The Pasteur effect

The Pasteur effect describes the phenomenon whereby fermentation is suppressed by the presence of oxygen. In reality, what this means from the perspective of the yeast cell is that the rate of glycolysis is slower under aerobic conditions than anaerobic conditions (due to regulation of phos- phofructokinase within the pathway). This ensures that bottlenecks are not encountered and that all carbon can be metabolized via the Krebs cycle and through oxidative phosphorylation, which is an efficient means of maximizing ATP yield. Arguably of equal or greater significance is that this also means that glycolysis proceeds at a faster rate under anaerobic conditions. This is necessitated due to the reduction in ATP yield which is encountered through switching from respiration to fermentation; the cell must increase glycolytic rate to counter this effect and to ensure that ATP demands are met. One caveat for this phenomenon is that the level of glucose must be relatively low (Lagunas, 1979) and that other nutrients (including nitrogen) are also limiting. Interestingly, these criteria are relatively flexible which means that yeasts can differ in their capacity to implement the Pasteur effect. Saccharo- myces yeasts only exhibit the Pasteur effect under quite stringent conditions and when cells are in a ‘resting' state (i.e. not actively growing). In contrast, yeasts such as Candida and Pichia are highly susceptible to this effect. The reason why there is such discrepancy is that the Pasteur effect can

Table 11.4 Regulatory mechanisms for sugar metabolism in yeast

Metabolic

effect

Description

Notable yeast species

Pasteur

effect

Inhibition of fermentation pathways in

the presence of oxygen

Oxygen restricts ethanol production

Saccharomyces yeasts are not affected due to the Crabtree effect

Crabtree

effect

Suppression of respiration pathways in the presence of glucose When a threshold of glucose is reached, yeast will only utilize the fermentation pathway

Positive: Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces

Negative: Pichia, Kluyveromyces, Debaryomyces, Torulaspora Species-dependent: Candida, Hanseniaspora

Kluyver

effect

Inhibition of fermentation pathway in the presence of certain sugars Yeast can only utilize certain sugars aerobically

Saccharomyces strains are Kluyver effect negative. Most non- Saccharomyces beer-spoiling yeasts are Kluyver effect positive for various sugars

Custers

effect

Anaerobic conditions cause fermentation to be suppressed Yeast utilize the fermentation pathway when oxygen is present

Brettanomyces

be influenced by the presence of other metabolic regulatory phenomena. For example, in some yeast (e.g. brewing strains) the Pasteur effect is rendered insignificant due to the Crabtree effect (see below). Irrespective, increasing the fermentation rate in the absence of oxygen leads to the rapid production of ethanol that can procure a competitive advantage in the ‘natural' environment. This type of approach is often referred to as the ‘make-accumulate- consume' (MAC) strategy, whereby organisms produce a compound that accumulates within the environment, and which can be re-assimilated and used for energy production at a later point in time (Fig. 11.6). This strategy may provide a significant benefit to a population since competing organisms, including bacterial species, are typically less tolerant to ethanol than is yeast.

The Crabtree effect (glucose repression) Arguably one ofthe most important mechanisms for metabolic regulation in yeast species is the Crabtree effect, often referred to as simply ‘glucose repression' (Barnett and Entian, 2005). The Crabtree effect occurs irrespective of the presence of oxygen and specifies that if the concentration of sugar is above a certain threshold, cells will metabolize exclusively via fermentation. As the name suggests, this is primarily due to repression of the respiratory pathway caused by the presence of glucose, and occurs to the extent that the Pasteur effect is overridden (i.e. the rate of glycolysis is not restricted by oxygen). It should be noted that other sugars such as fructose may also play a similar role to glucose, since many yeasts are able to conduct aerobic fermentation in their presence. However, glucose is certainly the primary sugar involved. The Crabtree effect is observed in most fermentative beer-spoiling yeasts, including Saccharomyces, Brettanomyces and Zygo- saccharomyces. In these organisms, the Crabtree effect can be induced by as little as 0.2% glucose, although this limit can vary upwards. The extent to which the Crabtree effect is induced can also be species-, and to a lesser extent strain-dependent, as indicated by analysis of the ratio of glucose that is fermented/respired under defined conditions (De Deken, 1966; Hagman et al., 2014).

In yeasts, the Crabtree effect can be observed in the form of a ‘short-term' or ‘long-term' response. The short-term Crabtree effect occurs when the cell encounters a sudden increase in glucose concentration (Van Urk et al., 1989), which causes the respiratory pathway to become saturated at pyruvate, resulting in carbon being directed towards ethanol formation (overflow metabolism) (Fig. 11.6). In contrast, the long-term Crabtree effect, characterized by the response to steady-state conditions, indicates that glucose (or a product of glucose metabolism) actively functions to repress the synthesis of respiratory enzymes. It is known that the yeast cell is able to respond to the presence of glucose by a series of signal transduction pathways that are regulated based on the concentrations of extracellular and intracellular glucose, related metabolites, and flux through key glycolytic enzymes. This includes activation of a range of genes that function as transcriptional regulators, and those that are involved in glucose sensing and sugar uptake (for a comprehensive review see Conrad et al., 2014). Although many of the regulatory activities are believed to operate at the transcription regulation level, some may also act directly on specific respiratory enzymes, including those involved in gluconeogenesis, the Krebs cycle, and mitochondrial function and maintenance (Kappeli, 1986). It should be noted that despite effort in this direction, at the present time much is still unknown about glucose sensing and its regulatory mechanisms in S. cerevisiae, while in other yeast species it remains largely unexplored. However, it is likely that the underlying principles behind glucose repression are similar among yeasts, even if the precise mechanisms differ.

Although many yeasts are subject to the longterm Crabtree effect, the reason why this form of metabolism has evolved is difficult to explain, especially since fermentation has a significantly lower ATP yield than respiration. However, it has been suggested that the long-term Crabtree effect is essentially an evolutionary extension of overflow metabolism (Hagman and Piskur, 2015), while the acceleration of glycolysis and the preference for alcohol production is also likely to function to create a hostile environment for competitors as part of a MAC strategy. In addition, it is possible that rapid sugar uptake can be considered to be a form of glucose scavenging, inhibiting growth of other microbes by starvation, while others have argued that the Crabtree effect evolved simply due to the benefits associated with an overall increase in the rate of ATP production (Pfeiffer and Morley,

2014). In the context of beer-spoiling yeasts, the fact that many species are not Crabtree-positive, or that they exhibit either a short-term response or a ‘weaker' long-term response overall is significant. For example, purely respiring yeast species such as Kluyveromyces marxianus or Candida utilis may need to adopt other strategies to limit the glycolytic rate if a metabolic overflow is encountered. This may manifest in regulation of sugar uptake, formation of glycerol, or in the production of intracellular reserve carbohydrates (glycogen/trehalose). There is also likely to be an impact based on niche development, which may ultimately determine which species are likely to compete with production stains during fermentation. Crabtree-negative yeasts will inevitably be less competitive than production strains during fermentation and will only be able to survive at low concentrations, hence one of the reasons why they are typically associated with alternative processing stages.

 
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