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Crabtree effect
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The Crabtree effect, named after the English biochemist Herbert Grace Crabtree,[1] describes the phenomenon whereby the yeast, Saccharomyces cerevisiae, produces ethanol (alcohol) in aerobic conditions at high external glucose concentrations rather than producing biomass via the tricarboxylic acid (TCA) cycle, the usual process occurring aerobically in most yeasts e.g. Kluyveromyces spp.[2] This phenomenon is observed in most species of the Saccharomyces, Schizosaccharomyces, Debaryomyces, Brettanomyces, Torulopsis, Nematospora, and Nadsonia genera.[3] Increasing concentrations of glucose accelerates glycolysis (the breakdown of glucose) which results in the production of appreciable amounts of ATP through substrate-level phosphorylation. This reduces the need of oxidative phosphorylation done by the TCA cycle via the electron transport chain and therefore decreases oxygen consumption. The phenomenon is believed to have evolved as a competition mechanism (due to the antiseptic nature of ethanol) around the time when the first fruits on Earth fell from the trees.[2] The Crabtree effect works by repressing respiration by the fermentation pathway, dependent on the substrate.[4]
Ethanol formation in Crabtree-positive yeasts under strictly aerobic conditions was firstly thought to be caused by the inability of these organisms to increase the rate of respiration above a certain value. This critical value, above which alcoholic fermentation occurs, is dependent on the strain and the culture conditions.[5] More recent evidences demonstrated that the occurrence of alcoholic fermentation might not be primarily due to a limited respiratory capacity,[6] but could be caused by a limit in the cellular Gibbs energy dissipation rate.[7]
For S. cerevisiae in aerobic conditions,[8] glucose concentrations below 150 mg/L did not result in ethanol production. Above this value, ethanol was formed with rates increasing up to a glucose concentration of 1000 mg/L. Thus, above 150 mg/L glucose the organism exhibited a Crabtree effect.[9]
It was the study of tumor cells that led to the discovery of the Crabtree effect.[10] Tumor cells have a similar metabolism, the Warburg effect, in which they favor glycolysis over the oxidative phosphorylation pathway.[11]
References
[edit]- ^ Crabtree, HG (1929). "Observations on the carbohydrate metabolism of tumours". The Biochemical Journal. 23 (3): 536–45. doi:10.1042/bj0230536. PMC 1254097. PMID 16744238.
- ^ a b Thomson JM, Gaucher EA, Burgan MF, De Kee DW, Li T, Aris JP, Benner SA (2005). "Resurrecting ancestral alcohol dehydrogenases from yeast". Nat. Genet. 37 (6): 630–635. doi:10.1038/ng1553. PMC 3618678. PMID 15864308.
- ^ De Deken, R. H. (1966). "The Crabtree Effect: A Regulatory System in Yeast". J. Gen. Microbiol. 44 (2): 149–56. doi:10.1099/00221287-44-2-149. PMID 5969497.
- ^ De Deken, R. H. (1 August 1966). "The Crabtree Effect and its Relation to the Petite Mutation". Journal of General Microbiology. 44 (2): 157–165. doi:10.1099/00221287-44-2-157. PMID 5969498.
- ^ van Dijken and Scheffers, 1986 J.P. van Dijken, W.A. Scheffers; Redox balances in the metabolism of sugars by yeasts; FEMS Microbiol. Lett., 32 (3) (1986), pp. 199-224; https://doi.org/10.1016/0378-1097(86)90291-0
- ^ Postma, E; Verduyn, C; Scheffers, WA; Van Dijken, JP (February 1989). "Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae". Applied and Environmental Microbiology. 55 (2): 468–77. Bibcode:1989ApEnM..55..468P. doi:10.1128/AEM.55.2.468-477.1989. PMC 184133. PMID 2566299.
- ^ Heinemann, Matthias; Leupold, Simeon; Niebel, Bastian (January 2019). "An upper limit on Gibbs energy dissipation governs cellular metabolism" (PDF). Nature Metabolism. 1 (1): 125–132. doi:10.1038/s42255-018-0006-7. ISSN 2522-5812. PMID 32694810. S2CID 104433703.
- ^ Verduyn, C., Zomerdijk, T.P.L., van Dijken, J.P. et al. Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode. Appl Microbiol Biotechnol 19, 181–185 (1984). https://doi.org/10.1007/BF00256451
- ^ Verduyn, C., Zomerdijk, T.P.L., van Dijken, J.P. et al. Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode. Appl Microbiol Biotechnol 19, 181–185 (1984). https://doi.org/10.1007/BF00256451
- ^ Pfeiffer, T; Morley, A (2014). "An evolutionary perspective on the Crabtree effect". Frontiers in Molecular Biosciences. 1: 17. doi:10.3389/fmolb.2014.00017. PMC 4429655. PMID 25988158.
- ^ Diaz-Ruiz, Rodrigo; Rigoulet, Michel; Devin, Anne (June 2011). "The Warburg and Crabtree effects: On the origin of cancer cell energy metabolism and of yeast glucose repression". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1807 (6): 568–576. doi:10.1016/j.bbabio.2010.08.010. PMID 20804724.
Further reading
[edit]- Crabtree HG (1928). "The carbohydrate metabolism of certain pathological overgrowths". Biochem. J. 22 (5): 1289–98. doi:10.1042/bj0221289. PMC 1252256. PMID 16744142.
Crabtree effect
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Definition and Overview
Core Phenomenon
The Crabtree effect refers to the metabolic shift in yeast cells, notably Saccharomyces cerevisiae, toward aerobic alcoholic fermentation when exposed to high glucose concentrations, resulting in the production of ethanol and carbon dioxide rather than complete oxidation of glucose through respiration, even though oxygen is plentiful.[4] This phenomenon represents an overflow metabolism where excess carbon flux through glycolysis exceeds the capacity for respiratory processing, prioritizing rapid energy generation over maximal efficiency.[8] Key features of the Crabtree effect include a respiratory quotient (RQ) exceeding 1, reflecting greater CO₂ production than O₂ consumption due to the decarboxylation of pyruvate to ethanol.[9] The process yields only 2 ATP per glucose molecule via substrate-level phosphorylation in glycolysis, in contrast to approximately 18 ATP from oxidative phosphorylation in full respiration, underscoring the trade-off for faster growth rates.[4] Associated byproducts extend beyond ethanol to include glycerol and acetate, which arise from redox balancing and minor pathway diversions during the fermentative overflow.[10] Unlike strictly anaerobic fermentation, which requires oxygen absence, the Crabtree effect manifests under fully aerobic conditions, driven by glucose excess that represses respiratory enzymes and pathways.[4] In typical batch cultures with initial glucose levels above 0.2 g/L, cells exhibit rapid glucose uptake, leading to prompt ethanol accumulation that persists until substrate depletion, often resulting in a biphasic growth pattern with an initial fermentative phase followed by respiratory diauxic shift.[11][12]Affected Organisms
The Crabtree effect is predominantly exhibited by certain unicellular fungi, particularly yeast species within the Ascomycota phylum. Saccharomyces cerevisiae, commonly known as baker's or brewer's yeast, displays a robust Crabtree-positive phenotype, characterized by the repression of respiration and induction of aerobic fermentation in the presence of high extracellular glucose concentrations. This leads to ethanol production as a major metabolic outcome under oxygen-replete conditions.[8] Other Crabtree-positive yeasts include Schizosaccharomyces pombe, which secretes substantial ethanol (up to 42% of consumed glucose) during aerobic growth on excess glucose, and species from genera such as Zygosaccharomyces and Dekkera (e.g., Dekkera bruxellensis). In comparison, Crabtree-negative yeasts like Kluyveromyces marxianus, Debaryomyces hansenii, Scheffersomyces stipitis (formerly Pichia stipitis), and Candida utilis favor respiratory metabolism, fully oxidizing glucose with minimal byproduct formation even at elevated sugar levels, resulting in higher biomass yields.[8][2][13] Beyond yeasts, the Crabtree effect, often termed overflow metabolism in this context, occurs in select bacteria such as Escherichia coli, where high glucose flux under aerobic conditions exceeds respiratory capacity, prompting acetate excretion instead of complete oxidation. It is infrequently observed in filamentous fungi, though examples exist in species like Rhizopus oryzae, which produces ethanol aerobically when glucose is abundant. While primarily observed in unicellular organisms such as yeasts, the Crabtree effect has also been reported in specific mammalian cell types, including tumor cells and kidney proximal tubule epithelial cells, though it remains uncommon across higher eukaryotes as a whole.[14][15][16] For instance, a 2023 study demonstrated the Crabtree effect in normal kidney proximal tubule epithelial cells at physiological glucose levels.[16] Distinctions in the Crabtree effect include long-term and short-term manifestations. The long-term variant arises during sustained growth in high-glucose media, enforcing persistent fermentative metabolism. The short-term variant represents a rapid, transient switch to aerobic ethanol production following glucose pulses in low-sugar-adapted cells, as seen in S. cerevisiae and related species.[2] The onset of the Crabtree effect depends on a glucose concentration threshold that differs among strains; in S. cerevisiae, this is approximately 0.15 g/L for typical laboratory isolates, while industrial strains may trigger it at lower levels (around 0.05 g/L) due to enhanced glucose uptake capacities.[4]Historical Background
Discovery
The Crabtree effect was first described by English biochemist Herbert Grace Crabtree in 1929 as part of his investigations into the carbohydrate metabolism of tumor tissues. In experiments using slices of rat sarcoma and other malignant tissues suspended in buffered saline, Crabtree observed that adding glucose to the medium caused a marked decrease in oxygen consumption, despite ample oxygen availability, while simultaneously increasing lactic acid production through aerobic glycolysis. This repression of respiration by high sugar concentrations represented a key deviation from expected oxidative metabolism in normal tissues.[17] Crabtree's early experiments quantified this phenomenon by measuring oxygen uptake and carbon dioxide output using manometric techniques on tumor preparations with varying glucose levels, typically ranging from 0 to 0.2% concentration. At low glucose, respiration proceeded efficiently with a respiratory quotient near 1, indicating complete oxidation; however, at higher levels, the quotient rose above 1, signaling a shift to fermentative breakdown and reduced overall respiratory efficiency. These findings highlighted how excess glucose could inhibit mitochondrial respiration, favoring rapid glycolytic flux even under aerobic conditions.[17] The effect is named after Crabtree for his pioneering observations, though it built directly on Otto Warburg's 1920s studies of elevated glycolysis in tumors, which emphasized high lactate output but did not fully elucidate the glucose-mediated respiratory inhibition. While Crabtree's work focused on mammalian tumor cells, the phenomenon was soon recognized in microbial systems, particularly yeast, where analogous high-sugar repression of respiration leads to ethanol formation. Related early insights into yeast fermentation trace to Louis Pasteur's 19th-century demonstrations of sugar metabolism under aerobic conditions, but the Pasteur effect inversely describes how respiration suppresses fermentation at low sugar levels.[17]Subsequent Research
Following the initial observation of the Crabtree effect in the 1920s, research in the 1950s and 1960s advanced the understanding of glucose-mediated repression of respiratory enzymes in yeasts. Jacques Monod's work on diauxic growth and catabolite repression in bacteria during the 1940s and 1950s provided a conceptual framework that was applied to yeasts, where high glucose concentrations were shown to inhibit the synthesis of enzymes involved in alternative carbon source utilization. In 1966, R.H. De Deken demonstrated that the Crabtree effect functions as a regulatory system in Saccharomyces cerevisiae, where elevated glycolytic rates under aerobic conditions lead to repression of respiratory enzyme synthesis, thereby inhibiting oxygen consumption. During the 1970s, studies further delineated the temporal dynamics of the effect. Maria Lagunas and colleagues distinguished between short-term and long-term components: the short-term effect involves rapid inhibition of respiration upon glucose addition, often linked to transient ATP accumulation, while the long-term effect entails transcriptional repression of respiratory genes over hours. A key milestone was Helmut Holzer's 1967 work on catabolite repression in yeast, which highlighted the inactivation of enzymes like malate dehydrogenase in the presence of glucose, establishing a link between glucose signaling and metabolic enzyme turnover.[18] Experimental approaches evolved during this period from respirometry, which measured oxygen uptake rates to quantify respiratory inhibition, to isotopic labeling techniques using radiolabeled glucose (e.g., ^{14}C-glucose) to trace carbon fluxes through glycolysis and the tricarboxylic acid cycle. These methods allowed researchers to quantify the diversion of carbon toward ethanol production rather than complete oxidation, providing evidence for flux partitioning in Crabtree-positive yeasts. In the 1980s and 1990s, attention shifted to key regulatory enzymes controlling glycolytic flux. Studies identified hexokinase isozymes, particularly hexokinase PII, as central to glucose repression, with mutants showing reduced repression and altered respiratory capacity. Phosphofructokinase was similarly implicated in flux control, where its activation under high glucose promotes glycolytic overflow and limits mitochondrial entry of pyruvate. By the 1990s, research confirmed the involvement of mitochondria, revealing that Crabtree-positive yeasts possess inherently limited respiratory chain capacity, making them prone to fermentation even under aerobic conditions. In the 2000s and beyond, advances in genomics and systems biology have further elucidated the molecular mechanisms, identifying key signaling pathways such as Snf1/AMPK and TOR that mediate glucose repression, as confirmed in studies up to 2025. These developments built on earlier genetic tools to enable precise manipulation and causal analysis of regulatory networks.[1] Despite these advances, pre-2000 research highlighted persistent gaps, including the absence of genetic tools for direct manipulation of regulatory pathways, which hindered causal mechanistic studies. Early recognition of industrial implications emerged, particularly in ethanol production for brewing and bioenergy, where the effect's role in maximizing fermentative yields was noted but challenging to optimize without molecular interventions.Biochemical Mechanism
Metabolic Pathways
The Crabtree effect prominently features an accelerated flux through glycolysis, the initial metabolic pathway for glucose breakdown. In this process, one molecule of glucose is phosphorylated and cleaved into two molecules of pyruvate through 10 sequential enzymatic reactions, including key steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase. This pathway generates a net yield of 2 ATP and 2 NADH per glucose molecule under standard conditions. During the Crabtree effect in organisms like Saccharomyces cerevisiae, the glycolytic flux increases substantially—often more than 5-fold—enabling rapid ATP production to support growth despite the presence of oxygen.[10][19] Under high-glucose conditions, pyruvate is largely shunted away from oxidative metabolism toward fermentative pathways to maintain redox balance and high throughput. In yeast, the dominant route is alcoholic fermentation, where pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase, releasing CO₂, and then reduced to ethanol by alcohol dehydrogenase using NADH as the electron donor. This step regenerates NAD⁺, which is essential for the continued operation of glycolysis at elevated rates. The net biochemical transformation, which dominates despite aerobic availability, can be represented as:
[19][4]
Concomitantly, respiratory pathways are repressed, limiting the entry of pyruvate into mitochondria and reducing overall oxygen consumption. A key restriction occurs at pyruvate dehydrogenase (PDH), where activity is curtailed, preventing efficient conversion of pyruvate to acetyl-CoA and subsequent flux through the tricarboxylic acid (TCA) cycle. Further downstream, diminished activity of cytochrome c oxidase in the electron transport chain contributes to lowered O₂ utilization, favoring fermentation over complete oxidation.[19][20]
Minor branches from glycolysis also contribute to byproduct formation during overflow metabolism. For instance, dihydroxyacetone phosphate can be diverted to glycerol via reduction by glycerol-3-phosphate dehydrogenase and dephosphorylation, aiding in NADH reoxidation under osmotic stress or redox imbalance. Additionally, a small fraction of acetaldehyde may be oxidized to acetate by aldehyde dehydrogenase, serving as an alternative sink for excess carbon. These pathways ensure metabolic flexibility but yield lower energy efficiency compared to full respiration.[10]