A study on the fundamental mechanism and the evolutionary driving forces behind aerobic fermentation in yeast

Abstract

Baker's yeast Saccharomyces cerevisiae rapidly converts sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. The later phenomenon is called Crabtree effect and has been described in two forms, long-term and short-term effect. We have previously studied under fully controlled aerobic conditions forty yeast species for their central carbon metabolism and the presence of long-term Crabtree effect. We have also studied ten steady-state yeast cultures, pulsed them with glucose, and followed the central carbon metabolism and the appearance of ethanol at dynamic conditions. In this paper we analyzed those wet laboratory data to elucidate possible mechanisms that determine the fate of glucose in different yeast species that cover approximately 250 million years of evolutionary history. We determine overflow metabolism to be the fundamental mechanism behind both long- and short-term Crabtree effect, which originated approximately 125-150 million years ago in the Saccharomyces lineage. The "invention" of overflow metabolism was the first step in the evolution of aerobic fermentation in yeast. It provides a general strategy to increase energy production rates, which we show is positively correlated to growth. The "invention" of overflow has also simultaneously enabled rapid glucose consumption in yeast, which is a trait that could have been selected for, to "starve" competitors in nature. We also show that glucose repression of respiration is confined mainly among S. cerevisiae and closely related species that diverged after the whole genome duplication event, less than 100 million years ago. Thus, glucose repression of respiration was apparently "invented" as a second step to further increase overflow and ethanol production, to inhibit growth of other microbes. The driving force behind the initial evolutionary steps was most likely competition with other microbes to faster consume and convert sugar into biomass, in niches that were semi-anaerobic.

Fig 1. Yeast O₂ consumption and CO₂ production rates.

ATP is produced primarily from respiration, but also from fermentation. When yeast respires, O₂ will be consumed. CO₂ is produced from both respiration as well as fermentation. This figure illustrates that all short-term Crabtree positive yeasts have an initially higher energy metabolism that increases rapidly as compared to short-term Crabtree negative yeasts, as the steady-state cultures leave glucose limited growth and enter higher growth-rates in excess of glucose. The average O₂ consumption (left) and CO₂ production rates (right) between replicates are illustrated for cultures at steady-state (SS), time interval 5–20 minutes (Early-phase), and time interval after 60 minutes (Late-phase) while glucose was still present after a glucose-pulse. Error bars correspond to the standard deviation.

Fig 2. Yeast central carbon metabolic pathways at steady-state growth.

All short-term Crabtree positive yeasts possess an upregulated aerobic (blue) and anaerobic (red) glycolytic pathway, even under fully aerobic conditions, when energy and carbon-source is limiting. At low glucose uptake rates, yeasts cells are purely respiring and there is a carbon-flux only through glycolysis (GF) and respiration (RF). Upon a sudden glucose excess condition (glucose pulse), the glycolytic flux will exceed the respiratory flux, which results in a fermentative flux (FF) and ethanol production. In other words, it appears as if slowly dividing short-term Crabtree positive cells, with little food around are already equipped with a strong energy producing apparatus, for rapid glucose consumption and energy production.

Fig 3. Yeast glucose consumption rate and respiratory quotient (RQ).

Under purely respiratory metabolism at low biomass formation (growth) rates, roughly one CO₂ molecule will be produced for each O₂ molecule consumed. Fermentation does not require molecular O₂ as the final electron acceptor, and will only produce one CO₂ in the first decarboxylation step of each pyruvate. Hence, RQ ratios that are significantly greater than one indicate a fermentative activity. All ethanol-forming yeasts have RQ ratios significantly greater than one, while all non-ethanol forming yeasts have an RQ close to, or equal to one. The average glucose consumption rates (left) and RQ ratios (right) between biological replicates are illustrated for cultures at steady-state (SS), time interval 5–20 minutes (Early-phase) and time interval after 60 minutes (Late-phase) while glucose was still present after a glucose pulse. Error bars correspond to the standard deviation.

Fig 4. Carbon-flux balance.

Glucose was the main carbon and energy source for cell proliferation in all experiments. The sugar is taken up by cells, and metabolized to form biomolecules and release energy for growth. The flow of carbon in central carbon metabolism was estimated from the average formation rates in a time interval of end products such as, biomass, CO₂ from fermentation and respiration, and ethanol. These output rates should be lower than or roughly equal to the input rates of carbon into a cell, which is equivalent to the average glucose uptake rates. 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. lactis, which had a glycolytic capacity close to intermediate fermenting yeast, such as L. kluyverii, L. waltii and T. franciscae only fermented weakly, and thus appeared to have a retarded response to glucose. This can however be explained by the higher anabolic flux in K. lactis, which directs the carbon flow from fermentative pathways to biomass formation.

Fig 5. The origin of overflow precedes the origin of glucose repression of respiration.

These figures illustrate the difference in population means among different phylogenetic groups and parameters (see Table 1 for designations) with error bars corresponding to the 95% confidence interval. (A) No significant difference in O₂ 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.

Fig 6. Long-term glucose repression of respiration.

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. cerevisiae that it exhibits repressed respiration when cultivated at high growth-rates on glucose, even under aerobic batch conditions. This trait was named after it’s discoverer H.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. cerevisiae and sister-species, but surprisingly most of the other respiro-fermenting yeast species appear to lack this trait. 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. 5B and S10D Fig.) we cannot rule out the possibility that this peculiar trait might occur in any of the other early branching clades, nor the existence of several regulatory pathways that could govern glucose repression.

Fig 7. Pulse—Growth parameters in correlation with glucose uptake rates at early and late growth phases.

The average glucose uptake rates for all time intervals and investigated species [17] are plotted against the average of (A) RQ, (B) CO₂ production rates, (C) O₂ 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.), but become more apparent at later time intervals, when the cultures were more adapted to the new growth conditions (see also S4 Fig.).

Fig 8. Evolution of long-term Crabtree effect in the Saccharomyces-lineage.

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 evolution of the peculiar trait of Crabtree positive yeast species, which appears less energy-efficient as compared to their Crabtree negative counterpart have been discussed from an ecological aspect and explained by the “make-accumulate-consume” strategy [14].

Fig 9. Distribution of theoretical ATP production rates in yeast.

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. 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.

Fig 10. Evolutionary scenario for the origin of Crabtree effect in Saccharomycetales yeast.

This figure illustrates the capacity of central carbon metabolic pathways for the metabolic groups of yeast (as designated in Table 1), when grown on C₆-sugars such as glucose. Biomass formation rates have been left out, since no significant differences amongst groups could be observed (S2 Table). (A) Purely respiring yeasts, including Pichia, Debaromyces, Eremothecium and a majority of Kluyveromyces exhibited low glycolytic flux (GF), without any overflow metabolism (see also S10B Fig.). (B) Yeast that separated from the Eremothecium lineage, including some Kluyveromyces, and all Lachancea, Torulaspora, Zygotorulaspora and the majority of WGD yeasts possessed a greater glycolytic flux than respiratory flux (RF) capacity, what results in overflow metabolism. The upregulation of the anaerobic glycolysis has provided this group of yeast with a greater energy producing apparatus that can consume glucose more rapidly under aerobic conditions (see also S10C Fig.). (C) Our results can be interpreted as that traits such as overconsumption of glucose, and excess of energy producing capacity has enabled the development of a third metabolic group (including a majority of Kazachstania and Saccharomyces) that exhibit a trade-off between ethanol and energy production efficiency (see also S10D Fig.).