As expected, glucose uptake rates were high for short-term Crabtree positive yeasts, which is a necessity to maintain high carbon-flux through fermentative pathways. It is clear that fermenting yeasts possessed a high glycolytic flux and at least a basal activity of enzymes that constitute the fermentative pathway. It is also clear that K. This can however be explained by the higher anabolic flux in K. Our carbon-flux analysis Fig. Can the degree of unbalanced metabolism also explain the different degrees of short-term Crabtree effect in the fermenting yeast species?
Our results reveal that the level of short-term ethanol production is not only positively affected by smaller biomass flux i. These observations suggest that short-term Crabtree positive yeasts must possess a strong glucose uptake apparatus, even at glucose limiting conditions and at low growth rates. It appears as if glucose uptake is not strictly synchronized with growth and can therefore easily exceed the anabolic activity, which could be the underlying factor that triggered the observed short-term Crabtree effect in K.
These yeasts immediately take up glucose upon a glucose pulse, which is then catabolized to pyruvate. Because growth and respiration represents bottlenecks, pyruvate enters the available fermentative pathway. A fundamental trait that distinguishes S. Since long-term glucose pulse experiments could in theory be considered as equivalent to batch cultivation, a comparison between early and late time intervals after a glucose pulse should be able to reveal any long-term responses.
Our results show no such long-term repression of respiration in any of the investigated short-term Crabtree positive yeasts for the first 20 minutes as compared to time intervals after 60 minutes Fig. However, a new type of long-term glucose effect can be observed in all short-term Crabtree positive yeasts except K.
These yeasts exhibit increasing RQ-values at later time intervals after a pulse of glucose. Could it be that glucose repression of respiration is strictly associated with gene-regulation at high growth-rates? When we compared the O 2 consumption rates in forty different yeast species from batch cultures S1 Table , no significant differences between respiro-fermenting and purely respiring yeasts could be detected Fig. Hence, no long-term repression of respiration could be detected in at least the majority of respiro-fermenting yeasts, and the origin of aerobic fermentation does not coincide with the origin of glucose repression of respiration.
Could it then be that glucose repression of respiration is fully expressed only in S. Indeed, when we compared the O 2 consumption rates in S. These results suggest that the origin of strong glucose repression of respiration occurred relatively late in the yeast evolutionary history, likely after the whole genome duplication event WGD. Thus, the origin of glucose repression of respiration and Crabtree effect, as we know it in S. On the other hand, long-term glucose activation of the anaerobic glycolytic pathway appears to have evolved just before the divergence of the Lachancea and Saccharomyces lineages see also Fig.
This mechanism has to our knowledge never been demonstrated before. A No significant difference in O 2 consumption rates could be observed between purely respiring yeasts and respiro-fermenting yeasts. B Among all of the respiro-fermenting yeasts, only members of the Kazachstania and Saccharomyces clades appear to possess repression of respiration. C This figure is adapted from an earlier study [ 16 ], and illustrates how glucose consumption rates have evolved with the gradual increase of ethanol fermentation in the Saccharomyces lineage.
Purely respiring yeasts constitute group 1 and respiro-fermenting yeasts constitute groups 2, 3 and 4 as previously defined [ 16 ]. D Overflow metabolites other than ethanol, such as acetate, pyruvate, glycerol, lactate and succinate were readily detected in all respiro-fermenting yeasts as compared to purely respiring yeasts.
Thus, the origin of aerobic fermentation coincides with the origin of overflow metabolism in the Saccharomyces lineage. Oxygen and glucose consumption rates were determined from batch cultures of over forty yeast species at their exponential growth phase [ 16 ]. It is known from studies on S.
G Crabtree [ 1 ] and was originally associated with glucose repression of respiration in the mammal cell. Our results confirm the early observations made by de Deken for S. Although our results can confirm the existence of repression of respiration in a majority of yeast species that belong to the Saccharomyces and Kazachstania clade see also Fig. We have shown that a balance exist between glucose uptake rates and carbon-flux through anabolic and catabolic pathways in short-term Crabtree negative yeast species Fig.
Uncoordinated glucose uptake with growth causes imbalance between the catabolic and anabolic pathways that result in overflow of respiration, which further leads to increased carbon-flux through fermentative pathways in short-term Crabtree positive yeasts. The interrelationship between pathways is apparent in short-term responses to glucose, and is greatly affected by biomass formation rates. This can be observed in weak short-term Crabtree positive yeasts such as K. We have also shown that repression of respiration appears to occur only as a long-term response to glucose at high growth-rates, such as in batch cultures of S.
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Glucose repression of respiration is therefore not a major contributor to the observed imbalanced flow of carbon through the glycolytic and other anabolic and catabolic pathways in the majority of short-term Crabtree positive yeasts. In order to investigate for any evolutionary conserved interrelationship between fermentative and respiratory pathways among species, we expanded our analysis to interspecies comparison of I glucose uptake rate and II RQ, ethanol production rate, CO 2 production rate, O 2 consumption rate, and growth rate for all species and time intervals Fig.
The average glucose uptake rates for all time intervals and investigated species [ 17 ] are plotted against the average of A RQ, B CO 2 production rates, C O 2 consumption rates, D ethanol production rates, and E growth rates at the corresponding time intervals, while glucose is still present after a glucose-pulse. Yeast species can be grouped according to their glucose uptake rates and fermenting capacity. The critical glucose uptake rate GF crit is defined as the rate where overflow occurs, what separates fermenting from non-fermenting yeasts.
There is an interrelationship between fermentative and respiratory pathways that depend on glucose uptake rates among the investigated species. A linear correlation between ethanol fermentation and glucose uptake rates can be deduced from the data starting at GF crit. The linear correlations are highly variable at early time intervals see also S3 Fig. It can also be concluded that much of the variance in the data set was caused by an unstable growth in the transition from growth under glucose limiting- to glucose excess- conditions, which was primarily observed among respiro-fermenting yeast species within the first hour S3 Fig.
These conclusions are more apparent, when early time intervals are compared with later ones. At late time intervals, more coherent trends can be observed when the cultures were more adapted to new glucose rich conditions and growth sets in, also for respiro-fermenting yeasts S4 Fig. Overflow metabolism seem to be the fundamental mechanism behind short-term Crabtree effect in all investigated yeast species, and a delayed regulation of cell-growth as compared to an intrinsically upregulated anaerobic glycolysis, could transiently shift long-term Crabtree negative yeasts to short-term Crabtree positive yeasts, such as in the case of K.
Furthermore, overflow also appears to play an important role at later stages after a glucose pulse, and since long-term glucose pulse experiments are in principal equivalent to batch-cultivations, similar conclusions could be valid for both approaches. These results, together with the fact that the origin of short-term Crabtree effect coincides with the origin of long-term Crabtree effect [ 17 ], further suggests overflow to be the mechanism behind both traits.
To test this hypothesis, similar analyses were done on a dataset from a large-scale batch study of forty different yeast species [ 16 ], and the results are clear Fig. In these figures K. Simplified, when the glucose uptake rate reaches a certain rate, overflow in sugar metabolism occurs and results in the production of ethanol. The result reveals an almost perfect linear correlation between these parameters S6A Fig. It is also possible to introduce an additional parameter TOF into the model, which stand for Total Overflow Flux and is the sum of the formation of all other detected overflow metabolites acetate, glycerol, succinate, pyruvate and lactate.
The difference between the models is further illustrated by balancing out the carbon flows, and since the sum of several overflow products TOF is not significant as compared to the total flux, the addition of the variable TOF to model 1 does not improve it significantly under the applied growth conditions S6B and S7B Figs. We have previously shown that TCA-cycle intermediates such as succinate, the end product of glycolysis such as pyruvate, and intermediates in the pyruvate dehydrogenase bypass such as acetate are readily detected during growth in a majority of respiro-fermenting yeast species [ 16 ].
Here we show that the sum of all accumulated overflow metabolites are highly significant in respiro-fermenting yeasts, and absent in purely respiring yeasts Fig. This suggests that there are several potential bottlenecks for overflow to occur that might reside at pyruvate or beyond. An example of one such potential bottleneck is the consumption of TCA-cycle intermediates i. Another example is the observed short-term intrinsic limitation in oxygen consumption rates among respiro-fermenting yeasts Fig. Hence, our results suggest an universal maximal capacity of respiratory flux that can support a maximal capacity of biomass flux in our growth conditions for all investigated yeast species, with glycolytic flux as the important variable that determines overflow equation 1 and 2 above.
Our data and the linearity of our model predict a metabolic flux-control as the most likely fundamental mechanism to sense glucose and control its uptake [ 22 ], even if the onset of fermentation is due to overflow. It is known that respiro-fermenting yeasts possess many genes encoding low-affinity glucose transporters, which facilitate glucose uptake in an energy independent manner when glucose concentration is high.
It is also known that some long-term Crabtree negative yeast species such as K. An uncontrolled and relaxed glucose uptake and glycolysis that is not tightly coupled to respiration and growth, could explain the difference in short-term Crabtree effect between i. Another very interesting aspect of aerobic fermentation is the evolutionary background for the development of these regulatory mechanisms.
Recently several hypotheses have emerged mainly from in silico approaches, and many of the evolutionary events that have been determined from comparative genomics approaches have previously been verified by comparative physiology approaches and discussed in the evolutionary context [ 16 ]. The long-term Crabtree effect has hitherto been limited to few reference species only, and has often been quantified from yields of metabolites and biomass.
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Hence, it is not strange that the long-term Crabtree effect has appeared, from an energetically point of view, as a very peculiar trait illustrated in Fig. Nevertheless, the evolutionary background for the development of aerobic fermentation has since the discovery of the budding yeast and its biological activities by Louis Pasteur, more than a century ago, remained unsolved.
This figure illustrates an overview of the evolution of long-term Crabtree effect, what resulted in lower energy-yield in Saccharomyces yeast species that possess the respiro-fermentative lifestyle. Theoretical ATP yields from anaerobic glycolysis in blue and respiration in red with standard deviations from two biological replicates were calculated during exponential growth-phase, using already published data [ 16 ].
Yeast species are ordered along the horizontal plane, roughly according to their reported phylogenetic relationship [ 29 ]. The timing of several evolutionary events that are relevant for the modern traits, such as the loss of respiratory complex I, the horizontal transfer of URA1 , and the whole genome duplication WGD event are highlighted in red. The conclusions from our study revealed overflow as the fundamental mechanism behind both short- and long-term Crabtree effect in all investigated yeast species summarized by equation 1 and 2 above. We speculate that the fermentative lifestyle originally evolved as an advantageous trait in a readily increasing glucose-rich environment that coincides with the appearance of the first angiosperms in nature [ 16 ].
The strategy to increase anaerobic glycolysis even under aerobic conditions, for increased energy output rates is even more apparent when phylogeny is not taken into account S9 and S10A-C Figs. This figure illustrates an overview of the evolution of the theoretical ATP production rates from anaerobic glycolysis in blue and respiration in red in the Saccharomyces lineage.
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While the evolution of Crabtree effect has resulted in lower energy-yield in Saccharomyces yeast species that possess the respiro-fermentative lifestyle, the sum of ATP production rates remain fairly unchanged between the different groups of yeasts. If phylogeny is not taken into account, a positive correlation between overflow metabolism and ATP production rates can be observed see also S9 Fig. ATP production rates with standard deviations from two biological replicates were calculated during exponential growth-phase, using already published data [ 16 ]. Yeast species are ordered along the horizontal plane, roughly according to their phylogenetic relationship [ 29 ].
The timing of several evolutionary events that are relevant for the modern traits, such as the loss of respiratory complex I, the horizontal transfer of URA1 and the whole genome duplication WGD event, are highlighted in red. Only within the members of the Saccharomyces and Kazachstania clades can a trade-off between energy and ethanol production be detected S10D Fig. This is of course a consequence of lower oxygen consumption rates among these yeasts see also Fig. The strategy of fine-tuning the interrelationship between evolutionary conserved pathways, such as anaerobic glycolysis and respiration are highly compatible and flexible, since both pathways are energy producing Fig.
An increased anaerobic glycolytic activity would also enable recycling of NADH for glycolysis to proceed and simultaneously drive anabolic reactions by directing the overflow of carbon to less energy-rich metabolites such as ethanol Fig. One way to increase overflow would of course require the uncoupled regulation of respiration from glycolysis in respiro-fermenting yeasts [ 27 ], so that anaerobic glycolysis can be independently upregulated, even under aerobic condition.
In other words, glycolytic flux would be less tightly regulated with respiration and biomass formation. Another way to accomplish imbalance between respiration and glycolysis could also be from the gross duplication of glycolytic genes, what has been observed in WGD yeasts [ 28 ]. However, it should be noted that the origin of short-term Crabtree effect [ 17 ], and the long-term upregulation of anaerobic glycolysis predates the WGD event S2 Fig.
At growth-conditions that are rich enough in free sugars to support a high biomass content, other limiting factors will set in, i. It is under these semi-anaerobic conditions that overflow metabolites, such as ethanol can be accumulated in sufficient amount that they become toxic.
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