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Sugar uptake and metabolism of beer-spoiling yeasts

All yeasts are heterotrophic organisms that require a variety of nutrients in order to be able to produce energy, maintain cellular function, grow, and reproduce. A carbohydrate source is essential for the production of ATP, and to fulfil structural carbon requirements, while nitrogen is important for protein and amino acid synthesis. Phosphorus is required for energy transduction, and as a major component of membrane phospholipids and nucleic acid (DNA and RNA) synthesis. Metal ions, vitamins, growth factors, and other trace elements may also be required for a variety of structural and functional roles within the cell. The majority of beer-spoiling yeast share broadly the same nutritional requirements as production strains; however, there can be significant differences with regard to the preferred sources of individual nutrients and the ways in which they are assimilated and utilized. Many of these differences form the basis of tests for detection of specific groups of yeast (see section ‘Detection and identification'). There are also variations in the extent to which specific metabolic end products are formed; in some species carbon may be directed primarily towards biomass production rather than ethanol. In others, there may be a more diverse range of products, including a higher propensity to form glycerol as a mechanism for redox balance. Furthermore, nutritional requirements may lead to certain metabolic pathways being preferentially employed, leading to the production of compounds, such as diacetyl, that are produced as by-products of amino acid metabolism.

The mechanisms of sugar assimilation and breakdown form a major difference between brewing and beer-spoiling yeasts. At the basic level, some beer- spoiling yeasts may not be able to transport certain sugars into the cell, while others may efficiently use those that brewing strains cannot, one example being the utilization of the disaccharide lactose by Kluyveromyces. Some yeasts cannot (or prefer not to) undergo fermentation (i.e. non-fermentative yeast strains), while others may only utilize fermentation pathways in the presence or absence of certain sugars. Yeasts such as Zygosaccharomyces are highly effective at fermenting simple carbon sources, while some species (especially S. cerevisiae strains with diastatic properties) are able to metabolize complex long-chain sugars. The pattern of sugar assimilation and utilization displayed by individual beer-spoiling yeasts can therefore have an impact not only on ethanol yield, but in some instances on mouthfeel, due to the removal of dextrins from beer. The causative reasons for differences in sugar utilization are unknown but are likely to be evolutionarily driven, perhaps related to primary habitat or, in the case of brewing strains, artificial selection. Irrespective, the process of sugar assimilation is controlled both genetically and metabolically, allowing the cell to produce and regulate a series of enzymes involved in uptake and metabolic pathways. In Saccharomy- ces yeasts, glucose uptake principally occurs via facilitated diffusion with no expense of metabolic energy (ATP). However, in other types of yeast the transport mechanism varies; for example, Candida, Kluyveromyces and Pichia strains employ active transport as the primary mechanism for glucose uptake. In such systems ATP is required to expel hydrogen ions, which functions to create an electrochemical gradient allowing for the transport of sugars into the cell via proton symport. This is an effective method when glucose is in short supply since it allows for transport of sugar against a concentration gradient, but under optimum conditions the benefit of expending energy for the uptake of sugar is questionable. A potential reason for this divergence in uptake strategy is that for respiratory (i.e. non-fermentative) yeasts the ‘cost' is relatively insubstantial given that a theoretical yield of 38 ATP can be achieved from a single glucose molecule through aerobic metabolism. However, for yeasts that rely primarily on the fermentation pathway, yielding 2 ATP per glucose, this is a considerably higher proportion of cellular energy, hence other more cost-effective strategies are preferred (Griffin, 1994).

When yeasts are cultivated on sugar, a flux occurs through central carbon metabolism (Fig. 11.6). As alluded to above, yeasts have the potential to convert this sugar into energy through fermentation (glycolysis) or via respiration (Krebs cycle and the electron transport chain). Simplistically, it might be expected that under aerobic conditions yeast would preferentially utilize the respiratory pathway to produce ATP, carbon dioxide, and water. Likewise,

Yeast carbohydrate metabolism. Yeasts can employ two major pathways for ATP production from glucose

Figure 11.6 Yeast carbohydrate metabolism. Yeasts can employ two major pathways for ATP production from glucose: respiration and fermentation. Glycolysis forms the initial stage of each pathway, which involves the conversion of glucose to pyruvate with the net gain of two units of ATP. During fermentation, pyruvate is subsequently converted into ethanol. This process does not produce additional ATP but is favourable due to the recycling of NAD+, which regenerates the cellular pool of NADH for re-use in glycolysis. During respiration, pyruvate is oxidized to H2O and CO2 through the process of oxidative phosphorylation. This yields ATP and regenerates NAD+, but has an absolute requirement for oxygen. Ethanol (and acetic acid in some yeasts) are believed to be formed by yeast as part of a MAC strategy and can be recycled for ATP production if needed. This process yields less energy than the direct oxidation of pyruvate because the synthesis of Acetyl-CoA (through acetaldehyde for ethanol, but directly for acetic acid) requires ATP Pathways are often employed as a means of redox balance (NAD+/NADH ratio) and are partially regulated by oxygen (Pasteur and Custers effects), and the type and concentration of sugar (Kluyver and Crabtree effects).

under anaerobic conditions it might be expected that the fermentation pathway would be employed with acetaldehyde acting as the final electron acceptor to produce ATP, ethanol, and carbon dioxide. However, as described above, this is not always the case, with some yeasts preferentially exploiting one or other pathway (i.e. being fermentative or non-fermentative). In reality, many yeasts are respiro-fermentative and have evolved to utilize both pathways virtually simultaneously (Hagman and Piskur, 2015). The extent to which this happens is variable and the direction of carbon flux is influenced by a variety of parameters, including the presence/absence of oxygen, the presence/absence of sugars, as well as lesser effects based on the concentrations of inorganic phosphate, glycolytic intermediates, ammonium ions, and intracellular pH. Glucose in particular (although other sugars may be significant, albeit to a lesser extent) and oxygen act as metabolic triggers, causing pathways to be preferentially employed (Rolland etal., 2002). These regulatory mechanisms are summarized in Table 11.4 and include the Pasteur, Crabtree, Kluyver and Custers effects. With the exception of the Kluyver effect, adoption of these mechanisms is typically driven by the ability to metabolize faster under specific conditions, or as a consequence of the generation of specific end-products, as described below. Both of these strategies are likely to have evolved to provide strains with some form of competitive advantage in ‘natural' environments.

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