic acid was caused by overflow carbon metabolism at high glucose concentration (Crabtree effect), which led to low ambient pH value. The low ambient pH repressed sclerotiorin biosynthesis. In order to overcome the low pH caused by Crabtree effect, many influenced factors, such as phosphate buffer system, initial pH, biomass loading, slowly metabolized carbon resource, and mixed sugars as carbon resource, were investigated. Finally, the low pH caused by Crabtree effect was eliminated by taking the advantage of carbon metabolite repression via utilization of mixed sugars (glucose and lactose) as carbon resource. And then, an ideal pH approximately 3.8 was maintained and corresponding high concentration of sclerotiorin (150 absorbance unit at 365 nm, corresponding to 1.9 g/l) was achieved by cell suspension culture with a relatively high biomass loading 11.7 g/l DCW (dry cell weight). [ABSTRACT FROM AUTHOR]

When faced with environmental changes, microbes enter a lag phase during which cell growth is arrested, allowing cells to adapt to the new situation. The discovery of the lag phase started the field of gene regulation and led to the unraveling of underlying mechanisms. However, the factors determining the exact duration and dynamics of the lag phase remain largely elusive. Naively, one would expect that cells adapt as quickly as possible, so they can resume growth and compete with other organisms. However, recent studies show that the lag phase can last from several hours up to several days. Moreover, some cells within the same population take much longer than others, despite being genetically identical. In addition, the lag phase duration is also influenced by the past, with recent exposure to a given environment leading to a quicker adaptation when that environment returns. Genome-wide screens in Saccharomyces cerevisiae on carbon source shifts now suggest that the length of the lag phase, the heterogeneity in lag times of individual cells, and the history-dependent behavior are not determined by the time it takes to induce a few specific genes related to uptake and metabolism of a new carbon source. Instead, a major shift in general metabolism, and in particular a switch between fermentation and respiration, is the major bottleneck that determines lag duration. This suggests that there may be a fitness trade-off between complete adaptation of a cell's metabolism to a given environment, and a short lag phase when the environment changes. [ABSTRACT FROM AUTHOR]

The high cell density culture of baker's yeast FX‐2 was investigated in a 50 L(A) automatic bioreactor. Herein, it was found firstly that the Crabtree effect clearly existed in batch fermentation with higher glucose content, then the critical initial glucose content range (≤2.00 g L−1) was reasonably ascertained to effectively avoid Crabtree effect. In the next fed‐batch fermentations with different strategies, the second strategy (maintain ethanol concentration lower than 0.10% and pH around 4.80) was confirmed to be more beneficial to yeast growth than the first strategy (keep reducing sugar not more than 2.00 g L−1 and control steady Carbon/Nitrogen ratio 3.05:1.00). After that, one optimal control strategy (maintain pH around 4.80 and keep respiratory quotient in the range of 0.90–1.00) was constructed to further enhance cell yield. Under an optimal control strategy, four schemes with the aim of achieving pH‐stat were compared, and yeast extract instead of other alkaline materials was selected as a better regulator. As a result, 148.37 g L−1 dry cell weight, 38.25 × 108 mL−1 living cells, and 8.24 g L−1 h−1 productivity were harvested, which respectively elevated 23.74%, 135.38%, and 24.47% compared to that obtained under the traditional scheme (regulate pH with ammonia); meanwhile, the maximum oxygen uptake rate and carbon dioxide excretion rate were both more than 250.00 mmol L−1 min−1. [ABSTRACT FROM AUTHOR]

publisher‐imprint‐name Springer volume‐issue‐count 1 issue‐article‐count 0 issue‐toc‐levels 0 issue‐pricelist‐year 2018 issue‐copyright‐holder The Author(s) issue‐copyright‐year 2018 article‐contains‐esm No article‐numbering‐style Unnumbered article‐registration‐date‐year 2018 article‐registration‐date‐month 5 article‐registration‐date‐day 29 article‐toc‐levels 0 toc‐levels 0 volume‐type Regular journal‐product ArchiveJournal numbering‐style Unnumbered article‐grants‐type OpenChoice metadata‐grant OpenAccess abstract‐grant OpenAccess bodypdf‐grant OpenAccess bodyhtml‐grant OpenAccess bibliography‐grant OpenAccess esm‐grant OpenAccess online‐first false pdf‐file‐reference BodyRef/PDF/40169_2018_Article_193.pdf target‐type OnlinePDF issue‐type Regular article‐type OriginalPaper journal‐subject‐primary Medicine & Public Health journal‐subject‐secondary Medicine/Public Health, general journal‐subject‐collection Medicine open‐access true --> Maintenance of health involves a synchronized network of catabolic and anabolic signals among organs/tissues/cells that requires differential bioenergetics from mitochondria and glycolysis (biological laws or biorhythms). We defined biological circadian rhythms as Yin (tumoricidal) and Yang (tumorigenic) arms of acute inflammation (effective immunity) involving immune and non‐immune systems. Role of pathogens in altering immunity and inducing diseases and cancer has been documented for over a century. However, in 1955s decision makers in cancer/medical establishment allowed public (current baby boomers) to consume million doses of virus‐contaminated polio vaccines. The risk of cancer incidence and mortality sharply rose from 5% (rate of hereditary/genetic or innate disease) in 1900s, to its current scary status of 33% or 50% among women and men, respectively. Despite better hygiene, modern detection technologies and discovery of antibiotics, baby boomers and subsequent 2–3 generations are sicker than previous generations at same age. American health status ranks last among other developed nations while America invests highest amount of resources for healthcare. In this perspective we present evidence that cancer is an induced disease of twentieth century, facilitated by a great deception of cancer/medical establishment for huge corporate profits. Unlike popularized opinions that cancer is 100, 200 or 1000 diseases, we demonstrate that cancer is only one disease; the severe disturbances in biorhythms (differential bioenergetics) or loss of balance in Yin and Yang of effective immunity. Cancer projects that are promoted and funded by decision makers are reductionist approaches, wrong and unethical and resulted in loss of millions of precious lives and financial toxicity to society. Public vaccination with pathogen‐specific vaccines (e.g., flu, hepatitis, HPV, meningitis, measles) weakens, not promotes, immunity. Results of irresponsible projects on cancer sciences or vaccines are increased population of drug‐dependent sick society. Outcome failure rates of claimed 'targeted' drugs, 'precision' or 'personalized' medicine are 90% (± 5) for solid tumors. We demonstrate that aging, frequent exposures to environmental hazards, infections and pathogen‐specific vaccines and ingredients are 'antigen overload' for immune system, skewing the Yin and Yang response profiles and leading to induction of 'mild', 'moderate' or 'severe' immune disorders. Induction of decoy or pattern recognition receptors (e.g., PRRs), such as IRAK‐M or IL‐1dRs ('designer' molecules) and associated genomic instability and over‐expression of growth promoting factors (e.g., pyruvate kinases, mTOR and PI3Ks, histamine, PGE2, VEGF) could lead to immune tolerance, facilitating cancer cells to hijack anabolic machinery of immunity (Yang) for their increased growth requirements. Expression of constituent embryonic factors would negatively regulate differentiation of tumor cells through epithelial–mesenchymal‐transition and create "dual negative feedback loop" that influence tissue metabolism under hypoxic conditions. It is further hypothesized that induction of tolerance creates 'dark energy' and increased entropy and temperature in cancer microenvironment allowing disorderly cancer proliferation and mitosis along with increased glucose metabolism via Crabtree and Pasteur Effects, under mitophagy and ribophagy, conditions that are toxic to host survival. Effective translational medicine into treatment requires systematic and logical studies of complex interactions of tumor cells with host environment that dictate clinical outcomes. Promoting effective immunity (biological circadian rhythms) are fundamental steps in correcting host differential bioenergetics and controlling cancer growth, preventing or delaying onset of diseases and maintaining public health. The author urges independent professionals and policy makers to take a closer look at cancer dilemma and stop the 'scientific/medical ponzi schemes' of a powerful group that control a drug‐dependent sick society before all hopes for promoting public health evaporate. [ABSTRACT FROM AUTHOR]

In this article, some new aspects of unified cell bioenergetics are presented. From the perspective of unified cell bioenergetics certain subsequent stages of cancer development, from initiation stage, through transformation to metastasis, are analyzed. Here we show that after transformation, cancer cells are permanently exposed to reactive oxygen species, that causes continual random DNA mutations and as a result genome and chromosomal destabilizations. The modern cancer attractor hypothesis has been extended in explaining cancer development. Discussion is conducted in light of current cancerogenesis research, including bioenergetic cancer initiation, the somatic mutation theory and the tissue organization field theory. In the article reasons complicating the discovery of patterns of cancer genome changes and cancer evolution are presented. In addition certain cancer therapeutic aspects are given attention to. [ABSTRACT FROM AUTHOR]

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter. [ABSTRACT FROM AUTHOR]

Industrial diploid strains of Saccharomyces cerevisiae are selected from natural populations and then domesticated by optimizing the preferred properties for producing products such as bread, wine, and sake. In this study, for comparing the fermentation performance of various industrial yeasts, seven diploid strains of S. cerevisiae, namely, BY4947 (laboratory yeast derived from S288C), Kyokai7 and Kyokai9 (sake yeasts), Red Star and NBRC0555 (bread yeasts), and QA23 and EC1118 (wine yeasts), were cultivated in a synthetic medium. The fermentation profiles of the seven yeast strains showed significant differences. The specific ethanol production rates of sake yeasts (Kyokai7 and Kyokai9) and wine strains (QA23 and EC1118) were higher and lower than those of laboratory strains, respectively. Targeted proteome analysis was also conducted to investigate the variation in the expression of metabolism-related enzymes. The expression profiles of central metabolism-related enzymes showed considerable variations among the industrial strains. Upregulation of the TCA cycle in wine strains was observed both in the synthetic and grape-juice media. These results suggested that these variations should be consequences of complex interactions between the domestication process, genetic polymorphism, and environmental factors such as the fermentation conditions. [ABSTRACT FROM AUTHOR]

The budding yeast Saccharomyces cerevisiae is an important microorganism for fermentation and the food industry. However, during production, S. cerevisiae commonly uses the ethanol fermentation pathway for glucose utilization if excess sugar is present, even in the presence of sufficient oxygen levels. This aerobic ethanol fermentation, referred to as the Crabtree effect, is one of the most significant reasons for low cell yield. To weaken the Crabtree effect in fed-batch and continuous culture, sugar flow should be limited. In addition, in continuous culture, the dilution rate must be reduced to avoid washing out cells. However, under such conditions, production speed might be sacrificed. It is difficult to solve this problem with the tradeoff between cell yield and production speed by using conventional tactics. However, a metabolomics approach may be an effective way to search for clues regarding metabolic modulation. Therefore, the purpose of this study was to reduce ethanol production in continuous culture of S. cerevisiae at a higher dilution rate through a metabolomics approach. We used a metabolomics analysis to identify metabolites that were drastically increased or decreased in continuous culture when the dilution rate shifted from biomass formation to ethanol fermentation. The individual addition of two of the selected metabolites, fumaric acid and malic acid, reduced ethanol production at a higher dilution rate. This result demonstrates the potential for using metabolomics approaches to identify metabolites that reduce ethanol production in continuous culture at high dilution rates. [ABSTRACT FROM AUTHOR]

Pyruvate decarboxylase (Pdc) is a cytosolic enzyme located at the branch point between fermentative and respiratory sugar catabolism. Here, we identified and functionally characterized KmPDC1 and KmPDC5 encoding two homologs of Pdc in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555. Despite the conservation of important Pdc domains, a few amino acid sequences essential for enzymatic activity are not conserved in KmPdc5p. Deletion of KmPDC1 alone eliminated most of Pdc activity, but the growth of the Kmpdc1Δ strain on glucose was comparable to that of the wild type (WT) strain under aerobic conditions. In contrast to the WT, Kmpdc1Δ could not grow on glucose under oxygen-limited conditions. The KmPDC5 deletion did not generate any apparent change in Pdc activity or growth patterns under several tested conditions. Whereas the expression of KmPDC1 was enhanced by glucose, the basic expression levels of KmPDC5 were very low, without a detectable difference between glucose and nonfermentable carbon sources. Moreover, KmPDC5 overexpression was unable to complement the growth defect of Kmpdc1Δ in the presence of antimycin A, and the purified recombinant KmPdc5p was inactive in Pdc activity assay, supporting the notion that KmPdc5p may lack Pdc enzymatic activity. Notably, compared to the WT, Kmpdc1Δ single and Kmpdc1Δpdc5Δ double mutants produced significantly less glycerol, acetate, and ethanol while accumulating pyruvate. Altogether, our data indicate that a single deletion of KmPDC1 is sufficient in Crabtree-negative K. marxianus strains to generate a starting host strain for engineering of production of high-value biomaterials derived from pyruvate without byproduct formation. [ABSTRACT FROM AUTHOR]

Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functional mitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggesting that excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect are displayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstrated that expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, we demonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis when cells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumour cells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosis regardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygen uptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se does not prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cells should undergo apoptosis or not. [ABSTRACT FROM AUTHOR]

Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as “Crabtree effect”, has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S . cerevisiae , resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD + . Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD + (1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP. [ABSTRACT FROM AUTHOR]

An increase in glucose concentration in the medium rapidly decreases respiration rate in many cell types, including tumor cells. The molecular mechanism of this phenomenon, the Crabtree effect, is still unclear. It was shown earlier that adding the intermediate product of glycolysis fructose-1,6-bisphosphate to isolated mitochondria suppresses their respiration. To study possible roles of glycolytic intermediates in the Crabtree effect, we used a model organism, the yeast Saccharomyces cerevisiae. To have the option to rapidly increase intracellular concentrations of certain glycolytic intermediates, we used mutant cells with glycolysis blocked at different stages. We studied fast effects of glucose addition on the respiration rate in such cells. We found that addition of glucose affected cells with deleted phosphoglycerate mutase (strain gpm1-delta) more strongly than ones with inactivated aldolase or phosphofructokinase. In the case of preincubation of gpm1-delta cells with 2-deoxyglucose, which blocks glycolysis at the stage of 2-deoxyglucosephosphate formation, the effect of glucose addition was absent. This suggests that triosephosphates are intermediates of the Crabtree effect. Apart from this, the incubation of gpm1-delta cells in galactose-containing medium appeared to cause a large increase in their size. It was previously shown that galactose addition did not have any short-term effect on respiration rate of gpm1-delta cells and, at the same time, strongly suppressed their growth rate. Apparently, the influence of increasing triosephosphate concentration on yeast physiology is not limited to the activation of the Crabtree effect. [ABSTRACT FROM AUTHOR]

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development. [ABSTRACT FROM AUTHOR]

Background: Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. Results: Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. Conclusions: REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines. [ABSTRACT FROM AUTHOR]

S -Adenosyl- l -methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, 13 C-metabolic flux analysis ( 13 C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. 13 C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the 13 C-based metabolic flux analysis ( 13 C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h −1 ) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden–Meyerhof–Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth. [ABSTRACT FROM AUTHOR]

Evidence suggests that AMP protein kinase (AMPK) is the main target of the phytochemical resveratrol (RSV) in mammalian cells. Data also indicates that RSV stimulates glucose metabolism; however, the molecular link between RSV and glucose uptake remains unknown. Herein, we provide evidence indicating that RSV stimulates glycolysis via sucrose non-fermenting 1 gene ( SNF1, Saccharomyces cerevisiae orthologous of AMPK). S. cerevisiae cultures treated with 30 μM RSV showed an increase in extracellular acidification rate compared to untreated cells, indicating an elevated glycolytic flux. Also, RSV treatment increased transcription levels of two key glycolytic genes, hexokinase 2 ( HXK2) and phosphofructokinase 1 ( PFK1), as well as production of NADH. Moreover, RSV treatment inhibited mitochondrial respiration when glucose was used as a carbon source. Importantly, the effects of RSV on glycolysis were dependent of SNF1. Taken together, these findings suggest that SNF1 (AMPK in mammalian systems) is the molecular target of RSV in S. cerevisiae. [ABSTRACT FROM AUTHOR]

There are two main pathways of ATP biosynthesis: glycolysis and oxidative phosphorylation. As a rule, the two pathways are not fully active in a single cell. In this review, we discuss mechanisms of glycolytic inhibition of respiration (Warburg and Crabtree effects). What are the reasons for the existence of this negative feedback? It is known that maximal activation of both processes can cause generation of reactive oxygen species. Oxidative phosphorylation is more efficient from the energy point of view, while glycolysis is safer and favors biomass synthesis. This might be the reason why quiescent cells are mainly using oxidative phosphorylation, while the quickly proliferating ones - glycolysis. [ABSTRACT FROM AUTHOR]

Different species are usually thought to have specific adaptations, which allow them to occupy different ecological niches. But recent neutral ecology theory suggests that species diversity can simply be the result of random sampling, due to finite population sizes and limited dispersal. Neutral models predict that species are not necessarily adapted to specific niches, but are functionally equivalent across a range of habitats. Here, we evaluate the ecology of Saccharomyces cerevisiae, one of the most important microbial species in human history. The artificial collection, concentration and fermentation of large volumes of fruit for alcohol production produce an environment in which S. cerevisiae thrives, and therefore it is assumed that fruit is the ecological niche that S. cerevisiae inhabits and has adapted to. We find very little direct evidence that S. cerevisiae is adapted to fruit, or indeed to any other specific niche. We propose instead a neutral nomad model for S. cerevisiae, which we believe should be used as the starting hypothesis in attempting to unravel the ecology of this important microbe. [ABSTRACT FROM AUTHOR]

Poly d -lactic acid is an important polymer because it improves the thermostability of poly l -lactic acid by stereo complex formation. To demonstrate potency of continuous fermentation using a membrane-integrated fermentation reactor (MFR) system, continuous fermentation using genetically modified Saccharomyces cerevisiae which produces d -lactic acid was performed at the low pH and microaerobic conditions. d -Lactic acid continuous fermentation using the MFR system by genetically modified yeast increased production rate by 11-fold compared with batch fermentation. In addition, the carbon yield of d -lactic acid in continuous fermentation was improved to 74.6 ± 2.3% compared to 39.0 ± 1.7% with batch fermentation. This dramatic improvement in carbon yield could not be explained by a reduction in carbon consumption to form cells compared to batch fermentation. Further detailed analysis at batch fermentation revealed that the carbon yield increased to 76.8% at late stationary phase. S. cerevisiae , which exhibits the Crabtree-positive effect, demonstrated significant changes in metabolic activities at low sugar concentrations (Rossignol et al., Yeast, 20, 1369–1385, 2003). Moreover, lactate-producing S. cerevisiae requires ATP supplied not only from the glycolytic pathway but also from the TCA cycle (van Maris et al., Appl. Environ. Microbiol., 70, 2898–2905, 2004). Our finding was revealed that continuous fermentation, which can maintain the conditions of both a low sugar concentration and air supply, results in Crabtree-positive and lactate-producing S. cerevisiae for suitable conditions of d -lactic acid production with respect to redox balance and ATP generation because of releasing the yeast from the Crabtree effect. [ABSTRACT FROM AUTHOR]

The short-term Crabtree effect is defined as the immediate occurrence of aerobic alcoholic fermentation in response to provision of a pulse of excess sugar to sugar-limited yeast cultures. Here we have characterized ten yeast species with a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and we studied their general carbon metabolism in response to a glucose pulse. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that the Pichia, Debaryomyces, Eremothecium and Kluyveromyces marxianus yeasts do not exhibit any significant ethanol formation, while Kluyveromyces lactis behaves as an intermediate yeast, and Lachancea, Torulaspora, Vanderwaltozyma and Saccharomyces yeasts exhibit rapid ethanol accumulation. Based on the present data and our previous data relating to the presence of the long-term Crabtree effect in over 40 yeast species, we speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, occurring ~ 150 million years ago. [ABSTRACT FROM AUTHOR]

Constraint-based modeling is largely used in computational studies of metabolism. We propose here a novel approach that aims to identify ensembles of flux distributions that comply with one or more target phenotype(s). The methodology has been tested on a small-scale model of yeast energy metabolism. The target phenotypes describe the differential pattern of ethanol production and O consumption observed in 'Crabtree-positive' and 'Crabtree-negative' yeasts in changing environment (i.e., when the upper limit of glucose uptake is varied). The ensembles were obtained either by selection among sampled flux distributions or by means of a search heuristic (genetic algorithm). The former approach provided indication about the probability to observe a given phenotype, but the resulting ensembles could not be unambiguously partitioned into 'Crabtree-positive' and 'Crabtree-negative' clusters. On the contrary well-separated clusters were obtained with the latter method. The cluster analysis further allowed identification of distinct groups within each target phenotype. The method may thus prove useful in characterizing the design principles underlying metabolic plasticity arising from evolving physio-pathological or developmental constraints. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae addition of glucose inhibits oxygen consumption, i.e. S. cerevisiae is Crabtree-positive. During active glycolysis hexoses-phosphate accumulate, and probably interact with mitochondria. In an effort to understand the mechanism underlying the Crabtree effect, the effect of two glycolysis-derived hexoses-phosphate was tested on the S. cerevisiae mitochondrial unspecific channel ( Sc MUC). Glucose-6-phosphate (G6P) promoted partial opening of Sc MUC, which led to proton leakage and uncoupling which in turn resulted in, accelerated oxygen consumption. In contrast, fructose-1,6-bisphosphate (F1,6BP) closed Sc MUC and thus inhibited the rate of oxygen consumption. When added together, F1,6BP reverted the mild G6P-induced effects. F1,6BP is proposed to be an important modulator of Sc MUC, whose closure contributes to the “Crabtree effect”. [ABSTRACT FROM AUTHOR]

By performing an integrated comparative analysis on the physiology and transcriptome of four different S. cerevisiae strains growing on galactose and glucose, it was inferred that the transcription factors Bas1p, Pho2p, and Gcn4p play a central role in the regulatory events causing the Crabtree effect in S. cerevisiae. The analysis also revealed that a point mutation in the RAS2 observed in a galactose-adapted strain causes a lower Crabtree effect and growth rate on glucose by decreasing the activity of Gcn4p while at the same time is at the origin of higher growth rate on galactose due to a lower activity of the transcriptional repressor Sok2p. The role of Gcn4p on the trade-off effect observed on glucose was confirmed experimentally. This was done by showing that the point mutation in RAS2 does not result in a lower growth rate on glucose if it is introduced in a GCN4-negative background. [ABSTRACT FROM AUTHOR]

Fumaric acid is widely used in the food and beverage, pharmaceutical and polyester resin industries. Rhizopus oryzae is the most successful microorganism at excreting fumaric acid compared to all known natural and genetically modified organisms. It has previously been discovered that careful control of the glucose feed rate can eliminate the by-product formation of ethanol. Two key parameters affecting fumaric acid excretion were identified, namely the medium pH and the urea feed rate. A continuous fermentation with immobilised R. oryzae was utilised to determine the effect of these parameters. It was found that the selectivity for fumaric acid production increased at high glucose consumption rates for a pH of 4, different from the trend for pH 5 and 6, achieving a yield of 0.93   gg − 1 . This yield is higher than previously reported in the literature. Varying the urea feed rate to 0.255   m g L − 1 h − 1 improved the yield of fumaric acid but experienced a lower glucose uptake rate compared to higher urea feed rates. An optimum region has been found for fumaric acid production at pH 4, a urea feed rate of 0.625   m g L − 1 h − 1 and a glucose feed rate of 0.329   g L − 1 h − 1 . [ABSTRACT FROM AUTHOR]

The yeast Kluyveromyces marxianus has been pointed out as a promising microorganism for a variety of industrial bioprocesses. Although genetic tools have been developed for this yeast and different potential applications have been investigated, quantitative physiological studies have rarely been reported. Here, we report and discuss the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 during aerobic batch bioreactor cultivations, using a defined medium with different sugars as sole carbon and energy source, at 30 and 37 °C. Cultivations were carried out both on single sugars and on binary sugar mixtures. Carbon balances closed within 95 to 101 % in all experiments. Biomass and CO were the main products of cell metabolism, whereas by-products were always present in very low proportion (<3 % of the carbon consumed), as long as full aerobiosis was guaranteed. On all sugars tested as sole carbon and energy source (glucose, fructose, sucrose, lactose, and galactose), the maximum specific growth rate remained between 0.39 and 0.49 h, except for galactose at 37 °C, which only supported growth at 0.31 h. Different growth behaviors were observed on the binary sugar mixtures investigated (glucose and lactose, glucose and galactose, lactose and galactose, glucose and fructose, galactose and fructose, fructose and lactose), and the observations were in agreement with previously published data on the sugar transport systems in K. marxianus. We conclude that K. marxianus CBS 6556 does not present any special nutritional requirements; grows well in the range of 30 to 37 °C on different sugars; is capable of growing on sugar mixtures in a shorter period of time than Saccharomyces cerevisiae, which is interesting from an industrial point of view; and deviates tiny amounts of carbon towards metabolite formation, as long as full aerobiosis is maintained. [ABSTRACT FROM AUTHOR]

Abstract: Polyploidy, or whole-genome duplication (WGD), is a recurrent mutation both in cell lineages and over evolutionary time. By globally changing the relationship between gene copy number and other cellular entities, it can induce dramatic changes at the cellular and phenotypic level. Perhaps surprisingly, then, the insights that these events can bring to understanding other cellular features are not as well appreciated as they could be. In this review, we draw on examples of polyploidy from animals, plants and yeast to explore how investigations of polyploid cells have improved our understanding of the cell cycle, biological network complexity, metabolic phenotypes and tumor biology. We argue that the study of polyploidy across organisms, cell types, and time scales serves not only as a window into basic cell biology, but also as a basis for a predictive biology with applications ranging from crop improvement to treating cancer. [Copyright &y& Elsevier]

Abstract: Background: Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. Methods: We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. Results: Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. Conclusions: These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration. General significance: Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches. [Copyright &y& Elsevier]

Dekkera bruxellensis has been described as the major contaminant yeast of industrial ethanol production, although little is known about its physiology. The aim of this study was to investigate the growth of this yeast in diverse carbon sources and involved conducting shake-flask and glucose- or sucrose-limited chemostats experiments, and from the chemostat data, the stoichiome-try of biomass formation during aerobic growth was established. As a result of the shake-flask experiments with hexoses or disaccharides, the specific growth rates were calculated, and a different behavior in rich and mineral medium was observed concerning to profile of acetate and ethanol production. In C-limited chemostats conditions, the metabolism of this yeast was completely respiratory, and the biomass yields reached values of 0.62 gDW gS-1. In addition, glucose pulses were applied to the glucose- or sucrose-limited chemostats. These results showed that D. bruxellensis has a short-term Crabtree effect. While the glucose pulse was at the sucrose-limited chemostat, sucrose accumulated at the reactor, indicating the presence of a glucose repression mechanism in D. bruxellensis. [ABSTRACT FROM AUTHOR]

Dekkera bruxellensis has been described as the major contaminant yeast of industrial ethanol production, although little is known about its physiology. The aim of this study was to investigate the growth of this yeast in diverse carbon sources and involved conducting shake-flask and glucose- or sucrose-limited chemostats experiments, and from the chemostat data, the stoichiometry of biomass formation during aerobic growth was established. As a result of the shake-flask experiments with hexoses or disaccharides, the specific growth rates were calculated, and a different behavior in rich and mineral medium was observed concerning to profile of acetate and ethanol production. In C-limited chemostats conditions, the metabolism of this yeast was completely respiratory, and the biomass yields reached values of 0.62 gDW gS−1. In addition, glucose pulses were applied to the glucose- or sucrose-limited chemostats. These results showed that D. bruxellensis has a short-term Crabtree effect. While the glucose pulse was at the sucrose-limited chemostat, sucrose accumulated at the reactor, indicating the presence of a glucose repression mechanism in D. bruxellensis. [ABSTRACT FROM AUTHOR]

Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed "extended" overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis. [ABSTRACT FROM AUTHOR]

Abstract: During the last decades a considerable amount of research has been focused on cancer. A number of genetic and signaling defects have been identified. This has allowed the design and screening of a number of anti-tumor drugs for therapeutic use. One of the main challenges of anti-cancer therapy is to specifically target these drugs to malignant cells. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display an enhanced glycolytic activity and oxidative phosphorylation down-regulation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy. Nonetheless, the concept of aerobic glycolysis as the paradigm of tumor cell metabolism has been challenged, as some tumor cells use oxidative phosphorylation. Mitochondria are of special interest in cancer cell energy metabolism, as their physiology is linked to the Warburg effect. Besides, their central role in apoptosis makes these organelles a promising “dual hit target” for selectively eliminate tumor cells. Thus, it is desirable to have an easy-to-use and reliable model in order to do the screening for energy metabolism-inhibiting drugs to be used in cancer therapy. From a metabolic point of view, the fermenting yeast Saccharomyces cerevisiae and tumor cells share several features. In this paper we will review these common metabolic properties and we will discuss the possibility of using S. cerevisiae as an early screening test in the research for novel anti-tumor compounds used for the inhibition of tumor cell metabolism. [Copyright &y& Elsevier]

The harmonics of an electric current passing through a yeast suspension exhibit particular patterns according to the biological activity of the cells, constituting the nonlinear dielectric response of yeast cells, and the measurement of these patterns is a technique for determining the activity of living cells. The concentrations of glucose and oxygen in yeast culture medium influence whether fermentation or respiration of the yeast cells occurs. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically at sufficient glucose concentrations (aerobic and anaerobic fermentation), and also under aerobic conditions at a limited glucose concentration (respiration). The results showed that the harmonics were barely detectable for yeast cells in aerobic fermentation and respiration, but that cells in anaerobic fermentation produced substantial third and fifth harmonics. It is inferred that environmental conditions affect yeast cells' nonlinear dielectric response; furthermore, it appears that measurement of these nonlinear properties may be an effective means of determining the activity of yeast cells adjusted to the conditions of their cultivation. © 2009 Wiley Periodicals, Inc. Electron Comm Jpn, 92(9): 17–23, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10094 [ABSTRACT FROM AUTHOR]

Abstract: For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast. [Copyright &y& Elsevier]

The theory of niche construction suggests that organisms may engineer environments via their activities. Despite the potential of this phenomenon being realized by Darwin, the capability of niche construction to generally unite ecological and evolutionary biology has never been empirically quantified. Here I quantify the fitness effects of Saccharomyces cerevisiae's ecosystem engineering in a natural ferment in order to understand the interaction between ecological and evolutionary processes. I show that S. cerevisiae eventually dominates in fruit niches, where it is naturally initially rare, by modifying the environment through fermentation (the Crabtree effect) in ways which extend beyond just considering ethanol production. These data show that an additional cause of S. cercvisiae's competitive advantage over the other yeasts in the community is due to the production of heat via fermentation. Even though fermentation is less energetically efficient than respiration, it seems that this trait has been selected for because its net effect provides roughly a 7% fitness advantage over the other members of the community. These data provide an elegant example of niche construction because this trait clearly modifies the environment and therefore the selection pressures to which S. cerevisiae, and other organisms that access the fruit resource, including humans, are exposed to. [ABSTRACT FROM AUTHOR]