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Background: Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. Results: High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. Conclusions: These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii. [ABSTRACT FROM AUTHOR]

Abstract Background Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. Results High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. Conclusions These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.

Abstract 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.

Hexokinase II (Hxk2) is a master protein in glucose-mediated transcriptional repression signaling pathway. Degrading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene (nerolidol) production at gram-per-liter levels in Saccharomyces cerevisiae. Global transcriptomics/proteomics profiles in Hxk2-deficient background are important to understanding genetic and molecular mechanisms for improved nerolidol production and guiding further strain optimization. Here, proteomic responses to Hxk2 depletion are investigated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway, at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δ backgrounds. Carbon metabolic pathways and amino acid metabolic pathways show diversified responses to Hxk2 depletion and growth on ethanol, including upregulation of alternative carbon catabolism and respiration as well as downregulation of amino acid synthesis. De-repression of GAL genes may contribute to improved nerolidol production in Hxk2-depleted strains. Seventeen transcription factors associated with upregulated genes are enriched. Validating Ash1-mediated repression on the RIM4 promoter shows the variation on the regulatory effects of different Ash1-binding sites and the synergistic effect of Ash1 and Hxk2-mediated repression. Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δ background, but much weaker than those in HXK2-wildtype background. In summary, inactivating HXK2 may relieve glucose repression on respiration and GAL promoters for improved bioproduction under aerobic conditions in S. cerevisiae. The proteomics profiles provide a better genetics overview for a better metabolic engineering design in Hxk2-deficient backgrounds.

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]

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.

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]

Since Jacob and Monod’s discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as “metabolic reprogramming.” The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of E. coli to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible.

This paper proposes a general approach for building a mechanistic yeast model able to predict the shift of metabolic pathways. The mechanistic model accounts for the coexistence of several metabolic pathways (aerobic fermentation, glucose respiration, anaerobic fermentation and ethanol respiration) whose activation depends on growth conditions. This general approach is applied to a commercial strain of Saccharomyces cerevisiae. Stoichiometry and yeast kinetics were mostly determined from aerobic and completely anaerobic experiments. Known parameters were taken from the literature, and the remaining parameters were estimated by inverse analysis using the particle swarm optimization method. The optimized set of parameters allows the concentrations to be accurately determined over time, reporting global mean relative errors for all variables of less than 7 and 11% under completely anaerobic and aerobic conditions, respectively. Different affinities of yeast for glucose and ethanol tolerance under aerobic and anaerobic conditions were obtained. Finally, the model was successfully validated by simulating a different experiment, a batch fermentation process without gas injection, with an overall mean relative error of 7%. This model represents a useful tool for the control and optimization of yeast fermentation systems. More generally, the modeling framework proposed here is intended to be used as a building block of a digital twin of any bioproduction process. [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]

Exposed to changes in their environment, microorganisms will adapt their phenotype, including metabolism, to ensure survival. To understand the adaptation principles, resource allocation-based approaches were successfully applied to predict an optimal proteome allocation under (quasi) steady-state conditions. Nevertheless, for a general, dynamic environment, enzyme kinetics will have to be taken into account which was not included in the linear resource allocation models. To this end, a resource-dependent kinetic model was developed and applied to the model organism Saccharomyces cerevisiae by combining published kinetic models and calibrating the model parameters to published proteomics and fluxomics datasets. Using this approach, we were able to predict specific proteomes at different dilution rates under chemostat conditions. Interestingly, the approach suggests that the occurrence of aerobic fermentation (Crabtree effect) in S. cerevisiae is not caused by space limitation in the total proteome but rather an effect of constraints on the mitochondria. When exposing the approach to repetitive, dynamic substrate conditions, the proteome space was allocated differently. Less space was predicted to be available for non-essential enzymes (reserve space). This could indicate that the perceived “overcapacity” present in experimentally measured proteomes may very likely serve a purpose in increasing the robustness of a cell to dynamic conditions, especially an increase of proteome space for the growth reaction as well as of the trehalose cycle that was shown to be essential in providing robustness upon stronger substrate perturbations. The model predictions of proteome adaptation to dynamic conditions were additionally evaluated against respective experimentally measured proteomes, which highlighted the model’s ability to accurately predict major proteome adaptation trends. This proof of principle for the approach can be extended to production organisms and applied for both understanding metabolic adaptation and improving industrial process design.

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]

Abstract 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 $$0.07\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$ 0.07 g L - 1 h - 1 . The production phase behaved in a similar manner, where glucose addition of $$0.197\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$ 0.197 g L - 1 h - 1 resulted in fumarate production of $$0.150\,\hbox {g}\,\hbox {L}^{-1}\,\hbox {h}^{-1}$$ 0.150 g L - 1 h - 1 and a yield of $$0.802\,\hbox {g}\,\hbox {g}^{-1}$$ 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 $$0.395\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$ 0.395 g g - 1 h - 1 on biomass, while the maximum glucose uptake rate was established to be between 0.426 and $$0.533\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$ 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 $$\hbox {CO}_{2}$$ CO 2 . The highest biomass-based “homofumarate production”: rate of $$0.243\,\hbox {g}\,\hbox {g}^{-1}\,\hbox {h}^{-1}$$ 0.243 g g - 1 h - 1 achieved a yield of $$0.802\,\hbox {g}\,\hbox {g}^{-1}$$ 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.

This paper proposes a general approach for building a mechanistic yeast model able to predict the shift of metabolic pathways. The mechanistic model accounts for the coexistence of several metabolic pathways (aerobic fermentation, glucose respiration, anaerobic fermentation and ethanol respiration) whose activation depends on growth conditions. This general approach is applied to a commercial strain of Saccharomyces cerevisiae. Stoichiometry and yeast kinetics were mostly determined from aerobic and completely anaerobic experiments. Known parameters were taken from the literature, and the remaining parameters were estimated by inverse analysis using the particle swarm optimization method. The optimized set of parameters allows the concentrations to be accurately determined over time, reporting global mean relative errors for all variables of less than 7 and 11% under completely anaerobic and aerobic conditions, respectively. Different affinities of yeast for glucose and ethanol tolerance under aerobic and anaerobic conditions were obtained. Finally, the model was successfully validated by simulating a different experiment, a batch fermentation process without gas injection, with an overall mean relative error of 7%. This model represents a useful tool for the control and optimization of yeast fermentation systems. More generally, the modeling framework proposed here is intended to be used as a building block of a digital twin of any bioproduction process.

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.

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.

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 mgL−1h−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 mgL−1h−1 and a glucose feed rate of 0.329 gL−1h−1.

The yeast trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phosphate (T6P) in trehalose synthesis. Besides, Tps1 plays a key role in carbon and energy homeostasis in this microbial cell, as shown by the well documented loss of ATP and hyper accumulation of sugar phosphates in response to glucose addition in a mutant defective in this protein. The inability of a Saccharomyces cerevisiae tps1 mutant to cope with fermentable sugars is still a matter of debate. We reexamined this question through a quantitative analysis of the capability of TPS1 homologues from different origins to complement phenotypic defects of this mutant. Our results allowed to classify this complementation in three groups. A first group enclosed TPS1 of Klyveromyces lactis with that of S. cerevisiae as their expression in Sctps1 cells fully recovered wild type metabolic patterns and fermentation capacity in response to glucose. At the opposite was the group with TPS1 homologues from the bacteria Escherichia coli and Ralstonia solanacearum, the plant Arabidopsis thaliana and the insect Drosophila melanogaster whose metabolic profiles were comparable to those of a tps1 mutant, notably with almost no accumulation of T6P, strong impairment of ATP recovery and potent reduction of fermentation capacity, albeit these homologous genes were able to rescue growth of Sctps1 on glucose. In between was a group consisting of TPS1 homologues from other yeast species and filamentous fungi characterized by 5 to 10 times lower accumulation of T6P, a weaker recovery of ATP and a 3-times lower fermentation capacity than wild type. Finally, we found that glucose repression of gluconeogenic genes was strongly dependent on T6P. Altogether, our results suggest that the TPS protein is indispensable for growth on fermentable sugars, and points to a critical role of T6P as a sensing molecule that promotes sugar fermentation and glucose repression..

Abstract Background The yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive S. cerevisiae invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound. Results By searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by S. cerevisiae. To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography–tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g−1 glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L−1) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source. Conclusions We demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive S. cerevisiae. High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by S. cerevisiae.

Abstract Background Growth conditions that bring about stress on Phaffia rhodozyma cells encourage the synthesis of astaxanthin, an antioxidant carotenoid, which protects cells against oxidative damage. Using P. rhodozyma cultures performed with and without copper limitation, we examined the kinetics of astaxanthin synthesis along with the expression of asy, the key astaxanthin synthesis gene, as well as aox, which encodes an alternative oxidase protein. Results Copper deficiency had a detrimental effect on the rates of oxygen consumption and ethanol reassimilation at the diauxic shift. In contrast, copper deficiency prompted alcoholic fermentation under aerobic conditions and had a favorable effect on the astaxanthin content of cells, as well as on aox expression. Both cultures exhibited strong aox expression while consuming ethanol, but particularly when copper was absent. Conclusion We show that the induction of either astaxanthin production, aox expression, or aerobic fermentation exemplifies the crucial role that redox imbalance plays in triggering any of these phenomena. Based on our own results and data from others, we propose a mechanism that rationalizes the central role played by changes of respiratory activity, which lead to redox imbalances, in triggering both the short-term antioxidant response as well as fermentation in yeasts and other cell types.

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]

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]

Biofilm formation is a survival strategy for microorganisms facing a hostile environment. Under biofilm, bacteria are better protected against antibacterial drugs and the immune response, increasing treatment difficulty, as persistent populations recalcitrant to chemotherapy are promoted. Deciphering mechanisms leading to biofilms could, thus, be beneficial to obtain new antibacterial drug candidates. Here, we show that mycobacterial biofilm formation is linked to excess glycerol adaptation and the concomitant establishment of the Crabtree effect. This effect is characterized by respiratory reprogramming, ATP downregulation, and secretion of various metabolites including pyruvate, acetate, succinate, and glutamate. Interestingly, the Crabtree effect was abnormal in a mycobacterial strain deficient for Cpn60.1 (GroEL1). Indeed, this mutant strain had a compromised ability to downregulate ATP and secreted more pyruvate, acetate, succinate, and glutamate in the culture medium. Importantly, the mutant strain had higher intracellular pyruvate and produced more toxic methylglyoxal, suggesting a glycolytic stress leading to growth stasis and consequently biofilm failure. This study demonstrates, for the first time, the link between mycobacterial biofilm formation and the Crabtree effect.

Biofilm formation is a survival strategy for microorganisms facing a hostile environment. Under biofilm, bacteria are better protected against antibacterial drugs and the immune response, increasing treatment difficulty, as persistent populations recalcitrant to chemotherapy are promoted. Deciphering mechanisms leading to biofilms could, thus, be beneficial to obtain new antibacterial drug candidates. Here, we show that mycobacterial biofilm formation is linked to excess glycerol adaptation and the concomitant establishment of the Crabtree effect. This effect is characterized by respiratory reprogramming, ATP downregulation, and secretion of various metabolites including pyruvate, acetate, succinate, and glutamate. Interestingly, the Crabtree effect was abnormal in a mycobacterial strain deficient for Cpn60.1 (GroEL1). Indeed, this mutant strain had a compromised ability to downregulate ATP and secreted more pyruvate, acetate, succinate, and glutamate in the culture medium. Importantly, the mutant strain had higher intracellular pyruvate and produced more toxic methylglyoxal, suggesting a glycolytic stress leading to growth stasis and consequently biofilm failure. This study demonstrates, for the first time, the link between mycobacterial biofilm formation and the Crabtree effect. [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]

ABSTRACT When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the genetic network that determines the duration of the lag phase has not been studied in much detail. Here, we performed a genome-wide Bar-Seq screen to identify genetic determinants of the Saccharomyces cerevisiae glucose-to-galactose lag phase. The results show that genes involved in respiration, and specifically those encoding complexes III and IV of the electron transport chain, are needed for efficient growth resumption after the lag phase. Anaerobic growth experiments confirmed the importance of respiratory energy conversion in determining the lag phase duration. Moreover, overexpression of the central regulator of respiration, HAP4, leads to significantly shorter lag phases. Together, these results suggest that the glucose-induced repression of respiration, known as the Crabtree effect, is a major determinant of microbial fitness in fluctuating carbon environments. IMPORTANCE The lag phase is arguably one of the prime characteristics of microbial growth. Longer lag phases result in lower competitive fitness in variable environments, and the duration of the lag phase is also important in many industrial processes where long lag phases lead to sluggish, less efficient fermentations. Despite the immense importance of the lag phase, surprisingly little is known about the exact molecular processes that determine its duration. Our study uses the molecular toolbox of S. cerevisiae combined with detailed growth experiments to reveal how the transition from fermentative to respirative metabolism is a key bottleneck for cells to overcome the lag phase. Together, our findings not only yield insight into the key molecular processes and genes that influence lag duration but also open routes to increase the efficiency of industrial fermentations and offer an experimental framework to study other types of lag behavior.

ABSTRACT Dengue virus (DENV) is among the most important human arboviruses and is clinically and experimentally associated with lipid metabolism disorders. Using high-resolution respirometry, we analyzed the metabolic switches induced by DENV in a human hepatic cell line. This experimental approach allowed us to determine the contribution of fatty acids, glutamine, glucose, and pyruvate to mitochondrial bioenergetics, shedding light on the mechanisms involved in DENV-induced metabolic alterations. We found that while infection strongly inhibits glutamine oxidation, it increases the cellular capacity of metabolizing glucose; remarkably, though, this substrate, instead being used as an energy source, performs an anaplerotic role in the oxidation of endogenous lipids. Fatty acids become the main energetic substrate in infected cell, and through the pharmacological modulation of β-oxidation we demonstrated that this pathway is essential for virus replication. Interestingly, infected cells were much less susceptible to the Crabtree effect, i.e., the glucose-mediated inhibition of mitochondrial oxygen consumption, suggesting that infection favors cellular respiration by increasing ADP availability. IMPORTANCE Dengue virus infection is a major cause of human arbovirosis, for which clinical and experimental evidence supports the idea that liver dysfunction and lipid metabolism disorders are characteristics of severe disease. Analyzing mitochondrial bioenergetics, here we show that infection of hepatic cells with dengue virus favors the cellular capacity of metabolizing glucose, impairing the normal metabolic flexibility that allows the oxidative machinery to switch among the main energetic substrates. However, instead of being used as an energy source, glucose performs an anaplerotic role in the oxidation of endogenous fatty acids, which become the main energetic substrate during infection. Taken together, the results shed light on metabolic mechanisms that may explain the profound alterations in lipid metabolism for severe dengue patients, contributing to the understanding of dengue physiopathology.

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]

At specific growth rates above a particular critical value, Crabtree-positive microorganisms exceed their respiratory capacity and enter diauxic growth metabolism. Excess substrate is converted reductively to an overflow metabolite, resulting in decreased biomass yield and productivity. To prevent this scenario, the cells can be cultivated in a fed-batch mode at a growth rate maintained below the critical value, µcrit. This approach entails two major challenges: accurately estimating the current specific growth rate and controlling it successfully over the course of the fermentation. In this work, the specific growth rate of S. cerevisiae and E. coli was estimated from enhanced on-line biomass concentration measurements obtained with dielectric spectroscopy and turbidity. A feedforward-feedback control scheme was implemented to maintain the specific growth rate at a setpoint below µcrit, while on-line FTIR measurements provided the early detection of the overflow metabolites. The proposed approach is in line with the principles of Bioprocess Analytical Technology (BioPAT), and provides a means to increase the productivity of Crabtree-positive microorganisms. [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]

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]

The rate of biological aging varies cyclically and episodically in response to changing environmental conditions and the developmentally-controlled biological systems that sense and respond to those changes. Mitochondria and metabolism are fundamental regulators, and the cell is the fundamental unit of aging. However, aging occurs at all anatomical levels. At levels above the cell, aging in different tissues is qualitatively, quantitatively, and chronologically distinct. For example, the heart can age faster and differently than the kidney and vice versa. Two multicellular features of aging that are universal are: (1) a decrease in physiologic reserve capacity, and (2) a decline in the functional communication between cells and organ systems, leading to death. Decreases in reserve capacity and communication impose kinetic limits on the rate of healing after new injuries, resulting in dyssynchronous and incomplete healing. Exercise mitigates against these losses, but recovery times continue to increase with age. Reinjury before complete healing results in the stacking of incomplete cycles of healing. Developmentally delayed and arrested cells accumulate in the three stages of the cell danger response (CDR1, 2, and 3) that make up the healing cycle. Cells stuck in the CDR create physical and metabolic separation—buffer zones of reduced communication—between previously adjoining, synergistic, and metabolically interdependent cells. Mis-repairs and senescent cells accumulate, and repeated iterations of incomplete cycles of healing lead to progressively dysfunctional cellular mosaics in aging tissues. Metabolic cross-talk between mitochondria and the nucleus, and between neighboring and distant cells via signaling molecules called metabokines regulates the completeness of healing. Purinergic signaling and sphingolipids play key roles in this process. When viewed against the backdrop of the molecular features of the healing cycle, the incomplete healing model provides a new framework for understanding the hallmarks of aging and generates a number of testable hypotheses for new treatments.

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]

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.

Since Jacob and Monod's discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as "metabolic reprogramming." The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of E. coli to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rothman, Moore and Shulman.)

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]